ISEV2025 Abstract Book.

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Nicolas Kuszla 1 , Lucile Alexandre 1 , Laura Fouassier 2 , Florence Gazeau 1 , Stéphanie Mangenot 1 1 Université Paris Cité, NABI Laboratory, CNRS UMR 8175, INSERM U1334, Paris, France; 2 Sorbonne Université, Centre de Recherche Saint‐Antoine, INSERM UMR S938, Paris, France Abstract unavailable

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Presenter: Kaitlyn E. Bunn Vanderbilt University, USA Introduction : T cells orchestrate diverse immune responses, from infections to autoimmunity and allergy. To effectively coordinate tissue inflammation, T cells communicate with other cells in the local microenvironment through cell contact mediated and secreted signals, including extracellular vesicles (EVs). EVs have emerged as mediators of communication between immune cells, but the functions of T cell EVs in communicating signals in tissue inflammation are incompletely understood. In this study, we investigate how T cell EVs contribute to inflammation through their interactions with eosinophils, critical effectors of allergic inflammation, in the pathologic context of allergic asthma. Methods : We leveraged T cell‐specific membrane tagging and single vesicle flow cytometry to quantify T cell EVs in the airways during induced allergic inflammation in mice. We further treated in vitro differentiated eosinophils with EVs isolated from primary T cell culture media and performed cell death assays and RNA sequencing. Additionally, we utilized small molecule inhibitor, blocking antibody, and enzyme immunoassay studies to identify the active cargo carried by T cell EVs. Finally, we administered T cell EVs into the airways of mice with allergic inflammation and assessed lung eosinophilia. Results : T cell EVs are increased >10‐fold in lung lining fluid during allergic inflammation in mouse models of asthma. In vitro, T cell EVs promote eosinophil survival and induce pro‐survival, activation, and metabolic gene programs in eosinophils. T cell EVs carry the surface cargo IL‐3, which inhibits apoptosis in eosinophils through the activation of Jak/Stat signalling. The effect of EV‐associated IL‐3 is more potent at lower doses and longer lasting than that of soluble IL‐3. This effect depends on T cell activation state, as only T cells activated through the T cell receptor secrete EVs carrying IL‐3 that support eosinophil survival. In vivo, instillation of activated T cell EVs into the airways is sufficient to prolong eosinophilia during an allergic reaction. Summary/Conclusion : This study supports a role for T cell EVs and their cytokine cargoes as a critical signalling axis in allergic lung inflammation. As cytokine‐based communication is ubiquitous during immune responses, the capacity of EVs to serve as vehicles for cytokines may be a fundamental mechanism for inflammation‐associated pathology.

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Presenter: Chun Liu The University of Queensland, Australia Introduction : Bone regeneration remains a critical focus in regenerative medicine, with an increasing demand for innovative approaches to enhance bone healing and repair. Among additive manufacturing techniques, 3D bioprinting stands out as a cutting‐edge technology for fabricating 3D biological constructs, enabling precise control over hierarchical architecture and shapes through the layer‐by‐layer addition of bioinks and biological materials. Recent advancements have highlighted the potential of combining 3D bioprinting and small extracellular vesicles (sEVs) as a promising ‘cell‐free’ strategy for regenerative medicine applications. Our approach combines gelatin methacryloyl (GelMA) with small extracellular vesicles (sEVs) derived from primary human osteoblasts (hOBs) and gingival fibroblasts (hGFs) to create a 3D bioprinted scaffold model aimed at improving osteogenic differentiation and bone regeneration in vitro and in vivo. Methods : sEVs were isolated from primary hOBs and hGFs using size exclusion chromatography (SEC) and characterized by cyto‐TEM, NTA, BCA, FTIR, and multiple cytokine FACS. The sEVs were encapsulated in 6% GelMA and 3D bioprinted via microextrusion. sEV release from the constructs was validated using DiO‐labelling, confocal imaging, NTA, and CD9 ELISA. hOB attachment and osteogenic differentiation were assessed on GelMA scaffolds, with and without hOBs‐sEVs and hGFs‐sEVs, using qRT‐PCR, ALP staining, and Alizarin Red staining. In vivo analysis utilized a 26‐rat skull defect model, where bone regeneration was evaluated through micro‐CT, H&E staining, and immunohistochemical markers: Bone Sialoprotein (BSP), Collagen Alpha‐1(I) Amino‐Propeptide (CAAP), Osteopontin, Periostin, and von Willebrand Factor (vWF). Results : hOBs cultured on GelMA/hOBs‐sEV constructs demonstrated significantly improved cell attachment and focal adhesion, with upregulation of genes associated with mechanotransduction (YAP, Rac1, TAZ, FAK, and RhoA) and osteogenic differentiation (BSP, OSX, OPN, ALP, and OCN) compared to GelMA/hGFs‐sEV and GelMA‐only groups. After 2 weeks in vitro, ALP activity and Alizarin Red staining were markedly increased in hOBs on GelMA/hOBs‐sEV constructs. In vivo, GelMA/hOBs‐sEV constructs showed enhanced bone regeneration, with H&E and immunohistochemical analyses (BSP, CAAP, vWF, osteopontin, and periostin) confirming increased osteogenic marker expression and bone matrix formation compared to controls. Summary/Conclusion : Our findings indicate that 3D bioprinted GelMA scaffolds embedded with hOB‐derived sEVs significantly promote osteogenic differentiation and bone regeneration in vitro and in vivo.

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Presenter: Bonhan Koo Yonsei University, Seoul, Republic of Korea Introduction : Colorectal cancer (CRC) is a leading cause of cancer‐related mortality worldwide, primarily due to late‐stage diagnosis. Early detection is crucial for improving survival rates, but current methods like colonoscopy are invasive and have limited patient adherence. Liquid biopsy through blood‐derived extracellular vesicle (EV) analysis offers a promising non‐invasive alternative. However, existing EV isolation techniques are time‐consuming and yield low purity, limiting their clinical use. Methods : We developed a novel system that integrates an EV isolation platform based on an amine‐functionalized zeolite and homobifunctional hydrazide with artificial intelligence (AI)‐driven analysis to enhance CRC diagnostics. This platform efficiently isolates and concentrates EVs from blood plasma within 15 min, enabling immediate extraction of EV‐derived miRNAs and circRNAs without the need for separate lysis steps required by traditional methods. From an initial pool of 11 miRNAs and 3 circRNAs, four miRNAs (miR‐23a‐3p, miR‐92a‐3p, miR‐125a‐3p, and miR‐150‐5p) were identified and combined with the conventional marker carcinoembryonic antigen (CEA). AI was employed to evaluate 31 biomarker combinations to determine the optimal set for CRC diagnosis. Results : The optimal biomarker combination—miR‐23a‐3p, miR‐92a‐3p, miR‐150‐5p, and CEA—achieved an area under the curve (AUC) of 0.9861 for overall CRC detection, with a sensitivity of 95.83% and specificity of 100%. This performance significantly surpassed traditional CEA testing and the use of individual markers. The system demonstrated exceptional diagnostic accuracy across various CRC stages. For early‐stage CRC (stages 0–2), it identified optimal biomarker sets achieving an AUC of 0.9861, with a sensitivity of 91.67% and specificity of 100%. Specifically, for stages 0–1, certain biomarker sets achieved perfect diagnostic performance with an AUC of 1.0. In stage 2, the system achieved an AUC of 0.9722, with a sensitivity of 83.33% and specificity of 100%. Summary/Conclusion : Our system provides a rapid, reliable, and non‐invasive tool for CRC diagnostics, enhancing early detection through AI‐driven biomarker combination analysis. By integrating an efficient EV isolation platform with deep learning, this system offers substantial clinical potential for improving CRC outcomes.

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Federico Vannuccini 1 , Yari Ciani 1 , Vera Mugoni 1 , Orsetta Quaini 1 , Caterina Nardella 1 , Francesco Orlando 1 , Veronica Weber 1 , Alessia Marinelli 1 , Irene Casanova‐Salas 2,3 , Fabio Pessina 4 , Alessandro Vannini 4 , Ugo De Giorgi 5 , Orazio Caffo 6 , Consuelo Buttigliero 7 , Umberto Basso 8 , Joaquin Mateo 2,3 , Gerhardt Attard 9,10 , Francesca Demichelis 1 1 Computational and Functional Oncology Laboratory; University of Trento, Trento, Italy; 2 Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain; 3 Vall d'Hebron University Hospital, Barcelona, Spain; 4 Structural biology research centre, Human Technopole, Milan, Italy; 5 Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori”‐ IRST, Meldola, Italy; 6 Department of Medical Oncology, Santa Chiara Hospital Trento, Trento, Italy; 7 Department of Oncology, University of Torino, Torino, Italy; 8 IOV‐Istituto Oncologico Veneto, Padova, Italy; 9 Department of Medical Oncology, University College London Hospitals, London, UK; 10 UCL Cancer Institute, University College London, London, UK Introduction : Liquid biopsies offer a minimally invasive complement to tissue biopsies for monitoring cancer by capturing multiple molecular components circulating in body fluids, including cell‐free (cfDNA) and extracellular vesicles (EVs). Plasma‐derived coding and non‐coding EV‐ associated RNAs (EV‐RNA) offer insights into EV cellular origin, informing about disease status and ongoing cellular processes. Methods : In the framework of the multi‐institutional research program PRIME for the development of multi‐modal liquid‐biopsy assays for prostate cancer, we characterized plasma‐derived EVs and the EV‐total RNA cargo from longitudinal samples from 54 metastatic castration‐resistant prostate cancer (mCRPC) patients treated with an Androgen Receptor signalling inhibitor. Plasma samples were processed using ONCE, an in‐house developed approach for isolating EVs and cfDNA from a single plasma aliquot. EVs were isolated through ultracentrifugation, RNA libraries were obtained using the Takara Bio SMARTer smRNA‐Seq kit and then sequenced on NovaSeq 6000 with an SR150 protocol. cfDNA was profiled using the targeted PCF_SELECT assay. Results : We identified differences in EV physical properties and RNA cargo when stratifying patients based on prognosis. Worse prognosis patient plasmas contain larger EVs than better prognosis patients and healthy donors (HD; median diameter of 189, 158, and 119 nm, respectively) and exhibit stronger evidence of prostate‐related transcriptional signals. EV‐RNA cargo deconvolution indicates i) enrichment for epithelial cells and prostate luminal KLK3+ cells signal in patients versus HDs and ii) marked differences in immune cells contribution based on patients’ prognosis. Specifically, polarized Macrophages and prostate‐related EV components are associated with prognosis ( p value < 0.0001 for both) and are independent prognostic factors when accounting for circulating tumour DNA signal. Coherent trends are observed in a second mCRPC cohort ( n = 21). Summary/Conclusion : EV‐RNA profiling has the potential to contribute to mCRPC prognostic assessments by revealing tumour and immune cell components associated with patient prognosis. Funding : Accelerator Award 20218 funded by Cancer Research UK (A26822) and Fondazione AIRC per la ricerca sul cancro ETS (22792).

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Presenter: Nadin Bedikyan Koç University, Istanbul, Turkey Introduction : Exosomes are vesicles that vary in size from 30 to 100 nm and contribute significantly to the progress of cancer by including a large amount of released cell DNA and RNA. The biodistribution of tumour‐derived exosomes are critical for understanding their role in cancer progression and metastasis. Exosomes, are nano‐sized vesicles secreted by tumour cells, carrying bioactive molecules like proteins, lipids, and so forth, which can modulate the tumour microenvironment and influence distant organs. This study investigates the whole‐body biodistribution of fluorescent labelled tumour‐derived exosomes following intravenous injection in mice, leverageing advanced imaging techniques for comprehensive analysis. First, appropriate amounts of fluorescently stained MDA‐MB‐231 (breast cancer cell line) and U87 (GB cancer cell line) tumour‐derived exosomes were injected intravenously into C57BL6 mice. The injected tumour‐derived exosomes will then be used for imaging with IVIS. In our in vivo experiments, non‐toxic exosomes obtained from HEK293T will be used as a control, and MDA‐MB‐231 and U87 will be used, and the organs in which they accumulate their biodistribution will be compared, and micrometastasis will be examined in our future experiments. This comparison may provide insight into how different tumour‐derived exosomes contribute to organ‐specific metastasis and provide clues for targeted therapy, as well as the opportunity to examine in which organs non‐toxic HEK‐derived exosomes and tumour‐derived exosomes accumulate. Methods : Cell culture experiment exosome isolation exosome labelling with Alexa Fluor 647 exosome characterization, IVIS imagaing, and analysis of the biodistributıon. Results : In the study, successful isolation of non‐toxic exosomes from HEK cells was achieved. The isolation method resulted in high efficiency while preserving the biological activity of exosomes. The results obtained with the IVIS imaging system show that injected exosomes significantly The images revealed how the distribution of exosomes in different organs changed over time, which is important for understanding their behaviour in vivo and accumulation in certain organs, especially in organs such as the liver, spleen and lung. This suggests that exosomes have organotropic properties. Summary/Conclusion : our study will investigate whether injected tumour‐derived exosomes promote the formation of secondary tumours or affect the tumour microenvironment in specific organs, which will be the subject of subsequent research in vivo after biodistribution is observed.

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Presenter: Maria Pia Savoca Nottingham Trent University, Nottingham, UK Abstract unavailable

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Presenter: Shue Jin West China Hospital of Sichuan University, China (People's Republic) Introduction : During biomaterials‐mediated foreign body response (FBR), an abnormality in any of the interaction between implant materials and host tissue cells, such as a disturbance in the temporal or spatial of the cells involved in FBR, can delay or aggravate the FBR, and ultimately cause implant failure. Fortunately, manipulating exosomes secreted by macrophages is a verified strategy to mitigate FBR during tissue remodelling. Methods : In this study, the janus scaffold was prepared by continuous electrospinning and the macrophage‐derived exosomes induced by IL‐4/IL‐13 (M2‐Exo) were extracted by ultracentrifugation. Subsequently, the M2‐Exo was immobilized on the janus scaffold via surface modification using a cationic compound polyethyleneimine. Finally, the immunomodulatory effects of M2‐Exo‐functionalized janus fibrous scaffolds on bone and muscle tissue remodelling were evaluated by rat muscle injury model and cranial bone defect model. Results : Western blot results verified that the combination of IL‐4 and IL‐13 stimulation of bone marrow‐derived macrophages (BMDMs) can obtain regenerative M2‐Exo with stable and high expression of IL‐4. In vitro cell experiments showed that M2‐Exo could be internalized by L6 myoblast, bone mesenchymal stem cells (BMSCs), human umbilical vein endothelial cells (HUVECs), and BMDMs to promote cell migration, tube formation, myogenic differentiation, osteogenic differentiation, and anti‐inflammatory macrophage polarization. Animal experiments results indicated that M2‐Exo‐immobilized janus scaffolds accelerated vascularized bone remodelling and muscle regeneration. Histological analysis showed that M2‐Exo could regulate FBR by alleviating the excessive fibrosis caused by scaffold implantation, promoting neovascularization, and activating more pro‐regenerative CD206+ macrophages during bone and muscle remodelling. Summary/Conclusion : M2‐Exo‐functionalized janus electrospun fibrous scaffolds have great application potential in bone and muscle remodelling. We believe that these findings will contribute to the design and new paradigm of scaffolds in orthopedics and maxillofacial surgery.

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Manon Jammes 1 , Monika Sypecka 2,3 , Yedizza Rautavaara 4 , Seyedmohammad Moosavizadeh 1,5 , Trung Bach 1 , Abbas Tabasi 1 , Jiemin Wang 1 , Neil Lagali 4 , Thomas Ritter 1,5 1 Regenerative Medicine Institute, School of Medicine, University of Galway, Galway, Ireland; 2 Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland; 3 Doctoral School of Translational Medicine, Centre of Postgraduate Medical Education, Warsaw, Poland; 4 Department of Biomedical and Clinical Sciences, Faculty of Medicine, Linköping University, Linköping, Sweden; 5 CURAM Centre for Research in Medical Devices, University of Galway, Galway, Ireland Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs; MSC‐EVs) recently emerged as a promising approach to address corneal diseases, leveraging the anti‐inflammatory, immunomodulatory, and pro‐regenerative properties of MSCs while bypassing the challenges inherent to cell therapy. Exploring the uptake and biodistribution of MSC‐EVs within the tissue typically involves using lipophilic membrane dyes, like PKH, inevitably increasing EV size and modifying their surface composition which potentially affect the interactions with their targets. In this context, using intraluminal dyes presents a suitable alternative to standard protocols to ensure the preservation of EV mechanism of action and the reliability of generated data. Methods : EVs were isolated from human bone marrow MSCs using size exclusion chromatography. Fresh EVs were labelled with CFSE or CellTrace Far Red (CTFR), and the stability of the fluorescence signal was investigated by flow cytometry after ‐80°C storage (three different timepoints) followed by 37°C incubation to mimic in vivo administration. Human corneal epithelial cells (CEpiCs) and keratocytes were treated with CFSE‐ or CTFR‐EVs at several concentrations and the EV uptake was followed by fluorescence live imaging over four days. Results : The EV size and morphology remained unchanged after labelling with both dyes. Although most of the fluorescence was preserved, CFSE‐EVs exhibited signal reduction after 37°C incubation, while CTFR‐EVs were impacted by −80°C storage. Given the natural strong green fluorescence background in cell cultures, CFSE‐EV uptake was more arduous to track in vitro than CTFR‐EVs. Summary/Conclusion : CTFR staining is an effective method to label EVs and track them in vitro, avoiding the structural alterations associated with PKH‐like dyes. EV tracking on corneal explants after CTFR‐EV topical or subconjunctival administration is currently investigated to ensure the translation of the method to in vivo studies.

Dna

Katharina Maria Richter 1,2* , Smiths S. Lueong 3,4,5,6* , Marcel Kemper 1,2 , Allegra Angenendt 1,2 , Uwe Hansen 7 , Georg Evers 1,2 , Michael Mohr 1,2 , Georg Lenz 1,2 , Balazs Hegedüs 5,6,8 , Martin Metzenmacher 5,6,9 , Martin Schuler 5,6,9 , Annalen Bleckmann 1,2* , Kerstin Menck 1,2* 1 University of Münster, Dept. of Medicine A, Münster, Germany; 2 University Hospital Münster, West German Cancer Center, Münster, Germany; 3 University Hospital Essen, Bridge Institute for Experimental Tumor Therapy, Essen, Germany; 4 University Hospital Essen, DKTK Division of Solid Tumor Translational Oncology, Essen, Germany; 5 West German Cancer Center, Essen, Germany; 6 University Duisburg‐Essen, Medical Faculty, Essen, Germany; 7 University of Münster, Institute of Musculoskeletal Medicine, Münster, Germany; 8 University Medicine Essen—Ruhrlandklinik, Dept. of Thoracic Surgery, Essen, Germany; 9 University Hospital Essen, Dept. of Medical Oncology, Essen, Germany *These authors contributed equally . Introduction : Liquid biopsies offer a minimally invasive method for detecting genetic alterations in cancer patients, with promising implications for personalized treatment. Traditionally, circulating tumour DNA (ctDNA) is analysed from cell‐free DNA (cfDNA) in plasma, but recent findings suggest that extracellular vesicles (EVs) could provide a more informative source of ctDNA. This study aimed to compare the effectiveness of EV‐DNA and cfDNA in detecting tumour mutations in plasma samples from patients with non‐small cell lung cancer (NSCLC). Methods : Small and large EVs (sEVs/lEVs) were isolated from NSCLC cell culture supernatants or patient‐derived plasma samples (with informed consent) using differential ultracentrifugation. EV subpopulations were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting and/or mass spectrometry, highlighting differences in size and cargo. DNA was extracted with commercially available kits, isolating EV‐DNA from cell culture supernatant and, in the case of plasma, isolating both EV‐DNA and paired cfDNA from a single plasma sample. DNA quality and quantity were assessed with fluorometry and electrophoresis, and tumour mutations were detected by next‐generation sequencing (NGS). Results : Comparative analysis showed that lEVs contained significantly more DNA than sEVs and differed in their proteomic cargo. Both EV populations carried a significant amount of DNA‐binding proteins, albeit with quantitative differences regarding the specific enrichment of histones. Treatment of lEVs with DNase I and proteinase K resulted in reduced lEV‐DNA levels, indicating that the DNA may be partially bound to protein complexes on the vesicle surface. In NSCLC plasma samples ( n = 44), total DNA yield was lower for EV‐DNA than cfDNA. However, lEV‐DNA displayed larger fragment sizes than both sEV‐DNA and cfDNA, suggesting a unique DNA profile. The larger fragment size may enhance mutation detection, increasing the diagnostic potential of EV‐DNA. Indeed, NGS analysis demonstrated that EV‐DNA yielded higher allelic frequencies and a relatively greater number of oncogenic variants compared to cfDNA, suggesting a higher sensitivity for mutation detection. Summary/Conclusion : This study highlights lEVs as a promising source of ctDNA for mutation analysis in liquid biopsies. Further studies correlating these findings with primary tumour mutation profiles and clinical data will help define the role of EV‐DNA in molecular diagnostics, potentially improving tumour mutation detection and monitoring.

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Pratiksha Mahadik 1 , Sejal Patwardhan 1,2 1 Advanced Centre for Treatment Research Education in Cancer (ACTREC) Tata Memorial Centre, India; 2 Homi Bhabha National Institute, India Introduction : Introduction: Extracellular matrix (ECM) stiffening owing to excess deposition and crosslinking of collagen fibres is instrumental in breast cancer progression. ECM stiffness influences tumour behaviour and fate through direct and indirect effects. Recent reports highlight the profound role of ECM‐stiffness‐tuned exosomes in promoting tumour growth and invasion. However, the exact mechanism of ECM‐stiffness‐induced biogenesis and release of extracellular vesicles (EVs) remains largely elusive. Methods : Breast cancer cells (MCF‐7 and MDA‐MB‐231) were cultured on stiffness‐tunable hydrogels of stiffness mimicking normal mammary stroma (soft ECM) and metastatic breast tumour (stiff ECM). EVs were isolated from culture supernatant by a kit‐based method and characterised by western blotting, scanning electron microscopy, and nanoparticle tracking analyser (NTA). The number and pattern of multivesicular bodies (MVBs) in cells were studied using transmission electron microscopy (TEM) and immunofluorescence for CD63, lysobisphosphatidic acid (LBPA). Specific inhibitors or shRNA, co‐immunoprecipation and proximity ligation assays were employed to gain mechanistic insights. Results : The quality and purity of EV preparation were confirmed by the enrichment of EV‐specific markers (CD9, flotillin‐1) and the absence of cytosolic markers (cytochrome‐c, calreticulin) in both soft and stiff matrices. EV samples from both the substrate stiffness showed typical cup‐shaped morphology and size ranging from 70 to 130 nm. Interestingly, cells cultured on stiff matrices exhibited higher secretion of EVs as probed by NTA. Cells grown on stiff‐ECM displayed excess CD63 and LBPA puncta, suggesting a boost in EV biogenesis. TEM analysis revealed alterations in MVBs with increased intraluminal vesicles (ILVs) per MVB in stiff ECM conditions, which was abolished upon blebbistatin treatment. We mapped the role of various ESCRT pathways and associated proteins (e.g., Alix, STAM1, TSG101, VPS4, etc.), among which only TSG101 was found to be differentially expressed. Further investigation revealed the role of HSP90, since stiff ECM harboured high TSG101‐HSP90 interactions, and cells treated with 17AAG, a specific inhibitor of HSP90 abrogated ECM‐stiffness‐induced TSG101 levels with a concurrent decrease in LBPA spots, ILVs per MVBs and consequent reduction in EV secretion. Summary/Conclusion : Taken together, our findings establish a link between mechanotransduction and EV secretion, which unveils the novel role of the TSG101‐HSP90 axis in ECM stiffness‐driven biogenesis of EVs.

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Edina Bugyik 1 , Gábor Valcz 2,3 , Júlia Opra 1 , Nóra Fekete 1 , András Försönits 1 , Krisztina V. Vukman 1 , Melinda Rezeli 4,5 , Sándor Paku 6 , Katalin Dezső 6 , Edit I Buzás 1,2,7 1 Semmelweis University, Institute of Genetics, Cell‐ and Immunobiology, Budapest, Hungary, 2 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Budapest, Hungary, 3 Image Analysis Group, 3DHISTECH Ltd., Budapest, Hungary, 4 Department of Biomedical Engineering, Lund University, Lund, Sweden, 5 Swedish National Infrastructure for Biological Mass Spectrometry (BioMS), Lund University, Lund, Sweden, 6 Semmelweis University, I st Department of Pathology and Experimental Cancer Research, Budapest, Hungary, 7 HCEMM‐SU Extracellular Vesicle Research Group, Budapest, Hungary. Introduction : Tumour anorexia‐cachexia syndrome is a multifaceted condition characterized by decreased skeletal muscle mass, generalized weakness, diminished quality of life, and poor prognosis. It occurs in 50‐80% of cancer patients and is estimated to be responsible for 20‐30% of all cancer‐related deaths. Although cardiac atrophy is a significant component of this syndrome, early detection of cardiac changes is still a clinical unmet need due to the focus on treating the underlying disease and limitations in diagnostic methods. Methods : The C26 colorectal adenocarcinoma primary tumour model was used to develop tumour anorexia‐cachexia syndrome in vivo. The weight, tumour size, heart weight and tibia length of the mice were recorded upon termination. Large and small extracellular vesicles (lEVs and sEVs) were isolated from the blood samples using differential centrifugation alone or combined with size‐exclusion chromatography and were characterized using standard EV methodologies following the MISEV2023 guidelines. Wheat Germ Agglutinin (WGA‐Alexa‐488) labelling was applied for histology in order to confirm the presence of cardiac atrophy. Mass spectrometry was performed from the hearts, PFP and EV samples of healthy and diseased animals. Results : There was a significant difference in the heart weight and body weight of control and tumour bearing groups. We confirmed a decrease in the size of cardiomyocytes in the tumour group. A total of 4993 proteins were detected during the proteomic analysis. We determined the differentially expressed proteins between the control and tumour groups. These differentially expressed proteins are mainly involved in acute phase reaction, immunity, metabolic and cell adhesion processes, apoptosis and cytoskeletal organization. The differential presence of potential heart‐derived proteins was detectable (Myh6, Myh7, Tnni3). Summary/Conclusion : These findings provide valuable insights into the molecular changes associated with tumour‐induced cardiac atrophy. Further exploration of these proteins may lead to the development of a specific biomarker panel for early detection and intervention. Funding : This research was funded by NVKP_16‐1‐2016‐0004, VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003, 2019‐2.1.7‐ERA‐NET‐2021‐00015. EU's Horizon 2020 No. 739593.

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Presenter: Irena Koutna St. Anne's University Hospital Brno, Czech Republic Introduction : Extracellular vesicles (EVs), particularly exosomes, have garnered significant interest in recent years due to their potential as therapeutic agents. Mesenchymal stem cells (MSCs), with their regenerative and immunomodulatory properties, are a promising source of EVs for therapeutic applications. Among various sources of MSCs, the umbilical cord (UC) offers a non‐invasive and rich resource for isolation. UC‐MSCs secrete EVs that have been shown to promote tissue repair, modulate immune responses, and enhance cell survival and could be utilized for treatment of neurodegenerative disorders. Methods : 1.Isolation of UC‐MSCs: Human UCs were obtained from healthy full‐term births with informed consent. UC‐MSCs were isolated using enzymatic digestion followed by plating in MSC growth media. Cells were expanded until reaching confluence and characterized by flow cytometry for MSC surface markers, immunosuppression properties and tri‐lineage differentiation. 2.EV Isolation: EVs were isolated from the conditioned media of UC‐MSCs which was first centrifuged at 300 × g to remove cells and debris. The supernatant was then subjected to TFF followed by SEC. 3.Characterization of EVs: The size distribution and concentration of isolated EVs were determined by nanoparticle tracking analysis (NTA). Cryo‐electron microscopy (CEM) was used to observe the morphology of the EVs. Western blot analysis was performed to detect exosome markers. 4.Functional Assays: The biological activity of UC‐MSC‐derived EVs was assessed through cell proliferation assays (MTT assay), migration assays (wound healing assay), and immunomodulatory effects on immune cells (T cell suppression assay). Results : The UC‐MSCs cultured in a GMP EMA‐approved scheme is an excellent source for EVs because from one UC we are able to harvest up to 10 L of conditioned medium. Summary/Conclusion : UC‐MSCs are a promising source of biologically active vesicles with significant potential for therapeutic applications for neurodegenerative disorders. Funding : Supported by project nr. LX22NPO5107 (MEYS): Financed by European Union—Next Generation EU. Supported by the European Regional Development Fund—project CZECRIN_PHARMTECH, no. CZ.02.01.01/00/23_015/0008194. Created in collabouration with MED MUNI through the CZECRIN project (LM2023049), supported by the national budget through MEYS.

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Angelika Sysak 1,3 , Dominika Kozakiewicz 1 , Dagmar Šrůtková 2 , Martin Schwarzer 2 , Sabina Górska 1 1 Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Poland. 2 Institute of Microbiology, Czech Academy of Sciences, Czech Republic. 3 Department of Pharmacology and Toxicology, Wroclaw University of Environmental and Life Sciences, Poland. Introduction : Allergic diseases are becoming increasingly common across the globe, making them the most prevalent chronic illnesses in children. Probiotic bacteria represent a highly promising approach for primary prevention, with significant research confirming their potential. Several recent studies indicate that probiotics might help lower the risk of allergic conditions by modifying the natural microbiota, affecting infants' immune function. In addition to whole bacteria, researchers are exploring their components and bioactive molecules to develop new preventive and therapeutic strategies. Recently, there has been growing interest in extracellular vesicles (EVs) released by bacterial cells. This study aimed to investigate the physicochemical characteristics and immunomodulatory effects of Bifidobacterium adolescentis CCDM373 (Bad 373) derived EVs. Methods : Anaerobic bacteria cultures of B. adolescentis CCDM373 were used for EVs isolation. After centrifugation, supernatant filtration, and concentration with the pressure‐based Amicon device, EVs were isolated by ultracentrifugation (3 h at 4°C and 150,000 × g ). Obtained EVs were dissolved in 500 µL of sterile HEPES in 0.9% NaCl and purified on the Izon size‐exclusion column. Properties of EVs were investigated by Dynamic Light Scattering (DLS) method using Zetasizer (Malvern Panalytical), and transmission electron microscope (TEM, JEOL JEM F‐200). Three size‐based fractions of EVs were further investigated on human embryonal kidney cells (HEK293), stably transfected with TLR receptors, mouse bone marrow‐derived dendritic cells (BMDCs), and mouse splenocytes to determine their immunomodulatory properties. The influence of EVs on the production of cytokines was determined by the ELISA method. Flow cytometry analysis was used to investigate the effects of EVs on the expression of co‐stimulatory molecules (MHCII, CD40, CD80, CD86) in BMDCs. Results : DLS analysis revealed the batch‐to‐batch similarity of isolated EVs. In addition, they stimulated the TLR2 receptors in HEK293 cells, caused increased expression of co‐stimulatory molecules in BMDCs, and modulated the production of cytokines in mouse splenocytes. Summary/Conclusion : The results obtained allowed us, for the first time to our knowledge, to carry out physicochemical characterisation of Bad 373‐derived EVs. Furthermore, their immunomodulatory properties confirmed their valuable potential in the search for new preventive and therapeutic approaches for allergic diseases. Funding : This study was supported by the National Science Centre of Poland (UMO‐2021/43/I/NZ7/00693).

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Rachel Butler 1 , Yunyue Zhang 2 , Driton Vllasaliu 2 , Alan Goddard 1 1 Aston University, UK. 2 Kings College London, UK Introduction : Oral delivery of biologics, like nucleic acid therapies, could transform treatment through non‐invasiveness and ease of administration, making it more cost‐effective and accessible. However, progress remains limited by enzymatic degradation and poor gastrointestinal absorption. Current platforms, such as lipid nanoparticles and liposomes, suffer from low encapsulation efficiency and instability. Extracellular vesicles (EVs) are promising alternatives due to their ability to cross biological barriers. Among these, milk‐derived EVs (mEVs) demonstrate superior stability and transport across gut epithelium compared to other EVs and synthetic systems. This work aims to uncover the mechanisms enabling mEVs' efficient gut epithelial transport. Methods : mEVs were isolated from raw bovine milk according to MISEV2023 guidelines using ultracentrifugation and size‐exclusion chromatography, with a chymosin‐incubation step to eliminate casein micelles and improve vesicle purity. mEVs were harvested from the apical and basal sides of an in vitro intestinal epithelium model (Caco‐2 cell monolayer). Predicting the mEV surface's role in transport, we isolated mEV membranes to characterise membrane‐associated proteins. Mass spectrometry identified key components of the mEV lipidome and proteome that may be involved in intestinal permeability. Results : Comparative analysis of permeating ‘basal’ and non‐permeating ‘apical’ mEV populations revealed distinct molecular features associated with enhanced gut permeability. Basal mEVs were enriched in certain fatty acids and acyl chains, suggesting altered mechanical membrane properties could promote transcytosis. Proteomic analysis showed a depletion of membrane proteins in basal mEVs, indicating that overall membrane characteristics, rather than specific ligand‐receptor interactions, may govern gastrointestinal stability and transport across the intestinal epithelium. Furthermore, whilst mEVs are thought to derive from a diverse mixture of cell types, basal mEVs were depleted in immune markers, hinting that basal mEVs may originate from a distinct population of non‐immune cells, such as mammary epithelial cells. Summary/Conclusion : By identifying key intestinal‐permeating components, we provide insight for engineering gut‐permeable EVs. Future work will optimise a method of enriching the permeating mEV population by affinity purification to increase the efficiency of drug delivery for future engineered mEV therapies. This study demonstrates the potential of mEVs as scalable and cost‐effective platforms, paving the way for next‐generation oral delivery of biologics. Funding : UKRI Engineering Biology Mission Award (BB/Y008065/1)

Key

Presenter: Zehong Peng Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, China (People's Republic) Introduction : The PI‐RADS scoring system is based on magnetic resonance to evaluate prostate cancer (PCa) lesions, which are divided into grades 1‐5. In clinical practice, prostate biopsy is recommended for patients with PI‐RADS ≥ 3 lesions, but the positive rate is not satisfactory, with more than half of patients undergoing unnecessary biopsy. Therefore, developing a non‐invasive method for accurately identifying PCa from patients with PI‐RADS ≥ 3 lesions is crucial. We aim to achieve accurate diagnosis of PCa patients with PI‐RADS ≥ 3 lesions by screening key metabolites from serum extracellular vesicles (EVs) metabolic fingerprints (MFs). Methods : We used size exclusion chromatography to separate EVs and EV‐free serum from 120 patients and obtained their MFs using a ferric nanoparticle‐assisted laser desorption/ionization mass spectrometry detection platform. Subsequently, we compared the diagnostic value of EVs and EVs‐free serum MFs while screening key metabolites using machine learning and differential analysis. Furthermore, we combined key metabolites in EVs with PSA to construct a multi‐modal diagnostic model. Finally, we compared the different roles of EVs and EVs‐free serum in Gleason Score (GS ≥ 8) PCa‐related metabolic pathways through pathway enrichment analysis. Results : We successfully constructed MFs of EVs and EVs‐free serum. Then we identified 11 key metabolites from EVs and 2 from EVs‐free serum. We found that the diagnostic ability of EV key metabolites was significantly better than that of EVs‐free serum, with area under the curve (AUC) values of 0.74 and 0.57. By further combining key metabolites in EVs with PSA, we obtained an AUC value of 0.85 for both the training and testing groups, further enhancing the diagnostic value of EVs; Finally, we found that EVs and EVs‐free serum mainly regulated five independent metabolic pathways in GS ≥ 8 PCa, suggesting that they may play different roles in related metabolic processes. Summary/Conclusion : In this study, we achieved non‐invasive and accurate diagnosis of PCa patients with PI‐RADS ≥ 3 lesions. Among them, EVs have a better diagnostic ability, indicating their potential application in wider PCa populations in the future. Funding : This work was financially supported by the National Natural Science Foundation of China (82103485, 82227801).

New

Angelos Vrynas 1 , Salime Bazban‐Shotorbani 1 , Sara Arfan 2 , Karishma Satia 3,4,5 , Brian Cunningham 1 , Gauhar Sagindykova 1 , Mymuna Ashna 1 , Aoyu Zhang 1 , Diana Visan 1 , Aisher Chen 1 , Teige Matthews‐Palmer 6 , Mathew Carter 3,4,7 , Fiona Blackhall 4,7 , Kathryn L. Simpson 3,4,5 , Caroline Dive 3,4,5 , Paul Huang 2, , Sam H. Au 1 1 Imperial College London, United Kingdom. 2 The Institute of Cancer Research,, United Kingdom. 3 University of Manchester, United Kingdom. 4 Cancer Research UK Lung Cancer Centre of Excellence, United Kingdom. 5 SCLC Biology Group, Cancer Research UK Manchester Institute, United Kingdom. 6 Division of Structural Biology The Institute of Cancer Research, United Kingdom. 7 Christie Hospital National Health Service (NHS) Foundation Trust, United Kingdom. Introduction : It is well‐established that primary tumours release extracellular vesicles (EVs) modifying the microenvironment of distant organs to facilitate later colonization by circulating tumour cells (CTCs). However, it remains poorly understood if entrapped CTCs can directly alter their local microenvironment. In this work, we have identified and characterized a new class of EV released by patient small cell lung cancer CTCs in response to shear stress in capillary‐sized bifurcations that influences critical cellular regulators of metastasis. Methods : We emulated in vivo geometries and physiological pressures in engineered microfluidic capillary‐sized bifurcated microchannels. Transiting primary CTCs, CTC‐derived xenografts and six cancer cell lines were observed to shed large nuclei‐free fragments by live imaging. Purified fragments were characterized via immunofluorescence staining and proteomic analyses via mass‐spectrometry. Fragments were co‐cultured with human umbilical vein endothelial cells (HUVECs) or THP‐1 monocytes (1:3 ratio). Phenotypic changes were validated via immunofluorescence and PCR after 24hr exposure. Endothelium permeability by tumour cells and microparticles on transwell and microfluidic 3D endothelial networks was fluorescently quantified. Results : All tested cells shed large extracellular vesicles (LEVs) (1.17‐11.32 µm, mean:4.9±1.9 µm) with up to 90% frequencies. LEV biogenesis depended on fluid shear stress and constricted transit through bifurcations, distinguishing them from other EVs like large oncosomes (LOs). LEVs matched the MISEV2018 requirements for EVs validation including transmembrane and cytosolic proteins, nuclei absence and visual characterization by two complementary techniques. Our proteomics approach identified the presence of 3382 distinct proteins within LEVs samples, about 8‐fold higher compared to distinct proteins in LOs. LEVs were ingested by monocytes and HUVECs and elicited diverse signalling relevant to pre‐metastatic niches. LEVs polarized monocytes towards CD206+ M2 tumour‐associated macrophage lineages. LEVs also disrupted vascular endothelial junctions and increased permeability of tumour cells and microparticles. Finally, LEVs activated HUVECs promoting co‐interactions with monocytes. Summary/Conclusion : Concluding, we identified and characterized a new class of LEVs whose biogenesis is driven by tumour cells traversing capillary bifurcations under shear stress, which we named ‘shearosomes’. Their local generation in microvasculature at CTCs entrapment regions, their vast proteome and co‐interactions with immune and endothelial cells may serve to explain how CTCs form pre‐metastatic niches that enable their successful metastasis.

Rab

Presenter: Luke T. Nelson University of Hawaii, USA Introduction : In response to exercise, skeletal muscle (SkM) cells are known to enhance the secretion of small (∼50‐150 nm) extracellular vesicles (EVs). These EVs facilitate inter‐organ communication by carrying specialized molecular cargo that likely mediate the metabolic benefits of physical activity. However, the mechanisms underlying contraction‐induced EV secretion from SkM remain poorly understood. We investigated the role of Rab27a, a Rab GTPase, in regulating EV release using exercise‐mimetic treatments on cultured SkM cells in vitro. Methods : Immunofluorescence and biochemical assays were employed to study the localization and molecular associations of EV‐containing intracellular vesicles (known as multivesicular bodies (MVBs)) during this process. We analysed intracellular protein‐protein associations using proximity ligation assays (PLA) in fixed SkM cells. For characterization of EVs produced by SkM, we utilized Tunable Resistive Pulse Sensing (TRPS), Western blotting, and Transmission Electron Microscopy (TEM). Results : Our findings reveal that Rab27a plays a role in the translocation of MVBs to the plasma membrane in response to contraction‐signalling. Additionally, we identified the octameric exocyst protein complex, which is responsible for vesicle tethering prior to membrane fusion, as part of the intracellular trafficking mechanism that facilitates critical steps for EV secretion. Summary/Conclusion : This study highlights Rab27a and the exocyst complex as key regulators of contraction‐induced EV release in SkM. Our findings provide insights into the molecular basis of SkM‐derived EV secretion and its potential role in mediating interorgan signalling during exercise. These insights could pave the way for further exploration of SkM EVs as therapeutic targets. Funding : NIH OLA Hawaii #5U54MD007601

Red

Presenter: Elena Cecotti University of Torino, Turin, Italy Introduction : Liver fibrosis is a wound‐healing response that results from an imbalance in extracellular matrix (ECM) synthesis and degradation following liver injury. Several studies evaluated the anti‐fibrotic capacity of extracellular vesicles (EVs) derived from different sources and highlighted the correlation with the EV‐miRNA content. Red blood cell‐derived EVs (RBC‐EVs) represent a natural delivery system with several advantages over EVs from other sources. They are biocompatible, obtained in a cost‐effective way and with low horizontal gene transfer. In this study, we evaluate the effects of RBC‐EVs loaded with specific anti‐fibrotic miRNA mimics in bi‐dimensional (2D) and three‐dimensional (3D) models of hepatic fibrosis. Methods : RBCs were obtained from healthy donors, and EVs were purified by ultracentrifugation and characterized in accordance with ISEV guidelines through NTA, transmission electron microscopy, flow cytometry and western blot. RBC‐EVs were loaded by electroporation with anti‐fibrotic miRNA mimics and their therapeutic effect was tested on TGF‐β1 (10 ng/mL)‐activated human hepatic stellate cells (LX‐2) and in a 3D model of liver spheroids. Different RBC‐EV preparations were obtained by loading a single anti‐fibrotic miRNA mimic, and the EV treatment was performed with a single EV dose (5000 EV/cell, 10,000 EV/cell, 20,000 EV/cell) or with a combination of two RBC‐EV preparations loaded with different miRNA mimics. By real‐time PCR and western blotting, we evaluated the expression of the pro‐fibrotic markers alpha‐SMA and COL1A1. Results : By real‐time PCR, we assessed the presence of anti‐fibrotic miRNA mimics inside electroporated RBC‐EVs, confirming the efficacy of the loading protocol. In activated LX‐2 cells and in 3D liver spheroids, engineered RBC‐EVs significantly downregulated the expression of the pro‐fibrotic markers alpha‐SMA and COL1A1 both at RNA and protein levels. In particular, the treatment with specific combinations of RBC‐EVs seems to augment their anti‐fibrotic potential with respect to the treatment with the single EV preparation. Summary/Conclusion : Engineered RBC‐EVs represent a natural RNA‐delivery vehicle that can be directly transferred to the target cells, modulating their activated pro‐fibrotic phenotype and opening new perspectives for future clinical application. Funding : This study was funded by the European Union—Next Generation EU, Mission 4, Component 2, Investment line 1.4, CUP B93D21010860004.

Rna

Plenary Presenter: Jan Lötvall University of Gothenburg, Sweden

Sex

Presenter: Changrim Lee Harvard Medical School, Boston, Massachusetts, USA Introduction : Specialized pro‐resolving mediators (SPMs) and proinflammatory mediators are endogenous small molecules playing an essential role in health and disease. Biosynthesis and secretion of mediators from polymorphonuclear leukocytes are well understood, but those from epithelial cells still need to be studied. Methods : Using histamine‐mediated allergic inflammation in ex vivo cultures of human conjunctival goblet cells (HCjGCs) as a disease model of allergic conjunctivitis, we performed ELISA‐based quantification on seven representative lipid mediators (prostaglandin E2 (PGE2), leukotriene B4 (LTB4), lipoxin A4 (LXA4), resolvin E1 (RvE1), resolvin D1 (RvD1), maresin 1 (MaR1), and protectin D1 (PD1)) in three types of samples (extracellular vesicles (EVs), EV‐free media, and cell lysates) collected under three conditions (homeostasis, mild, and severe allergic inflammation). Then the results were compared between and within male HCjGCs (‘males’) and females. Results : Females biosynthesized and secreted higher levels of mediators than males in all three types of samples in all three conditions. Females had more than 50% of the proinflammatory mediators (PGE2 and LTB4) and SPMs (LXA4, RvE1, RvD1, MaR1, and PD1) in the extracellular space. This was also the case for SPMs in males; however, more than 50% of the proinflammatory mediators were retained in cells of males. In all samples, PD1 level was notably higher than the sum of the other four SPMs (LXA4, RvE1, RvD1, and MaR1). Summary/Conclusion : We conclude that the profile of intra‐ and extra‐cellular lipid mediators of HCjGCs is sex dependent. Lower levels of synthesis and secretion of lipid mediators in males than females during homeostasis and allergic conditions may be linked to the male predisposition to severe types of allergic conjunctivitis. Funding : This work is supported by National Institutes of Health (NIH) R01EY029789, EY029789 , T32EY007145, F32EY035529, P30EY003790; Korean‐American Scientists and Engineers Association (KSEA) Young Investigator Grant (C.L.).

The

Agustin Enciso‐Martinez 1,2 , Caroline Løppke 3 , Joyce J. Koene †1 , Jade Ebbelaar †2 , Meike van der Geest 1 , Mandy Los 2 , Robert Dagil 3 , Tobias Gustavsson 3,4 , Nick van Es 2,6 , Ton van Leeuwen 2 , Roman I. Koning 5 , Ali Salanti 3 , Edwin van der Pol 2 , Rienk Nieuwland 2 , Mette Ø. Agerbæk 3,7 , Peter ten Dijke 1 1 Oncode Institute and Leiden University Medical Center, Leiden, The Netherlands; 2 Amsterdam University Medical Center, Amsterdam, The Netherlands; 3 University of Copenhagen, Copenhagen, Denmark; 4 VAR2 Pharmaceuticals ApS, Copenhagen, Denmark; 5 Leiden University Medical Center, Leiden, The Netherlands, 6 Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands; 7 VarCT Diagnostics, Copenhagen, Denmark Edwin van der Pol, Rienk Nieuwland, Mette Ø. Agerbæk, and Peter ten Dijke are co‐senior authors Introduction : Despite the promise of tumour‐derived extracellular vesicles (tdEVs) as cancer biomarkers, straightforward identification of tdEVs is difficult due to the lack of tumour‐specific markers. We demonstrate that tdEVs present on their surface proteoglycans displaying oncofoetal Chrondroitin Sulphate (ofCS), a malignancy‐associated glycosaminoglycan modification, which enables robust and specific identification of tdEVs. Using the VAR2CSA protein, which selectively binds to ofCS, we successfully identified tdEVs in blood plasma samples of pancreatic adenocarcinoma (PDAC) patients. Methods : The recombinant VAR2CSA protein (rVAR2) was fluorescently labelled, and used to detect tdEVs from cancer cell lines, tdEVs spiked into healthy plasma, and tdEVs in plasma from advanced‐stage PDAC patients. For validation, we used orthogonal single EV detection techniques, including calibrated flow cytometry and super‐resolution fluorescence microscopy. The staining specificity was validated using a non‐ofCS‐binding rVAR2 mutant, and enzymatic digestion of ofCS chains by chondroitinase treatment prior to staining. Results : We identified ofCS‐positive tdEVs in conditioned media from multiple cancer cell lines. Furthermore, we detected tdEVs spiked into healthy donor plasma, as well as ofCS‐positive tdEVs in plasma from PDAC patients. In contrast, only minimal rVAR2 levels were detectable in plasma samples from healthy controls. These findings were consistent across the different methods, and the ofCS specificity of the rVAR2 staining was validated using the rVAR2‐mutant controls and chondroitinase treatment, both of which led to a significant decrease in particle counts and fluorescence signal. Summary/Conclusion : Our study demonstrates that tdEVs display ofCS on their surface, allowing for selective targeting by the malarial VAR2CSA protein. This approach enables the detection of tdEVs in blood plasma, offering a promising strategy for liquid biopsy applications. Funding : A.E.M. acknowledges funding from Oncode Institute and the Dutch Cancer Society (KWF) grant 15079. A.E.M. and E.v.d.P. acknowledge funding from the Dutch Research Council (NWO), VIDI grant 19724. C.L. and M.Ø.A. were funded by the Innovation Fund Denmark (grant number 9090‐00024B).

Use

Julio Reinecke Practice Wehling & Partner, Düsseldorf, Düsseldorf, Germany Introduction : Customary medications for pain and inflammation obstruct physiologic healing responses and increase the risk of chronification. ACS contains a complex composition of mediators released upon whole blood incubation. Animal and human studies show that this autologous secretome improves acute pain and inflammation, kicks off tissue regeneration and counteracts the notorious chronifying effects of glucocorticoid and NSAID treatment. In addition to, or subsequent to, ACS, numerous techniques have surfaced that uniformLy claim to exploit physiological self‐healing mechanisms. Such include, but are not limited to, fat or bone marrow derived cell or cell fragments and platelet‐rich plasma. The multitude of preparations is as heterogeneous as are the clinical results. Allogenic mesenchymal stem cells as pharmaceutical products have also been developed. The unifying principle of such approaches is the fact that not the cells, but their releasates or secretomes are now believed to be the main active principle. As a therapeutic autologous secretome, ACS has a high standard of reproducibility, reflecting in consistently good clinical efficacy. Methods : ACS results from in vitro coagulation and incubation of whole patient blood at physiologic temperature for > 3 h employing a CE‐marked medical device. It can be routinely filtered by 0.22 µm, is routinely aliquoted and can be kept frozen for up to 12 months at −20°C or colder. ACS strategy does not aim for a purified single component but employs a complex mixture released by coagulating whole blood. Results : NTA (Zeta View) determined ACS particle counts regularly are between 10 9 and 10 13 per mL and sizes range from 30 to 200 nm. Composition of ACS secretome (proteins, EV, lipid mediators) differs significantly from baseline blood plasma. We summarize treatment options and results for ACS and illustrate feasible application routes. We present a graphic comparison of processing techniques for the autologous and allogenous preparations described above, demonstrating the simplicity and safety of the method. Summary/Conclusion : Compared to some other technologies ACS is simple and safe to generate. Treatment efficacy has been demonstrated in numerous studies. One strength is that the therapeutic mechanism is based on multiple mediators, including EV.

Why

Presenter: Brian S. Dobosh Emory University, USA Introduction : EVs reflect the state of the cell they originate from and exert significant effects on recipient cells due to their cargo. However, this cargo can sometimes also be found in a soluble (non‐EV) fraction. The ‘carrier effects’ of EVs describe how cargo function changes as a result of being associated with an EV. The carrier effects include (1) concentrating signalling molecules; (2) protecting cargo; (3) targeting specific cell subsets; and (4) altering the functional properties of the cargo. Here, we set up a demonstration using designer EVs with epidermal growth factor (EGF), green fluorescent protein (EGFP), EGFP mRNA, or neutrophil elastase protein (a protease) to illustrate the carrier effects of EVs isolated from HEK293T cells. Methods : EVs were purified by 300 kDa tangential flow filtration. Structure, size, concentration, and contaminating factors were evaluated by transmission electron microscopy, ELISA, nanoparticle tracking analysis, western blotting, and qRT‐PCR following MISEV2023 guidelines. Results : (1) To generate EVs with EGF in the protein corona, HEK293T cells were transfected with a plasmid expressing EGF fused to the N‐terminus of PTGFRN, a protein known to localize to EVs. Soluble EGF or EGF+ EVs were applied to EGFR reporter cells. Despite the same concentration of EGF being used, EGF+ EVs showed 2 to 13 times as much signalling as soluble EGF. (2) EVs were loaded with EGFP mRNA using a CD63‐L7Ae system with in vitro transcribed EGFP as a control. Following RNase treatment, the EVs protected up to 95% of the EGFP mRNA, but the non‐EV‐bound EGFP mRNA was not detectable. 3) Single‐chain variable fragments (scFv) targeting CD14 were fused to PTGFRN‐EGFP and given to neutrophils (CD14+). EVs with anti‐CD14 scFv showed at least double the delivery efficiency compared to EVs without anti‐CD14 or expressing a control scFv. (4) Neutrophil elastase (NE) was incubated with HEK293T EVs to place it on the corona or used in a soluble form. NE+ EVs had 3 times as much specific activity compared to soluble NE, demonstrating that the EV changed the kinetics of the proteins in its corona. Summary/Conclusion : Studying these carrier effects is essential to elucidate the mechanisms by which EV‐associated biomarkers exert their effects.

Wnt

Nadine Winkler 1 , Antonia Schubert 1,2,3 , Lukas Schik 1 , Oksana Voloshanenko 1,2 , Dominic Helm 1 , Michael Boutros 1,2 1 German Cancer Research Center (DKFZ), Germany; 2 Heidelberg University, Germany; 3 National Center for Tumor Diseases (NCT), University Hospital Heidelberg, Germany Introduction : Wnt signalling is pivotal for stem cell behaviour and cell differentiation. While mutations in Wnt pathway components are found in several cancer types, for example, colorectal cancer (CRC), little is known about the different modes of Wnt ligand trafficking and their effect on cancer pathophysiology. To travel extracellularly, Wnt ligands are secreted on carriers, such as extracellular vesicles (EVs). EV‐bound Wnt ligands have been shown to promote recipient cells' proliferation and invasiveness. However, it remains unclear how oncogenic Wnt mutations affect the CRC secretome, particularly EV secretion and cargo, and which mechanisms are mediated through EV‐bound versus non‐EV‐bound Wnt ligands. Methods : Using CRISPR/Cas9, we generated two cell lines from the CRC line HCT116: APCtrunc with a clinically relevant truncation of APC, causing constitutive Wnt pathway activation, and EVIKO, a knock‐out of the Wnt cargo receptor EVI/Wntless, rendering the pathway inactive. Wnt ligand secretion from HCT116 wild‐type, APCtrunc, and EVIKO cells was characterized by western blots. EV fractions were enriched from the HCT116 lines, the CRC line SW480, and healthy colon fibroblasts with differential centrifugation. The effect of Wnt mutations on particle quantity and size was investigated using nanoparticle tracking analysis and electron microscopy. The proteomic profiles of EV fractions and cell lysates were identified by mass spectrometry. Results : We established and characterized cell lines with Wnt‐specific mutations, HCT116 APCtrunc and EVIKO, observing a differential secretion of selected Wnt ligands. Two distinct particle fractions with vesicle morphology were reproducibly enriched from cell supernatants. All fractions contained classical EV markers, while different Wnt ligands and Wnt pathway receptors were detected on HCT116 APCtrunc‐ and EVIKO‐derived large and small EVs. Furthermore, the Wnt mutational background altered the number of particles secreted per mL of conditioned medium. We will further assess the influence of the isolated EV and non‐EV fractions on cell migration and tumour invasiveness. Summary/Conclusion : By investigating the role of Wnt signalling mutations in EV biogenesis, we will improve our understanding of how Wnt signalling modulates oncogenic signal distribution via EVs in CRC. Funding : This work is funded by the DKFZ‐MOST Cooperation Program in Cancer Research of the German Cancer Research Center and the Else Kröner‐Fresenius‐Stiftung (2022_EKEA.189).

Aloe

Presenter: Lei Zhang Nankai University, Tianjin, People's Republic of China Introduction : This study aimed to investigate whether exosomes derived from Aloe vera (Aloe‐Exo) could accelerate the healing of skin wounds in diabetic patients. Specifically, we evaluated the therapeutic potential of Aloe‐Exo when used as a bioactive dressing to treat skin injuries associated with diabetes. Methods : Exosomes were isolated from Aloe vera extracts and characterized for their size, morphology, and protein markers using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting. Aloe‐Exo was incorporated into a hydrogel‐based dressing and applied to diabetic mouse models with skin wounds. Wound healing progression, inflammation reduction, and tissue repair were evaluated through histological analysis, immunohistochemistry, and biochemical assays. In vitro, human dermal fibroblasts and keratinocytes were treated with Aloe‐Exo to assess cellular proliferation, migration, and anti‐inflammatory responses using cell viability assays, scratch tests, and ELISA. Results : Aloe‐Exo significantly promoted faster wound closure and enhanced collagen deposition in diabetic mice compared to control groups. Inflammatory cytokine levels were reduced, while the proliferation and migration of skin cells were markedly increased. Aloe‐Exo demonstrated strong antioxidative and anti‐inflammatory properties, aiding in the reduction of oxidative stress and inflammation at the wound site. In vitro studies further revealed that Aloe‐Exo stimulated fibroblast proliferation and keratinocyte migration, both critical for effective wound healing. Summary/Conclusion : Aloe vera‐derived exosomes provide a promising therapeutic approach for treating skin injuries in diabetic patients. By enhancing wound healing through anti‐inflammatory and antioxidative mechanisms, Aloe‐Exo offers a potential alternative as bioactive dressings for diabetic wound care. Funding : This work was supported by the Natural Science Foundation of Tianjin Science and Technology Bureau, China [grant number 21JCZDJC01050]; Tianjin Key Medical Discipline (Speciality) Construction Project, China [grant number TJYXZDXK‐047A]; and Tianjin Municipal Health Science and Technology Project, China [grant number TJWJ2021ZD003].

Anti

Presenter: Anat Aharon Tel Aviv University, Tel Aviv, Israel Introduction : Chimeric antigen receptor (CAR)‐T cells are genetically engineered T cells, directed against tumour‐associated antigens. Extracellular vesicles (EVs), derived from CAR‐T cells (CAR‐T EVs), preserve CAR‐T function and may overcome CAR‐T cell therapy challenges in patients with solid tumours by better infiltration and less cytokine storm. This study explored whether CAR‐T EVs can be used for effective immunotherapy for solid tumours. For that, EVs obtained from CAR‐T cells, stimulated by HER2 expressing cells or by Her2 recombinant‐coated beads, were compared. Methods : Anti‐HER2 CAR‐T cells were produced and stimulated by SKOV‐HER2+ cells or by recombinant HER2+coated beads. EVs were isolated from the cell media at multiple time points and compared with EVs of un‐transduced (UT) cells. All EVs types were characterized for their size, concentration, proteins content and functions, using Nano tracking analysis, protein array, and western blot (WB). EVs cytotoxic effects were assessed on breast cancer cells (SKBR‐HER2+), ovarian cells (SKOV‐HER2+ and OVCAR‐HER2‐), glioblastoma cells (U251‐HER2+), and on primary healthy cell culture (kidney, lung, skin, bone marrow mesenchymal stem cells). Cytotoxic effects were measured by IFNγ secretion level (ELISA), methylene blue killing assay and caspase 3/7 activity assay, using live cell imaging (Incucyte). Results : CAR‐T EVs contained a mixture of small and large EVs. Higher CAR‐T EVs concentrations were found in SKOV‐stimulated cells, compared to CAR‐T EVs of Her2+ coated beads. This is related to the fact that EVs of SKOV‐stimulated cells include a mixture of EVs derived from the cancer cells (∼60%) and CAR–T EVs (∼40%), while EVs of Her2+ coated beads include only CAR‐T EVs. EVs obtained after CAR‐T cells stimulation with Her2+ coated beads contained higher levels of cytokines (e.g., GM‐CSF, I‐309, IL‐13, IL‐17, RANTES) and induced significantly higher death of Her2+cancer cells, compared to UT EVs. That is similar to the cytotoxic effects that were induced by SKOV stimulated CAR T EVs. Moreover, EVs from CAR‐T cells stimulated by Her2+ coated bead did not affect the viability of healthy cells. Summary/Conclusion : CAR T EVs produced by Her2+coated beads may provide a novel, pure and potent immunotherapy approach, with an effective cytotoxicity and without effect on healthy cells. Funding : This study was supported by the KAMIN‐ Israel innovation Authority‐68014.

Arg1

Presenter: Minghao Sun Capricor Therapeutics, Inc., USA Introduction : Arginase 1 deficiency (ARG1‐D) is a rare genetic disorder characterized by the loss of arginase 1 (Arg1), the final enzyme in the urea cycle. In patients with ARG1‐D, hepatocytes are unable to convert ammonia into urea, leading to elevated levels of arginine and ammonia in the blood and cerebrospinal fluid, which in turn results in progressive neurological symptoms. Current therapeutic strategies primarily focus on managing plasma arginine and ammonia levels, but long‐term outcomes remain poor. As of now, there is no approved treatment specifically for ARG1‐D available in the United States. Methods : Utilizing Capricor's StealthX platform, researchers engineered the human Arg1 enzyme for expression on the exosome membrane in either intra‐ or extra‐exosomal orientations. The resulting Arg1‐exosomes were evaluated for their in vitro functionality as a disease‐specific therapeutic approach for ARG1‐D. Remarkably, regardless of their localization on the exosome membrane, Arg1 exhibited biological activity and effectively converted arginine into urea. Notably, only nanograms of Arg1 delivered via exosomes proved as potent as micrograms of human recombinant Arg1 protein. Additionally, engineered exosomes containing intra‐exosomal Arg1 were capable of delivering the Arg1 protein into 293F and HepG2 cells in a time‐ and dose‐dependent manner. Results : These findings suggest that Arg1 exosomes not only catalyse arginine conversion efficiently at lower doses but also protect the Arg1 protein during delivery to target cells, maintaining its enzymatic activity in vitro. In further investigations in vivo, a single dose of Arg1‐exosomes was able to restore Arg1 activity in the liver and reduce plasma arginine levels to those comparable to wild‐type mice within 2 h. Repeated dosing extended the lifespan of Arg1 knockout mice by 50% in preliminary tests. Summary/Conclusion : Together, these results indicate that StealthX platform‐engineered Arg1 exosomes have the potential to serve as an enzyme replacement therapy, delivering Arg1 to target hepatocytes and possibly providing broader clinical benefits for patients with ARG1‐D.

Back

Heikki Saari 1,2 , Jacopo Zini 2 , Julia Monola 2 , Riina Harjumäki 2 , Saara Laitinen 1 1 Finnish Red Cross Blood Service, Helsinki/Vantaa, Finland; 2 University of Helsinki, Faculty of Pharmacy, Helsinki, Finland Introduction : Protein quantification is an extremely basic part of biochemical research, including those regarding extracellular vesicles (EVs). Total protein concentration is a useful characteristic of EV samples, which can be used for normalizing samples for functional assays or molecular characterizations such as western blotting. However, since EVs are a complex, heterogeneous nanoparticles containing a mixture of proteins and other biomolecules that vary between different cell types, accurate protein quantification can be less straightforward than expected. As the exact protein composition of EVs cannot be provided, only nonspecific assays are applicable, with their inherent estimations and errors. In this study, selected colourimetric and spectroscopic methods were compared for assessing protein concentration from platelet‐ and PC‐3 prostate cancer EVs. Methods : EVs were purified from expiring platelet concentrates from donated blood or PC‐3 conditioned cell culture medium by ultracentrifugation and size exclusion chromatography. The EVs were characterized by Nanoparticle Tracking Analysis (NTA), cryo‐TEM and western blotting. Compared protein concentration methods were UV absorbance (Nanodrop) with 205 nm, 280 nm or the Scopes method, and BCA assay with and without SDS. Purified egg ovalbumin was used as a known concentration reference protein. Results : Based on the egg ovalbumin reference, the Scopes method gave the most accurate estimate for protein concentration. It is based on peptide bond absorbance at 205 nm, correcting for aromatic amino acid overlap. Compared to Scopes method, uncorrected A205 was naturally very close, but A280 significantly overestimated the EV protein concentration about 150% for platelet EVs and 240% for PC‐3 EVs. BCA assay on the other hand underestimated the protein concentrations to about 50% of the Scopes method, and addition of SDS slightly increased the estimate to about 60%. Summary/Conclusion : In our study, we found that there is significant variation between EV protein quantification methods, which may either over‐ or underestimate the protein concentration in an EV source‐dependent fashion. We suggest that the Scopes method is the most reliable protein quantification method for EVs and recommend it for the EV community. Funding : This study was supported by the Instruct‐ERIC R&R pilot funding #2729.

Bone

Evangelia Bochti 1 , Monica Tsimbouri 1 , Biswajoy Bagchi 2 , Xin Li 2 , Matthew Dalby 1 , Catherine Berry 1 Centre for the Cellular Microenvironment, University of Glasgow, Glasgow, USA 1 ; Medical Research Scotland, Sphere Bio, Cambridge, UK 2 Introduction : Bone tissue is the second most transplanted tissue worldwide, with over 4 million annual transplants. Bone marrow‐derived stromal cells (BMSCs) are used in bone tissue engineering due to their osteoblastic lineage. The beneficial effects of BMSCs are potentially mediated through extracellular vesicles (EVs). In collabouration with Sphere Fluidics Limited, this project aims to manufacture a novel BMSC‐EV microenvironment, encapsulating osteogenic‐primed EVs within hydrogel beads. The osteogenesis‐inducing beads could be used for localised, cell‐free bone regeneration and repair therapies. Methods : BMSCs were osteogenically primed for 28 days using two distinct approaches: chemical induction and mechanical induction, via nanoscale vibrational displacements. Osteogenic differentiation of BMSCs was verified via protein analysis and histological staining. BMSC‐derived EVs were successfully isolated and characterised by size and concentration. The bioactive cargo of osteogenic EVs’ is undergoing analysis at cytokine, metabolomic, proteomic and RNA levels. Osteogenic EVs were encapsulated in GelMA and alginate hydrogels and will soon be encapsulated with naive BMSCs in 3D hydrogel beads using Sphere Fluidics’ microfluidic technology. The viability of naive BMSCs within the hydrogel beads along with the osteogenic potency of the EVs will be assessed using Alamar Blue (viability assay) and at a gene level (qRT‐PCR) for early osteogenic markers (RUNX2 and Osterix). Results : Both chemical and mechanical inductions induced osteogenesis in BMSCs. EV isolation was verified, and EVs were consistently sized (60–250 nm). Differences in EV cargo are noted in the cytokine profile, with ongoing studies at a metabolomic, proteomic and RNA level between the osteogenic induction methods. Osteogenic EVs can be encapsulated within GelMA and alginate hydrogels, and their osteogenic potency in naïve BMSCs is to be investigated. Summary/Conclusion : BMSC analysis indicated osteogenesis. Ongoing characterisation of BMSC‐EVs aims to determine any differences in osteogenic‐derived EVs cargo, both compared to the negative control and between the induction approaches. Encapsulation of naive BMSCs with osteogenic EVs in hydrogel beads will indicate the ability of the BMSCs to uptake EVs in a 3D environment, emphasising the osteogenic potency of the EVs’ bioactive cargo on naive BMSCs. Funding : This PhD research project is funded by Medical Research Scotland (MRS) and Sphere Fluidics Limited, studentship ref. number: PHD‐50388‐2021.

Burn

De Taddeo M. 1,2 , Valade G. 1,2 , Grosbot M. 1,2 , Nivet M. 1,2 , Langle C. 1,2 , Goulinet S. 2 , Mauduit P. 2 , Jung V. 3 , Guerrera C. 3 , Banzet S. 1,2 , Peltzer J. 1,2 , Trouillas M . 1,2 1 French Armed‐Forces Biomedical Research Institute (IRBA), Clamart, France; 2 INSERM U1197, Villejuif, France; 3 Necker Proteomics, Paris, France Introduction : Despite the great diversity of approaches in regenerative medicine, severe burns still lead to delayed healing and abnormal scarring. The secretome of mesenchymal stromal cells (MSC) has strong potential in tissue repair, with immunomodulatory and remodelling properties similar to MSC themselves. Composed of extracellular vesicles (EV) and soluble factors (SF), its composition is modified by environmental stimuli. Our team leveraged this property by priming MSC with IL‐1β to enhance burn wound healing. However, the specific secretome components with pro‐repair properties and their mechanism of action remain elusive. This study aims to identify key components of the IL‐1β‐primed MSC secretome that promote cell proliferation, migration, and remodelling in vitro. Methods : MSC were primed with IL‐1β, and the conditioned medium was collected after 72 h. EV or EV + SF fractions were isolated and concentrated using tangential flow filtration with filter cutoffs at 500 or 10 kDa, respectively. Characterization was performed by cryo‐electron microscopy, NTA, flow cytometry phenotyping and mass spectrometry analysis. Various doses of these products, reflecting MSC numbers producing the secretome, were tested in proliferation, migration and extracellular matrix production assays involving keratinocytes and dermal fibroblasts under serum‐deprived conditions. PCR analyses were performed to assess myofibroblast activation. Results : EV or EV + SF fractions were characterized according to MISEV guidelines. Our results indicated no difference in particle concentration and size. Both fractions expressed EV markers (CD9, CD63, CD81) and MSC markers (CD90). Mass spectrometry analysis revealed differences between EV and EV+SF but few differences among priming doses. In functional assays, the EV + SF fraction more effectively promoted proliferation and migration of keratinocytes and dermal fibroblasts under stressed conditions than the EV fraction, regardless of priming dose. Nucleic acid and protein compositions with related reactomes supported these results, revealing more growth factors, interleukins, chemokines linked to matrix remodelling. Summary/Conclusion : Our research highlights the role of both soluble factors and extracellular vesicles in wound healing. We aim to validate these findings using an in vivo model. This study will help optimize scalable production methods for a product designed to improve severe burn healing.

Cdk6

Belinda S. Maw 1* , Elisabeth Gamper 1* , Nathalie Havranek 1 , Astrid Digruber 2 , Martin Maw 3 , Silvio Kau‐Strebinger 4 , Markus Zojer 1 , Gerwin Heller 5 , Zuzanna Kieron 1 , Jonatan Kendler 1 , Karin Hummel 6 , Ingrid Walter 3 , Waltraud Tschulenk 3 , Monika Ehling‐Schulz 2 , Veronika Sexl 1 , Thorsten Klampfl 1 , Karoline Kollmann 1 1 Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria; 2 Institute of Microbiology, University of Veterinary Medicine, Vienna, Austria; 3 Independent researcher 4 Institute of Morphology, University of Veterinary Medicine, Vienna, Austria; 5 Department of Medicine I, Division of Oncology, Medical University of Vienna, Vienna, Austria; 6 Vetcore facility for proteomics, University of Veterinary Medicine, Vienna, Austria Introduction : Extracellular vesicles (EVs) are linked to various diseases with implications in the progression, immune activation, biomarkers and therapy. Acute myeloid leukaemia (AML) is a highly aggressive disease with elevated EVs in the serum of patients. One crucial regulator and prognostic biomarker for AML is the cell cycle regulator cyclin‐dependent kinase 6 (CDK6). Palbociclib, a CDK4/6 kinase inhibitor, is already FDA‐approved for breast cancer and in clinical trials for AML; however, the exact mechanisms that are induced by the inhibition of both cell‐cycle kinases and potential combinatorial treatments remain to be elucidated. Methods : Utilizing transformed HPCLSK cell lines as a novel murine in vitro model of AML, we analysed the effects of CDK4/6 inhibition on cellular states. Transcriptomic changes were measured by RNA‐Seq experiments, and CDK6 interaction partners were identified by mass spectrometry and western blots. To pinpoint the single role of CDK6, we compared CDK4/6 inhibited cells with cells lacking CDK6. We measured EVs by nanoparticle tracking analysis, electron microscopy and mass spectrometry and validated our findings in human AML cell lines. Results : Human cancer cells are co‐dependent on CDK6 and components of intracellular vesicle pathways. CDK4/6 kinase inhibition in AML results in deregulated expression of genes assigned to intracellular vesicles and vesicle trafficking. Inhibition of CDK4/6 increases EV release of KMT2A‐MLLT3+ AML cells and alters the EV size distribution. We identified several proteins of the endosomal‐exosomal pathways as CDK6 interactors and detected high levels of CDK6 at endosomes, lysosomes and on EVs. Summary/Conclusion : These data identify a novel function of CDK6 at intracellular and extracellular vesicles and propose an important role of CDK6 as a regulator of intercellular communication in AML.

Cx43

Presenter: María D. Mayán Santos Universidade de Vigo, Vigo, Spain Introduction : BRAF and MEK inhibitors (BRAF/MEKi) have radically changed the treatment landscape for advanced BRAF mutation‐positive tumours. However, limited efficacy and the emergence of drug resistance remain major challenges for successful treatment. Methods : Here, using relevant preclinical models, we found that Connexin43 (Cx43), a protein involved in cell‐to‐cell communication, enhances the effectiveness of BRAF/MEKi by recruiting DNA repair complexes to lamin‐associated domains, thereby promoting persistent DNA damage and cellular senescence. Results : The nuclear compartmentalization induced by Cx43 contributes to genome instability and synthetic lethality due to excessive DNA damage, suggesting a novel therapeutic approach to overcoming drug resistance in these tumours. Based on these findings, we designed an innovative drug combination using small extracellular vesicles (sEVs) to deliver Cx43 mRNA in combination with BRAF/MEKi. Summary/Conclusion : This study demonstrates that this strategy enhances Cx43's antitumoural activity, increases the synergy of BRAF/MEKi, and resensitizes double‐resistant cells to cell death. These findings highlight the therapeutic potential of this approach in overcoming the limitations of current therapies and improving treatment outcomes for patients with advanced BRAF mutant tumours.

Dual

Presenter: Lingxin Zhu Wuhan University, China (People's Republic) Introduction : Myeloid‐lineage osteoclasts actively remodel both the mineral and proteinaceous components of bone during normal growth and development as well as pathologic states ranging from osteoporosis to bone metastasis. The cysteine proteinase cathepsin K confers osteoclasts with potent type I collagenolytic activity; however, cathepsin K‐null mice, as well as cathepsin K‐mutant humans, continue to remodel bone and degrade collagen by as‐yet‐undefined effectors. Herein, we identify a cathepsin K‐independent collagenolytic system in osteoclasts that is composed of a functionally redundant network of the secreted matrix metalloproteinase MMP9 and the membrane‐anchored matrix metalloproteinase MMP14. Methods : We generated myeloid‐specific Mmp14 conditional knockout mice in either wild‐type or Mmp9 null backgrounds. Primary bone marrow‐derived macrophages were isolated and differentiated into osteoclasts. Bone resorption was assessed in osteoclast‐bone wafers in vitro or in a calvaria organ culture model ex vivo. Results : Unexpectedly, whereas deleting either of the proteinases individually leaves bone resorption intact, we find that Mmp9/Mmp14 conditional double‐knockout osteoclasts display major defects in bone resorption in vitro. In vivo, Mmp9/Mmp14 conditional double‐knockout mice exhibited marked increases in bone density and displayed a highly protected status against either parathyroid hormone‐ or ovariectomy‐induced pathologic bone loss. Wild‐type osteoclasts inefficiently resorbed of Col1ar/r bone ex vivo, while Col1ar/r mice femurs exhibited an increase in trabecular bone density. In an effort to define the signalling mechanisms underlying a combined requirement for MMP9 and MMP14, bioinformatic analysis illustrates that double‐knockout osteoclasts—as well as MMP‐inhibited human osteoclasts—unexpectedly display major changes in transcriptional programs in tandem with compromised RhoA activation, sealing zone formation and bone resorption. Further study revealed that osteoclast function is dependent on the ability of MMP9 and MMP14 to cooperatively proteolyze the β‐galactoside–binding lectin, galectin‐3, on the cell surface. Mass spectrometry identified the galectin‐3 receptor as low‐density lipoprotein‐related protein‐1. Summary/Conclusion : Together, these studies characterize a previously unsuspected role for dual proteolytic signalling in regulating the turnover of galectin‐3 lattice to govern osteoclast activation in tandem with the ability of the dual metalloproteases to mediate bone collagenolytic effects, and identify the myeloid‐derived dual metalloproteases axis as a potential target for therapeutic interventions for bone‐wasting disease states. Funding : NSFC grants 82370914 and 81970919.

Fcn3

Presenter: Rocio Castillo Sánchez National Polytechnic Institute (Cinvestav), Mexico Introduction : Breast cancer is the second most common cancer among women worldwide. The development of predictive biomarkers for breast cancer would allow the identification of patients that will benefit from early treatment. The liquid biopsy emerges as a proposal for a non‐invasive method in the diagnosis and prognosis of breast cancer. There are several cell components that can be analysed in a liquid biopsy, such as circulating tumour DNA (ctDNA), circulating tumour cells (CCTs) and extracellular vesicles (EVs). EVs refers to particles that are released from cells, are delimited by a lipid bilayer, and cannot replicate on their own. EVs cargo is an indicator of its cell of origin, containing biomolecules such as lipids, genetic material and proteins. EVs secretion in breast cancer has been well‐described, making it an effective tool for diagnosis. Methods : Protein expression in EVs from plasma of breast cancer women and healthy donors was analysed using flow cytometry, western blot and HPLC mass spectrometry. All studied participants signed informed consent, and the protocol was approved by the ethics and research committee of INCan, Mexico (CI/600/15; CEI/710/15). Results : Proteomic analysis revealed that EVs contain proteins associated with different cellular processes, including ficolin (FCN3), which is a protein of the complement activation lectin pathway, and the inter‐α trypsin inhibitor heavy chains (ITIH2), which are proteins that play an important role in inflammation and carcinogenesis. Our result showed that FCN3 and ITIH2 were differentially expressed in the EVs from plasma of healthy donors and breast cancer patients. Moreover, expression of proteins was analysed by using Cox proportional hazard regression and the Kaplan–Meier model, and results demonstrated that FCN3 and ITIH2 are both independent prognostic biomarkers for overall survival in breast cancer patients. Summary/Conclusion : FCN3 and ITIH2 in the EVs are protective factors, because a decrease in their expression is an adverse prognosis in breast cancer.

Flow

Domenica Giannandrea, Ehsan Soleymaninejadian, Valentina Citro, Natalia Platonova, Clara Bernardelli, Piera Selvaggio, Monica Miozzo, Paola Ciceri, Mario Cozzolino, Lavinia Casati, Elena Lesma, Raffaella Chiaramonte Università degli Studi di Milano, Milan, Italy Domenica Giannandrea and Ehsan Soleymaninejadian are co‐first authors . Elena Lesma and Raffaella Chiaramonte are co‐senior authors . Introduction : Extracellular vesicles (EVs) are recognised as valuable biomarkers for disease due to their ability to be released by various cell types into body fluids, including urine, plasma, cerebrospinal fluid, semen, and saliva. The number, size and cargo of circulating EVs correlate with the progression of autoimmune diseases, making them promising diagnostic tools for diseases like multiple sclerosis (MS) and type 1 diabetes (T1D). Specifically, small EVs (sEVs) from platelets, immune, endothelial, and central nervous system (CNS) cells can help the characterization of MS subtypes and provide insights into disease activity and treatment responses. Additionally, exosomes may serve as tissue‐specific biomarkers for diagnosing T1D progression. Apoptotic bodies (ApoBds) are another type of cell‐derived particle that may indicate disease progression. Their presence in circulation reflects apoptotic death in damaged tissues, with studies showing elevated plasma concentrations of ApoBds in neurological patients compared to healthy donors. Flow cytometry is a rapid, minimally invasive technique for analysing these vesicles, requiring only small sample sizes. So, starting from this contest, in this study, flow cytometry strategies were developed to assess the role of sEVs and ApoBds as future biomarkers in MS and T1D. Methods : For sEV characterization, up to 37 epitopes were analysed on vesicles derived from plasma samples of healthy donors using the MACSPlex Kit. Blood was processed to isolate plasma, which was then ultracentrifuged to obtain sEVs, characterized and quantified by nanoparticle tracking analysis (NTA). For ApoBds characterization, they were isolated in vitro from the OPM2 cell line after bortezomib treatment. Three centrifugation protocols were evaluated, with the most effective being 700 × g for 10 min followed by 3000 ×  g for 30 min, achieving optimal purity and yield. Results : The data obtained report the characterization of plasma sEVs for endothelial, immune and platelet markers. Additionally, results showed a significant increase in ApoBds from bortezomib‐treated cells. Summary/Conclusion : This study establishes a robust methodology for the flow cytometric characterization of sEVs and ApoBds, paving the way for future investigations into their potential as biomarkers for T1D and MS. Funding : This study was supported by the Project PNC 0000001 D3 4 Health, CUP B53C22006080001, Funded by NextGenerationEU.

From

Roland Prielhofer, Claudia Lindner, Melanie Reininger, Ingrid Hartl, Klaus Graumann Phoenestra GmbH (Linz, Austria) Introduction : Extracellular vesicles (EV) from Mesenchymal Stromal Cells (MSC) hold significant potential for regenerative medicine and other therapeutic applications. As the vesicular fraction of the secretome has been shown to achieve similar biological effects as the cells themselves, MSC‐derived EV are being extensively investigated for clinical use. However, large‐scale production of EV in consistent quality and sufficient quantities for large‐scale studies has proven challenging. To address this, innovative technologies for EV manufacturing are essential to achieve scalability and reproducibility, paving the way for standardized, GMP‐compliant production processes suitable for broader clinical and industrial applications. Methods : Phoenestra has developed a scalable bioreactor manufacturing setup based on a proprietary basket‐impeller design, that holds macrocarriers, enabling efficient cultivation of adherent cells. It operates in perfusion mode, allowing significant volumes of conditioned media to be harvested continuously—currently tested for up to 49 days. Following harvest, conditioned media is processed by depth filtration and low‐shear ultra‐/diafiltration. The resulting EV preparations were extensively characterized through Nanoparticle Tracking Analysis for particle number, size and distribution, and nanoflow cytometry for lipid particle and EV content as well as MSC marker expression. Variability is closely monitored across and within bioreactor runs by comparing samples from different time points, ensuring consistency in EV quality and yield. Results : Utilizing stable cell lines over extended cultivation times and the advantage of xeno‐free conditions in perfusion bioreactor cultivation, we achieved consistent EV quality across manufacturing runs. Further process optimization for key cultivation process parameters resulted in a 2‐3‐fold EV productivity increase to at least 2.5‐7.5 E+11 EV per day and yielded an improved MSC‐EV phenotype. Following MSC culture optimizations and scalable isolation and purification methods, we produced EV of consistent high quality in ample quantities already at the 250 mL bioreactor scale—sufficient to meet the demand of small clinical studies. Summary/Conclusion : Our proprietary manufacturing system addresses the demand for consistent EV quality to meet the requirements for clinical supply. Thus, our innovations will significantly facilitate and accelerate the translation of EV products into therapeutic applications.

High

Presenter: Domenico De Bellis University “G. d'Annunzio” of Chieti‐Pescara, Chieti‐Pescara, Italy Introduction : Pancreatic cancer (PC) is one of the most aggressive malignancies, ranking among the leading causes of cancer‐related mortality globally. Despite advances in understanding tumour biology, PC's asymptomatic progression and poor response to therapy result in limited survival outcomes. Therefore, identifying robust biomarkers is crucial to improving diagnostic accuracy, prognostication, and therapeutic management. Leukocyte‐derived extracellular vesicles (LEVs), which mediate intercellular communication, have shown potential as indicators of immune response and disease progression. This study investigates whether circulating EVs derived from leukocytes serve as predictive biomarkers for clinical outcomes in PC patients, potentially guiding personalized treatment approaches. Methods : This prospective study included 56 patients with histologically confirmed PC and 48 age‐ and sex‐matched healthy controls. Blood samples were collected, and a patented, advanced flow cytometry protocol was applied to quantify leukocyte‐derived (CD45+) and immune checkpoint molecule‐expressing PD‐L1+ EVs. The relationship between EVs levels and clinical outcomes, including overall survival (OS), progression‐free survival (PFS), and disease control, was evaluated, focusing particularly on patients with advanced and unresectable disease undergoing chemotherapy. Results : Analysis revealed that PC patients exhibited significantly elevated levels of total EVs, CD45+ EVs, and PD‐L1+ EVs compared to healthy controls ( p < 0.05). Elevated baseline levels of CD45+ EVs were strongly associated with favourable survival outcomes, independently predicting improved OS (HR = 0.17; p = 0.02) and PFS in advanced PC cases. Notably, patients whose CD45+ EV levels remained stable or decreased during chemotherapy achieved higher rates of disease control, whereas increases in EV levels were indicative of progressive disease. These findings highlight the dynamic role of leukocyte‐derived EVs in monitoring therapeutic efficacy and disease progression. Summary/Conclusion : The study underscores the clinical utility of blood‐derived CD45+ EVs as a novel, non‐invasive biomarker for prognostic and predictive purposes in pancreatic cancer. By providing real‐time insights into immune status and treatment response, leukocyte‐derived EVs could enhance decision‐making in clinical oncology, supporting tailored treatment regimens for patients with PC. This investigation opens avenues for further research on EV‐based liquid biopsies.

Host

Presenter: Dominik Fleischhacker University of Graz, Graz, Austria Introduction : Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. As a facultative pathogen, V. cholerae ’s lifecycle is marked by environmental persistence in aquatic reservoirs as well as colonization of the human intestinal tract. Recent research suggests that in the presence of antimicrobial agents, such as mitomycin C, a new type of vesicle is being formed, which relies on the cell death of a sub‐population. My research focuses on finding natural inducers for this type of vesicle and their physiological role. Methods : Protein quantification (Bradford assay) Nucleic acid quantification (SYTO 9 Green‐Fluorescent Nucleic Acid Stain) Lipopolysaccharide (LPS) quantification (FM 4–64 assay) Size and nanoparticle amount measurement (Zetasizer Nano ZS90 and NanoSight NS300) Transmission electron microscopy Qualitative and quantitative proteomic analyses (LC‐MS), DNA sequencing and analyses (Illumina). Horizontal gene transfer experiment in vitro and in vivo (murine model). Results : Our results show that V. cholerae produces explosive‐type BEVs in the presence of host‐derived antimicrobial stressors, that is, bile. State‐of‐the‐art BEV content characterization, morphological analyses, and OMIC‐based analyses identified several qualitative and quantitative differences between blebbing‐type and explosive‐type BEVs. Most importantly, the explosive‐type BEVs contained substantial amounts of cytoplasmic proteins and nucleic acids. The incubation of explosive BEVs derived from a tetracycline‐resistant V. cholerae isolate with a tetracycline‐sensitive wild‐type strain resulted in vesicle‐mediated HGT of the antibiotic resistance cassette in vitro and in vivo. Downstream experiments using diverse mutants of the natural competence system identified periplasmic, inner membrane and cytoplasmic components to be essential for vesicle‐mediated HGT, while outer membrane or exterior parts are dispensable. Our data suggest the possibility of vesicle‐mediated HGT during intestinal colonization facilitating the spread of virulence factors or antimicrobial resistance genes. Summary/Conclusion : By applying the antimicrobial substances MMC and bile, vesicles containing significantly higher amounts of protein biomass and nucleic acids are being formed. These vesicles also have a higher degree of complexity and more closely resemble the WCL compared with the condition without additional stressors. These vesicles are also able to promote HGT of a chromosomal element in a ComEA‐dependent but PilA‐independent pathway both in vitro and in vivo (murine model). Funding : FWF

Know

Melanie Reininger, Ingrid Hartl, Claudia Lindner, Roland Prielhofer, Klaus Graumann Phoenestra GmbH, Austria Introduction : Investigating the composition‐function relationship of mesenchymal stromal cell (MSC)‐derived extracellular vesicles (EVs) is complex due to their inherent heterogeneity and lack of process reproducibility. To address the challenge, we strive for process standardization to minimize product variability. Our approach involves the use of telomerized MSC lines, xeno‐ and hPL‐free media and a controlled, scalable stirred tank bioreactor setup. This strategy enables us to reproducibly establish a solid data foundation towards product definition and supports the identification of true cell line‐specific commonalities and differences in EV characteristics. Methods : EVs were produced from five telomerized MSC lines derived from different human sources (adipose tissue, chorionic plate, Wharton's Jelly, bone marrow, and placenta) in our patented stirred tank bioreactor setup. Conditioned media was harvested, filtered, concentrated and washed via tangential flow filtration using a low‐shear membrane pump. These EV preparations were analysed in multiple ways: particle concentration and size distribution were determined by nanoparticle tracking analysis (NTA), protein content was measured by bicinchoninic acid assay (BCA), surface marker composition was analysed via nanoflow analysis, miRNA profiles were established by small RNA sequencing, and their biological function was assessed in several different cell‐based bioassays. The results from each analysis method were collectively correlated with biological activity. Results : Several cell line‐specific differences in EV composition were found: although cultured in identical media conditions, the protein‐to‐particle ratio demonstrated to be highly cell line‐dependent, ranging between 3 and 23 mg/1E + 10 particles. Further, we were able to confirm the presence of relevant EV markers and lipids and show that a broader marker profile as well as the bioactivity in different assays are determined by cell source and cultivation time. In‐depth miRNA analysis showed that the 10 most prevalent miRNAs are mostly consistent in identity across different cell lines, though they vary in abundance and ranking. Summary/Conclusion : Our reproducible process enables the generation of standardized EV preparations that facilitate comprehensive analysis and characterization. Thereby, we are paving the way for better product understanding and, eventually, definition of critical quality attributes and key contributors to biological functionality. Ultimately, it allows us to select and tailor EV preparations to meet specific (pre‐)clinical needs.

Long

Presenter: Marco Loria University of Palermo, Palermo, Italy Introduction : Cancer progression relies heavily on intercellular communication within the tumour microenvironment, and tumour‐derived small extracellular vesicles (TD_SEVs) play a crucial role in this interaction. It has been demonstrated that TD_SEVs enhance tumour growth, allow pre‐metastatic niche formation and promote epithelial‐to‐mesenchymal transition (EMT). The EMT is a developmental process through which cells lose their epithelial identity and acquire a mesenchymal phenotype. In cancer, this process is prodromal to tissue fibrosis, thus favouring organ colonization by metastatic cells. Colorectal cancer (CRC) is one of the most common tumours, and almost 50% of the undiagnosed patients will develop liver metastases during the disease. Recent evidence highlighted the role of alternative splicing (AS) in the gene expression rearrangement required for EMT. In CRC, however, there is currently insufficient data relating TD_SEVs to AS. Our studies aim to dissect how the cargo of colorectal cancer‐derived small extracellular vesicles (CRC_SEVs) promotes AS related to EMT in hepatocytes, to shed light on the molecular mechanisms driving liver metastases of CRC. Methods : CRC_SEVs were isolated from the conditioned media of SW480, SW620 and HCT‐116 cells through ultracentrifugation. The SEVs were resuspended in PBS, RNA lysis buffer or RIPA buffer. RNA and protein content were assessed through qRT‐PCR and western Blot, respectively. A human healthy hepatocyte cell line, the THLE‐2, was treated with the CRC_SEVs, and the biological effects were investigated through qRT‐PCR, immunofluorescence and RNA in situ. Results : our data demonstrated that, in CRC, the long non‐coding RNA H19 binds RBFOX2, an RNA‐binding protein involved in pro‐EMT AS, leading this to its mRNA targets. Here we found that CRC_SEVs transport both lncH19 and RBFOX2 and the treatment of the THLE‐2 with the CRC_SEVs promotes AS of RBFOX2, target mRNAs involved in cell shape remodelling. Interestingly, vesicles deprived of lncH19 also show lower levels of RBFOX2 and cannot induce the same AS mechanisms in the hepatocytes. Summary/Conclusion : our data indicated, for the first time to our knowledge, that lncH19 carries splicing factors inside EVs, affecting AS processes in recipient cells and enforcing a pre‐metastatic microenvironment.

Loss

Presenter: Jingwen Yang Wuhan University, China (People's Republic) Introduction : Cranial sutures function as growth centres for calvarial bones. Abnormal suture closure will cause permanent cranium deformities. MMP9 is a member of the gelatinases that degrades components of the extracellular matrix. MMP9 has been reported to regulate bone development and remodelling. However, the function of MMP9 in cranial suture and cranium development is still unknown. Methods : We used Mmp9 global knockout mice and analysed the cranial suture and cranium via micro‐CT and histological staining. The underlying mechanisms were determined using unbiased RNA‐Seq analysis and confirmed via ex vivo 3D culture and in vivo inhibitor application. Results : We identified that the expression of Mmp9 was specifically elevated during fusion of posterior frontal (PF) suture compared with other patent sutures in mice. Interestingly, inhibition of MMP9 ex vivo or knockout of Mmp9 in mice (Mmp9‐/‐) disturbed the fusion of PF suture. Histological analysis showed that knockout of Mmp9 resulted in wider distance between osteogenic fronts, suppressed cell condensation and endocranial bone formation in PF suture. Proliferation, chondrogenesis and osteogenesis of suture cells were decreased in Mmp9‐/‐ mice, leading to the PF suture defects. Moreover, transcriptome analysis of PF suture revealed upregulated ribosome biogenesis and downregulated IGF signalling associated with abnormal closure of PF suture in Mmp9‐/‐ mice. Inhibition of the ribosome biogenesis partially rescued PF suture defects caused by Mmp9 knockout. Summary/Conclusion : These results indicate that MMP9 is critical for the fusion of cranial sutures, thus suggesting MMP9 as a potential therapeutic target for cranial suture diseases. Funding : This work was supported by the National Natural Science Foundation of China (No. 82170915 and No. 31500788).

Milk

Presenter: Tehila Ben Ziv Tel Aviv University, Israel ABSTRACT UNAVAILABLE

Mstn

Presenter: Sitong Lyu Yanbian University, Yanji, People's Republic of China Introduction : Myostatin (MSTN) is an inhibitor of skeletal muscle growth and development. MSTN mutant pigs mainly exhibit a phenotype of increased muscle and decreased fat deposition, which has great potential in the treatment of obesity. Exosomes are nanovesicles with characteristics of donor cells and are important mediators of intercellular communication. It is worth investigating whether MSTN‐mutated porcine exosomes (MSTN‐Exos) have similar metabolic benefits on obesity. Methods : In this study, obese mice were established by high‐fat diet (HFD). Serum exosomes were isolated from MSTN mutant pigs and injected into adipose tissue of mice. The effects of serum exosomes on adipose tissue morphology and biochemical indexes were observed. At the same time, the 3T3‐L1 cell line was used to treat adipose differentiation with MSTN‐Exos. The effect of MSTN‐Exos on adipose differentiation was evaluated by measuring cell differentiation degree and triglyceride content. In addition, we performed biological sequencing analysis of MSTN‐Exos to reveal its underlying molecular mechanism. Results : The results showed that after local injection of MSTN‐Exos into adipose tissue, fat deposition was significantly reduced in HFD mice. Meanwhile, an obvious browning trend was observed in white adipose tissue, which was manifested by significantly increased expression of brown adipose‐related genes PGC‐1α and UCP1. In vitro experiments showed that MSTN‐Exos significantly decreased the adipose differentiation ability of 3T3‐L1 cells, which was manifested as decreased triglyceride accumulation and decreased expression of CEBP‐α and PPAR‐α genes related to adipose differentiation. Through biological sequencing of MSTN‐Exos, the mechanism of MSTN‐Exos promoting browning of white fat was analysed. Summary/Conclusion : Our study shows that MSTN‐Exos significantly reduces fat deposition in HFD mice by promoting browning of white fat, which has the potential to promote fat metabolism and improve obesity. The detailed elucidation of the reaction molecules and their mechanisms of action will provide new insights into the fight against obesity and related diseases.

Nano

Presenter: Carley D. Ross Beckman Coulter Inc., Brea, California, USA Introduction : Biological EVs occur in low concentrations as heterogeneous populations making these biological particles challenging to study and analyse. To overcome this challenge, ultracentrifugation was used to concentrate cell culture EVs from green fluorescent protein (GFP) expressing Jurkat and Chinese Hamster Ovary (CHO) cells. The concentrated EVs were subjected to rate zonal density gradient ultracentrifugation (DGUC) and fractions collected allowing further characterization and analysis using nano flow cytometry and sedimentation velocity analytical ultracentrifugation (SV‐AUC). Methods : Extracellular vesicles derived from CHO and Jurkat cells expressing CD81‐GFP were purified and concentrated using two cycles of ultracentrifugal pelleting and resuspension. EVs from both purification cycles were characterized by nano flow cytometry and sedimentation velocity analytical ultracentrifugation (SV‐AUC). To separate EV subpopulations, rate zonal DGUC was used and resulting bands were fractionated and dialyzed. These fractions were analysed by both SV‐AUC and nano flow cytometry. Results : Distinct populations of EVs were observed by nano flow cytometry analysis on scatter and fluorescent channels. The AUC results suggest that the first purification cycle resulted in a mixture of small and potentially low‐density species, with the majority sedimenting below 20 Svedberg units (S). The second purification cycle resulted in a wider distribution of larger and potentially higher‐density species, with the majority sedimenting below 100 S. Thus, the second cycle of pelleting/resuspension appears to have enriched the proportion of larger and denser species. Rate zonal DGUC separated the heterogeneous pellet populations into distinct subpopulations. Summary/Conclusion : The rate zonal ultracentrifugation step reduced EV purification time from overnight to 90 min and separated unique subpopulations. These subpopulations were further characterized by nano flow cytometry and SV‐AUC. Funding : This study was supported by the Beckman Coulter Life Sciences.

Oral

Mona Belaid 1,2 , Wei Heng Chng 2 , Ram Pravin Kumar Muthuramalingam 2 , Yun Wei Lim 3 , Bertrand Czarny 3 , Giorgia Pastorin 2 , Driton Vllasaliu 1 1 King's College London, United Kingdom; 2 National University of Singapore, Singapore; 3 Nanyang Technological University, Singapore Introduction : Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. IBD affects over 10 million people worldwide, and current treatments are associated with poor clinical outcomes and severe systemic side effects. Our previous work in vitro showed that mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) are promising therapeutics for IBD due to their regenerative potential and immunomodulatory properties. The aim of this study is to explore the therapeutic potential of MSC‐EVs to treat IBD in vivo. MSC‐EVs are most commonly administered by injection; however, the oral route offers advantages in treating IBD, as it can directly target the site of action and is a more convenient route of administration. Here, we report a novel formulation of MSC‐EVs for oral delivery, and we investigate the therapeutic effect in an in vivo model of IBD. Methods : MSC‐EVs were isolated by ultracentrifugation on a sucrose cushion and characterized for particle size, concentration, protein content, marker analysis and morphology. The stability of MSC‐EVs was evaluated in gastric and intestinal fluids using NTA, nanoflow cytometry, and cryo‐EM. A double‐coating formulation consisting of chitosan and Eudragit S100 was developed to improve the stability of MSC‐EVs for oral administration and target colon delivery in a dextran sodium sulphate (DSS)‐induced colitis mouse model. C57BL/6J mice received 3% DSS in drinking water to induce colitis, and MSC‐EVs were administered daily by oral gavage (1E9 particles/dose) for 5 days. Mice were scored daily for disease activity, and at necropsy, colon tissue was collected for histological examination and quantification of inflammatory markers. Results : The double coating protected MSC‐EVs from enzymatic degradation and facilitated the release of intact EVs in the colonic fluid. Coated MSC‐EVs significantly reduced the disease activity index and inflammation in the colon of DSS mice, while non‐formulated MSC‐EVs showed similar results to the negative control group. Summary/Conclusion : A novel formulation of MSC‐EVs was developed for oral delivery and reduced inflammation in mice with DSS‐induced colitis. This study highlights the potential of MSC‐EVs as novel therapeutics for IBD and suggests that oral delivery could be an effective route of administration. Funding : The in vivo work was supported by NAMIC grant A‐8001601‐00‐00.

Over

Presenter: Edward H. Park Recharge Biomedical, USA Introduction : Because of the lack of specific MHC self‐identity antigens on MSC exosomes, there is reason to believe that exogenously‐sourced EVs would work similarly to endogenously‐produced ones and potentially help repair and restore function in vivo. Methods : After informed consent, including the clear understanding that the use of MSC exosomes was not FDA‐approved, nor the standard of care, over 500 patients, in over 1000 encounters, underwent over 2500 procedures. Results : The presenter will review the procedures, complications, general response rates, and possible mechanisms of action for these powerful and naturally regenerative EVs. The presentation will include a cursory review of the omics of the EV product that was used. Summary/Conclusion : With multiple trials in the USA and Europe of MSC exosomes for various indications, reviewing the common reactions, complications, and effects of a therapeutic modality that may soon be available for off‐label use in the clinic is valuable. The presenter's clinical experience will demonstrate high safety, efficacy, and predictable benefits from the use of exogenous MSC exosomes.

Role

Juçara Gastaldi Cominal 1 , Larwsk Hayann 1 , Maryanne Trafanni Mello 1 , Luiz Henrique da Silva Andrilli 1,2 , Ana Paula Ramos 1 , Saida Mebarek 3 , Massimo Bottini 2,4 , José Luís Millán 2 , Pietro Ciancaglini 1 1 University of Sao Paulo—Faculty of Philosophy, Sciences and Letters at Ribeirao Preto, Chemistry Department, Ribeirao Preto, Brazil; 2 Sanford Burnham Prebys Medical Discovery Institute—Sanford Children's Health Research Center, La Jolla, California, USA; 3 Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR CNRS 5246, Université Claude Bernard Lyon 1, Villeurbanne, France; 4 Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy Introduction : Mineralizing cells release a special class of extracellular vesicles, known as matrix vesicles (MVs), with the unique property to bind to collagen fibres and contribute to mineralization. Following their release, MVs bind to the extracellular matrix (ECM), where their highly specialized enzymatic machinery facilitates the formation of seed minerals within the vesicles’ lumen, subsequently releasing the minerals onto the ECM. However, how MVs propagate minerals onto the collagenous ECM remains unclear. We address these questions by exploring the “protein corona” paradigm whereby nanoparticles entering a biological milieu become cloaked by a corona of soluble proteins modifying their biological functions. Methods : Here, we isolated native MVs from the growth plates of chicken embryos. After removing the protein corona from the native MVs using high ionic strength buffer, we obtained shaved MVs. Reconstituted MVs were produced by incubating shaved MVs with the removed protein corona constituents. MVs were obtained by different ultracentrifugation steps and characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), scanning electron microscopy (SEM). The protein content was assessed by electrophoresis and proteomic analyses. Results : Our results showed that both the removal and reconstitution of the protein corona significantly affect the biochemical and physicochemical properties of MVs, resulting in three well‐defined groups. Shaved MVs exhibited an increase in the activity of tissue non‐specific alkaline phosphatase (TNAP) and a decrease in mineral deposition compared to native MVs. Reconstituted MVs partially recovered these functions, showing a reduction of TNAP activity and mineral deposition compared to native MVs. Furthermore, changes in the protein corona affect MV ability to anchor to the collagenous ECM, which is crucial for initiating the propagation of the mineral phase within this organic matrix. Proteomic analyses revealed changes in the protein profile of the MVs resulting from the removal of the protein corona, indicating that shaved proteins were primarily related to external structural and ECM organization and catabolism. Summary/Conclusion : These findings underscore the role of the protein corona in modulating the mineralization capabilities of MVs. Understanding these interactions could lead to new therapeutic strategies for enhancing bone repair and regeneration (https://academic.oup.com/jbmrplus/advance‐article/doi/10.1093/jbmrpl/ziae168/7931791). Funding : FAPESP 2019/08568‐2; CNPq 305426/2021‐4.

Rose

Barone A. 1 , Ciriolo L. 2 , Pellegrini B. 3 , Fiore D. 1 , Silletta A. 3 , Tolomeo A.M. 5 , Panza S. 1˒4 , Paolino D. 1˒4 1 Department of Experimental and Clinical Medicine; 2 Department of Health Science; 3 Department of Medical and Surgical Sciences; 4 Research Center “ProHealth Translational Hub”; “Magna Graecia” University of Catanzaro, Italy; 5 Department of Cardiac, Thoracic and Vascular Science and Public Health, University of Padova, Italy Panza S. and Paolino D. are co‐senior authors . Introduction : Plant‐derived nanovesicles (PDNVs) are emerging as promising nanocarriers for bioactive compounds. Their natural origin, biocompatibility, and potential therapeutic properties make them ideal candidates for biomedical applications. Compared to the mammalian‐derived EVs, PDNVs offer ethical sourcing and environmental benefits. The aim of this work is to investigate the physicochemical properties, stability, antioxidant activity and therapeutic potential of rose petal‐NVs (RPNVs), with a focus on their application in wound healing. Methods : RPNVs were extracted by triturating the rose petals for 5 min, followed by sequential centrifugation, ultracentrifugation, 0.2 µm filtration and size‐exclusion chromatography, through Akta Prime system. NTA characterized the particle size and concentration, as well as TEM analysis, while Dynamic Light Scattering assessed the zeta potential. Long‐term stability was evaluated using Turbiscan Lab Expert at 25°C and 32°C. Antioxidant activity was tested using DPPH and ABTS assays, and in vitro wound healing efficacy was evaluated on BJ fibroblasts by scratch assays and RT‐PCR. Biosafety was assessed in vitro and in vivo on healthy human volunteers (Ethical Approval number: 392/2019). Informed consent was obtained from all subjects. Results : Purification of RPNVs was optimized using Sepharose CL‐2B resin, resulting in homogeneous particles with a size range of 50‐200 nm. The RPNVs showed a negative zeta potential of ‐23 mV, ensuring stability in suspension. Long‐term stability studies confirmed no destabilization phenomena and storage after freeze drying method preserved EV integrity. Antioxidant assays showed significant radical scavenging activity. RPNVs promote an epithelial phenotype by increasing E‐Cadherin and decreasing N‐Cadherin, Alpha‐SMA and Collagenase A1 levels. Furthermore, the upregulation of MMP‐9 alongside minimal modulation of MMP‐2 suggests selective extracellular matrix remodelling. Tests on human volunteers revealed no adverse effects on skin barrier function. Summary/Conclusion : RPNVs show potential for enhancing wound healing, demonstrated by their antioxidant activity and positive effects on key markers of tissue repair. The RPNVs remain stable under suitable storage conditions, ensuring their long‐term efficacy. In vitro and in vivo biosafety assessments confirmed their safety, supporting their potential for future dermatological applications. Funding : Next Generation EU—Italian NRRP, (Directorial Decree n. 2021/3277)—project Tech4You—Technologies for climate change adaptation and quality of life improvement, n. ECS0000009.

Smac

Jordan J. Yin 1,2,3,4 , Daniel Panting 1,2,3,4 , Maria Dimancheva 1,2,3,5 , Mikaela van der Merwe 1,2,3,4 , Mariam Hakoum 1,2,3,4 , Martine St‐Jean 1 , Nathalie Earl 1 , Eric LaCasse 1,2 , Robert Korneluk 1,4 , Dylan Burger 6,7 , Rostyslav Horbay, 1,2,3,4 , Shawn Beug 1,2,3,4 1 Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Canada; 2 Centre for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, Canada; 3 Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Canada; 4 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada; 5 Department of Biology, University of Ottawa, Ottawa, Canada; 6 Kidney Research Centre, The Ottawa Hospital Research Institute and University of Ottawa, Ottawa, Canada; 7 Department of Cellular and Molecular Medicine and School of Pharmaceutical Sciences, University of Ottawa, Ottawa, Canada Introduction : The inhibitor of apoptosis proteins (IAPs) are crucial in regulating programmed cell death by directly inhibiting caspases and promoting cell survival. These proteins are key negative regulators of the alternative NF‐κB pathway and its downstream, pro‐inflammatory and immune responses. A class of drugs termed SMAC mimetic compounds (SMCs) has been developed to antagonize the IAP proteins. Consequently, these SMCs can promote cell death and stimulate immune responses. This study aims to evaluate the ability of SMCs to promote anti‐tumour immune responses driven by the intercellular transfer of antigen‐presenting small extracellular vesicles (sEVs). Methods : sEVs were isolated from cancer cells treated with SMCs via ultracentrifugation and characterized per the latest ISEV recommendations via Zetaview NTA, immunoblotting and NTA. Subsequently, dendritic cells were treated with different combinations of sEVs and SMCs and analysed via flow cytometry for immune activation and tumour‐antigen presentation. SMC and sEV‐treated dendritic cells were also co‐cultured with SMC‐treated, antigen‐specific CD8+ T cells, which were analysed for activation with flow cytometry. Results : The release of sEVs from SMC‐treated cancer cells was increased by ∼30‐fold compared to control groups. These sEVs were positive for syntenin, a classical ESCRT‐dependent sEV biogenesis biomarker. Furthermore, the sEVs extracted measured between 50 and 200 nm according to NTA results. Dendritic cells co‐treated with SMC and sEVs from SMC‐treated cancer cells showed increased tumour‐antigen presentation and activation compared to control groups. Upon co‐culturing tumour antigen‐specific CD8+ T cells with these activated dendritic cells and SMCs, T cell activation was increased over ∼2‐fold when compared to controls. Summary/Conclusion : This research provides insight into the potential synergistic effects of SMCs and sEVs on a specific, host‐mediated immune response against cancer cells. Future directions include investigating the mechanism linking SMC and sEV release, as well as determining if these anti‐tumour responses can be replicated upon SMC and sEV treatments in vivo. Funding : This study was supported by CIHR (#PJT‐169126) and CRS (#935202)

Stem

Parinita Agrawal 1 , Nisha Rajendran 1 , Ritu Raj 1 , Suvro K. Chowdhury 1 , Namit Dey 1 , Alka Bhat 1 , Sabyasachi Chattopadhyay 1 , Imrankhan Pathan 1 , Mujib Ullah 2 , Rita Mahapatra 1 , Abha Gour 3 , Anil Tiwari 3 , Virender S. Sangwan 3 , Arun Chandru 1 , Tuhin Bhowmick 1,2 1 Pandorum Technologies Pvt. Ltd., Bangalore, India; 2 Pandorum International Inc., Boston, Massachusetts, USA; 3 Dr. Shroff's Charity Eye Hospital, New Delhi, India Introduction : Corneal blindness affects millions of people worldwide, and its treatment is impacted by the limited availability and sub‐par quality of donor cornea tissues. Mesenchymal stem cells (MSCs)‐derived extracellular vesicles (EVs), drawing attention for the treatment of various disorders, have been explored here as therapeutics for corneal wound healing. We developed bioprinted corneal lenticules as a means for targeted and sustained delivery of EVs while studying corneal regeneration. Methods : EVs isolated from MSCs by ultracentrifugation and purified by density gradient separation were evaluated by nanoparticle tracking analysis and electron microscopy for size estimation and western blot for protein profile following MISEV2023‐guidelines. Establishing therapeutic function in cell lines, the study investigated their encapsulation in a cornea‐mimetic biopolymeric ink by bioprinting corneal lenticules using the digital light processing technique. Their therapeutic application and regenerative potential were explored by release profile and in vitro functional studies. Results : EVs, with a size of 70–150 nm, appeared as cup‐shaped vesicles in transmission electron microscopy. Expressed characteristic markers CD9, CD63, CD81, TSG101, Flotillin, and Alix, with remarkable re‐epithelialization, anti‐inflammatory, anti‐fibrosis, and neurogenesis activities. The bioprinted lenticule exhibited cornea mimetic properties, with light transmittance > 85% and compressive modulus comparable to that of a native cornea. The average release rate of 22% over 120 days, showcased their potential as a sustained EV delivery platform. Demonstrating 98% compatibility with human corneal stromal cells, this system proved to conserve EVs activity and support corneal regeneration. Summary/Conclusion : MSCs derived EVs emerged as promising bioactive agents for stimulating re‐epithelization and supporting corneal regeneration. Their delivery mediated by bioprinted corneal lenticules ensures regeneration, preserving structural and functional requirements of the tissue. The study describes a promising strategy for clinical translation of EVs towards the treatment of corneal keratopathy involving inflammation and removing the dependency on donor corneas for transplants.

They

Invited Speaker: Janis A. Müller Philipps University Marburg, Germany

This

Presenter: Florian Flo Rachenne University of Montpellier, France Introduction : The dengue virus (DENV) is an arbovirus transmitted by Aedes mosquitoes through saliva during a bite on a vertebrate host, playing a key role in viral infection. Dengue affects nearly 390 million people annually and with the absence of an effective treatment, understanding saliva's role in transmission could reveal new therapeutic targets. My research team identified extracellular vesicles (EVs) in Aedes aegypti saliva. These EVs contain subgenomic flavivirus RNA (sfRNA), a non‐coding RNA from the degradation of viral genomic RNA by a cellular enzyme. sfRNA interacts with immune proteins in human. Methods : Using molecular biology and proteomics, I showed that syntenin in the yellow fever mosquito is involved in the secretion of DENV sfRNA in saliva. Using RNA affinity chromatography, I isolated proteins from the Aag2 cell line involved in extracellular vesicle (EV) biogenesis, focusing on syntenin. I confirmed this interaction via immunoprecipitation and quantitative PCR. Western blot and PCR assessed the virus's impact on EV secretion, while nanoparticle tracking analysis (NTA) quantified EV size and number during infection. Finally, syntenin knock‐down in infected Aedes aegypti mosquitoes confirmed syntenin's role in sfRNA secretion. Results : Syntenin and DENV sfRNA interaction: I showed that syntenin from the yellow fever mosquito interacts with DENV sfRNA. Using RNA affinity chromatography, syntenin was isolated with in vitro‐produced sfRNA. Immunoprecipitation from infected Aag2 cells revealed enrichment of both sfRNA and viral genomic RNA. Impact of DENV infection on EV secretion: I demonstrated that DENV infection increases secretion of two EV markers (syntenin and CD63 orthologue) into Aag2 cell supernatant, correlating with larger and more numerous EVs. Syntenin levels were also elevated in the carcasses and salivary glands of infected mosquitoes. Finally, syntenin knock‐down in infected female mosquitoes reduced DENV sfRNA secretion in saliva, suggesting syntenin's role in sfRNA secretion. Summary/Conclusion : In conclusion, my thesis highlights syntenin's role in dengue virus sfRNA secretion in mosquito saliva. My results show an interaction between syntenin and sfRNA, explaining how sfRNA is internalized into extracellular vesicles (EVs). Synhenin knock‐down in vivo reduced sfRNA secretion. This work enhances our understanding of how viruses hijack EV biogenesis in mosquitoes to boost transmission to humans.

Time

Ola Tuvesson, Agnes Zimmer, Isa Lindgren NorthX Biologics, Matfors, Sweden Introduction : Extracellular vesicles (EVs) have lately raised as an attractive new therapeutic modality for both immunotherapy and as vectors for various cargos such as nucleic acids and proteins. This new class of therapies is emerging as a transformative area of biopharmaceutical innovation, with potential applications in therapeutics, drug delivery, and vaccines. As for all new modalities the translation from research to clinical and commercial use is not without challenges, particularly in developing effective manufacturing processes and qualified analytical methods meeting the CMC requirements of Good Manufacturing Practices (GMP). This talk aims to put the light on some of the challenges that need to be addressed for progressing the scientific novelty of bacterial EVs and the practicalities of modern GMP‐compliant manufacturing. Methods : Drawing on the extensive GMP expertise of a NorthX Biologics, a leading CDMO with several EV projects in development, this presentation will outline some of the principles of GMP as applied to the production of bacterial EVs, emphasizing the distinct challenges and considerations unique to this field. Topics will include the development of robust and reproducible upstream processes for vesicle production, scalable purification strategies tailored to the specific properties of EVs, and strategies to characterize the complex products to verify consistency, safety, and efficacy. Results : Special focus will be given to: Describing the road map for the translation from bench to bedside. Critical quality attributes (CQAs) for EVs and their alignment with regulatory expectations. Analytical challenges in characterizing EVs for purity, potency, and sterility. • Risk management and contamination control strategies tailored to bacterial systems. Summary/Conclusion : By addressing these key topics, this presentation seeks to give inventors working in the EV field some useful practical insights into the GMP landscape for bacterial EVs. As the field advances, leverageing GMP expertise and collabourative partnerships early in the product development journey will be essential to translating the promise of bacterial EVs into real‐world therapeutic breakthroughs.

Tiny

Tosin Opadokun, Sophie Chauvin‐Bose, Petra Rohrbach McGill University, Canada Introduction : Plasmodium falciparum infected red blood cells (iRBCs) release extracellular vesicles (EVs) during their asexual stages (ring, trophozoite, schizont). These EVs carry biomolecular cargo that influence parasite development. We hypothesize that EVs from stage specific‐iRBCs carry cargo that induces gametocytogenesis, the differentiation of asexual parasites into sexual stages. We isolated stage specific EVs from conditioned media and tested their effect on gametocyte development in P. falciparum cultures. Methods : Stage‐specific EVs were isolated from P.falciparum blood stages using differential centrifugation. Nanoparticle tracking analysis was used to measure EV concentration and size, and transmission electron microscopy confirmed EV size and purity. Western blotting detected stage‐specific proteins, including Band 3, Glycophorin A, flotillin 1 and 2. Proteomic analysis was performed by LC‐MS/MS, with peptides analysed using a Thermo Orbitrap Fusion mass spectrometer at 120,000 resolution (MS1) and HCD sequencing (15,000 resolution) at top speed for peptides with a charge of 2+ or greater. Cell culture assays assessed gametocyte development after stage specific EVs were added to infected RBC cultures. Results : We identified eight proteins potentially involved in gametocytogenesis, including GEXP02, GEXP12, plasmepsin IV, RESA, and StAR‐related lipid transfer protein. To assess EV function, we treated P. falciparum cultures with stage specific EVs on various days. The highest gametocyte production occurred 72 h post‐treatment. The priming effect of EVs was further confirmed by treating uninfected RBCs with EVs prior to adding them to parasite cultures. RBCs primed in this manner produced similar levels of gametocytes to those treated directly with EVs, indicating that EVs do not necessarily interact directly with the parasite but rather prime the RBCs, enabling them to support parasite invasion and differentiation into sexual stages. Summary/Conclusion : Treating cultures with P. falciparum‐derived EVs induced gametocyte production, demonstrating that these EVs carry cargo capable of triggering gametocytogenesis. Uninfected RBCs primed with EVs also facilitated gametocyte development, suggesting that EVs prime RBCs for parasite invasion and differentiation into gametocytes. These findings have significant implications for malaria transmission and potential intervention strategies. Future studies will focus on mechanistic insights and MS to compare protein profiles of EV‐treated cultures. Funding : Natural Sciences and Engineering Research Council of Canada (NSERC).

Tips

Sinziana Popescu 1 , Haley Stang 2 , Richard Day 2 , Costanza Emanueli 1 1 National Heart and Lung Institute, Imperial College London, London, UK; 2 Department of Ageing, Rheumatology & Regenerative Medicine, UCL Division of Medicine, University College London, London, UK Introduction : Small extracellular vesicles (sEVs) produced by cultured mesenchymal stem cells (MSCs) are currently investigated as advanced therapy medicinal products in cardiology. Among several cardioprotective activities, MSC‐sEVs pro‐angiogenic properties are of interest for treating ischaemic vascular disease. Environmental cues, such as the attachment substrate, modulate the MSC secretome. Here, we developed polymeric microspheres with highly tunable physical properties to act as cell substrates for 3D cultures and explored how the microcarrier size range impacts the MSC‐sEV yield, formulation and in vitro pro‐angiogenic activity. Methods : Porous microcarriers were produced from poly(lactic‐co‐glycollic) acid using thermally induced phase separation (TIPS) within three size ranges: small = 250–355 µm, medium = 425–500 µm, and large = 600–710 µm. TIPS ultrastructural features, porosity, and morphology pre‐ and post‐cell attachment were examined (scanning electron microscopy and Morphologi‐G3, Malvern). Suspension cultures of MSCs attached to the microcarriers were maintained in vertical‐wheel bioreactors (PBS Biotech) for up to 26 days. For sequential MSC‐secretome harvesting (at days 6, 12, 21 and 26), the culture media was temporarily switched (24 h) to serum‐free. MSC‐sEVs were isolated by size exclusion chromatography (qEV columns; Izon). MSC‐sEV size‐distribution and concentration were determined by Nanosight (Malvern). The tetraspanins expression profile of the MSC‐sEVs was assessed by Exoview‐R100 (NanoView). The MSC‐sEV proangiogenic potential was assessed by studying their capacity to induce proliferation (WST‐1 assay, Roche) of human primary cardiac microvascular endothelial cells (ECs) prepared from both male and female donors. Results : Ultrastructural analysis of the microcarrier surfaces revealed similar hierarchical structures for all microcarrier groups. Particle diameters were not significantly affected by the culture. Similar MSC‐sEV yields were recovered from all microcarriers, with diameter peaks at 100–117 nm. Tetraspanin expression changed with the TIPS size: at Day‐6, CD63 expression was 74.5% in small, 58.8% in medium and 65.6% in large TIPS; CD81: 13.2% in small, 8.9% in medium and 5.2% in large TIPS; CD9: 12.5% in small, 29.8% in medium and 20.9% in large TIPS. All MSC‐sEVs preparations induced EC proliferation, with maximal potency observed for MSCs‐EVs collected at Day‐6 (80.6% for Large TIPS) and Day‐12 (88.8% for small TIPS). Summary/Conclusion : The growth of MSCs on TIPS microcarriers increases the pro‐angiogenic properties of MSC‐derived sEVs.

Tp53

Martina Presicci 1 , A. E. Martinez 2 , C. Di Battista 3 , M. Gironella 3 , J. C. Bourdon 4 , T. van Wezel 1 1 Dept of Pathology, Leiden University Medical Center (LUMC), Leiden, The Netherlands; 2 Dept of Cell and Chemical Biology, Leiden University Medical Center (LUMC) Leiden, The Netherlands, 3 Gastrointestinal & Pancreatic Oncology group, FRCB‐IDIBAPS; Experimental Pathology Dept. IIBB‐CSIC; Facultat de Medicina i Ciencies de la salut, Universitat de Barcelona, Spain, 4 School of Medicine, Cancer Division, University of Dundee, Dundee, Scotland, UK Introduction : Colorectal cancer (CRC) is one of the leading causes of cancer‐related deaths worldwide, emphasizing the need for non‐invasive biomarkers to enable early detection and improve survival outcomes. Extracellular vesicles (EVs) carry proteins, RNA, and other biomolecules that reflect the functional state and molecular profile of their cells of origin. These unique properties make EVs promising candidates for biomarker discovery. Mutations in TP53, a critical tumour suppressor gene, are frequently implicated in CRC and are linked to cancer progression and resistance to therapy. This study investigates the presence of TP53 isoform proteins and mRNAs in EVs isolated from the conditioned media of lymphocyte B cell lines derived from individuals with hereditary cancer predisposition disorder. Our goal is to evaluate their potential as biomarkers for familial CRC detection. Methods : EVs were isolated and purified from serum‐free conditioned media of lymphocyte B cells derived from individuals with hereditary cancer predisposition disorder. EVs were characterized via nanoparticle tracking analysis (NTA) and Western blotting for positive and negative EVs markers. TP53 isoform proteins were detected using validated TP53 isoform‐specific antibodies, while mRNA was analysed using reverse transcription (RT) and droplet digital PCR (ddPCR) with probes specific for different TP53 splice variant transcripts. Results : Western blot analysis confirmed the presence of EV markers in size exclusion chromatography (SEC)‐enriched fractions, validating the isolation protocol. Preliminary findings revealed TP53 isoform protein enriched in EVs. RT‐ddPCR analysis identified specific TP53 splice variant mRNA in EV‐enriched fractions. Notably, the abundance of TP53 splice variant mRNA and p53 protein isoforms was higher in EVs derived from CRC cell lines associated with Li‐Fraumeni syndrome compared to those from sporadic CRC cell lines. Summary/Conclusion : This study indicates the presence of specific TP53 isoform proteins and mRNAs in EVs, underscoring their potential as biomarkers for CRC. Our findings suggest that EVs act as carriers of some TP53 splice variants and isoform‐specific signals. This highlights the intricate nature of this tumour suppressor gene, whose complexity has been observed in various cancer types but is not yet fully understood. Funding : Funded from the European Union's Horizon Europe research and innovation programme under the Marie Skłodowska‐Curie Doctoral Network Grant Agreement No. 101072448.

Type

Aiden Blumer, Julian Krause, Silvio Kau Strebinger University of Veterinary Medicine Vienna, Austria Introduction : Type 2 inflammation, driven by Th2 cytokines such as interleukin‐4 (IL‐4) and IL‐13, is implicated in chronic inflammatory diseases affecting respiratory and cutaneous tissues. This immune pathway, notably driven by IL‐4 and IL‐13 via JAK/STAT signalling, has recently been linked to skin fibrosis in secondary lymphedema—focus area of our investigation. Given that IL‐4 and IL‐13 can alter endosomal‐lysosomal traits, we hypothesize that genes regulated by STAT proteins, or their physical interactions influence extracellular vesicle (EV) dynamics in lymphedema, affecting both local and systemic disease processes. We regard fibroblasts as an underestimated cell type in this context, which may reveal novel insights into tissue remodelling and immune modulation. Methods : We performed a comprehensive database search to compile the STAT1/3/6 interactome and identify genes regulated by these proteins. Data were cross‐referenced with Gene Ontoloy (GO) annotations to pinpoint connections of IL‐4/IL‐13‐STAT signalling with EV biogenesis. An extensive literature review assessed previously identified direct STAT interactions to uncover associations with EV biognesis. To pre‐investigate EV dynamics in fibroblasts, NIH‐3T3 cells were treated with IL‐4, IL‐13, and their combination at various time points up to 24 hs. Cleared culture supernatant (500 and 1500 g) was analysed using NTA, flow cytometry, and TEM. Particle counts were normalized to cell numbers. Results : Preliminary data reveal many STAT interactions impacting EV‐related processes in human and murine cells. These interactions extend beyond transcriptional targets to include direct protein‐protein associations, suggesting a multi‐faceted role of STATs in modulating EV biogenesis within inflamed tissues. Type 2 inflammation was found to tune EV release in murine fibroblasts, which reflects in silico results. Summary/Conclusion : This study highlights a potential mechanistic link between dysregulated Type 2 immune responses and EV dynamics in fibroblasts, which may underlie local progression of lymphedema and its systemic manifestation. Further experiments are necessary to explore the precise molecular underpinnings of this association.

What

Krisztina V. Vukman 1 , Kelsey Fletcher 1 , Tamás Visnovitz 1,2 , Péter Lőrincz 3 , Tünde Bárkai 1 , Dorina Lenzinger 1 , Edit I.Buzás 1,4,5 1 Semmelweis University, Department of Genetics, Cell‐ and Immunobiology, Nagyvárad tér 4. 1089 Budapest, Hungary; 2 ELTE Eötvös Loránd University, Department of Plant Physiology and Molecular Plant Biology, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary 3 ELTE Eötvös Loránd University, Department of Anatomy, Cell and Developmental Biology, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary 4 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Nagyvárad tér 4. 1089 Budapest, Hungary; 5 HCEMM‐SU Extracellular Vesicle Research Group, Hungary, Nagyvárad tér 4. 1089 Budapest, Hungary Introduction : Mast cells are best known for their ability to release granules during allergic reactions upon cross‐linking of Fcε‐bound IgE molecules by allergens. On the other hand, mast cell‐derived extracellular vesicles (EVs) were among the first EVs shown to carry RNA molecules (Valadi H et al., 2007). Methods : The aim of our study was to clearly distinguish, for the first time, different types of mast cell‐derived extracellular particles (EPs) including extracellular granules (EGs) and EVs. We studied cultured mast cells from mouse bone marrow, mouse peritoneal mast cells, and the mouse mast cell line (MC/9). Mast cells were cultured in the presence and absence of IgE‐DNP, LPS, and Ca++ ionophore A23187. To separate and characterize the released EPs, we employed differential centrifugation, density gradient centrifugation, size‐exclusion chromatography, immunoaffinity techniques, high‐resolution flow cytometry, nanoparticle tracking analysis, confocal and electron microscopy. Results : We observed differences in EP secretion patterns during Th1 responses (upon LPS stimulation) as well as in IgE‐dependent and IgE‐independent (Ca++ ionophore A23187‐induced) mast cell degranulation. Our data suggest that mast cells predominantly secrete EVs during Th1 responses, while produce mainly EGs during Th2‐associated degranulation. Surprisingly, during IgE‐independent degranulation, mast cells secreted a mixed population of both EVs and EGs. Since the calcium ionophore A23187 is routinely used in both mast cell research to study degranulation and in EV research to induce EV secretion, our findings indicate a need to reinterpret previous results in both fields. Importantly, we successfully identified molecular markers (such as histamine) that can help distinguish and separate mast cell‐derived EP populations. Summary/Conclusion : In summary, this study represents an important step toward understanding the distinct roles of mast cell‐derived EP types (EGs and EVs) in the complex immune‐orchestrating roles of mast cells. Funding : NVKP_16‐1‐2016‐0004 grant of the Hungarian National Research, Development and Innovation Office (NKFIH), VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, the Therapeutic Thematic Program TKP2021‐EGA‐23. RRF‐2.3.121‐2022‐00003 (National Cardiovascular Laboratory Program), 2019‐2.1.7‐ERA‐NET‐2021‐00015, EU's Horizon 2020 Research and Innovation Program (No. 739593) and János Bolyai Research Scholarship of the Hungarian Academy of Sciences. National Research, Development and Innovation Fund ( FK138851 ), Hungarian Academy of Sciences (LP2022‐13/2022), Eötvös Loránd University Excellence Fund (EKA 2022/045‐P101‐2)

When

Presenter: Olesia Gololobova Johns Hopkins Medicine, USA Introduction : Extracellular vesicles (EVs) have the inherent ability to transfer bioactive molecules, which has positioned them as promising candidates for therapeutic applications, including targeted drug delivery, regenerative medicine, and vaccine development. However, the clinical translation of EV‐based therapies necessitates a thorough understanding of their biocompatibility and immunogenicity, especially when EVs are derived from different cell types and administered across species barriers. Methods : In this study, we evaluated the immunogenicity of EVs derived from three distinct human cell types: Expi293F cells (a derivative of the 293 human embryonic kidney cell line), umbilical cord mesenchymal stem cells (MSCs), and red blood cells (RBCs). EVs were enriched and purified using a combination of differential centrifugation, gel‐exclusion chromatography, and ultrafiltration techniques. EVs were administered intravenously to Macaca nemestrina (pig‐tailed macaque). Particles were characterized using nanoflow cytometry (NFCM), nanoparticle tracking analysis, microfluidic resistive pulse sensing (MRPS), SDS‐PAGE and immunoblotting, SP‐IRIS, transmission electron microscopy (TEM), and single‐molecule localization microscopy. Immune responses were measured by assessing the levels of IgG responses against intact EVs in macaque plasma. Cytokine production in plasma was checked using an MSD assay. Results : Characterization of EVs confirmed that Expi293F‐ and MSC‐derived EVs exhibited typical morphology, size distribution, and expression of characteristic EV proteins, as assessed by TEM, NFCM, MRPS, and immunoblotting. However, RBC‐derived EVs showed co‐isolation of likely platelet‐derived EVs and displayed a bimodal size distribution, indicating a heterogeneous population. All three EV types elicited measurable immune responses in the macaque models, as evidenced by the production of anti‐IgG antibodies. This uniform immunogenicity across different EV sources indicates that EVs from diverse cellular origins can trigger adaptive immune responses when administered across species barriers. Additionally, ex vivo PBMC biodistribution studies revealed that EVs interacted preferentially with specific blood cell populations, particularly B‐cells (for Expi293F and MSC EVs), but no interaction was found for RBC EVs. Summary/Conclusion : We demonstrated that EVs derived from Expi293F cells, MSCs, and RBCs can induce immune responses in a non‐human primate model. This underscores the importance of thoroughly evaluating the immunogenicity of EV‐based therapies, especially when considering cross‐species applications and careful consideration in the clinical translation of EV‐based therapeutics.

About

With a membership of nearly 2000 individuals spanning the globe, the International Society for Extracellular Vesicles (ISEV) stands as the premier professional organization for scientists and researchers engaged in the exploration of extracellular vesicles (EVs). Established in 2012 in Sweden, ISEV subsequently relocated its headquarters to New Jersey, USA. ISEV is dedicated to fostering global consistency and robustness in EV research, as underscored by the MISEV guidelines of 2014 and 2018 and the update in 2023. The society facilitates this mission through an array of initiatives, including educational offerings, task forces, special interest groups, workshops, and summer schools, while also managing two peer‐reviewed, gold open access journals—the Journal of Extracellular Vesicles and the Journal of Extracellular Biology. A cornerstone of ISEV's activities is its flagship annual gathering, a focal point that provides a crucial avenue for knowledge exchange. By means of its comprehensive programmes and services, ISEV plays an indispensable role in delivering vital training and research prospects for those immersed in the realm of EV research. To provide global leadership in the extracellular vesicle field through research, education, translation, and fostering partnerships. Advancing EV research and applications worldwide.

Acute

Judith Eberz 1 , Elsa Kratz 1 , Lea Walther 1 , Alexandra Brahmer 1 , Carsten Geiß 1 , Tim Ebert 2 , Nils Gassen 2 , Marianne Müller 3 , Eva‐Maria Krämer‐Albers 1 1 Johannes Gutenberg University Mainz, Germany, 2 University Hospital Bonn, Germany, 3 University Medical Center Mainz, Germany Introduction : Extracellular vesicles (EVs) circulating in the bloodstream potentially cross the blood‐brain barrier (BBB), facilitating interaction between the brain and periphery. The composition and kinetics of circulating EVs can correlate with acute brain states and provide insight into the dynamics of neurological disorders, including stress‐related diseases. Since physiological stress in form of acute exercise is known to trigger the release of EVs into the circulation, we asked whether acute mental stress elicits a similar EV response. To this end, we assessed the dynamics of circulating EVs in a cohort of first‐time bungee jumpers and performed multiparametric profiling of EVs in plasma samples. Methods : Blood plasma was collected longitudinally at two time points prior and four time points after bungee jump (−1 week, ‐20 mins, +6 mins, +1 h, +2 h, +4 h, +1 week) and analysed for platelet background and hemolysis according to the MIBlood recommendations. CD63+ EVs were separated from plasma by immuno‐isolation. Phenotyping of CD63+ EVs was performed by multiplex bead‐based flow cytometry using the MACS‐Plex EV‐Immuno and EV‐Neuro assay (Miltenyi Biotec). Data were expressed as fold‐change post versus prior to the jump. Results : Bungee jumping was accompanied by an increase of different EV populations up to 4 h after the jump, followed by a decline to baseline after 1 week. EVs following these kinetics were carrying integrins (CD29, CD49e) and other surface markers characteristic of lymphocytes (CD3, CD45), platelets (CD41b, CD42a) or endothelial cells (CD31, CD105). Brain‐specific markers were not detected among increasing EVs. To reveal the physiological background and trigger of EV release during acute mental stress, we are currently investigating EV kinetics in another cohort subjected to dexamethasone injection and sleep deprivation. Summary/Conclusion : Acute mental stress, as occurring during bungee‐jumping, leads to a transient increase of circulating EVs originating from peripheral cell sources. Monitoring circulating EV profiles could thus inform about distinct stress states. Current and future studies include different stress models to explore relevant EV populations and their functional role in stress signalling and adaptation. Funding : This research was funded by the Leibniz‐Wissenschaftscampus NanoBrain. We thank Miltenyi Biotec for providing us with a prototype of the Neuro‐EV platform.

Apoe4

Presenter: Zhengrong Zhang Mayo Clinic, USA Introduction : Extracellular vesicles (EVs) are key mediators in transferring pathological proteins associated with Alzheimer's disease (AD). The apolipoprotein E (APOE) gene, particularly the ε4 allele, is a major genetic risk factor for late‐onset AD. However, the influence of APOE genotype on the biological characteristics and cargo composition of brain‐derived extracellular vesicles (BDEVs) in AD remains poorly understood. Methods : In this study, BDEVs were isolated from human AD temporal cortex with APOE3/3 and APOE4/4 genotypes ( N = 3–20 per group). Nanoflow cytometry and super‐resolution microscopy were used to evaluate the tau load within these BDEVs. EV‐mediated tau transmission was assessed in aged human Tau KI and APPNL‐G‐F: Tau KI mice in vivo. BDEV uptake and tau seeding were evaluated in iPSC‐derived neuronees through live imaging. Additionally, shotgun lipidomics and data independent acquisition proteomics were analysed for the lipidome and proteome of BDEVs, respectively. Key molecules linked to APOE genotypes were identified through integrated weighted gene co‐expression network and trait correlation analysis. Results : APOE4/4 BDEVs exhibited a higher proportion of phosphorylated tau (pS396) at single EV levels. Upon injection of 300 pg tau of BDEVs into aged Tau KI and APPNL‐G‐F: Tau KI mice, APOE4/4 BDEVs significantly increased AT8+ phosphorylated tau levels and induced neuroinflammation in vivo. Moreover, APOE4/4 BDEVs demonstrated stronger neuronal uptake and tau transfer compared to APOE3/3 BDEVs, impairing the neuronal activity of iPSC‐derived neuronees. Lipidomics revealed that APOE4/4 BDEVs were significantly enriched in pro‐inflammatory long‐chain polyunsaturated fatty acids (PUFA) compared to APOE3/3 with Braak stage association. Furthermore, gene ontology pathway analysis using differentially expressed proteins in APOE4/4 BDEVs showed increased activity in oxidative phosphorylation and sterol‐binding. Multi‐omics integrated data revealed that upregulation of PUFA was significantly associated with elevated cell adhesion molecules and ATPase transporter activity, with neural cell adhesion molecule as one of the key molecules. Notably, neutralizing antibodies against the molecule effectively reduced APOE4/4 BDEV internalization and mitigated EV‐mediated tau pathology in recipient cells. Summary/Conclusion : Our comprehensive analysis suggested that AD APOE4/4 BDEVs may enhance transmissibility of tau pathology and neuroinflammation via enriched pro‐inflammatory PUFA and cell adhesion molecules. Targeting these pathways presents a promising therapeutic strategy to mitigate disease progression, particularly in APOE4 individuals.

Blood

Hilal N. Sensoy *1 , Michelle Tsang‐A‐Sjoe 2 , Niala Masoumi 1 , Monique A. J. van Eijndhoven 1 , Leontine Bosch 1 , Cristina Gomez‐Martin 1 , Joris J. T. H. Roelofs 3 , Yongsoo Kim 1 , Ekaterina S. Jordanova 4 , Kerstin M. Heutinck 5 , Kenny de Wildt 1 , Nils J. Groenewegen 1 , Ineke R. J. ten Berge 5 , Katrien Grunberg 6 , Raymond M. Schiffelers 7 , Jan R. T. van Weering 8 , Sandra A. W. M. Verkuijlen 1 , Irene E. Bultink 2 , Jaap M. Middeldorp 1 , Alexandre E. Voskuyl 2 , D. Michiel Pegtel 1 , and S. Rubina Baglio 1 * Presenting author, 1 Department of Pathology, Amsterdam UMC location Vrije Universiteit Amsterdam, 2 Department of Rheumatology and Clinical Immunology, Amsterdam UMC location Vrije Universiteit, 3 Department of Pathology, Academic Medical Center, University of Amsterdam, 4 Center for Gynaecologic Oncology Amsterdam, Department of Gynaecology, VU University Medical Center, 5 Department of Experimental Immunology, Academic Medical Center, 6 Department of Pathology, Radboudumc, 7 Department of Clinical Chemistry & Hematology, University Medical Center Utrecht, 8 Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University Introduction : Proliferative lupus nephritis (LN) is a severe condition causing impaired kidney function that occurs in 40%–60% of systemic lupus erythematosus (SLE) patients. LN is characterized by a marked interferon (IFN) response in tubular epithelial cells (TEC), but the biological trigger has remained unclear. We previously showed that extracellular vesicles (EVs) released by B cells are enriched in polymerase III (Pol III) transcripts of endogenous and viral (EBV) origin. These transcripts carry pathogen‐associated molecular patterns (PAMPs) that can be recognized by innate RNA sensors. We hypothesize that in LN, due to glomerular damage, high amounts of inflammatory EVs reach the tubular epithelium, inducing an IFN response that exacerbates the disease. Methods : Gene set enrichment analysis was used to identify key pathways and cell signatures in proliferative LN. RNA‐seq was used to define the pol‐III transcriptome of B cell EVs. In situ hybridization was employed to reveal the presence of EBV transcripts in human tissues. In vitro assays were used to uncover the mechanisms of internalization and the functional effect of B cell EVs on TEC cells. Results : scRNA‐seq analysis of proliferative LN identified enrichment of blood cell transcripts, including (EBV‐infected) B lymphocyte RNAs in TEC cells. Notably, the same blood cell signatures were enriched in plasma EVs. SLE patient plasma and purified EVs contained the EBV‐encoded pol III‐transcript EBER1, which selectively accumulated in the cytoplasm of TEC in lupus but not IgA nephritis biopsies. Functional assays showed uptake of B cell EVs by primary TECs in a phosphatidylserine (PtdSer)‐dependent manner. Importantly, B cell EVs deliver EBERs into endosomes of primary TECs, driving IFN gamma, TNF alpha and IL6 production through TLR3 activation. Summary/Conclusion : In SLE patients, blood cell‐derived inflammatory EVs target TEC cells via binding to PtdSer receptors, inducing an IFN‐mediated inflammatory response.

Brain

Presenter: Paniz Shirmast Griffith University, Gold Coast, Australia Introduction : Brain infections and neurological disorders are difficult to treat due to the blood‐brain barrier (BBB), which excludes most drug delivery systems, including lipid nanoparticles (LNPs). Extracellular vesicles (EVs), nanometre‐sized lipid bilayers, offer protection to their cargo and have shown promise in delivering therapeutics safely and efficiently. In this study, we aimed to develop a new class of EV‐based nanoparticles capable of achieving brain biodistribution, with the goal of delivering therapeutic molecules for potential treatment of neurodegenerative diseases. Methods : EVs were collected from HEK293, NSC, and B.END3 cell lines and purified using tangential flow filtration (TFF) with 500 kDa molecular weight cutoff (MWCO) pores. They were further concentrated with Amicon Ultra centrifugal filter tubes (10 kDa MWCO). EVs were characterized following MISEV guidelines using western blot, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). Lipids were extracted from the EVs for lipidomics analysis, and new EVs were formed using the lipids. A GFP‐expressing DNA plasmid was encapsulated using microfluidics. The newly formed EVs were characterized by DLS, transmission electron microscopy (TEM), western blot, and NTA. Packageing efficiency was assessed using PicoGreen and protection assays. Transfection efficiency was measured using flow cytometry, while confocal microscopy tracked nanoparticle entry into cells. Results : Western blot confirmed the presence of EV markers CD63, CD9, and CD81. DLS showed that HEK293, NSC, and B.END3 EVs had sizes of 30.82, 36.23, and 33.26 nm, respectively. After packageing GFP, sizes increased to 72.45, 67.28, and 79.57 nm, respectively. DLS and NTA results were consistent. Western blot revealed changes in the EVs' protein corona, with surface markers removed during lipid‐based EV formation. The PicoGreen assay showed 94.7% packageing efficiency after adding helper lipids. Intravenous injection of the new EVs demonstrated successful brain biodistribution despite changes to the protein corona. Summary/Conclusion : Our results show that the newly formed packaged EVs adhere to MISEV guidelines, effectively preserving their cargo and successfully transfecting it into cells. Additionally, our findings suggest that the EVs can reach the brain, with their biodistribution likely influenced by their lipid profile rather than their protein corona. These EVs hold great potential for the treatment of brain‐related diseases. Funding : Griffith University.

Cargo

Presenter: Seong‐A Kim Korea Institute of Science and Technology, Republic of Korea Introduction : The cGAS‐STING pathway responds to cytosolic DNA within cells in the context of tumour immunity, infection, and various inflammatory diseases, inducing the recruitment and activation of immune cells. DNA damage accumulation leads to the formation of micronuclei and cytosolic material, activating cGAS‐STING. In tumours, this cytosolic material is engulfed by macrophages and dendritic cells, promoting their maturation and activation. Additionally, cGAS‐STING activation in non‐phagocytic cells enhances antitumour immunity. Intrinsic activation of STING within CD8 T cells is essential for generating and sustaining CD8 T cells with a stem‐like phenotype, contributing to durable immune responses. Here, we developed the cargo‐loading strategy into tumour‐derived EVs and investigated how they affected an antitumour effect. Methods : Tumour‐derived extracellular vesicles (EVs) were isolated to study the impact of tumour‐derived EVs on T cell activation and stemness following the MISEV 2023 guidelines. The content within secreted tumour‐derived EVs was quantified after EV isolation. Flow cytometry was conducted to evaluate the activation of the cGAS‐STING pathway in tumour cells and T cells in response to cargo‐loaded tumour‐derived EV both in vitro and in vivo. Modulation of EV secretion in vivo, achieved through blocking EV generation, tumour EV injection, or synthetic cargo complexes, was analysed to determine the impact of EVs on enhancing T cell stemness in vivo. Results : We observed an increase in the cargo following the induction of cargo loading in tumour cells. The cargo‐loaded EVs activated the cGAS‐STING pathway and promoted immune response in tumour cells, an effect not observed with EVs without inducing cargo loading. The cargo‐loaded tumour‐derived EVs enhanced the proportion of cGAS‐STING‐activated TCF1+ CD8 T cells in tumour microenvironment. Inhibition of EV secretion reduced immune cell uptake of tumour‐derived EVs, particularly by CD8 T cells, and decreased the proportion of TCF1+ CD8 T cells in vivo. Injection of cargo‐loaded tumour EVs promoted cGAS‐STING activation and increased TCF1+ CD8 T cells within the tumour. Summary/Conclusion : The induction of cargo loading in tumour increased the cargo loaded into tumour‐derived EVs. The cargo‐loaded tumour‐derived EVs enhanced antitumour immune response by activating the cGAS‐STING pathway and upregulating TCF1 expression in CD8 T cells.

Cd169

Marialaura Fanelli 1 , Vita Petrone 1 , Rossella Chirico 1 , Luigi Coppola 2 , Chiara Sorace 2 , Chiara Cipriani 1 , Martina Giudice 1 , Claudia M. Radu 3 , Elisabetta Teti 2 , Vincenzo Malagnino 2,4 , Marco Iannetta 2,4 , Sergio Bernardini 1 , Emanuela Balestrieri 1 , Loredana Sarmati 2,4 , Sandro Grelli 1,5 , Antonella Minutolo 1* , Claudia Matteucci 1* 1 Department of Experimental Medicine, University of Rome Tor Vergata, Rome, 00133, Italy; 2 Infectious Diseases Clinic, Policlinic of Tor Vergata, Rome, 00133, Italy; 3 Thrombotic and Hemorrhagic Diseases Unit Department of Medicine—DIMED University of Padua, Italy; 4 Department of Systems Medicine, University of Rome Tor Vergata, Rome, 00133, Italy; 5 Virology Unit, Policlinic of Tor Vergata, Rome, 00133, Italy. Introduction : Elevated inflammation and immune dysregulation are main consequences of SARS‐CoV‐2 infection. The dysregulated inflammatory state persists after COVID‐19, generating the post‐acute sequelae of SARS‐CoV‐2 infection in Long COVID individuals (LC). The role of CD169 as a marker in the early diagnosis of SARS‐CoV‐2 infection and its association with severity and clinical outcome was demonstrated in COVID‐19 patients (COV). We evaluated the expression of CD169 and HLA‐DR on leukocytes subpopulations and on circulating extracellular vesicles (EVs) from COV and LC to better elucidate their involvement in immunological dysfunction. Methods : Blood samples from COV, LC, and Healthy Donors (HDs) were collected at Tor Vergata University Hospital of Rome. Leukocytes subpopulations and circulating EVs were characterized for HLA‐DR and CD169 expression in COV, LC and HD through flow cytometry (CytoFLEX). The different markers analysed were associated with clinical and biochemical parameters in COV and LC. Results : CD169 RMFI was found significantly higher in COV than in HDs and LC, resulting in a good marker of viral infection. Whereas the percentage of CD169+ monocytes was high in COV and LC, and the percentage of HLA‐DR+ monocytes was low with respect to HD. Notably, the percentage of activated monocytes CD169+HLA‐DR+ were high in COV and persisted in LC. The percentage of HLA‐DR+CD169+ monocytes correlated inversely with a‐PTT ratio in COV, and directly with the numbers of platelets in LC. The percentage and number of HLA‐DR+CD169+ EVs were significantly elevated in COV and, although lower, persist in LC compared to HD. The analyses of different size EVs in COV, LC and HD showed a different expression of HLA‐DR and CD169 markers. Summary/Conclusion : CD169 RMFI and myeloid activation markers were confirmed as predictive markers of COVID‐19, and these markers persisted in LC. This data opens a new possibilities for studying the use of CD169 and HLA‐DR at cellular and EVs level as markers during acute infection or in post‐infection sequelae. Funding : Funded by HERVCOV project (GA101057302) HORIZON‐HLTH‐2021‐DISEASE‐04 Personalised medicine and infectious diseases: understanding the individual host response to viruses.

Donor

Presenter: Karyna Tarasova Veterinary Medicine University of Vienna, Austria Introduction : Mesenchymal stem cells (MSCs) demonstrate promising therapeutic effects for osteoarthritis, predominantly mediated by paracrine signals. Notably, foetal and perinatal cells exhibit greater efficacy than adult cells. This study evaluates the therapeutic efficacy of extracellular vesicles (EVs) derived from foetal and perinatal MSCs on inflamed adult ovine chondrocytes and synoviocytes in vitro. Methods : EVs were isolated from the supernatant of ovine foetal umbilical cord‐derived MSCs (fMSCs) and chondrocytes (fCCs) ( n = 4 biological replicates) and immortalized human perinatal MSCs cell lines (Wharton's jelly (WJ‐MSC/TERT273) and amniotic membrane (P‐MSC/TERT308)), which had been cultured in serum‐free medium in hollow fibre bioreactors, using tangential flow filtration (300 kDa cut‐off). EVs were characterized for particle size distribution and number and marker proteins. The therapeutic effects of foetal and perinatal‐derived EVs (1 × 10 9 particles/mL) were assessed on inflamed (1 ng/mL TNF‐α and IL‐1β) chondrocytes in 2D and 3D culture. Healthy, inflamed untreated and inflamed dexamethasone‐treated chondrocytes served as controls. Multi‐omics analyses (RNA‐Seq, miRNA‐Seq, mass spectrometry proteomics) were employed as readouts. Results : All 4 EV types were effectively internalized by inflamed and healthy chondrocytes, with no difference between allogeneic and xenogeneic EVs. Inflammation significantly increased uptake of all 4 EV types. Notably, fCC‐derived EVs exhibited the highest uptake. All treatments significantly reduced the inflammatory response at 24 h post‐treatment, comparable to dexamethasone. Ingenuity pathway analysis revealed that while all 4 EVs inhibited ‘HIF1A signaling,’ foetal and perinatal EVs showed distinct pathway modulation. Foetal EVs uniquely downregulated the ‘neutrophil extracellular trap signalling,’ ‘HOTAIR regulatory,’ and ‘leukocyte extravasation signalling’ pathways, with fCC‐EVs leading to the highest activation of ‘sirtuin signaling.’ Foetal MSCs and fCCs derived EVs showed a superior pro‐regenerative effect compared to perinatal‐derived EVs with significant upregulation of several mRNAs (aggrecan (ACAN) and collagen type II alpha 1 (COL2A1)). Summary/Conclusion : Treatment outcomes demonstrated clear distinctions based on donor age (foetal vs. perinatal) and cell differentiation (MSC vs. articular chondrocytes). Selection of EVs sources tailored to specific clinical needs may thus optimize therapeutic efficacy in osteoarthritis treatment.

Drugs

G. Cerrotti 1 , S. Buratta 1,2 , R. M. Pellegrino 1 , H. Alabed 1 , P. Gorello 1,3 , E. Calzoni 1 , C. Emiliani 1,2,3 , L. Urbanelli 1,2,3 1 Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; 2 Extracellular Vesicles network (EV‐net) of the University of Perugia, Italy; 3 CEMIN – Center of Excellence for Innovative Nanostructured Material, University of Perugia, Perugia, Italy Introduction : The endolysosomal system plays an important role in the biogenesis and secretion of EVs. We analysed EVs release of cell treatment with drugs impairing the autophagic/lysosomal system by administering to cells (1) chloroquine (CQ), commonly used to impair autophagy by autophagosome‐lysosome fusion blockade; (2) bafilomycin A1 (BafA1), known to disrupt autophagic flux by independently inhibiting V‐ATPase‐dependent lysosomal acidification and Ca‐P60A/SERCA‐dependent ER calcium transport. Methods : EVs were isolated from cell medium by differential ultracentrifugation and further separated by density gradient. Small EVs were collected, characterized morphologically by SEM, quantified by NTA, and their markers assessed by IB. Their lipid content was analysed by LC/MS. The biological anti‐inflammatory effects of these vesicles on THP‐1 were also assayed. Results : BafA1 and CQ both induce the release of EVs enriched in autophagy markers in several cell types, but interestingly only BafA1 induced the release of a remarkably higher number of small EVs. Baf A1‐EVs were also less dense, and their lipid composition was consistent with an enrichment of the general lipid content. Therefore, lysosomal alkalinization, induced by both drugs, is associated with the release of EVs carrying autophagy markers, but only Baf A1, an inhibitor of the ER calcium pump SERCA, increases the number of EVs. We extended our search for autophagy markers associated with EVs to biological fluids. EVs from plasma of healthy subjects and individuals with sickle cell and thalassaemia traits revealed the presence of autophagy markers. Summary/Conclusion : Although Baf A1 and CQ are drugs commonly used to inhibit autophagy, their role in prompting EVs release is usually not considered but may have implications in pathologies characterized by impairment of the autophagic/lysosomal system, such as age‐related neurodegenerative disorders. Funding : This work has been funded by the European Union‐NextGenerationEU under the Italian MUR National Innovation Ecosystem grant ECS00000041 – VITALITY (to Carla Emiliani). The authors acknowledge Università degli Studi di Perugia and MUR for support within the project VITALITY. This manuscript reflects only the authors’ views and opinions; neither the European Union nor the European Commission can be considered responsible for them.

Early

Presenter: Juan P. Hinestrosa Xzom USA Abstract unavailable

First

Kameswari Priyanka Devarakonda 1 , Csenge Szász 2 , Ani Barbulova 1 , Angela Mary Joseph 1 , Immacolata Fiume 1 , Veronika Kralj‐Iglic 3 , Anna Romolo 3 , Lilla Turiák 4 , Mirjam Balbisi 4 , Gabriella Pocsfalvi 1 1 National Research Council of Italy, Institute of Biosciences and BioResources, Italy. 2 Pediatric Center, MTA Center of Excellence, Semmelweis University, Hungary. 3 University of Ljubljana, Slovenia. 4 HUN‐REN Research Centre for Natural Sciences, Hungary. Introduction : Plant cell suspension culture (CSC) derived extracellular vesicles (EVs) less studied than mammalian EVs in vitro cultures. The outstanding plasticity of plant cells makes plant EVs an interesting system to study from the point of view of EV secretion. Here, we optimised the EV isolation from the model Nicotiana tabacum Bright Yellow‐ 2 (BY2) plant CSC to determine the molecular cargo and improve understanding on the biogenesis of EVs. Methods : The cellular growth of BY2 cells was studied by counting, fluorescein diacetate (FDA) based metabolic viability measurements and packed cell volume. EVs were harvested from conditioned cell media at initial exponential phase of growth at maximum metabolic activity. BY2 EVs isolation was performed by differential ultracentrifugation by optimising different buffers and conditions to achieve maximum EV yield. The isolate was purified by size exclusion chromatography or tangential flow filtration. BY2 EVs were separated into BY2 EVs and EVs fractions by Density gradient ultracentrifugation (DGUC) later were characterised by protein content, particle size and distribution by interferometric light microscopy, morphology by cryoTEM. Mass spectrometry based proteomic analysis and bioinformatic analyses were performed to identify proteins. Results : Out of four different buffers and 50 mM citrate pH 5.8 resulted in good yield and less copurifying extracellular matter. Three different scales of BY2 isolation resulted in lowest volume scale (5 mL starting volume) with the highest EV yield. DGUC led to two main EV populations: the low density at 1.06‐1.08 g/mL and high density at 1.12‐1.13 g/mL. Initial proteomic analysis identified 50 proteins. Interestingly most of the proteins were enzymes, amongst these, top ranking proteins were beta‐xylosidase, tetrahydrocannabinolic acid synthase‐like and subtilisin‐like protease SBT1.8. Summary/Conclusion : Three different scales of BY2 isolation will facilitate different aspects of plant EV structural, biological and molecular studies. We speculate on possible secretion mechanisms based on the putative roles of the protein content on the EVs. The optimised conditions let us to isolate BY2 EV at 1ug/mL with 2.33x108particles/mL at the lowest scale. Proteomic and bioinformatics studies let us to hypothise interesting insights into the EV secretion. Funding : This work was supported by the European Union's Horizon 2020 “nutriEVs” grant agreement N. 101109583.

Frail

Presenter: Magdalena Sepulveda University of Talca, Talca, Chile Introduction : During ageing, several physiological systems are altered, with emerging frailty representing a significant cardiovascular disease risk factor. Among the scales to determine frailty are the Fried Phenotype and Frailty Trail Scale (FTS), but it is also important to evaluate the presence of procoagulant platelets and extracellular vesicles. This study aimed to determine the levels of procoagulant platelets and the concentration of extracellular vesicles in frail and non‐frail older women. Methods : A blood sample with anticoagulant sodium citrate 3.2% of 50 older women was taken and centrifuged to obtain platelet‐rich plasma (PRP) and platelet‐poor plasma (PPP). PRP was used to analyse the exposure of phosphatidylserine (PS) and intracellular calcium levels. PPP was used for the analysis of extracellular vesicles by ultracentrifugations. The concentration of extracellular vesicles was determined using the Nieuwland's formula. These activation markers were measured using fluorescently labelled antibodies and flow cytometry and expressed as Median Fluorescence Intensity (MFI). Results : In the studied population, the average age was 73.46 years, and 66% of the women had abdominal obesity. According to the 25‐point cut‐off, 74% were non‐frail and 26% were frail, whereas with the 7‐point cut‐off, 26% were non‐frail and 74% were frail. When the cut‐off score was 25 points for FTS‐5, no significant differences were found in levels of PS and calcium levels, procoagulant platelets and extracellular vesicles. There were significant differences in the concentration of extracellular vesicles positive for Annexin V/P‐Selectin/Mitochondria (AV+/CD62P+/Mitochondria+) ( p value 0.0084), positive for Annexin V/GPIIb/Mitochondria (AV+/CD41a+/Mitochondria+) ( p value 0.0151) and positive for Annexin V/GPIIIa/Mitochondria (AV+/CD61+/Mitochondria+) ( p value 0.0294), where it can be observed that older women with cut‐off ≥ 7 points have a higher concentration of extracellular vesicles. Summary/Conclusion : A cut‐off score ≥ 7 points for FTS‐5 could represent a novel tool for evaluating the behaviour of platelets in older people, which present procoagulant platelets and higher concentrations of extracellular vesicles than cut‐off < 7 points, which could constitute a cardiovascular risk factor. Funding : This research was funded by ANID Scholarship grant N° 21230657, ANID/FONDECYT grant N° 1220339 and was also supported by ANID‐Anillo ACT210097 (MIBI: Interdisciplinary Group on Mitochondrial Targeting and Bioenergetics). Also, we thank ANID/FONDEQUIP N° EQM200049 (Flow Cytometry) and ANID/FOVI240035.

Fully

Presenter: Bifeng Liu Huazhong University of Science and Technology, Wuhan, People's Republic of China Introduction : Cancer is a leading cause of global mortality. Recent studies have demonstrated that EVs play an irreplaceable role in cancer progression. To develop an efficient method for EVs analysis would be greatly helpful for cancer identification in clinics. The emerging microfluidic technology is anticipated to become an ideal approach for EVs analysis due to its advantages in miniaturization, automation, integration, and so forth. In this presentation, we interestingly demonstrated fully integrated microfluidic systems for EVs analysis. Coupled with AI, those methods have been successfully applied to cancer identification. Methods : Here, we present a novel fully integrated lab‐on‐a‐disc (iExoDisc) that isolates exosomes directly from blood samples. This platform consists of three integrated modules: plasma separation, highly abundant protein removal, and nanopore membrane‐based total isolation module. We applied iExoDisc to isolate exosomes from clinical samples and validated its performance by high‐throughput MALDI‐TOF‐MS and lectin microarrays for glycan profiling of plasma exosomes. For enhancing EVs detection, a rapid and visually detectable method termed freeze‐thaw‐induced floating patterns of gold nanoparticles (FTFPA) is proposed, which surpasses current state‐of‐the‐art technologies by achieving a 100‐fold improvement in the limit of detection of EVs. Results : The findings from the microfluidic systems indicated that galactosylation and sialylation exhibit potential as diagnostic indicators for triple‐negative breast cancer (TNBC). FTFPA could accurately identify EVs derived from subtypes of breast cancers with AI algorithms. Intriguingly, learning the freezing‐thawing‐microstructures of EVs with a random forest algorithm is not only able to distinguish their original cell lines (with an accuracy of 95.56%) but also succeeds in processing clinical samples ( n = 156) to identify EVs by their healthy donors, breast lump and breast cancer subtypes (Luminal A, triple‐negative breast cancer, and Luminal B) with an accuracy of 83.33%. Summary/Conclusion : The iExoDisc for Evs analysis paves a new avenue to early cancer detection and point‐of‐care diagnosis. The AI‐empowered micro‐visualization method establishes a rapid and precise point‐of‐care platform that is applicable to both fundamental research and clinical settings. Funding : National Key Research and Development Program of China (2021YFA1101500, 2023YFF1205002); National Natural Science Foundation of China (22074047, 32171248, 12102142).

Hairy

Filippo Maltoni 1,2 , Alessandro Broccoli 1,2 , Ghazal Narimanfar 1 , Simone Tiberi 1 , Roberto Maria Pellegrino 3 , Martina Barone 2 , Dorian Forte 1 , Giulia Gabrielli 1,2 , Lisa Argnani 1 , Carla Emiliani 3 , Pier Luigi Zinzani 1,2 , Lucia Catani 1,2 1 University of Bologna, Italy; 2 IRCCS Azienda Ospedaliero‐Universitaria di Bologna, Italy; 3 University of Perugia, Italy Introduction : Hairy cell leukaemia (HCL) is a rare and incurable lymphoproliferative B‐cell tumour characterized by infiltration of malignant hairy cells (HCs) within bone marrow. No disease‐specific biomarkers have yet penetrated clinical practice. Small (S‐) and large (L‐) extracellular vesicles (EVs) are particles promoting intercellular communication with a role in the ‘education’ of tumour niche. However, their function in HCL is unknown. Moreover, studies integrating protein, lipid and mRNA cargo of different plasma EV populations from (rare) cancer patients are completely lacking. Methods : L‐ and S‐EVs were isolated from plasma of 8 HCL patients and 10 healthy donors (HD) by ultracentrifugation and characterized for surface protein expression (flow cytometry), lipid (mass spectrometry) and mRNA cargo (sequencing). Results : Comparing HCL S‐ and L‐EVs, the former were enriched in immune (CD8, CD25), cancer/stem cell (CD326, CD133‐1), and EV (CD9, CD63, CD81) markers, while the latter resulted enriched in CD42a (platelet marker). Notably, CD25 is an HC marker. L‐EVs were also significantly enriched in phosphatidylethanolamine (PE), plasmenyl phosphatidylethanolamine (PE‐P), phosphatidylserine (PS), and phosphatidylinositol (PI) lipid classes. Compared to HD EVs, HCL S‐ and L‐EVs overexpress B (CD19, CD20) and T (CD8) cell markers and the CD44 adhesion molecule. HCL S‐EVs are enriched in HC (CD25), monocyte (CD14), and endothelial (CD105) markers. Lipidomic analysis revealed a significant increase in PE, PE‐P, and PS in both HCL EV subpopulations, with HCL L‐EVs also enriched in sphingomyelin. Exploring mRNA content, we found clear separation between S‐ and L‐EVs and between HCL and HD samples and revealed that S‐EVs carry transcripts of various lengths, while L‐EVs rarely express the shortest ones. Gene Ontology analysis showed that, comparing HCL S‐ and L‐EVs, the latter are enriched in transcripts encoding membrane structures. Comparing patients and HD, we found that HCL L‐EVs were enriched in transcripts related to HCL B‐cell origin/function and vesicle biogenesis, while HCL S‐EVs showed higher expression of transcripts related to cytoplasmic components, RNA splicing and B‐cell origin/function. Summary/Conclusion : We identified a tumour‐associated signature of circulating HCL EVs that provides pathogenesis‐related information and highlights EVs for future liquid biopsy approaches in rare tumours. Funding : The work was funded by the Italian Ministry of Health, RC‐2025‐2797531.

Human

Juliana Fortes Di Iorio 1 , Anna Julia Pietrobon 2 , Nadjania Saraiva de Lira Silva 1 , Maria Notomi Sato 2 , Ana Claudia Torrecilhas 1 1 Universidade Federal de São Paulo (UNIFESP); Instituto de Ciências Ambientais, Químicas e Farmacêuticas; Departamento de Ciências Farmacêuticas; Labouratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. 2 Laboratory of Dermatology and Immunodeficiencies, LIM‐56, Department of Dermatology, Tropical Medicine Institute of São Paulo, University of São Paulo Medical School, São Paulo, Brazil Introduction : Chagas disease is caused by T. cruzi affects approximately 6 to 7 million people in America. Diagnosis, treatment, and control of the disease offer significant difficulties to eliminating Neglected Tropical Diseases in endemic areas. T. cruzi shed EVs at different phases of the life cycle, that affect the innate immune via TLR2 in host cells. Monocytes and macrophages play an important role in starting innate as well as adaptive immune responses during T. cruzi infection. Methods : THP‐1 cells incubated with EVs‐Tcruzi for 24 and 48 h, fixed and using scanning electron microscopy (SEM). The EVs labelled with PKH26 and after interaction with THP‐1 cells at 0, 5, 15, 30 min, 1, 24, and 48 h (confocal microscopy and flow cytometry‐Amnis ImageStream). Human monocyte isolated from peripheral blood PBMCs and treated with EVs‐Tcruzi and performed by flow cytometry to detected markers (CD14, CD16, CD80, CD86, CD69, HLA‐DR). The invasion assay, monocytes incubated with or without EVs and used to two distinct parasite concentration and measured the number of infected cells and intracellular parasites. Results : EVs‐Tcruzi enhance intracellular parasites and differentiation of monocytes in macrophages. In parallel, we found NF‐kB translocation using confocal microscopy, as demonstrated by endogenous GFP expression. NF‐kB pathway activation observed 30 min after interaction, which was consistent with ImageStream's results. We found an established connection between EV uptake and NF‐kB activation as the cells. We detected that monocytes uptake more EV‐Tcruzi than lymphocytes after 24 h. In parallel, we observed changes in the monocyte activation profile and increased expression of CD14, CD16, CD80, CD86, CD69 and HLA‐DR performed by flow cytometry. Summary/Conclusion : Our results demonstrate that T. cruzi‐derived EVs interact with monocytes, activating the NF‐kB pathway and stimulating an increase in cell area and complexity that results in monocytes differentiation into macrophages. These data provide new evidence that EVs shed by T. cruzi interact with host cells, acting as a booster in infection, modulating the immune response. Funding : Support by CAPES, CPNq e FAPESP 2020/07870‐4

Icam5

Presenter: Manish Bhomia Uniformed Services University of the Health Sciences, USA Introduction : Currently, markers reported in literature to isolate neuronal ‐derived extracellular vesicles (NEVs) lack specificity to the human central nervous system (CNS). Additionally, a proportion of these markers may exist as soluble proteins, further limiting their value. To address this research gap, we have identified a novel neuronal marker, intercellular cell adhesion molecule‐5 (ICAM5), that is highly specific to the CNS and can be used to isolate specific NEVs from human biofluids. Methods : Mass spectrometry (MS) was performed using EVs isolated from primary human neuronal cell cultures. Novel EV markers identified from this screen were validated in control human serum and CSF (BioIVT Inc.). Size exclusion chromatography (SEC) was performed, and EVs were probed for CD63, CD81 and novel NEV markers. Electron microscopy (EM) was performed to confirm the NEVs in immunoprecipitated samples using a novel marker. Super‐resolution microscopy (SRM) was performed to demonstrate the surface presence of the NEV marker. Clinical TBI samples were analysed to quantify neurological injury markers in immunoprecipitated NEVs. Results : The MS screen identified 63 proteins that were highly enriched and specific for the human brain, and ICAM‐5 was a potential NEV marker. Size exclusion chromatography confirmed the presence of ICAM‐5 in EVs in the same fractions as EV‐associated tetraspanins (CD‐63 and CD‐9) in human serum and cerebrospinal fluid samples. Further, EVs immunoprecipitated with ICAM‐5 antibody and confirmed with EM showed the presence of neuronal markers such as tau and neuronal specific enolase (NSE) and were negative for glial markers on a western blot. SRM analysis confirmed the presence of ICAM5 on the surface of EVs in both CSF and serum samples and colocalizing with tetraspanins. Finally, ICAM‐5 NEVs showed enhanced expression of UCH‐L1 in serum samples from patients with mild TBI. Summary/Conclusion : Our data demonstrates that ICAM‐5 is present on the surface of EVs and is a novel and specific marker for enrichment of NEVs from human biofluid. Funding: Disclaimer The opinions expressed herein are those of the author(s) and are not necessarily representative of those of the Uniformed Services University of the Health Sciences, the Department of Defense, or the United States Army, Navy, or Air Force. Funding : National Institutes of Health.

Lipid

Alissa Rumin 1 , T. Chase White 1 , Dinah Loerke 1 , Ann M. Wehman 1,2 1 University of Denver, Denver, Colourado, USA; 2 Texas A&M University, College Station, Texas, USA Introduction : Microvesicle or ectosome budding from the plasma membrane is poorly understood. We previously discovered a conserved inhibitor of extracellular vesicle (EV) release from the plasma membrane, the C. elegans P4‐ATPase TAT‐5. TAT‐5 acts as a lipid flippase to maintain phosphatidylethanolamine (PE) asymmetry in the membrane bilayer. When TAT‐5 activity is lost, PE is exposed on the surface of cells, ESCRT proteins are recruited to the cytosolic face of the plasma membrane, and EV budding increases a 100‐fold. TAT‐5's essential flippase activity is required for embryonic development and fertility, but how PE lipids regulate EV release or other physiological processes is unclear. Methods : PE lipids are often polyunsaturated, which can alter physical properties of the membrane, such as increasing membrane fluidity. Therefore, we asked whether disrupting lipid desaturases that form double bonds in fatty acid tails would alter EV release. We also performed temperature shift experiments to alter membrane fluidity. Results : We found a three‐fold decrease in EV release after disrupting several lipid desaturases, suggesting that long‐chain (C20) or polyunsaturated lipids promote EV release. As these lipids promote membrane fluidity, these findings raise the possibility that PE exposure could alter membrane fluidity by decreasing the saturation of the outer leaflet of the membrane bilayer. Furthermore, worms raised at 23°C release significantly fewer EVs after a 4‐h shift to 20°C and significantly more EVs after a 4‐h shift to 26°C. Summary/Conclusion : The temperature shift experiments and lipid desaturate knockdown experiments are consistent with changes in membrane fluidity altering plasma membrane budding. These studies also highlight the importance of maintaining consistent feeding and temperature conditions for quantitative studies of EV release. Funding : Allen Distinguished Investigator grant, NIH R35GM152234.

Lmtk3

Presenter: Mark Samuels Zhejiang Chinese Medical University, China (People's Republic) Introduction : Breast cancer (BC) remains a leading cause of cancer mortality, with extracellular vesicle (EV)‐mediated communication between BC cells and the innate immune system playing a crucial role in all aspects of the disease. Lemur tail kinase 3 (LMTK3) is an oncogene frequently upregulated in BC, driving therapy resistance, invasion and metastasis, ultimately worsening patient outcomes. Building on preliminary data linking LMTK3 to endosomal trafficking and immunomodulation, this study investigates the roles of LMTK3 in EV biogenesis and EV‐mediated communication between BC cells and the innate immune system. Methods : EVs were collected from control and LMTK3‐overexpressing BC cells using differential ultracentrifugation, then their proteins and physical characteristics were thoroughly characterised on a single particle and population level. Biochemical assays and microscopy techniques were used to identify how LMTK3 affects EV biogenesis. EVs were also used in 3D coculture models to assess how LMTK3‐dependent EV cargo alters monocyte infiltration, macrophage polarisation and anti‐cancer activity. Bioinformatics analysis of TCGA datasets validated results using clinical data. Results : LMTK3 upregulation induces the production of large EVs enriched in oncogenic and immunoregulatory proteins. Single particle analysis revealed changes in the proportions of different EV subpopulations as measured by the abundance of tetraspanins. We identified Rab7 as an in vivo substrate of LMTK3 where its phosphorylation results in altered late endosomal trafficking, inhibiting degradation of cargo and favouring release of EVs. Functionally, EVs from LMTK3‐overexpressing but not control BC cells caused metabolic changes in monocytes, shifting macrophages towards and M2‐like polarisation and reducing monocyte infiltration. Summary/Conclusion : LMTK3 overexpression in BC correlates with poor prognosis and reduced overall survival. This may partly be due to macrophage M2 polarisation and exclusion of M1 macrophages from the tumour microenvironment, mediated by LMTK3‐dependent alterations in EV cargo. Furthermore, we demonstrate a novel mechanism of Rab7 regulation by LMTK3, impacting EV cargo selection and multivesicular body trafficking dynamics.

Mirna

Chamilani Nikapitiya, E. H. T. Thulshan Jayathilaka, Mawalle Kankanamge Hasitha Madhawa Dias, Hyun‐Jin Shin, and Mahanama De Zoysa College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea Introduction : EVs have been divided into medium/large EVs (>200 nm) and small EVs (< 200 nm) by the minimal information for studies of extracellular vehicles (EVs) 2018/2023 guidelines. World Organization for Animal Health (OIE) has listed viral haemorrhagic septicaemia (VHS) as an important contagious viral disease that influences wild or farmed fish worldwide. The etiological agent of VHS is the viral hemorrhagic septicemia virus (VHSV). Understanding the molecular mechanisms involved in VHSV infection is important for identifying new therapeutic targets against the infection. This study aimed to characterize (physicochemical and molecular level) the plasma‐derived exosomes from olive flounder (Paralichthys olivaceus) upon VHSV challenge and phosphate buffered saline (PBS) injection (VHSV‐Exo and PBS‐Exo, respectively) to find the VHSV effect on the exosomes content. Methods : The exosomes were isolated using the differential ultracentrifugation method and characterized from PBS injected and VHSV challenged olive flounder plasma. Total RNA was isolated, small RNA sequencing libraries were constructed and libraries were sequenced. To profile the miRNAs expression patterns of VHSV‐Exo and PBS‐Exo, first the expression levels of miRNAs were normalized and then differentially expressed (DE) miRNAs were analysed using DESeq2. DE miRNAs were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses for functional and pathway enrichment analysis. Results : Nanoparticle tracking analysis revealed a lower particle size and higher concentration of VHSV‐Exo compared to the PBS‐Exo group. Both comprised exosome markers (CD9, CD81, and CD63). We identified 48 known miRNAs and the highly abundant miRNA in both groups was pol‐miR‐21‐5p/hhi‐miR‐21. Among 48, one miRNA (hhi‐miR‐1788) was specific to the VHSV‐Exo group. Furthermore, six miRNAs were upregulated while seven were downregulated (fold change > 1.2 or < 0.83; q value < 0.05). Enrichment analysis indicated that VHSV mainly regulates transcription, cell‐cell adhesion, cell‐matrix adhesion, and cytokine‐mediated signalling pathways. Summary/Conclusion : Our findings indicated that VHSV infection influenced the miRNA expression profile, and identified DE miRNAs could be considered for the diagnosis of VHSV infection in fish. Further validation of these miRNAs assist in developing novel engineered therapeutics together with exosomes as drug delivery carriers in the future. Funding : This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) 2021R1A2C1004431 and RS‐2024‐00347938.

Model

Presenter: Joyce Rops Amsterdam UMC, Amsterdam, The Netherlands Introduction : Flow cytometry is a widely used method to measure concentrations of extracellular vesicles (EVs) in blood plasma and other biofluids. With flow cytometry, fluorescent labelling is used to identify EVs in plasma, but non‐specific binding of labels to lipoproteins may result in overestimation of the measured EV concentration. Here we developed a model to correct the measured EV concentration for non‐specific binding to lipoproteins. Methods : A logistic regression model was developed and fitted to flow cytometry data from the Antiplatelet Therapy Effect on Extracellular Vesicles (AFFECT EV) study, which uses the labels cluster of differentiation (CD)31, CD45, CD61, CD62p, CD146, CD235a, lactadherin and fibrinogen. To validate the model, EVs and lipoproteins were identified using the flow cytometry scatter ratio (Flow‐SR), a label‐free method that can distinguish EVs from lipoproteins based on their difference in refractive index (RI). Results : We tested the model on all data from the AFFECT EV study. Here, preliminary data from CD45‐positive particles will be used as a representative example. CD45‐positive EV concentration values, corrected for false‐positive events using both the model and Flow‐SR, were in close agreement with each other (0.98 ± 0.38). Of all CD45‐positive particles with a diameter > 200 nm that were identified as CD45‐EVs, 70% were classified as EVs by the model and 72% according to Flow‐SR. The results confirm that concentrations of labelled particles in plasma should be corrected for false‐positive events. Summary/Conclusion : Commonly used fluorescent labels, including monoclonal antibodies, do not only label EVs but also lipoproteins in plasma. The developed model corrects for false‐positive events and can therefore be used to improve the accuracy of EV concentration measurements with flow cytometry. In contrast to Flow‐SR, which can only be applied to EVs > 200 nm using a limited number of flow cytometers, our new model can be applied to all EV sizes measured by all flow cytometers. After validation using spike‐in measurements with lipoproteins, this model can be used in clinical research studies and possibly applied to other biofluids containing EVs and non‐EV particles. Funding : The authors acknowledge the Dutch Heart Foundation for funding this work.

Nat10

Presenter: Zheng Tong The Third‐affiliated Hospital of Sun Yat‐sen University, China (People's Republic) Introduction : Chronic kidney disease (CKD), derived from acute kidney injury (AKI), causes irreversible renal failure. Therefore, inhibiting AKI‐CKD transition is clinically vital. Vascular rarefaction, induced by apoptosis of vascular endothelial cell (VEC), is a key contributor of AKI‐CKD transition. Unfortunately, the mechanism of vascular rarefaction following AKI remains unknown. Methods : The in vitro model of TEC(tubular epithelial cell)‐VEC communication was used to discover the role of exosome in TEC‐VEC kill chain. In vitro screen with epigenetic inhibitor library was performed to identify the decisive mediator of TEC‐exosome‐VEC kill chain. Conditional knockout mice was used to verify the in vivo role of NAT10 in vascular rarefaction and AKI‐CKD transition. acRIP‐seq and RNA‐seq were applied to investigate the mechanisms of NAT10. Results : The injured TEC induced VEC apoptosis depending on exosomes. More importantly, targeting NAT10 significantly breaks the TEC‐exosome‐VEC kill chain. Conditional NAT10 depletion in TEC reversed vascular rarefaction and AKI‐CKD transition. Mechanistically, NAT10 constructed the TEC‐exosome‐VEC kill chain via strongly elevating the exosomal abundance of THBS1 and FBN1, the pro‐apoptotic protein of VEC. Specifically, NAT10 upregulated THBS1 and FBN1 via catalyzing mRNA ac4C. Meanwhile, NAT10 favoured package of FBN1 protein into exosomes via catalyzing protein acetylation. In addition, we found that TGF‐β and stiffening stabilized mRNA and protein of NAT10, upregulating NAT10 in succession after AKI. Summary/Conclusion : After AKI, the injured TEC causes vascular rarefaction and AKI‐CKD transition via exosomes. NAT10, which is upregulated by TGF‐β and stiffening in injure TEC, initiates and strengthens the TEC‐exosome‐VEC kill chain via increasing exosomal abundance of THBS1 and FBN1. NAT10 orchestrates mRNA ac4C modification and protein acetylation to upregulated THBS1 and FBN1 and to favour FBN1 loading into exosomes. Therefore, targeting NAT10 with Remodelin is a promising strategy for vascular rarefaction in AKI‐CKD transition. Funding : National Natural Science Foundation of China (No. 82170771, No. 82370756, No. 82100799)

Novel

Gabriela Zavala 1 ; Pablo Berríos 2 ; Felipe Sandoval 1 ; Sebastian Aguayo 2,3 ; Christina Schuh 1 1 Centro de Medicina Regenerativa, Facultad de Medicina, Clínica Alemana‐Universidad del Desarrollo, Santiago, Chile 2 School of Dentistry, Faculty of Medicine, UC Chile. 3 Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, UC Chile Introduction : Neuroinflammation is caused by everyday noxae such as air pollution, trauma or SARS‐COV‐2, among others. Continuous exposure results in the activation of microglia, leading to changes in membrane fluidity, increased motility and secretion of pro‐inflammatory cytokines, which can result in neuronal damage. Recently, our group discovered EVs in Royal Jelly (RJEVs)‐ a bee product with known anti‐neuroinflammatory properties. Until now, it is not known how Royal Jelly exerts its anti‐inflammatory effects. Aim of this study was to investigate the effects of RJEVs on LPS‐activated microglia, focusing on mechanical properties, motility and modulation of the inflammatory response. Methods : RJEVs (isolation: Vivaspin (100 kDa), ultracentrifugation) were characterized with NTA, TEM and proteomics. RJEV uptake into ctrl and LPS‐stimulated HMC‐3 microglia were assessed with inhibitors and flow cytometry. Secretion of pro‐inflammatory cytokines (10‐2500 RJEV/cell) was measured with ELISA. Young's modulus of HMC‐3 cells was probed with living‐cell atomic force microscopy force spectroscopy. Motility was assessed with a PHIOcell watcher, tracing HMC‐3 cells for 24 h after stimulation. Results : Proteomics of RJEV (130 nm, mean size) revealed the presence of bee‐derived proteins such as MRJP‐1, Jelleins, Defensins, apolipoproteins, CD63, and Synthenin. LPS exposure altered uptake mechanics: 33% more RJEVs were internalized and the main mechanism changed to clathrin‐dependent endocytosis and macropinocytosis, shifting away lipid raft endocytosis or membrane fusion. RJEVs significantly decreased IL1b, IL6 and TNFa in a dose‐dependent manner. LPS decreased the Youngs modulus by 44% and RJEVs by 28%, indicating a more flexible cell membrane. RJEVs in LPS‐stimulated HMC‐3 cells increased Youngs modulus to ctrl levels. Also, RJEVs decreased LPS‐induced motility in a dose‐dependent manner. Summary/Conclusion : Honeybee RJEVs are able to integrate into human microglia and exhibit significant anti‐inflammatory effects, decreasing pro‐inflammatory cytokines, motility, and altering membrane mechanics. These findings highlight the potential of RJEVs to modulate neuroinflammatory pathways and cell mechanics, offering insights into interkingdom communication with therapeutic implications. Funding : Funding: ANID FONDECYT 1220803

Outer

Jana Klimentova, Ivona Pavkova, Jan Bavlovic, Jiri Stulik Military Faculty of Medicine, University of Defense, Czech Republic Introduction : Francisella tularensis is a gram‐negative facultative intracellular bacterium causing a severe disease called tularemia. Being one of the most infectious pathogenic bacteria and because there is no licensed vaccine, it is classified as a potential biological warfare agent. Francisella releases vesicles of unusual tubular shape highly enriched in immunomodulatory proteins and virulence factors. Methods : The vesicles were isolated from the culture supernatants of F. tularensis subsp. holarctica fully virulent or mutant strains by ultrafiltration followed by ultracentrifugation and purified by density gradient centrifugation. For their characterization we used TEM, SEM and proteomic analyses. In in vitro studies we used the isolated vesicles to treat murine primary bone marrow macrophages and evaluated their response by antibody microarrays, cytokine ELISA, TEM and fluorescence microscopy. The release of vesicles from the bacteria during the interaction with macrophages was visualized by TEM. In in vitro studies mice were immunized by the isolated vesicles and then challenged by the lethal dose of the parent strain. The sera of the immunized animals were probed for cytokine and antibody response by the antibody microarrays and western blot. All animal experiments were approved by the Animal Care and Use Committee of the Military Faculty of Medicine, University of Defense. Results : The vesicles entered rapidly into macrophages and accumulated inside. They were non‐toxic for the macrophages, slightly prolonged their viability and elicited strong proinflammatory and antimicrobial response. The nanotubules were formed mainly during the entry of the bacterium into the host cell. In mice the in vivo protective effect was significant but the survival time was only slightly prolonged. We have found several hypervesiculating strains among mutants with disrupted O‐antigen. Summary/Conclusion : Due to their origin and content and regarding their rich interaction with the host cells, the membrane vesicles of Francisella present an interesting material from the point of view of their protective potential. Currently, we also search for mutant strains, especially the hypervesiculating ones and those with enhanced protectivity, which could serve as a source of vesicles more suitable for the protective studies. Funding : The work was supported by the Ministry of Defense of the Czech Republic (project no: DZRO‐FVZ22‐ZHN II).

Phase

Kaixiang Zhang, Gaoge Sun, Hang Hubert Yin, Ying Zhang Tsinghua University, China Introduction : Exosomes are EVs derived from multivesicular bodies. Multiple mechanisms are involved in the generation of exosomes and make contributions to the sorting and transportation of exosomal cargos, including mRNAs. Here we have identified that the phase separation of MVB‐related proteins, such as ESCRT family proteins, can induce the bubbling of exosomes. The RNA‐binding protein cargo NPM1 can be involved in this condensate and further transport its binding RNAs into exosomes. This work reveals a new mechanism for exosome generation and cargo transportation. Methods : EVs from cultured cells were collected with ultracentrifugation. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot (WB) were carried out for the characterization of EVs according to MISEV2023 guidelines. Immunofluorescence microscopy was used to observe the proteins' subcellular locolization and the formation of phase separation condensate. Fluorescence recovery after photobleaching (FRAP) was used for the validation of condensate. In vitro experiments were used to simulate the relationship between phase separation and exosome generation. RNA‐binding protein cargos in EVs were identified by proteomics. Next‐generation sequencing was employed to identify total mRNAs in EVs. Results : In this work, we found that some ESCRT family proteins can form phase separation condensate in cells and co‐localize with CD63‐marked MVBs. We have also identified NPM1 as an RNA‐binding protein that accounts for the sorting of mRNA into EVs. NPM1 can be involved in the phase separation condensate with the binding of RNAs in MVB, which can be further covered and released by EVs into the extracellular environment. Utilizing computational analysis, we identified motifs in mRNA that are specifically recognized by the NPM1 protein and facilitate the sorting into EVs. Summary/Conclusion : In conclusion, our findings reveal that the phase separation process is important in the generation and cargo sorting process of exosomes. The RNA‐binding protein NPM1 is a cargo involved in this phase separation condensate. This mechanism can contribute to the sorting of protein and RNA cargos into exosomes, which can have further functions in intercellular communications. Funding : This work was funded by the National Key R&D Program of China (no. 2023YFC3605400, 2021YFE0109300), the National Natural Science Foundation of China (no. 22137004, 21977061), and the Beijing Natural Science Foundation (IS23100, IS23107).

Ptov1

Piyush Kumar, Indranil Banerjee, Yuba Raj Pokharel Faculty of Life Sciences & Biotechnology, South Asian University, SAARC Nations, New Delhi, 110068, India Introduction : Breast Cancer (BrCa) is a complex, heterogeneous disease based on receptor status. Subtype TNBC is highly metastatic and shows recurrent characteristics. Exosomes are 150 nm cargo‐loaded extracellular vesicles that have been reported to promote cancer progression and metastasis. The translational significance of these vesicles can address the lack of early diagnostic and limited treatment options for TNBC patients. PTOV1, a conserved adaptor protein is associated with high‐grade breast tumours. It affects Wnt signalling by suppressing DKK‐1 activity and interacts with genes involved in modulating exosome biology. This could be particularly relevant if PTOV1 is directly or indirectly involved in exosome‐mediated BrCa pathogenesis. Methods : Stable PTOV1 knockout and overexpression in MDA‐MB‐231 and MCF7 cells were created using pLJM‐1 and LentiCRISPRv2 strategy. For preparing exosomes, the conditioned medium was filtered through a 0.22‐µm and ultracentrifuged at 110,000 g for 70 min at 4 degrees C and pellets were resuspended in PBS. Exosomes were characterized by DLS (size < 150 nm) and exosome‐associated markers (CD9, CD63, and FLOT‐1, Negative marker BCL‐2) by western blot. The protein profile of cell lysates and exosome proteins was visualized by the Coomassie brilliant blue staining. Changes in exosomal cargo were studied through western blot and miRNA Real‐time PCR. The transformation potential of the exosomal fractionate on the mammary non‐tumourigenic epithelial cells (MCF10A) was studied after treatment with 80ug /experiment exosomal fractionate. Results : Findings suggest that PTOV1 influences the exosome cargo selection and release. Moreover, Micro BCA Assay data indicate that PTOV1 expression influences the release of exosomal protein fractionates with no change in exosome size. This change in the exosomal landscape affects the activity of Akt1/2 and proliferation, cell cycle, 3 D spheroids formation, migration, and invasion properties in mammary non‐tumourigenic epithelial cells. The same trend was observed in studies with the serum samples from TNBC patients. Summary/Conclusion : Conclusively, findings demonstrate that PTOV1 overexpression induces changes in the EMT process in MCF10A cells via changes in the exosomes‐mediated horizontal transfer of bioactive molecules. Funding : Indian Council of Medical Research (ICMR).

Raman

Silvia Picciolini 1 , Alice Gualerzi 1 , Martina Gerli 1 , Francesca Rodà 1,2 , Aurora Mangolini 1 , Zachary Clemens 3 , Aaron Barchowsky 4 , Amrita Sahu 3,4,5 , Marzia Bedoni 1 1 IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy; 2 Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy; 3 Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 4 Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 5 McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Introduction : Arsenic (As) exposure impairs tissue function resulting in reduced mobility and overall quality of life. Although there still exists a dearth of interventions aimed to restore As‐impairment, preliminary findings from the Sahu and Barchowsky labouratories have demonstrated that neuromuscular electrical stimulation (E‐stim) and pharmacological intervention with mitochondrially targetted peptide‐SS‐31 improves As‐impaired functional regeneration of skeletal muscle. It has also been demonstrated that As disrupts extracellular vesicle (EV) mediated skeletal muscle signalling in regenerating muscle fibres. However, the regulatory cargoes within the EVs driving As‐effects are yet to be investigated. Herein, we propose to determine the effect of E‐stim and SS‐31 intervention on circulating biochemical signatures of EVs in As‐exposed animals by using Raman Spectroscopy. Methods : Blood EVs were isolated by size‐exclusion chromatography and ultracentrifugation. Size distribution and concentration have been assessed by nanoparticle tracking analysis. EV samples were analysed by means of a Raman microspectroscope equipped with 532 laser line and multivariate statistical analysis were performed in order to compare the Raman fingerprint of EVs from As‐exposed mice with and without E‐Stim or SS‐31 treatment. Results : We found that EVs size and concentrations are not affected by As exposure or the interventions. Raman data showed that EV fingerprints are significantly different between As and treated mice with the main modifications in spectral regions related to lipids and proteins. We also observed some spectra differences that could be related to changes in the presence and conformations of specific molecules involved in oxidative stress condition (cytochrome, glutathione and nucleic acids). Summary/Conclusion : Our findings demonstrated that both rehabilitative and pharmacological treatments alter the biochemical composition of circulating EVs of As‐exposed mice, suggesting that they could improve As‐impaired autocrine, paracrine, and endocrine signalling capabilities mediated through EVs. Moreover, our results showed that Raman EV characterization can represent a valuable method to identify potential involvement of molecules in oxidative stress. Funding : This research was funded by the Italian Ministry of Health, The Pittsburgh Foundation, USA and National Institute of Environmental Health, USA.

Rapid

Presenter: Casey Huang National Taiwan University of Science and Technology, Taipei, Taiwan (Republic of China) Introduction : Extracellular vesicles (EVs) are key mediators of intercellular communication with potential as biomarkers and therapeutics but isolating them from conditioned cell culture medium (CCM) remains challenging. Traditional methods, such as ultracentrifugation (UC), ultrafiltration, and size‐exclusion chromatography (SEC), have limitations in speed, purity, and ease of use. To address these challenges, we developed the EV‐Osmoprocessor (EVOs), a novel device leverageing osmosis‐driven filtration for rapid, efficient EV isolation with minimal user input. Methods : The EVOs device was fabricated with a dual‐layer PVDF membrane system, housed between custom‐designed poly(methyl methacrylate) (PMMA) layers and immersed in a polyethylene oxide (PEO) solution to drive osmosis. The process reduced sample volume by 50‐fold in under two h, recovering concentrated EVs from a 200 µL reservoir. Characterization of the isolated EVs included cryo‐electron microscopy to confirm intact morphology, along with detection of key EV markers (CD81, CD63, TSG101, ALIX). Cardioprotective assays demonstrated that EVOs‐isolated EVs retained functional bioactivity comparable to those obtained by UC and ExoQuick (EQ). Results : The EVOs device, composed of a dual‐layer PVDF membrane system, processes 10 mL of CCM in less than 2 h, concentrating it 50‐fold to 200 µL. Osmosis effectively removed small particles while retaining 75.6% of 100 nm and 81.5% of 200 nm EV‐sized particles. When used with CCM, EVOs reduced 99.7% of albumin and improved particle‐to‐protein ratios (3.3 × 10⁷ particles/µg). Cryo‐electron microscopy confirmed intact EV morphology, and key EV markers (CD81, CD63, TSG101, ALIX) were detected. Cardioprotective assays demonstrated that EVOs‐isolated EVs retained functional bioactivity comparable to those obtained by UC and EQ. While EVOs alone resulted in slightly lower purity than UC, combining it with SEC enhanced EV purity to 3.6 × 10⁹ particles/µg, exceeding UC performance. Summary/Conclusion : Our findings suggest that EVOs is a promising new tool for rapid, high‐yield EV isolation with potential applications in biomarker discovery and therapeutic development. Further optimization of the filtration membrane could enhance its performance for broader applications.

Serum

Alice Gualerzi 1 , Martina Gerli 1 , Gemma Lombardi 2,3 , Silvia Picciolini 1 , Aurora Mangolin 1 , Valentina Mangolini 1,4 , Francesca Rodà 1,5 , Luana Forleo 1,6 , Rita Martino 1 , Stefano Faggiano 1 , Silvia Ramat 7 , Stefano Giuseppe Doronzio 3,8 , Diego Longo 3,8 , Marco Baccini 3 , Francesca Cecchi 3,8 , Marzia Bedoni 1 1 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milano, Italy; 2 Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Università di Firenze, Florence, Italy; 3 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Firenze, Italy; 4 Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, Brescia, Italy; 5 Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy; 6 Medical and Surgical Pathophysiology and Transplantation Department, University of Milan, Italy; 7 Parkinson Unit, Azienda Ospedaliera Universitaria Careggi, Firenze, Italy; 8 Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Università di Firenze, Florence, Italy Introduction : Extracellular vesicles (EVs) are nanoparticles secreted by all body cells and released in biofluids. In Parkinson's disease (PD), EVs seem to represent a vehicle of disease‐related molecules, such as α‐synuclein. Also, blood EV biochemical modifications have been correlated with people with PD (pwPD) clinical profiling. In the present study, we hypothesize EV involvement in both pathogenic mechanisms occurring at PD onset, along with biological processes occurring in the brain during disease progression and therapeutic treatment in pwPD. We expect EVs to be mediators of brain remodelling induced by motor rehabilitation, which is crucial in the treatment of pwPD by ameliorating motor and non‐motor impairments and improving their quality of life. Methods : After providing informed consent, a cohort of 44 pwPD was recruited. Blood samples, as well as clinical, neuropsychological, and instrumental variables, were collected for each patient before and after a treadmill‐based rehabilitation programme (8 weeks). EVs were isolated from serum by combining size exclusion chromatography and ultracentrifugation methods. Biochemical characterization of EV samples was obtained by Raman spectroscopy to evaluate spectral differences in EV fingerprints before and after rehabilitation. Raman data were subsequently correlated with clinical and motor parameters to evaluate the potential of Raman spectra as predictive biomarkers of rehabilitation efficacy. Results : EVs were successfully isolated from the serum of pwPD through a reproducible method. No differences in size distribution and protein biomarkers were observed. Raman analysis highlighted significant differences in samples before and after rehabilitation in multiple spectral regions. For both groups, Raman spectra clearly showed peaks attributable to α‐syn and carotenoids with variable intensities, suggesting that motor‐cognitive rehabilitation can induce significant biochemical differences in circulating EVs. Summary/Conclusion : In conclusion, our data suggest a possible EV contribution in restorative mechanisms induced by intensive rehabilitation and confirm that EVs could provide an accessible and measurable biomarker source for neurodegeneration and rehabilitation‐induced brain remodelling. Thus, EV characterization by Raman spectroscopy can be used as a support tool in the diagnosis and monitoring of PD progression, as well as the effectiveness of rehabilitation programmes, allowing for the personalization of rehabilitation strategies. Funding : This study was funded by Fondazione Cassa di Risparmio di Firenze.

Shiga

Markus Wendler, Alexandra Gerogianni, Ida Arvidsson, and Diana Karpman Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden Introduction : Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is a pathogen that causes severe gastroenteritis and haemolytic uremic syndrome characterized by non‐immune haemolytic anaemia, thrombocytopenia, and acute kidney injury. EHEC is non‐invasive and releases virulence factors in the gut, including Shiga toxin, that are crucial for strain pathogenicity. Our group has shown that blood cells release EVs containing the toxin, which thereby circulates and reaches the kidney, where the toxin is endocytosed. The aim of this study was to investigate if Shiga toxin‐positive EVs exhibit altered potency compared to free toxin. Methods : HeLa cells were stimulated with Shiga toxin 2 (10 ng/mL) or PBS in 1% Exo‐free foetal bovine serum for 24 h to induce vesicle secretion. EVs were isolated by size exclusion using a 450 nm syringe driven filter unit (PES membrane). The EV suspension was purified with a centrifugal filter at 100 kDa to remove free proteins. EVs were characterized by immunoblotting and nanoparticle tracking. EVs were 100–450 nm (median 140 nm), and were identified by positivity for CD63, TSG101, and Alix. Cytotoxicity of the pure toxin and within vesicles was confirmed by performing a Vero cell assay. BalbC mice were injected iv with Shiga toxin 2 at 126 ng/kg, the same toxin concentration in EVs, EVs alone or PBS. Results : All mice treated with toxin‐positive EVs ( n = 9) developed clinical signs of disease by day 3, whereas 4 mice treated with free toxin ( n = 6) developed signs of disease by day 6 and the remaining 2 survived until Day 7. Mice treated with EVs alone ( n = 6) or PBS ( n = 5) remained unaffected. Urea levels were highest in mice challenged with toxin‐positive EVs, indicating kidney injury. Kidney pathology and fibrinogen staining are being performed as well as specific staining for vesicles and toxin in target organs. Summary/Conclusion : Shiga toxin 2‐positive EVs are more potent than free Shiga toxin 2 when delivered iv in a mouse model of EHEC virulence. The results suggest that bacterial toxin packaged within vesicles is capable of reaching target organs, possibly by evading a host response.

Small

Presenter: Andrie Koutsoulidou The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus Introduction : Myotonic Dystrophy Type 1 (DM1) is a hereditary disorder and the most common adult‐onset muscular dystrophy worldwide. DM1 hallmark symptoms are myotonia, muscle weakness, and wasting which steadily worsen over time. We previously reported that small extracellular vesicles (EVs) isolated from serum samples of DM1 patients contain elevated levels of muscle‐specific miRNAs, miR‐1, miR‐133a, miR‐133b, and miR‐206, compared to healthy participants. Importantly, the levels of the four muscle‐specific miRNAs encapsulated within small EVs were found to be associated with the progression of muscle wasting in DM1 patients. Moreover, we showed that these miRNAs were increased in EVs circulating in the serum of a DM1 animal model. In this study, we aimed to investigate miRNAs in small EVs that could potentially differentiate between stable and progressive DM1 patients. Methods : Serum samples from DM1 patients were obtained from the ‘PhenoDM1’ study. Patients were categorized as progressive or stable at the time of blood collection based on their outcome measures. Small EVs were isolated using size‐exclusion chromatography method. EV characterisation was confirmed with TEM imaging and small‐particle flow cytometry. Total RNA, including small RNA, was extracted from EV samples followed by transcriptomics using small RNA next‐generation sequencing (NGS). Bioinformatics analysis allowed for the identification of deregulated miRNA cargo encapsulated in EVs. The best candidate miRNAs were selected for validation using Real‐Time PCR. Results : A total of 69 progressive and stable DM1 and healthy serum samples were used for small EV isolation followed by small RNA‐NGS. The total miRNA population was identified and analysed with bioinformatics tools. Heatmaps between progressive and stable DM1 patients and healthy controls were used to assess the expression signatures of miRNAs within small EVs. Several novel EV‐miRNAs were detected showing altered levels and were associated with the disease progression. Validation of the top miRNAs was performed with RT‐qPCR. Summary/Conclusion : This study suggests that circulating EVs encapsulate differential miRNA profile between stable and progressive DM1 patients and healthy individuals. This strongly highlights the promising use of EVs and their miRNA content as biomarkers for disease progression in patients with DM1. Funding : This work was funded by AFM Téléthon France and A. G. Leventis Foundation.

Study

Presenter: Ting Lan School of Medicine, Southeast University,China (People's Republic) Introduction : Human umbilical cord mesenchymal stem cells (hUC‐MSCs) are pluripotent stem cells with significant clinical potential. The extracellular vesicles (hUC‐MSC‐EVs) secreted by these cells carry RNA and proteins beneficial in various disease models. However, traditional two‐dimensional culture methods for hUC‐MSCs face limitations, including restricted growth area and inconsistent cell quality. Scaffold‐free spheroid culture offers a more physiologically relevant microenvironment, preserving cell characteristics and optimizing culture space. Despite its advantages, challenges such as spontaneous spheroid formation and EV yield remain. This study explores how CT enhances MSC spheroid formation and the role of secreted EVs in treating skin injuries. Methods : (1) The impact of CT on MSCs was evaluated using CCK8 assays and paracrine functional tests. (2) MSCs underwent scaffold‐free spheroid culture with CT intervention, with morphology documented over time. (3) Second‐generation transcriptome sequencing elucidated mechanisms underlying CT's effect on MSC spheroid formation. (4) PCR assessed EV production‐related gene expression post‐CT intervention; cell supernatants were collected for EV isolation and NTA detection. (5) CT‐3D MSC‐EVs were utilized in HUVEC scratch and tube formation assays. (6) A mouse skin injury model assessed the efficacy of CT‐3D MSC‐EVs. Results : CCK8 assays indicated that CT promotes MSC proliferation and upregulates paracrine factors. After 24 h of spheroid culture, CT enhanced MSC spheroid formation and elevated EV‐related genes (CD9, CD81, CD63, Alix) and stemness genes (Nanog, Sox2, Oct4). Transcriptome sequencing confirmed CT's role in promoting spheroid formation via upregulation of adhesion molecules. Scratch assays showed CT‐3D MSC‐EVs significantly improved HUVEC migration compared to 2D‐MSC‐EVs, and tube formation assays further validated their superior efficacy. In vitro experiments demonstrated the effectiveness of CT‐3D MSC‐EVs in repairing mouse skin injuries. Summary/Conclusion : This study highlights that CT promotes MSC spheroid formation by enhancing intercellular adhesion molecules and EV secretion, improving therapeutic effects in skin injury treatment.

Swarm

Presenter: Alan Chin Kinetic River Corp., Boston, Massachusetts, USA Introduction : Determining the fluorescent fraction of stained exosomes can be a relatively cumbersome task, requiring measurements of the total exosome concentration, the fluorescently tagged exosome concentration, and the background concentration along with processing of the data involving subtracting the background concentration from the total exosome concentration to accurately determine the fluorescent fraction of stained exosomes to total exosome population. Although swarm detection is now widely regarded as a situation to avoid when using flow cytometry to study exosomes, it is only necessary to avoid swarm detection if one requires single‐particle information. Because the fluorescent fraction (while obtainable via single‐particle statistics) is a bulk sample measurement (e.g., using nanoparticle tracking analysis), it is possible to use swarm detection to increase the scatter and fluorescence signals and obtain a rapid, accurate estimate of the fluorescent fraction. Methods : We utilized a custom‐built flow cytometer to perform measurements on samples with concentrations up into the swarm regime. We also utilized a nanoparticle tracking analyser to characterize each sample to obtain a baseline concentration as well as a measurement of the fluorescent fraction for each sample. Titrations were prepared ranging from the single‐particle regime to the swarm regime. Results : We examined fluorescent bead samples (40‐nm yellow‐green, 75‐nm MD655) with size and/or fluorescence below the detection limit of a flow cytometer. By increasing the concentration of the bead samples, we were able to confirm that both the scatter and fluorescence signals increase in concert to yield a rapid fluorescent fraction estimation without background subtraction. A fluorescent fraction of 100% can be achieved if an additional step with background subtraction is performed. We utilized the same procedure to measure the fluorescent fraction of fluorescently labelled exosomes (HEK293‐derived CD63‐EGFP) and were able to achieve a fluorescent fraction that matches the specified value. Summary/Conclusion : Our results demonstrate that swarm detection in flow cytometry can be a useful tool for rapidly and accurately determining the fluorescent fraction of exosomes. Funding : This work was made possible in part by US government support under award number 1R43GM155492‐01 from the NIH.

Tdp43

Presenter: Yi‐Ya Fang Prevail Therapeutics, New York, New York, USA Introduction : There are relatively few validated biomarkers that can be used to identify presymptomatic people who are at risk of developing amyotrophic lateral sclerosis (ALS), diagnose ALS, monitor the disease progression, or assess the efficacy of new therapies. Previously validated biomarkers, such as NfL and GFAP, only reflect downstream effects and do not specifically reflect the molecular pathology of ALS. TDP‐43 is a DNA‐binding protein which has been found to leave the nucleus and form aggregates in ALS patients. However, the ability to use TDP43 as a biomarker of disease is limited because detection of TDP‐43 in the blood and CSF of patients has not been possible. Extracellular vesicles (EVs), which are nano‐sized and lipid bilayer‐bound particles, play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control system for the cellular clearance of ND‐associated proteins. Methods : In this study, EVs were isolated from human plasma by precipitation, immunoaffinity, centrifugation, and size exclusion columns before applying on TDP‐43 single molecule array (Simoa) assay. Results : TDP‐43 signal was higher in unlysed EVs compared to lysed EVs, which suggests that TDP‐43 exists mostly in the lumen of EVs rather than the surface of EVs. In addition, we found that TDP‐43 signals in isolated EVs were significantly higher in ALS samples compared to age‐matched healthy samples. By further isolation of neuronally derived EVs using biotinylated L1CAM antibody and streptavidin resin, we observed the same trend in TDP43 signal as total EVs, which was higher TDP‐43 levels in ALS compared to healthy subjects. This isolation method combined with the Simoa assay has shown good reproducibility and linearity, and a freeze‐thaw cycle of isolated EVs did not impact the accuracy of the results. Summary/Conclusion : In summary, we developed a robust TDP‐43 assay that will enable the potential use of TDP‐43 as an ALS biomarker.

Tnfα

Manting Luo, Chong Zheng, Bin Yang, Jiwei Luo, Jianlong Li Department of Orthopedic, Nanfang Hospital, Southern Medical University, Guangzhou, China Introduction : Osteosarcoma‐derived extracellular vesicles and particles (EVPs) could induce liver metabolic dysregulation through Kupffer cells. Osteosarcoma chemotherapy faces significant challenges due to the severe systemic toxicity of chemotherapeutic agents like doxorubicin, which causes cardiac toxicity, hepatic dysfunction, and myelosuppression. These toxicities limit the dose and effectiveness of treatment. This study investigates a novel strategy involving TNFα inhibition, aimed at improving chemotherapy efficacy and mitigating the side effects associated with doxorubicin therapy. Methods : An orthotopic osteosarcoma model was established using K7M2 cells implanted in the tibia of BALB/cJ mice. Mice were treated with doxorubicin (25 mg/kg, ip) either alone or in combination with thalidomide (50 mg/kg, ip, daily for 7 days). The study evaluated tumour growth, chemotherapy‐induced toxicity, and hepatic metabolic functions. Key assessments included Bodipy staining for hepatic lipid accumulation, qPCR for cytochrome P450 (CYP) enzyme expression, echocardiography for cardiac function, and haematological and biochemical analyses to assess systemic effects. TEM, NTA, and WB were used to identify EVPs. Results : The osteosarcoma released EVPs, upregulated TNFα from Kupffer cells, causing hepatic lipid accumulation, and suppressed the expression of key CYP enzymes, disrupting normal drug metabolism. While doxorubicin alone hardly reduced tumour size, nor reduced the secretion of EVPs, it induced severe cardiotoxicity and metabolic dysfunction. In combination with the TNFα inhibitor, thalidomide restored CYP enzyme expression, alleviated hepatic lipid accumulation, and improved overall chemotherapy tolerance. This combination therapy resulted in enhanced tumour suppression and reduced toxicity, particularly in terms of heart function, compared to doxorubicin monotherapy. Summary/Conclusion : This study introduces a novel therapeutic strategy by combining TNFα inhibition with doxorubicin to enhance osteosarcoma chemotherapy sensitivity. The mechanism involves restoring hepatic metabolic balance and reducing inflammation‐induced dysfunction, thereby improving chemotherapy tolerance and efficacy. This approach not only offers a promising new direction for osteosarcoma therapy but also has the potential for broader applications in other malignancies, particularly those with high chemotherapy‐associated toxicity. Funding : [GuangDong Basic and Applied Basic Research Foundation (2024A1515030050) and Presidential Foundation of Nanfang Hospital, Southern Medical University (2023A031)].

Using

Nahide Zeren Arda Ozturk 1 , Oliwia Majchrzak 2 , Gianluca Ulivi 2 , Viorica Patrulea 2 , Petek Ballar Kirmizibayrak 3 , Gerrit Borchard 2 , Ozgen Ozer 3 1 Fenerbahce University, Turkiye, 2 University of Geneva, Switzerland, 3 Ege University, Turkiye Petek Ballar Kirmizibayrak, Gerrit Borchard and Ozgen Ozer are co‐senior authors . Introduction : Exosomes are extracellular vesicles that contribute to several kinds of biological processes. Their ability to provide intercellular communication, one of their most important functions, has inspired the use of exosomes as drug carriers. Fully synthetic exosome‐mimetics (FSEM) is a drug delivery technology that takes its inspiration from the natural exosome. FSEM membrane proteins and lipids enhance adhesion and fusion. They also have a low toxicity profile. It is crucial that the proteins used in FSEM retain their 3D structure and conformation in the membrane.TSP‐1 and CD9 proteins are membrane proteins that function during intercellular communication. The CD9 protein is inserted into the exosome membrane, while the TSP‐1 protein is bound to the outside of the exosome membrane. Different methods are used to produce synthetic exosome mimetics, depending on the position of the proteins in the membrane. Methods : The FSEMs were prepared using DMPC, DOPE, DOPS, sphingomylin and cholesterin. To incorporate CD9 protein, the liposomes were treated with Triton X‐100, followed by incubation with CD9 protein. Then the Triton X‐100 was removed using a detergent removal column and the membrane incorporation was completed. To bind the TSP‐1 protein, liposomes were prepared using a modified DOPE molecule.The binding of the TSP‐1 protein to the liposome membrane was achieved by the EDC‐NHS reaction. Results : Particle size, zeta potential and PDI measurements of FSEM were as targeted and compatible with natural exosomes. Protein presence and conformation in the formulations were established by SDS‐PAGE analysis and the bands were compatible with commercial proteins alone and markers. FSEM also showed higher cellular uptake in SK‐MEL3 cells compared to DOTAP and phosphatidylcholine liposomes. Summary/Conclusion : Enhanced cellular uptake is achieved by using lipids present in natural exosomes and liposomes functionalised with membrane proteins in the preparation of FSEM. This drug delivery system could provide a new alternative for non‐toxic drug and gene delivery. Funding : Ege University Office of Scientific Research Projects, University of Geneva, TUBITAK 2244 project

Acidic

Noemi Torriero 1 , Chiara Maceri 1 , Beatrice Crestani 1 , Valentina Moccia 1,2 , Guillaume Gilliard 3 , Valentina Zappulli 2 , Pietro Parisse 4 , Valeria Rondelli 3 , Davide Ferraro 1 1 Department of Physics and Astronomy, University of Padova, Padova, Italy 2 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy; 3 Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy; 4 Istituto Officina dei Materiali (CNR‐IOM), Consiglio Nazionale delle Ricerche, Trieste, Italy Introduction : Compared to other known methods used for extracellular vesicles (EVs) isolation, immunoaffinity with magnetic beads allows us to obtain EVs with a lower yield but much higher purity. However, one of the main issues related to this technique is the detachment of the EVs from the beads, which often results in the alteration of EVs structural integrity and the dissolution of surface markers, making them unsuitable for functional assays. Here we propose a pH‐based method for the elution of the EVs following CD9‐coated magnetic bead‐mediated immunoaffinity. Methods : EVs from the MDA‐MB‐231 cell line were previously isolated with ultrafiltration (UF) and characterized with nanoparticle tracking analysis (NTA), bicinchoninic acid (BCA) assay and western blot (WB). EVs were then incubated with beads functionalized with CD9 antibody for 3 h at room temperature in shaking conditions. To elute the EVs from the beads, an acidic solution of glycine‐HCl (pH 3) was added to the beads for 30 min on ice, followed by a basic tris‐HCl solution (pH 8). The beads were then extracted with a magnet, and the supernatant containing the EVs was evaluated with NTA, BCA, WB, transmission electron microscopy (TEM), atomic force microscopy (AFM), small angle X‐ray scattering (SAXS), and infrared spectroscopy (FT‐IR). Results : In spite of a lower yield of EVs compared to the pre‐enriched UF sample, analyses confirmed the elution from beads and recovery of intact EVs. Pre‐enriched UF‐derived samples had an initial concentration of 10 10 EVs/mL, whereas a final recovery of 10 9 EVs/mL followed detachment from the beads after the acidic treatment. Both TEM and AFM analyses confirmed the higher purity of the eluted EVs, in contrast with UF‐derived EVs, rich in co‐isolated proteins. Summary/Conclusion : This protocol allows us to detach EVs from magnetic beads following immunoaffinity isolation. Given the potentialities of EVs in several fields, this method represents a promising solution to isolate intact EVs and exploit them for subsequent functional assays. This protocol is also being applied in our Laboratory for the isolation of EVs from raw plasma in microfluidic devices, aiming at obtaining pure EVs for diagnostic purposes. Funding : Next Generation EU Mission4 Component1, for projects no. P2022J2ZC2 and 2022YYLSJ2.

Breast

Presenter: Mohammed Ismail Youssef Elmallah University of Tours, Tours, France Introduction : Tumours are well known to promote the induction of immunosuppressive microenvironments by recruiting the surrounding cells to serve their own survival and growth. This could be assigned to the release of soluble factors by tumour and large numbers of heterogenic extracellular vesicles (EVs) of different origins, including microvesicles and exosomes. Tumour‐derived EVs gain the attention of several research groups in terms of their immunomodulatory effect, as they encompass immunosuppressive proteins and microRNAs targeting suppressive pathways in recipient cells. Methods : EVs were isolated from MDA‐MB‐231 by differential ultracentrifugation then characterized by nanoparticle tracking analysis and western blot. PBMCs were isolated from the blood of healthy donors, and then CD14+ monocytes were selected. CD14+ cells were treated with EVs derived from the wild‐type MDA‐MB‐231 cells that express P2X4 (MDA‐EXO +/+P2X4). The cells were incubated for 7 days at 37°C and 5% CO 2 in the presence of IL‐4 and GM‐CSF. The change in the surface expression of both differentiation and maturation markers was monitored by flow cytometry. Results : Flow cytometric analysis revealed that CD14+ cells treated with MDA‐EVs ± P2X4 showed a significant reduction in the expression of the cell surface receptor HLA‐DR compared to the positive control (cells only treated with IL‐4 and GM‐CSF). In addition, no significant differences regarding the surface expression of CD86, CD33, and CD14 as well as the expression of the released cytokines were observed. Interestingly, we found that CD14+ cells treated with MDA‐EVs ± P2X4 displayed a significant reduction in the glycogen content compared to the positive control using electron microscopy. We are planning to test the same experiments by treating CD14+ cells with MDA‐EVs deprived of the P2X4 receptor (MDA‐EVs ‐/‐ P2X4) in order to investigate whether P2X4 plays a central role in the induction of an immunosuppressive environment and subsequent cancer progression and metastasis. Summary/Conclusion : MDA‐MB‐231‐derived EVs inhibit the maturation of monocyte‐derived dendritic cells by decreasing the cytoplasmic content of glycogen and decreasing the surface expression of HLA‐DR. we conclude that our results may provide new insight toward the immunosuppressive as well as toward the immunomodulatory role of tumour‐derived EVs. Funding : This study was supported by INCa PLBIO_16110 “PURINO4EXO” to S.R., Grant Region Centre‐val de Loire “EXOGRAL” to S.R.

Cancer

R. Ishihara 1 , H. Yokohari 1 , R. Ogata 1 , T. Nakajima 2 , E. Shimura 1 , and T. Baba 1 1 Juntendo University, Tokyo, Japan; 2 Yokohama City University, Yokohama, Japan Introduction : Extracellular vesicles (EVs) present in body fluids have garnered significant attention as novel biomarkers for various diseases, including cancers. However, the inherently low concentration of EVs in these fluids poses challenges for their detection. To facilitate facile and highly sensitive EV detection for cancer point‐of‐care diagnostics, we have developed portable surface‐functionalized power‐free poly(dimethylsiloxane) microfluidic chips (SF‐PF microchips). In this study, we present the development of a novel SF‐PF microchip and demonstrate its application in detecting cancer cell‐derived EVs. Methods : The SF‐PF microchip for EV detection was prepared in the following four steps: (1) A PDMS microchip was fabricated using a soft lithography technique. (2) The inner surfaces of the microchannels of the microchip were modified with poly(2‐aminoethyl methacrylate) (PAEMA) via UV graft polymerization. (3) Anti‐integrin β1 antibodies were subsequently immobilized into the grafted polymer phase. (4) The antibody‐immobilized microchip was attached to a glass slide and degassed. Subsequently, EVs were detected using laminar‐flow‐assisted dendritic amplification on the SF‐PF microchip. Results : The surface modification of PAEMA was confirmed via ATR‐FTIR spectroscopy. Anti‐integrin β1 antibody was selected as a targeting antibody for antigens highly expressed on EVs derived from malignant breast cancer cells. Immobilization of a sufficient amount of anti‐integrin β1 antibodies on the microchannel surface was verified using a fluorescent‐labelled secondary antibody. EV signals were evaluated based on the signal‐to‐blank ratio, which was significantly higher for EVs than for the control ( p < 0.05, two‐tailed t ‐test). Anti‐integrin β1 antibodies immobilized on the inner surface of the microchannel effectively recognized EV surface antigens, enabling selective EV detection. The limit of detection (LOD) of the SF‐PF microchip was calculated according to the 3σ criterion from a calibration curve, yielding an LOD of 2.1 × 10 10 EVs/mL. This value is comparable to the EV concentration in blood. The required sample volume and assay time were 2.0 µL and 20 min, respectively. Summary/Conclusion : A portable SF‐PF microchip that enables facile and highly sensitive EV detection was successfully developed. This SF‐PF microchip has the potential to contribute to the establishment of EV‐based point‐of‐care cancer diagnostics. Funding : This research was partly supported by JSPS KAKENHI (20K12697 and 23K11905).

Common

Darja Božič 1* , Katja Vrabec 1 , Valentina Novak 1 , Ivana Petrović Koshmak 1 , Andrej Raspor 1 , Nastja Bizjak Ternik 1 , Ana Železnik 1 , Maja Leskovec 1 , Matevž Arko 2 , Anna Romolo 2 , Alenka Nemec Svete 2 , Vladimira Erjavec 2 , Martina Gobec 2 , Nika Strašek Benedik 2 , Melanie Reininger 3 , Ingrid Hartl 3 , Tobias Tertel 4 , Bernd Giebel 4 , Klaus Graumann 3 , Veronika Kralj‐Iglič 2 , Aleš Štrancar 1 1 Sartorius BIA Separations, Slovenia; 2 University of Ljubljana, Slovenia; 3 Phoenestra Gmbh, Austria; 4 University of Duisburg‐Essen, Germany Introduction : The functional studies of extracellular vesicles (EVs) remain controversial due to the limited purity of preparations and inadequate analytics, which are typically focused on an isolated parameter (e.g., particle concentration, marker‐positive subpopulations, total protein/nucleic acid content). We believe that a better understanding of preparations’ composition is crucial for relating specific functionalities to different sample constituents. Methods : Size exclusion (SEC) and ion‐exchange chromatography (IEC) for characterizing EV preparations were explored using a PATfix system with incorporated detectors for light absorbance, fluorescence, and multi‐angular light scattering. Results : Strengths and weaknesses of SEC and iEC were evaluated in detail. Ultimately, a combined, two‐dimensional liquid chromatography (2D‐LC) method was developed, providing a detailed profile of sample components resolved by size and by charge. The approach is applicable to biological samples of varying complexity, such as cell culture conditioned media and various EV‐enriched preparations. Similar subpopulations were recognized in samples from very diverse sources, opening a new perspective on subclasses of EVs and non‐EV extracellular nanoparticles (EPs). Summary/Conclusion : Comprehensive chromatographic characterization of EV preparations provides the insights that can pave the way towards better understanding of the relationship between the structure and function of EVs and other EPs. Funding : The study was funded by Sartorius Bia Separations.

Cyclic

Presenter: Yeonju Lee Hanyang University, Seoul, Republic of Korea Introduction : Oral drug delivery offers a patient‐friendly and minimally invasive approach for treating inflammatory bowel disease (IBD). However, high immune tolerance and oxidative stress in the gastrointestinal (GI) tract pose significant challenges. While extracellular vesicles (EVs) are promising carriers for IBD therapeutics, engineered EVs loaded with multi‐regulatory proteins are essential for sustained anti‐inflammatory effects. Methods : Herein, we report a smart therapeutic approach using cyclic protein‐charged EVs (cPC‐EVs) to rapidly attenuate oxidation and inflammation in the gut. Taking advantage of the enhanced permeability and stability of cyclic proteins, we designed cyclic versions of three immune‐regulatory proteins (human metallothionein 1A, hMT1A; cytosolic domain of cytotoxic T‐lymphocyte antigen 4, ctCTLA4; T cell protein tyrosine phosphatase, TC‐PTP) by knotting their N‐ and C‐termini via a split intein‐mediated process, which function as anti‐inflammatory agents within EVs. We examined natural EVs ( Lactobaccillus plantarum ‐derived EVs, LpEVs, or plant‐derived EVs, pEV) in combination with N‐C cyclization of these proteins. Results : Through in silico and in vitro comprehensive analyses of enhanced green fluorescent protein (EGFP) with peptide tags, we identified optimal peptide tags for improved membrane permeability in cyclic proteins. Consequently, multifunctional cPC‐EVs containing three cyclic immune‐regulatory proteins demonstrated synergistic anti‐inflammatory effects in both IBD organoids and orally administered IBD mouse models, significantly outperforming naïve LpEV or pEVs. Summary/Conclusion : These findings suggest that cPC‐EVs are very useful as a versatile oral drug platform for various inflammatory diseases.

Direct

Presenter: Rikinari Hanayama Kanazawa University, Kanazawa, Japan Introduction : EVs are important mediators of cell–cell communication, including immune regulation. Despite the recent development of several EV‐based cancer immunotherapies, their clinical efficacy remains limited. Methods : We created antigen‐presenting EVs (AP‐EVs) to express peptide‐major histocompatibility complex class I, costimulatory molecule, and IL‐2. This enabled the selective delivery of multiple immune modulators to antigen‐specific CD8+ T cells, promoting their expansion in vivo without severe adverse effects. Results : AP‐EVs accumulated in the tumour microenvironment, increasing IFN‐γ+ CD8+ T cell and decreasing exhausted CD8+ T cell numbers, suggesting that AP‐EVs transformed the ‘cold’ tumour microenvironment into a ‘hot’ one. Combination therapy with AP‐EVs and anti‐PD‐1 demonstrated enhanced anticancer immunity against established tumours. Summary/Conclusion : Engineering EVs to co‐express multiple immunomodulators represents a promising method for cancer immunotherapy. Funding : This study was supported by the Japan Agency for Medical Research and Development

During

Adriana T. Larregina 1,2 , Juliana S. Powell 3 , Olga Tkacheva 1 , William J. Shufesky 3 , Beer Stolz D 4,5 , Sullivan M 4,5 , Simon C. Watkins 4,5 , and Adrian E. Morelli 2,3 . Departments of Dermatology 1 Immunology 2 Surgery 3  and Cell Biology and Physiology and Center for Biologic Imaging University of Pittsburgh. Pittsburgh, PA, USA. Introduction : Allergic contact dermatitis (ACD) is a severe inflammatory skin disease that affects up to 18% of the population in USA and Europe. The onset of ACD requires the activation of the inflammasome in keratinocytes resulting in the synthesis and secretion of IL‐1β. These mechanisms are potentiated by substance P (SP) a pro‐inflammatory neuropeptide that signals with high affinity the neurokinin 1 receptor (NK1R). The events by which keratinocytes that are targets of neurogenic inflammation promote ACD remain poorly understood. Because IL‐1β is a leaderless protein released by non‐conventional secretory pathways, we hypothesized that contact sensitizers activate the inflammasome in keratinocytes, which release IL‐1β in small extracellular vesicles (sEVs) and that these effects require SP. Methods : In vitro studies utilized mouse or human immortalized keratinocytes treated with the contact sensitizer 2,4‐dinitrochlorobenzene (DNCB) in the presence or not of SP. Increase of multivesicular bodies (MVB) was quantified by transmission electron microscopy and analysis of MVB fusion with the cell membrane was assessed by Total Internal Reflection Fluorescence Microscopy in keratinocytes stable transfected with pLenti‐Hluo_M153R‐CD63mCherry. Quantification and size of secreted EVs was evaluated by nanotracking particle analysis. In vivo studies induced ACD in K14‐NK1RKO or in K14‐Rab27aKO mice and their littermate controls. Results : Signalling mouse and human keratinocytes with SP stimulated the formation of MVBs that fused to cell membrane and increased the secretion of sEVs. These effects required the expression of NK1R and Rab27a since they were abrogated in keratinocytes from K14‐NK1Rfl/fl or K‐14‐Rab27aKO mice. sEVs secreted by DNCB and SP‐signaled keratinocytes expressed CD63, CD81 and TSG101 and contained IL‐1β. Molecular studies showed that keratinocytes treated with SP have increased transcripts of proteins involved in the activation of signals 1 and 2 of the inflammasome and augmented amount of IL‐1β. Induction of ACD in K14‐NK1Rfl/fl or in K14‐Rab27aKO mice was impared compared to their littermate controls. Summary/Conclusion : Our data demonstrating that SP signalling of keratinocytes exposed to allergen potentiates the secretion of sEVs that are necessary for ACD support the therapeutic potential of blocking the SP‐NK1R axis for the treatment of the disease. Funding : NIH R01 AI168142 and NIH R01 AI174273

Effect

Presenter: Mahsa Khodabakhshmajd Lehigh University, USA Introduction : Antimicrobial peptides are short, cationic, amphiphilic peptides that bind electrostatically to the negatively charged bacterial surface. Recently, evidence has emerged that bacterial extracellular vesicles, with their surface properties that resemble the surface of the parent bacterium, act as “decoys”, preventing the antibacterial activity of the peptides. The specific factors that regulate this decoy activity remain unclear, however. Methods : We compared the decoy activity of vesicles produced by several Gram‐negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Aggregatibacter actinomycetemcomitans) against LL‐37. To isolate the role of specific membrane properties, including charge and saturation, in this decoy activity, we compared our results to those obtained using liposomes of controlled lipid compositions. Results : We observed that all of the tested vesicles were protective against LL‐37 to some extent, with vesicles produced by P. aeruginosa displaying the strongest decoy activity. Liposomes with increased amounts of negatively charged phosphatidylglycerol (PG) were more protective than those composed of neutrally charged lipids; however, charge alone did not fully explain the results obtained with the bacterial vesicles, as P. aeruginosa were not the most highly charged vesicles used. In addition to charge, we have observed that lipid saturation and the resulting membrane fluidity impact the binding of LL‐37 and resulting decoy activity. Summary/Conclusion : Together, these results demonstrate that both lipid charge and saturation influence the ability of LL‐37 to bind to vesicles and therefore regulate the decoy activity of the vesicles. Funding : This work was supported by the United States National Institutes of Health and the Pennsylvania Department of Health.

Evring

Presenter: Rebecca Mayer University of California, Davis, California, USA Introduction : Effective surveillance for many cancers, including head and neck cancer (HNC) and ovarian cancer (OvCa), is limited by current biomarker assays, which often lack specificity and sensitivity, particularly in early‐stage cases. To address these limitations, we introduce EVring technology, enabling isolation‐free enrichment of extracellular vesicles (EVs) directly from whole biofluids by exploiting Marangoni flow during biofluid drop drying. This approach preserves EV heterogeneity and optimizes subpopulation analysis, advancing EV‐based diagnostics for cancer detection and surveillance. Methods : EVring leverages apparatus‐free coffee ring dynamics and Marangoni flow to enrich EVs directly from diluted patient serum (IRB‐approved protocol with informed consent), using only simple solution and surface treatments to control separation and enhance EV subpopulation resolution at the droplet edge. Raman spectroscopy was employed to analyse EV metabolite profiles within the ring, capturing protein, lipid, and metabolite profiles relevant to cancer progression and recurrence. Machine learning models trained on Raman spectral data classified EV features and linked them to metabolic pathways associated with cancer. Results : The EVring platform achieved size‐specific separation of EV subpopulations without any additional isolation. Control analyses using reference bead size standards within the size range of small EVs (50–200 nm) confirmed a high degree of spatial subfraction by size within the ring. Raman spectra revealed metabolic signatures in EVs associated with recurrence, including cholesterol, proline, and fatty acid esters. Subsequent machine learning models classified recurrence vs. remission states with 100% sensitivity and 94.3% specificity. Additionally, EVring demonstrated the ability to maintain EV integrity, providing direct insights into EV‐driven metabolic reprogramming in cancer. Summary/Conclusion : This approach represents a major advancement in EV analysis, enabling the first isolation‐free, clinically relevant analysis of EVs directly from whole biofluids. The isolation‐free approach dramatically simplifies clinical workflows by eliminating labour‐intensive purification, reducing sample processing time, and minimizing known artefacts associated with traditional isolation methods. With broader implications beyond cancer surveillance, this platform can be used for isolation‐free enrichment of EVs directly from human biofluids for numerous downstream applications across many indications.

Exovia

Presenter: Martin Sieber BIONET Corp, Taipei, Taiwan (Republic of China) Introduction : Hair loss is a significant concern for many individuals, prompting the exploration of innovative approaches for hair follicle revival. One promising strategy involves the use of exosomes derived from umbilical cord mesenchymal stem cells (UCMSCs), which are known to carry various bioactive factors that can influence cellular behaviour. This study investigates the potential of UCMSC‐derived exosomes to enhance the proliferation of hair cells and improve the overall health and function of hair follicles. We employ bioinformatics tools to analyse the biological pathways and molecular networks associated with these exosomes. Our findings not only enhance the understanding of the role of exosomes in hair regeneration but also provide valuable insights for the development of new therapeutic strategies. Methods : Cell proliferation assays were conducted using the MTS assay to assess cell viability. The mRNA gene expression in the cells was measured using real‐time PCR. Cell migration was evaluated using a scratch assay. The secretion levels of inflammatory factors were determined through ELISA. Additionally, ingenuity pathway analysis (IPA) (Qiagen) was employed to identify canonical pathways and upstream regulators. Results : UCMSC‐derived exosomes demonstrate a range of beneficial effects on hair follicle stem cells, hair follicle dermal papilla cells, and keratinocytes. They promote significant proliferation of hair follicle cells in a dose‐dependent manner and markedly enhance keratinocyte migration within 24 h. Additionally, these exosomes upregulate hair growth‐related genes, including VCAN, ALP, WNT5A, VEGFA, and SOD1, while downregulating the hair loss gene DKK1. Under oxidative stress conditions, UCMSC exosomes facilitate the restoration of cell numbers and reduce IFN‐γ secretion by 54% in stimulated PBMCs, indicating their anti‐inflammatory properties. Pathway analysis suggests that UCMSC exosomes are involved in key signalling pathways associated with hair growth, with IPA analysis revealing significant engagement in the PI3K/Akt signalling pathway. Summary/Conclusion : UCMSC‐derived exosomes demonstrate significant potential for revitalizing hair follicles by promoting the proliferation of various types of hair cells, enhancing their migratory capacity, and combating inflammation. Importantly, we have identified key molecular mechanisms that enhance our understanding of how UCMSC‐derived exosomes support hair regeneration. By harnessing these benefits, UCMSC‐derived exosomes emerge as a promising option for clinical applications in hair regeneration therapies.

Facile

Presenter: Cong M. Nguyen Griffith University, Brisbane, Queensland, Australia Introduction : Zinc‐based metal‐organic frameworks (ZnMOF) have gained significant interest for their tunable properties and potential applications in biology. In this study, we synthesized ZnMOFs using adenine as the organic linker and explored their potential for aptamer‐based targeting and extracellular vesicle (EVs) enrichment. Methods : The synthesized ZnMOF was characterized using UV‐Vis spectroscopy, dynamic light scattering, and zeta potential measurements. Post‐synthesis treatments, such as polystyrene sulfonate and sonication, were employed to modulate the particles' physicochemical properties. Functionalization of ZnMOF with an aptamer was assessed through fluorescence microscopy. Nanoparticle tracking analysis was used to evaluate the EV capture efficiency. Downstream analysis of captured EVs included western blotting and loop‐mediated isothermal amplification. Results : Among the tested nucleobases, Adenine was the only nucleobase spontaneously incorporated into ZnMOF, with Zn:Adenine ratio of 2:1. The particle size and zeta potential of ZnMOF could be modulated using polystyrene sulfonate treatment and sonication. The fluorescently‐tagged VCAM‐1 aptamer with poly adenine‐tailing was successfully incorporated into ZnMOF supramolecular structure, amplifying signal in our immunofluorescence assay. CD63‐aptamer incorporated ZnMOF efficiently cleared EVs from the input sample. The utility of reverse‐complement oligo could release EVs from ZnMOF particles. The rescued extracellular vesicles were confirmed to express endosomal markers (CD63 and CD9) and negative for markers of other sub‐cellular compartments. RNA samples from hypoxic SVEC endothelial EVs isolated with CD63‐aptamer@ZnMOF were significantly amplified, compared to the normoxic SVEC EVs. Summary/Conclusion : In conclusion, we successfully synthesized ZnMOF using adenine as a nucleobase, resulting in tunable particles that exhibit promising properties for biological applications. Our results demonstrated that post‐synthesis treatments, including polystyrene sulfonate (PSS) and sonication, can be employed to modulate the size and surface charge of the ZnMOF particles, providing versatility for various applications. The incorporation of aptamers such as VCAM‐1 and CD63 aptamers into ZnMOF particles allowed for the development of bio‐functionalized materials with enhanced capabilities in immunofluorescence and EVs isolation.

Gentle

Yu‐Hsin Chang 1 , Ani Barbulova 1 , Immacolata Fiume 1 , Priyanka Devarakonda 1 , Angela Mary Joseph 1 , Miriam Piccioni 1 , Greta Jasulaityte 2 , Stefania Crispi 1 , Gabriella Pocsfalvi 1 1 National Research Council of Italy, Institute of Biosciences and BioResources, Rome, Italy; 2 FORMULATRIX (UK) Ltd., Bedford, UK Stefania Crispi and Gabriella Pocsfalvi are co‐senior authors . Introduction : µPulse Tangential Flow Filtration system (Formulatrix) has already been successfully used for exosome harvesting from mammalian cultures. Here we optimized the setup of the µPulse TFF system to isolate EVs from the conditioned cell culture medium (CCM) of cell suspension cultures (CSCs) of different plant species and compared it with the ultracentrifugation (UC)‐based method. Nicotiana tabacum BY2 CSCs were used to optimize the system. Methods : Solanum lycopersicum, Phaseolus vulgaris, Humulus lupulus, Vicia faba and BY2 were grown in liquid nutrient media and sub‐cultured weekly. CCM of 7‐day‐old cultures was used as a source for EVs isolation. CCM was subjected to differential low‐velocity centrifugation to remove cells and cell debris. The supernatant of the 15000 × g (S15K) was used to harvest EVs by either UC or µPulse TFF. For TFF, disposable microfluidic filter chips containing three types of modified polyethersulfone (mPES) membrane with 300, 100, 50 kDa MWCF were tested at different return valve pressures and at different volumes. EV concentration, buffer exchange, labelling and loading experiments were optimised. The harvested EVs were characterized using the interferometric light microscopy (Videodrop, Myriade), by protein yield (Qubit) and by protein profile (SDS‐PAGE) analysis. BY2 CCM EVs were studied over 7 days alongside the growth curve. Results : µPulse TFF system provides an efficient and gentle way to isolate plant EVs from CSCs. S15K BY2 was used to optimize µPulse TFF parameters. We have found that 300 and 100 kDa filters yield similar yields, while the 50 kDa filter resulted in higher protein concentration, most probably due to the presence of co‐purifying proteins. The size and size distribution (200–300 nm diameter particles) of EVs obtained by µPulse TFF were similar to those harvested by UC. Summary/Conclusion : We have optimized a plant CSC‐based EV‐isolation platform on the µPulse TFF instrument. Funding : This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Staff Exchange project “FarmEVs” grant agreement N. 101131175 and PRIN PNRR 2022 n. P2022RLH39 and funded by the European Union HORIZON‐EIC‐2023‐PATHFINDERCHALLENGES‐01 nutriEV project under grant agreement N. 101161353.

Glycan

Hema Saranya Ilamathi 1,2 , Mariana Castañeda Mesa 1,2 , Oscar P. B. Wiklander 1,2 1 Department of Laboratory Medicine, Unit for Biomolecular and Cellular Medicine, Karolinska Institutet, Stockholm, Sweden; 2 Breast Center, Karolinska Comprehensive Cancer Center, Karolinska University Hospital, Stockholm, Sweden Introduction : The glycosylation profiles of lipids and proteins play crucial roles in cell signalling and immune response. In cancer, these patterns shift, driving tumour progression, metastasis, and immune evasion. Tumour cells release extracellular vesicles (EVs) containing various biomolecules that can serve as liquid biopsy biomarkers. However, the variation in glycan profiles of tumour‐derived EVs (tEVs) across different cancer stages remains unclear. Currently, there are limited quick and affordable technologies for glycan profiling. Our goal is to develop a rapid and cost‐effective technique to characterize these glycan profiles. Methods : We isolated EVs from non‐cancerous and cancerous cells using size exclusion chromatography and quantified particle numbers with a Zetaview analyser. To profile glycans, we developed an in‐house bead‐based protocol utilizing lectins. EVs were pulled down using lectin‐conjugated beads and analysed via flow cytometry. Results : Our findings reveal distinct glycan expressions on HEK EV surfaces. Using lectins such as ConA, WGA, and UEA, which target mannose, sialic acid, and fucose residues, respectively, we noted higher sialic acid levels on HEK EV surfaces with minimal mannose detection. Fluorescent‐tagged tetraspanin antibodies confirmed EV‐lectin binding. Interestingly, EVs from breast cancer cells showed altered glycan profiles with reduced sialic acid and fucose residues compared to non‐cancerous cells. Summary/Conclusion : Our in‐house lectin‐based EV profiling technique is faster (< 24 h) and more cost‐effective than traditional glycan profiling methods like microarrays and mass spectrometry. We aim to develop a multiplex lectin‐based bead panel to fingerprint cancer cell glycan profiles and identify potential glycan‐based biomarkers for future research. Funding : This project is funded by Cancerfonden, the Swedish Research Council fund (VR grant), and the CIMED grant.

Higher

Mariele Montanari 1 , Daniele Lopez 1 , Giovanna Panza 1 , Sara Biagiotti 1 , Faiza Abbas 1 , Rita Emili 2 , Michele Guescini 1 , Fabiana Fanelli 1 , Mauro De Santi 1 , Paola Lanuti 3 , Elena Barbieri 1 , Ludovica Di Fabrizio 1 , Giosuè Annibalini 1 , Barbara Canonico 1 1 Department of Biomolecular Sciences, University of Urbino Carlo Bo,Urbino, Italy 2 , U.O.C. Oncologia Medica, Ospedale Santa Maria della Misericordia, AST Pesaro Urbino, Urbino, Italy, 3 University “G.d'Annunzio,” Chieti‐Pescara, Italy Introduction : Among techniques for single EV analysis, flow cytometry (FC) has a high potential for clinical application due to its high throughput and multiplex fluorescence capability. Indeed, bead‐based FC has also been used to interrogate EV surface proteins. Particularly, IGF‐1R (CD221), a key member of the IGF axis, known for its oncogenic role in multiple cancer lineages, was investigated on both cells and EVs in blood from breast cancer survivors since the IGF‐1/IGF‐1R pathway is highly breast cancer subtype context‐dependent. Methods : An FC method for analysing the expression and activation of the IGF‐1 receptor is currently set up in our Laboratory, giving good results on myeloid cells. The plasma of donors and BC survivors was investigated at different time points for EV detection and CD221 positivity. BC patients are enrolled in the MoviS Trial (ClinicalTrials.gov: NCT04818359 ) and follow a 3‐month aerobic exercise and nutritional education programme. All reagents were spun at 21,000 ×  g , following general guidelines. We stained blood plasma (centrifuged at 2500 ×  g ) with a lipophilic cationic dye (LCD, BD Biosciences) recently identified as a generic EV tracer, highly useful when samples containing heterogeneous EV population need to be analysed or when EVs may not express the classical tetraspanin markers (e.g., CD9, CD81, CD63). Rosetta calibration beads and the application of either an SSC or fluorescent trigger threshold were employed. The capture bead technique was applied using Exostep (Immunostep) for bead‐based evaluation. Results : Preliminary results show higher percentages of CD221+ EVS in blood from BC survivors concerning age‐matched donors. Furthermore, these CD221+ EVS mainly belong to the pool of CD81 negative EVs. Although further tests are needed (also to increase adherence to the MISEV guidelines), this finding is confirmed by Exostep data. Other experiments are ongoing to confirm data and to add more MISEV technical approaches. Summary/Conclusion : The IGF‐1 receptor is an important immunomodulator, emerging as an attractive target for cancer therapy. These preliminary findings will be integrated with the characterization of the isolated EVs and nanoparticle tracking analysis, aiming at consolidating current results and coupling them with data on the expression and activation of the IGF‐1 receptor on immune cells.

Highly

Presenter: Yawen Fu Karolinska Institutet, Stockholm, Sweden Introduction : CRISPR‐Cas9 genome editing technology has revolutionized the landscape of gene therapy, immunotherapy, and regenerative medicine. Clinical trials using CRISPR‐Cas9 for gene therapy are currently in progress or approved, such as in the treatment of patients with hemoglobinopathies. However, repairing a large defect or inserting a whole gene is not possible using CRISPR‐Cas9, base editing, or prime editing. Hence, there is growing interest in homology‐directed repair (HDR)‐mediated precise gene knock‐in to correct loss‐of‐function mutations and ultimately cure genetic diseases. However, the HDR efficiency is currently lower than indels (insertions and deletions), which has become a bottleneck for the application. Methods : Here, we leverage advanced extracellular vesicle (EV) engineering methods to develop a novel modality for delivering Cas9‐ribonucleoproteins (RNPs) as well as templates of a gene to integrate the correcting‐cDNA at a specific locus by HDR simultaneously, all of which have been achieved by an ‘All‐in‐One’ Delivery Vehicle (AiDV). Results : Our highly efficient nanoparticles can deliver the complete HDR machinery to multiple clinically relevant cell types, including T cells and hematopoietic stem cells (HSCs). With a simple addition to cell cultures, we achieve robust HDR editing rates (e.g., 40% precise gene knock‐in in Jurkat cells). Notably, in combination with the small molecule compound M3814, which inhibits non‐homologous end‐joining (NHEJ) repair, HDR is enhanced to over 90% at one endogenous locus. Summary/Conclusion : This approach significantly improves HDR‐mediated gene knock‐in efficiency, making it applicable for clinical treatment. The straightforward operation and the possibility of in vivo application would make this treatment more economical than other options, paving the way for its development and improvement as an off‐the‐shelf treatment. Funding : This study was supported by the Svenska Sällskapet för Medi­cinsk Forskning (SSMF) Postdoc Grant 2024.

Immune

Xin Zhang 1,2 , Sisi Ma 3 , Syeda Iffat Naz 3 , Erik J. Soderblom 4 , Constantin Aliferis 3 , Virginia Byers Kraus 1,2,5 1 Duke Molecular Physiology Institute, Duke University School of Medicine, Duke University, Durham, North Carolina, USA; 2 Department of Orthopaedic Surgery, Duke University School of Medicine, Duke University, Durham, North Carolina, USA; Institute for Health Informatics, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA; 4 Duke Proteomics and Metabolomics Core Facility, Duke University School of Medicine, Duke University, Durham, North Carolina, USA; 5 Department of Medicine, Duke University School of Medicine, Duke University, Durham, North Carolina, USA Introduction : Extracellular vesicles (EVs) are important in many biological processes, including those related to ageing and longevity. Our causal analyses of adults aged 71 years and older from the prospective Duke Established Populations for Epidemiologic Studies of the Elderly (D‐EPESE) cohort ( n = 1507, PMID: 36182774) identified lymphocytes, physical function and activity, and regular exercise as key contributors to longevity, highlighting the vital roles of immune and muscle systems in extending lifespan. The objective of the present study was to identify circulating extracellular vesicle (EV) biomarkers that indicate longevity. Methods : The baseline plasma EV proteome ( n = 48 D‐EPESE participants, mean age 77.2 ± 1.7 years; 50% female, 50% Black, 50% < 2‐year survival, 50% ≥ 10‐year survival) was analysed using high‐resolution mass‐spectrometry and flow cytometry. Fresh EDTA blood specimens were centrifuged, and plasma was aliquoted and frozen at −80°C until analysis. After thawing, plasma was centrifuged to remove debris, followed by EV isolation using polymer‐based precipitation and characterization for size, lipid bilayer structure and surface markers. The capacity of EV peptides to predict 10‐year longevity was evaluated in a discovery dataset ( n = 32) and a validation dataset ( n = 16). Results : Longevity‐associated large EV (LEV) plasma subpopulations, defined by surface markers, were predominantly linked to immune and muscle cells. Of the 7960 identified plasma EV peptides (corresponding to 519 proteins), nearly half were related to the immune system and 10% to muscle. However, among the 756 peptides with higher abundance and 130 with lower abundance in long‐lived versus short‐lived participants, 437 were enriched in the immune system markers, while only 12 were enriched in muscle markers. Final predictive models incorporating 3–5 plasma EV peptides demonstrated high discriminative capacity for longevity, with receiver operating characteristic areas under the curve ranging from 0.91 to 1 in the hold‐out validation dataset. Summary/Conclusion : Our findings suggest that immune cells produce plasma EVs linked to longevity, shedding light on the key mechanisms that regulate healthy ageing. The EV predictors of longevity indicate that there could be advantages in targeting complement pathways to extend lifespan, potentially using one of the many complement inhibitors currently available or in clinical trials. Funding : This work was supported by the National Institute on Ageing R56AG060895, R01AG070146, and R01AG054840.

Impact

Sunyoung Ham 1,2 , Xuanxuan Li 1,2 , Yue Su 1, 2 , Su‐Ho Park 1, 2 , Andreas Möller 1,2 1 Chinese University of Hong Kong, Hong Kong SAR 2 Li Ka Shing Institute of Health Sciences, Hong Kong SAR Introduction : Ultracentrifugation is the most commonly employed method for enriching small extracellular vesicles (sEVs) due to its efficiency in pelleting small, buoyant particles. While there are established guidelines regarding optimal centrifugal force and time to prevent cell and organelle damage, limited data exist on the specific impacts of ultracentrifugation on sEV integrity. This study aims to investigate how different centrifugal forces affect the physical properties of sEVs. Methods : To estimate the forces exerted on sEVs and liposomes during centrifugation, theoretical calculations were conducted, focusing on potential limits for shape deformation and rupture based on membrane composition. Experimentally, sEVs from different natural sources and manufactured liposomes were labelled with two different colours and subjected to different centrifugal forces and durations. The samples were analysed through nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, and super‐resolution microscopy to evaluate changes in particle size and shape. Results : Theoretical calculations indicated that increased centrifugal forces could exceed the rupture threshold for lipid membranes. Moreover, the increased centrifugal forces are capable of altering sEV size and protein distribution, suggesting the hypothesised vesicle rupture or deformation occurring during ultracentrifugation. Notably, liposomes consisting of DOPC (Dipalmitoylphosphatidylcholine) and cholesterol demonstrated greater structural stability compared to sEVs across varied centrifugal forces and durations. Summary/Conclusion : Understanding the impact on vesicle integrity by ultracentrifugation will enable us to better define the limits of interpretation of sEVs generated by this method as well as optimise centrifugation conditions designed to preserve sEV integrity.

Killer

Isabel Doutor 1,2 , Cristiana Ulpiano 1,2 , Gabriel Costa 1,2 , Cláudia Lobato da Silva 1,2 , Ana Azevedo 1,2 , Ana Fernandes‐Platzgummer 1,2 1 Department of Bioengineering and iBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Portugal; 2 Associate Laboratory i4HB—Institute for Health and Bioeconomy, Portugal. Introduction : Umbilical cord blood (CB)‐derived natural killer (NK) cell immunotherapy offers significant potential against several malignancies, including blood cancers. This potential arises from the cytotoxic nature of NK cells, coupled with the allogeneic properties, low immunogenicity, and easy accessibility of CB. Another promising immunotherapy approach involves using extracellular vesicles (EVs), naturally secreted by different cell types, including NK cells, as a safer cell‐free alternative. However, the persistent challenge lies in obtaining the therapeutic quantities needed of functional NK(CB) cells and their derived EVs. Methods : To address this, CD56+ NK cells from CB samples underwent expansion in various interleukin‐2‐supplemented culture media, namely X‐VIVO 15, NK MACS (NKM) with 5% human serum (hS), CTS NK‐Xpander (NKX) with 5% hS, StemSpan SFEM II, PRIME‐XV NK Cell CDM, and GMP SCGM. NK(CB) cells were characterized by flow cytometry, and their activation was assessed through degranulation and cytotoxicity assays against the K562 cell line. A conditioning phase followed, utilizing NKX without hS or supplemented with exosome depleted human platelet lysate (hPL‐EVd). The culture medium was renewed every 24 or 48 h to evaluate the impact on cell viability and EV production. EVs were isolated by anion‐exchange chromatography, characterized according to MISEV2023 guidelines and tested in cytotoxicity assays. Results : NKM and NKX were the most favourable culture media for NK cell expansion, each maintaining a CD3‐CD56+ NK cell percentage above 90%. Enhanced NK cell activation was observed, demonstrated by heightened degranulation and cytotoxicity. Notably, hPL‐EVd‐supplemented NKX led to increased particle production during conditioning, while maintaining consistent cell viability over 96 h. No significant difference was observed between the two conditioning schedules tested. The optimized downstream process showed an enrichment of particles smaller than 200 nm with adequate morphology. Key proteins required for cytotoxic activity against cancer cells, such as perforin and granzyme B, were confirmed, along with the cytotoxic effects of NK(CB)‐EVs on K562 cells. Summary/Conclusion : These results are crucial for the optimal production of NK(CB) cells and NK(CB)‐EVs, laying the foundation for robust manufacturing processes. This study is the first to produce highly cytotoxic EVs from readily available NK(CB) cells for use in immunotherapy.

Liquid

Seungmin Kim 1,2# , Youbin Kang 3# , Hyunku Shin 4# , Eun Byul Lee 4 , Byung‐Joo Ham 3* and Yeonho Choi 1,2,4,5 * 1 Department of Biomedical Engineering, Korea University, Seoul, Republic of Korea; 2 Interdisciplinary Program in Precision Public Health, Korea University, Seoul, Republic of Korea; 3 Exopert Corporation, Seoul, Republic of Korea; 4 Department of Life Science, University of Seoul, Seoul, Republic of Korea; 5 School of Biomedical Engineering, Korea University, Seoul, Republic of Korea Introduction : The diagnosis of depression typically relies on subjective patient self‐reports and clinical observations, and treatment response can only be confirmed after a 5–7 week course of antidepressants. This highlights the need for diagnostic techniques using objective indicators of medical decision‐making. We developed a novel method to detect depression and predict treatment response by analysing plasma extracellular vesicles (EVs) using nanoplasmonic spectra and deep learning. Methods : We isolated plasma EVs, which can penetrate the blood‐brain barrier and carry psychiatric information. EV Raman signals were obtained to detect molecular information from depressive patients ( n = 98), and non‐depressive controls, including healthy controls ( n = 42) and panic disorder patients without depression ( n = 6). These signals were used to develop a deep learning‐based diagnostic and predictive algorithm. The algorithm consisted of two main steps: (1) diagnosing depression and (2) predicting treatment response. Results : Our algorithm successfully distinguished depression patients not only from healthy individuals but also from panic disorder patients with an area under the curve (AUC) accuracy, sensitivity, and specificity of 0.95, 0.91, and 0.95, respectively. Subsequently, our algorithm classified responders and non‐responders to antidepressants with a high AUC accuracy, sensitivity, and specificity of 0.91, 0.89, and 0.93, respectively. The clear identification of individuals demonstrates the potential of the model to proactively predict the treatment response before encountering medication failures. To confirm the basis of our diagnostic outcomes, we applied explainable AI (XAI) to the algorithm. Consequently, we identified the key decision‐positive values, allowing us to interpret the critical features driving the model's classification performance. Summary/Conclusion : Our technique demonstrates the potential of liquid biopsy‐based depression detection and prediction of antidepressant response for companion diagnostics. We address an unmet need in depression diagnosis by offering a more objective method and showcasing its potential for personalized treatment strategies. This approach reduces unsuccessful treatment attempts and improves therapeutic outcomes, enabling individualized strategies that maximize treatment efficacy. Funding : This study was supported by the National Research Foundation of Korea grant (RS‐2023‐00277719) and the Technology Development Program (RS‐2022‐TI016650) funded by the Ministry of SMEs and Startups.

Paving

Presenter: Giacomo Vacca Kinetic River Corp., Mountain View, USA Introduction : Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder leading to the development of fluid‐filled cysts in the collecting duct of the kidney. ADPKD greatly affects the quality and length of life of patients and can lead to renal failure. Most recent estimates show that ADPKD affects approximately 417,000 people in the United States alone. Despite this level of prevalence, current diagnostic methods for ADPKD are invasive, lacking sensitivity, or prohibitively expensive. Methods : Our preliminary work (Dr Ward's Laboratory) involved proteomic analysis on 2008 proteins expressed on extracellular‐like vesicles (ELVs) using label‐free mass spectroscopy on sucrose/D 2 O gradient‐purified urine‐derived samples from ADPKD patients (PKD1 mutation). Of these, four proteins were identified as candidate quantifiable biomarkers of disease: polycystin‐1 (PC1), polycystin‐2 (PC2), exosomal polycystin‐1 interacting protein (EPCIP), and a fibrocystin homolog (TMEM2). We developed a fluorescent antibody conjugate specific to one of these (PC1); we then used a flow cytometer optimized for nanoparticle detection (Kinetic River) to evaluate the effectiveness of this approach to rapidly detect and quantify the presence of PC1‐positive ELVs in urine. Samples from first‐void urine of 7 PKD patients and 7 normal individuals were prepared (2000 ×  g , 10 min), stained (PE anti‐PC1 from Dr Ward's Laboratory and membrane dye Aco‐490 from Acoerela), filtered using size‐exclusion centrifuge columns, and analysed on the nanoparticle analyser. Background (sheath fluid), unstained, membrane‐stain‐only, and antibody‐stain‐only controls were collected. Results : ELVs were identified against background events using a combination of scattering and fluorescence gates. Membrane‐stained, PC1‐expressing urine ELV populations were clearly resolved against non‐expressing ones for sizes down to approximately 150 nm. Summary/Conclusion : Our results demonstrate that a PC1‐based assay run on an ultrasensitive flow cytometer can detect PKD‐related ELVs in urine and differentiate between normal and ADPKD patients. We plan to build on this approach to incorporate additional ADPKD biomarkers to increase the fidelity of the diagnostic assay. Funding : This work was made possible in part by US government support under award number 1R43GM155492‐01 from the NIH.

Plasma

Presenter: Zhi‐Rou Zhou The First Affiliated Hospital of Sun Yat‐Sen University, Guangzhou, People's Republic of China Introduction : Group 2 innate lymphoid cells (ILC2s) are novel key players in the induction and maintenance of allergic inflammatory responses in airways. Various extracellular vesicles (EVs) are released into plasma in allergic airway inflammation, correlating with impaired airway function and severe inflammation. EV‐derived miRNAs are extensively involved in intracellular communication. However, the interaction between plasma EVs and immunocytes in this complex network remains to be further elucidated. Our study aimed to characterize the miRNA profiles in plasma EVs from healthy controls and AR patients, to evaluate the effect of plasma EVs on ILC2s and to identify miRNAs potentially contributing to ILC2 dysfunction in AR. Methods : Plasma EVs were isolated from healthy controls and AR patients by using size exclusion chromatography (SEC), and differential miRNA expression profiles were obtained through miRNA sequencing. Peripheral blood mononuclear cells (PBMCs) were exposed to plasma EVs and miRNA mimics and miRNA inhibitors to assess the effect of plasma EVs and the underlying mechanisms. Results : We found that EVs from HC and AR patients exhibited comparable characteristics in terms of concentration, structure, and EV marker expression. Both HC‐EVs and AR‐EVs were efficiently internalized by ILCs. AR‐EVs significantly promoted the differentiation and activation of ILC2s compared to HC‐EVs. Unique miRNA signatures of HC‐EVs and AR‐EVs were revealed by miRNA sequencing in diverse biological processes, among which miR‐150‐5p, miR‐144‐3p, miR‐10a‐5p, and miR‐10b‐5p were identified as potential contributors to AR‐EVs’ effects on ILC2s. The level of IL‐13+ ILC2s was only increased by mimics of the miR‐150‐5p and was significantly decreased after the treatment of miR‐150‐5p inhibitor. Summary/Conclusion : Our study demonstrated that plasma EVs from patients with AR exhibited a pronounced capacity to significantly enhance the differentiation and activation of ILC2s, which was correlated with an elevated expression of miR‐150‐5p in AR‐EVs. These findings contribute to the advancement of our comprehension of EVs in the pathogenesis of AR and hold the potential to unveil novel therapeutic targets for the treatment of AR. Funding : This study was supported by National Natural Science Foundation of China.

Rala/B

Kuang‐Jing Huang 1,2 , Louis Bochler 1,2,3,4,5 , Katerina Jerabkova‐Roda 1,2,3,4,5 , Valérie Demais 6 , Victor Hanss 2,7 , Danièle Spehner 8 , Annabel Larnicol 1,2,3,4,5 , Olivier Lefebvre 1,2,3,4,5 , Laetitia Paulen 1,2,3,4,5 , Jean‐Yves Rinckel 2.3,9 , Avais M. Daulat 10 , Jean‐Paul Borg 10 , Etienne Coyaud 11 , Florent Colin 1,2,3,4,5 , Patrick Schultz 2,7 , Jacky G Goetz 1,2,3,4,5* , Vincent Hyenne 1,2,3,4,5,6* 1 Tumor Biomechanics, INSERM UMR_S1109, Strasbourg, France; 2 Université de Strasbourg, Strasbourg, France; 3 Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France; 4 Equipe Labellisée Ligue Contre le Cancer, Nantes, France; 5 Centre de Recherche de Biomédecine de Strasbourg (CRBS), Université de Strasbourg, Strasbourg, France; 6 Plateforme Imagerie In Vitro, CNRS UAR 3156, Neuropôle, University of Strasbourg, Strasbourg, France; 7 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France; 8 CNRS, SNC5055, Strasbourg, France; 9 Etablissement francais du sang (EFS), INSERM UMR_S1255, Strasbourg, France; 10 Aix‐Marseille Université, CNRS, INSERM, Institut Paoli‐Calmettes, Centre de Recherche en Cancérologie de Marseille (CRCM), Marseille Protéomique, Marseille, France; 11 Department of Biology, INSERM UMR_S1192, Protéomique Réponse Inflammatoire Spectrométrie de Masse‐PRISM, Université de Lille, Lille, France Introduction : Extracellular vesicles (EVs) are membrane‐bound particles naturally released by cells and become crucial mediators of intercellular communication. There are two primary sources of EVs based on their biogenesis pathways: exosomes and ectosomes. Exosome secretion is a multi‐step process including endosome maturation into multi‐vesicular bodies (MVBs), intra‐luminal vesicle (ILV) budding, cargo loading, MVB transport, and fusion with the plasma membrane. Our previous studies revealed that Ral family GTPases regulate MVB maturation and exosome secretion (Hyenne et al. JCB 2015), influencing the protein and RNA composition of extracellular vesicles (EVs) from a mouse mammary cancer cell line, thus enhancing their pro‐metastatic function (Ghoroghi et al. eLife 2021). Here, we investigate the molecular mechanisms through which Ral proteins drive exosome secretion. Methods : Using BioID, we analysed the interactome of RalA/B, identifying 400 interacting proteins, including EHD4, a dynamin‐like protein that promotes membrane fission. We used CRISPR to knock out EHD4, examining its impact on MVB maturation and EV secretion with total internal reflection fluorescence (TIRF) microscopy to quantify MVB secretion, spinning‐disc confocal microscopy to observe MVB motility, and electron microscopy to study endosome ultrastructure. Live imaging with confocal microscopy captured Ral‐EHD4 interactions. Results : EHD4 is critical for EV secretion, a function restored by re‐expressing WT EHD4 or its paralog EHD1. Using high‐pressure freezing sample preparation and volume‐EM with FIB‐SEM, we revealed MVB morphology and found that EHD4 can shape the size of MVB, similarly to Ral GTPases. Live‐cell imaging showed that EHD4 KO impairs MVB mobility, highlighting EHD4's role in secretory MVB maturation. Rescue experiments using EHD4 mutants reveal its role in different steps of MVB maturation and identify essential EHD4 domains. In parallel, to assess the impact of EHD4 on tumour EVs function, we performed an orthotopic mammary tumour growth analysis in a syngeneic mouse model and observed that EHD4 knock‐out significantly delays primary tumour growth. Summary/Conclusion : EHD4 modulates EV secretion by interacting with Ral GTPases to regulate MVB maturation. Our study uncovers a novel ‘MVBs‐shaping’ mechanism that affects the biogenesis of tumour EVs and their oncogenic function.

Recent

Yasemen Kesimoğlu 1,2 , Mehmet Hikmet Üçışık 3 1 Yeditepe University, Istanbul, Türkiye; 2 İstanbul Topkapı University, Istanbul, Türkiye; 3 Istanbul Medipol University, Istanbul, Türkiye Introduction : Cardiovascular diseases are the leading cause of death in the world, followed by cancer. Among the deaths caused by cancer, lung, colon, stomach, breast, oesophagus, pancreas, liver and prostate are the most common, respectively. Extracellular vesicles are structures with a lipid double layer, which are synthesised by cells and contain components such as protein, RNA and DNA. Extracellular vesicles can be isolated from different cell sources such as mammals, plants, and bacteria and from different samples such as urine, blood, and saliva. Extracellular vesicles can also be used as biomarkers, vaccines, vesicular therapy, or drug delivery systems. In this study, the purpose of using extracellular vesicles in cancer research studies registered at clinical.trials.gov was investigated. Methods : The clinical trials website was searched using the keywords ‘cancer,’ ‘exosome,’ ‘extracellular vesicle.’ A total of 213 clinical trials were identified. Trials were identified by their clinicaltrials.gov ID to avoid re‐analysis. After removing trials that didn't fit topic of the trial, there were 194 clinical trials. The type of cancer, purpose of using extracellular vesicles, source, material analysed, activity status of trial, and location of trial were analysed. Results : Of the cancer clinical trials, 19.1% were conducted in lung cancer, 14.4% in prostate cancer, and 12.4% in breast cancer. Of patient samples, 72% were blood, and 15% were urine. Of extracellular vesicles isolated from different sources, 56.7% were investigated for biomarkers, 33.5% for drug efficacy, 1.5% for drug delivery studies. RNA was analysed in 33.5%, protein in 30.4% of the extracellular vesicle studies. In 26.8% of clinical trials, information about the material to be studied was not provided. When analysing the current status of clinical trials, 32.5% were recruiting, 21% were completed status. When analysing the location of the trials, 34% were conducted in USA, 23.2% in China. Summary/Conclusion : While lung cancer was the most commonly studied cancer, blood samples were most commonly used for extracellular vesicle isolation. While isolated extracellular vesicles were most often studied as biomarkers, RNA and protein analyses were most often preferred for extracellular vesicle samples. While one third of clinical research is still ongoing, the location of studies shows that one third of them are in the United States.

Sample

Estefanía Lozano‐Andrés 1 , Ye Tian 2 , Sten F. W. M. Libregts 1 , An Hendrix 3,4 , Xiaomei Yan 2 , Ger. J. A. Arkesteijn 1 , Marca H. M. Wauben 1 1 Utrecht University, Utrecht, The Netherlands; 2 Xiamen University, Xiamen, People's Republic of China; 3 Ghent University, Ghent, Belgium; 4. Cancer Research Institute Ghent, Ghent, Belgium Introduction : High‐sensitivity flow cytometry has evolved in the last decade to accommodate the need for single EV analysis. This resulted in a variety of instruments from different generations: from analogue high‐end instruments that are tailored for EV analysis to those used for both cell and EV measurements to instruments built for small particle analysis. Hence, it is important to evaluate how results acquired with distinct instruments compare. For this purpose, we used nanoparticles (NPs) and fluorescent biological recombinant EVs (rEVs) to investigate the performance of these instruments. Methods : We measured a range of different concentrations of non‐fluorescent silica NPs (siNP, sizes from 50 to 200 nm) by using light scatter thresholds and rEVs (average size 120 nm) by using both light scatter and fluorescence thresholds on the three different platforms, that is, a N30 nanoflow analyser (NF), an optimized BD Influx (IF) and a CytoFLEX LX (CF). Results : Using light scatter detection, we found significant differences among instruments in relation to background signals, optimal sample concentrations and limits of detection. Only the NF was able to detect the smallest siNP (68 nm). The IF provided reliable rw‐FSC information from the 91 nm siNPs with the least background interference, while the CF detected the 91 nm siNPs based on violet‐SSC with greater background interference. Based on light‐scattering (SSC and FSC), the rEVs overlapped with the background on all instruments. Only on the NF was the rEV fluorescence sufficient to visualize a separate population, while the IF and CF showed a continuous fluorescent population on top of the background. Summary/Conclusion : We found that with the light scatter threshold, the combination of background and low sample concentrations compromises the ability to discriminate particles of interest. The NF requires a higher sample concentration compared to the IF and CF. In addition, the CF is more affected by background interference and swarm detection compared to the IF. Furthermore, our results showed that rEVs can be used to define the optimal concentration range of a particular instrument, thereby ensuring reliable data interpretation. Therefore, the application of rEVs is useful when complex biological samples are compared by flow cytometric analysis.

Sex‐

Donna Elizabeth Sunny 1 *, Elke Hammer 1 , Rabea Schlüter 2 , Stephan Michalik 1 , Uwe Völker 1 , Matthias Heckmann 1 1 University of Medicine Greifswald, Greifswald, Germany; 2 University of Greifswald, Greifswald, Germany Introduction : Cerebral oxygenation differences in preterm infants due to exposure to high oxygen levels during intensive care and sex‐specific oxidative stress responses can disrupt oligodendrocyte maturation, affecting neuronal development and function differently in male and female brains. Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication and stress response in the brain. Here, we used a proteomic approach to identify and characterize the cargo composition of small (< 200 nm) and large (< 1000 nm) EVs released by primary, mouse‐derived oligodendrocyte progenitor cells (OPCs) of both male and female origins separately. We aim to pinpoint molecular alterations within this cargo, triggered by hyperoxic conditions, in a sex‐ and size‐specific manner. Methods : Male‐ and female‐derived OPC spheroids, obtained from neurosphere cultures, were treated with 80% oxygen for 24 h, and EVs were isolated using the precipitation method. This was followed by size separation using the filtration method. The EVs were characterized to meet the Minimal Information for Studies of EVs 2023, including electron microscopy and western blot analysis. The EVs subsequently underwent an untargeted mass spectrometric analysis on an ESI‐LC‐MS/MS system followed by initial data analysis using Spectronaut (Biognosys) and subsequent quantification of differences by the SpectroPipeR pipeline. Results : Our analysis identified distinct sex‐ and size‐specific protein signatures with only 17% and 22% overlap in large (lEVs) and small extracellular vesicles (sEVs) between males and females after hyperoxia. In response to hyperoxia, male‐derived lEVs showed a greater number of significantly altered proteins than female‐derived lEVs, whereas female‐derived sEVs exhibited more protein changes than those from males, suggesting enhanced stress response signalling in females via sEVs and a significant role for lEVs in male cells. Nanoparticle tracking analysis revealed a slight increase in sEV intensity in female samples post‐hyperoxia. Male lEVs showed an upregulation of mitochondrial proteins, including those involved in oxidative phosphorylation and ATP synthesis, while female sEVs showed an enrichment of proteasomal complex proteins, with ingenuity pathway analysis highlighting post‐translational protein phosphorylation pathways. Summary/Conclusion : Our results suggest that intercellular stress response signalling is mediated differentially by male and female OPCs via size‐specific secretion of EVs, whereby large and small EVs carry different protein cargo.

Single

Madeline Cramer, Stephen Lenzini, Elie Zakhem, Jon A. Rowley RoosterBio, Inc., Frederick, Maryland, USA Introduction : Heterogeneity of EV populations complicates their study, as traditional bulk analysis methods fail to capture individual differences in size, content, and surface markers. Single‐vesicle analysis offers a more precise approach, enabling detailed characterization of EV subpopulations. We developed protocols for calibrated NanoFlow cytometry to quantify single‐vesicle surface marker expression and applied them to purified MSC‐EVs from three sources. Methods : We generated purified MSC‐EVs through RoosterBio established production and purification protocols. EV tetraspanin identity markers (CD81, CD63) and MSC identity marker (CD73) were selected for single‐vesicle analysis. For each antibody, optimization steps were performed to identify the optimal staining protocol. Beads with known equivalent reference fluorophore (ERF) values were obtained from NanoFCM. Data was acquired on the NanoFCM Flow NanoAnalyzer and single‐vesicle analysis methods were established to quantify the number of CD molecules per particle. The lower limit of detection was determined by background fluorescence. We then applied this analysis to purified EVs from bone marrow (BM), umbilical cord (UC), and adipose (AD) MSC‐EVs. Results : Antibody titration was used to select a dilution of 800X for samples at 1E10 P/mL. A standard curve was generated from ERF beads to calibrate arbitrary fluorescence intensity to units of Alexa Fluor 488. The antibody‐specific limit of detection for positive expression was between 4 and 10 molecules per particle. Across all tissue sources, particles were 50%–70% CD81+, 40%–60% CD63+, and 15%–35% CD73+. Single‐vesicle analysis suggested CD81 and CD63 were enriched on AD‐EVs (40 CD81, 53 CD63 per particle) compared to UC‐EVs (33 CD81, 44 CD63 per particle) and BM‐EVs (30 CD81, 41 CD63 per particle). CD73 was preferentially expressed on larger EVs and showed ∼20 CD73 per particle for all tissue sources. Summary/Conclusion : We successfully developed single‐vesicle quantitation of EV surface markers by calibrated NanoFlow cytometry. Application of this novel analysis not only showed retention of CD81, CD63, and CD73 in our highly purified EVs, but also discerned tissue‐ and marker‐dependent differences in EV properties. This high‐resolution characterization of EVs can advance our understanding of EV diversity, aid interpretation of EV bioactivity, and support development of methods to enrich specific EV subtypes.

Stable

Presenter: Jelle van den Beukel Amsterdam University Medical Center, The Netherlands Introduction : Measuring concentrations of extracellular vesicles (EVs) is difficult due to their heterogeneity, which leads to incomparable and irreproducible data. To overcome this problem, nanospheres of known concentration could help track the dilution and volume of the EV‐containing sample during analysis. However, adding nanospheres to body fluids results in non‐specific adsorption of proteins, and with that, the formation of a protein corona. This interaction leads to a decrease in the concentration of nanospheres over time, resulting in unstable concentrations of nanospheres. Our aim is to develop nanospheres coated with anti‐fouling polymer brushes that can prevent non‐specific adsorption of proteins present in EV‐containing (bio) fluids. Methods : Polystyrene nanosphere cores were functionalized using surface‐initiated photoinduced electron transfer‐reversible addition‐fragmentation chain‐transfer polymerization (SI‐PET‐RAFT), with three different monomers: N‐(2‐hydroxypropyl)methacrylamide (HPMA), methacrylate phosphocholine (MPC), and carboxybetaine methacrylate (CBMA). Functionalization was monitored using dynamic light scattering (DLS), X‐ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The functionalized nanospheres were incubated in 6 mg/mL fluorescently labelled bovine serum albumin solution and fluorescently labelled human blood plasma to monitor their anti‐fouling properties and stability over time. Results : Functionalization of 220 nm polystyrene nanospheres increased their diameter to 260 nm (HPMA), 250 nm (MPC), and 350 nm (CBMA). XPS confirmed the chemical composition, and TEM showed core‐brush structures on the surface of all functionalized nanospheres. Anti‐fouling tests in two different fluorescent protein solutions demonstrated no binding of proteins to the HPMA‐coated nanospheres. In contrast, MPC nanospheres became slightly fluorescent in human blood plasma, and CBMA nanospheres became fluorescent in human blood plasma. Stability measurements in human blood plasma showed a stable concentration for the HPMA and MPC nanospheres up to 5 h, whereas the CBMA nanospheres decreased in concentration. Summary/Conclusion : Stable anti‐fouling nanospheres can be produced in size ranges of 250–260 nm. Concentrations of HPMA and MPC nanospheres are stable in undiluted human blood plasma for at least 5 h. This stability shows that coated nanospheres can be used as internal calibrants or quality controls for instruments used to detect nanoparticle concentrations in biofluids, such as flow cytometers. Funding : Dutch Research Council (NWO), VIDI 19724.

Tgfβ+

Katarzyna Piszczatowska 1 , Katarzyna Czerwaty 2 , Karolina Dżaman 2 , Nils Ludwig 1 , Mirosław J. Szczepański 1,2 1 Department of Biochemistry, Medical University of Warsaw, Poland, 2 Department of Otolaryngology, The Medical Centre of Postgraduate Education, Poland Introduction : Chronic rhinosinusitis (CRS) is an inflammatory disease affecting almost 5‐12% of the worldwide population and is categorized into two main subtypes: CRS with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP). One of the key aspects of CRS pathogenesis is tissue remodelling (TR). TGFβ–related pathways are widely known for their participation in TR not only in CRS, but also in various other diseases. In our recent study, we detected increased TGFβ expression levels in the upper respiratory epithelium of CRSwNP patients. The aim of this study was to characterize the quantity, quality and TGFβ cargo content in plasma–derived small extracellular vesicles (sEVs) obtained from patients diagnosed with CRSsNP or CRSwNP qualified for Functional Endoscopic Sinus Surgery (FESS). Methods : sEVs were isolated from plasma using pre‐clearing with differential centrifugation and Size Exclusion Chromatography (SEC). Subsequent characterization was performed by Nanoparticle Tracking Analysis (NTA), Cryo‐Electron Microscopy (Cryo‐EM), and western blotting (WB). Results : sEV sizes ranged from 83 to 109 nm and concentrations were determined as 1,90E+09 to 2,6E+11 particles/mL. We observed differences in the concentration value between patients. Cryo‐EM imaging revealed the characteristic shape of sEVs with bilayer membranes and sizes comparable to the NTA findings. WB demonstrated the presence of CD9 and CD63, TGFβ as well as the negative marker Grp94. Summary/Conclusion : In our study, we demonstrated the presence of TGFβ+ sEVs in the plasma obtained from CRS patients. Those sEVs might be involved in the CRS pathogenesis, however, further investigation with the use of further methodologies as well as wider patient cohorts are urgently needed. Funding : Young Investigator Grant from Medical University of Warsaw: 1WK/1/M/MB/N/23

Tissue

Pia Lenz 1 , Johannes Oesterreicher 1 , Johannes Grillari 1,3 , Franz‐Josef Nierscher 2 , Alexandra Pomberger 2 , Hubert Hetz 2 , Heinz Steltzer 2 , Roman Ullrich 2 , Carolina Zeller 1 , Wolfgang Holnthoner 1 , Stephan Müller‐Botz 1,4 , Marcin F. Osuchowski 1 , Gerhard Fritsch 1,4 , Johannes Zipperle 1 1 Ludwig Boltzmann Institute for Traumatology, the Research Center in cooperation with AUVA, Vienna, Austria; 2 Department of Anesthesiology and Intensive Care Medicine, AUVA Trauma Center Vienna, Vienna, Austria; 3 Institute for Molecular Biotechnology, BOKU University, Vienna, Austria; 4 Department of Anesthesiology and Intensive Care Medicine, AUVA Trauma Center Salzburg, Academic Teaching Hospital of the Paracelsus Medical University, Salzburg, Austria Introduction : Traumatic brain injury (TBI) exposes tissue factor (TF, CD142)‐rich neuronal tissue, triggering robust coagulation and immune responses. We examined whether TF‐bearing EVs in plasma could serve as a clinically relevant indicator for confirming TBI and whether temporal fluctuations in platelet‐, innate‐, and adaptive‐immune markers on EVs mirror the evolving host response to a major traumatic incident. Methods : We analysed 42 severely injured adults (Injury Severity Score > 15), enrolled in the bicentric PRIME study ( NCT06314841 ), classifying them as TBI ( n = 21) or non‐TBI ( n = 21) based on clinical and imaging assessments. Blood samples were obtained at admission (0 h), 24, 48, and 96 h. EV characterization in plasma used the MACSPlex EV Kit, measuring markers of haemostasis (CD41b, CD42a, CD62P, CD142), innate immunity (CD14, CD56), adaptive immunity (CD3, CD4, CD8, etc.), and cellular regulation (CD9, CD63, and CD81). Semi‐quantitative epitope‐specific signals were recorded as median fluorescence intensity (MFI). Kruskal–Wallis tests with Dunn's post hoc correction determined significance ( p < 0.05). Results : At 0 h, TBI patients showed higher CD142+ EV levels than non‐TBI (MFI, median [IQR]:1814 [1531–3354] versus 3450 [2047–5278], p = 0.024), and receiver operating characteristic (ROC) analysis confirmed moderate diagnostic value (AUC = 0.703) for TBI. No differences were observed in other antigens or at 24, 48, and 96 h. In all patients, platelet‐associated EV markers were elevated at admission, indicating an acute procoagulant state. Over 24–96 h, markers of innate immunity (e.g., myeloid, NK cell–associated signals) and later adaptive and regulatory pathways (T‐cell and antigen presentation) increased, reflecting a shift from immediate haemostatic activation toward inflammatory and reparative processes. Summary/Conclusion : A transient surge in TF‐bearing EVs at admission prompts an early procoagulant activation secondary to TBI. Subsequent changes in platelet, innate, and adaptive EV markers in the entire cohort suggest complex immune and regenerative dynamics following major trauma. EV‐based phenotyping may complement existing neuronal markers for rapid TBI detection and better characterize evolving host responses. Funding : This study was supported by the Austrian Workers’ Compensation Board (AUVA), institutional funds and Particle Metrix GmbH.

Tspan8

Rihab Ksouri 1 , Stanka Matic 1 , Siobhan King 2 , Irina Nazarenko 1 1 Institute for Infection Prevention and Control, Faculty of Medicine, Medical Center—University of Freiburg, Germany 2 ONI, Oxford, England, United Kingdom Introduction : Extracellular vesicles (EV) are nanoscale heterogeneous membrane structures released into the extracellular space by most cells and present in almost all biofluids. EVs are of remarkable importance due to their involvement in physiological and pathological processes; they contain diverse biomolecules, including proteins, lipids, metabolites and nucleic acids, including RNA and DNA. However, due to the small size and heterogeneity of EVs, it is still not known in detail which of the cargo is located inside the EV and which is associated with the EV surface, forming the so‐called EV biocorona, with DNA being the most prominent molecule identified as associated with EVs. Methods : A fibrosarcoma cell model consisting of 3 cell lines was used: HT1080, HT1080‐V stably transfected with a control lentiviral vector T2A‐PuroR, and HT1080‐Tspan8 transfected with the hTSPAN8‐T2A‐PuroR vector and expressing Tspan8. Tangential Flow Filtration (TFF) was used to isolate EVs that retained the EV biocorona. To obtain a comprehensive DNA and histone profile, the whole cell secretome was separated into three fractions: largeEV, smallEV and EV‐depleted using TFF. The isolated fractions were characterised using nanoparticle tracking analysis (NTA) to determine particle concentration. Integrity and quality control of isolated fractions were assessed using microBCA protein assays and dot blot for EV biomarker's presence. Histone presence was assessed by Western blot, and DNA content and concentration were measured using a QIAamp DNA Mini Kit, followed by fragment length measurement using an Agilent Bioanalyzer. Results : We observed that the particle concentration of small EV ranging between 50 and 150 nm, increased compared to large EV. Characterisation of small EV also showed the highest expression of EV biomarkers. increased DNA content was associated with small EV, with a variation of fragment sizes between 1500‐7000 bp. The presence of all histones was demonstrated by Western blot in our cell lines and small EV. Summary/Conclusion : During this work, we were able to establish our method for the isolation and quantification of EV‐associated DNA and histones. Our future work will focus on the specific localisation of DNA and histones within EVs and the associated protein corona.

Tumour

Presenter: Geuna Park Kyungpook National University, Daegu, Republic of Korea Introduction : NK cells are natural killer cells that originated from haematopoietic stem cells and are included in the innate lymphoid cells and have the ability to inhibit tumour progression. NK cell‐derived exosomes can mimic donor cells' characterization and induce target tumour cell death by biological killing mechanisms. RELT is a cell surface receptor that is upregulated in tumour cells such as lung tumour. In this study, we surface‐modified exosomes derived from NK cells with the RELT‐biding peptide (named RELT) to target RELT‐expressing tumour cells, and also with the KLAKLAKKLAKLAK pro‐apoptotic peptide (named KLA) to induce mitochondrial damage and cytotoxicity. Methods : NK cells were incubated in exosome‐free medium for 48 h for the isolation of exosomes. Following filtration, supernatants were pelleted by ultrahigh‐speed centrifugation twice, and exosomes were resuspended in phosphate‐buffered saline. Then, NK exosomes were surface‐modified with the RELT pep and KLA using the DOPE‐PEG‐NHS linker for 15 min at 37°C. Results : Immunofluorescence microscopy and flow cytometry showed that A549 lung tumour cells and Panc1 pancreatic tumour cells expressed high levels of RELT on the cell surface. Both unlabelled NK exosomes and RELT‐ and KLA‐labelled NK exosomes were efficiently internalized into A549 cells. Importantly, NK exosomes dual‐labelled with both RELT and KLA showed a more efficient cytotoxicity in A549 cells and Panc1 cells than unlabelled NK exosomes and NK exosomes labelled with either RELT or KLA alone. An in vivo study using tumour‐bearing mice showed that tumour growth was reduced by NK exosomes labelled with peptides more efficiently than by unlabelled NK exosomes. Summary/Conclusion : These results suggest that RELT‐targeted and KLA pro‐apoptotic peptide‐armed NK exosomes would be a promising tool for targeted therapy of RELT‐expressing tumours.

Adipose

Presenter: Dalia Ibarissen INSERM, Montpellier, France Introduction : Systemic sclerosis (SSc) is a rare autoimmune disease characterized by 3 pathological processes: vasculopathy, fibrosis and dysregulation of the immune response. SSc is potentially a lethal disease with no curative treatment to date. On the other hand, we have previously demonstrated that adipose tissue‐derived mesenchymal stromal cells (ASCs) as well as their derived extracellular vesicles (EVs) can slow the progression of the pathology in the HOCl‐induced SSc murine model. However, regarding clinical application, the use of autologous ASCs remains a topic of ongoing debate. Here, we compared the functional properties of ASCs from healthy donors (h‐ASCs) and scleroderma patients (SSc‐ASCs), focusing on their anti‐fibrotic and immunosuppressive effects. We also characterized the EVs released by these cells to better understand potential differences in their therapeutic potential. Methods : ASCs and their derived EVs were isolated from healthy donors and from diffuse SSc patients and then amplified. Their anti‐fibrotic effect was assessed in a TGF‐β1 induced model where the ASCs were cocultured with fibroblasts stimulated with 5 ng/mL of TGF‐β1 for 24 h. The myofibroblastic phenotype was assessed via RT‐qPCR. Their immunomodulatory effect was determined in a mixed lymphocyte reaction for 96 h. The size distribution of EV was evaluated by nanoparticle tracking analysis (NTA), expression of EV markers by flow nanocytometry and western blot, and the total amount of proteins and RNA using the Qubit technology. Proteomics analysis has been done on hEVs and SScEVs. Results : Both ASCs decrease the expression of pro‐fibrotic markers (αSMA, COL1A1, COL3A1) as well as increase anti‐fibrotic markers (MMP1, MMP3), but only SSc‐ASCs seem to have a pro‐apoptotic effect on stimulated fibroblasts. MLR results showed that h‐ASCs inhibit PBMC proliferation in a dose‐dependent way, thus underlying their immunomodulatory effects. However, with SSc‐ASCs, the degree of inhibition was markedly reduced. Similar results were obtained with EVs from the respective ASCs. Proteomic analysis showed that 28 proteins are differentially expressed between h‐EVs and SSc‐EVs, with 14 were upregulated in h‐ASCs. Summary/Conclusion : SSc‐ASCs and their EVs partially lose the properties of functional h‐ASCs and EVs. This underlines the interest of using allogenic ASCs and EVs for therapeutic applications in SSc.

Annexin

Presenter: Jamboor K. Vishwanatha University of North Texas Health Science Center, USA Introduction : Tumour‐derived extracellular vesicles (TEVs) promote cancer metastasis by interacting with distant recipient cells. In metastatic triple‐negative breast cancer (TNBC), the lungs are frequently affected, with metastasis occurring in 20‐30% of cases. Annexin A2 (AnxA2), a membrane‐associated protein, is notably elevated in TNBC tissues and EVs, leading to poor patient outcomes. It ranks among the top EV proteins and recruits cargo like microRNAs and proteins. Our lab's research shows reduced lung and brain metastasis with lowered small EV (sEV) AnxA2 levels in vivo. We aim to evaluate the functional role of AnxA2 in driving TNBC lung metastasis via sEVs. Methods : The lung metastatic MDA‐MB‐4175 (LM2) cells were used to stably downregulate AnxA2, with their metastatic potential evaluated via migration, invasion, and proliferation assays. sEVs were isolated using optimized differential ultracentrifugation and filtration method and characterized per MISEV2023 guidelines. The protein cargo of sEV was analysed through quantitative proteomics. Recipient cell uptake and downstream signalling modulation were also assessed. Results : AnxA2 downregulation reduced the metastatic potential of LM2 cells, as evidenced by decreased migratory, invasive, proliferative, and plasmin‐generating abilities. sEVs isolated from conditioned cell culture media were positive for ESCRT proteins, heat shock proteins, and tetraspanins (CD9, CD81), while negative for GM130, calnexin, and cytochrome c. Their size distribution ranged from 30 to 300 nm, displaying distinct double‐membraned vesicles under cryo‐electron microscopy. Proteomic profiling revealed a unique sEV proteomic landscape among groups, differentially impacting biological processes like tumour cell proliferation, cell viability, and migration of tumour cells and fibroblasts. Uptake studies of sEVs by non‐metastatic TNBC epithelial cells and lung fibroblasts showed altered uptake and changes in downstream functional processes. Summary/Conclusion : Lowering AnxA2 expression in TNBC cells reduces their metastatic potential by changing the sEV proteomic cargo, affecting their interactions with recipient cells at local and distant sites. Our findings reveal novel functionality of AnxA2 in the progression of TNBC metastasis through sEVs.

B7‐H3

Presenter: Rosalba Florio University “G. d'Annunzio”, Chieti‐Pescara, Italy Introduction : Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A new frontier of cell‐based immunotherapy in haematological malignancies is represented by chimeric antigen receptor (CAR)‐T cells. Unfortunately, their efficacy in solid tumours is still limited. Extracellular vesicles (EVs) emerged as regulators of the immune system. In this context, CAR‐T‐derived EVs represent a promising development of CAR‐T immunotherapy approaches. In this study, we explored the antitumour activity of CAR‐T‐derived EVs targeting B7‐H3, which is overexpressed in PC cells and resulted in safety in clinical trials. Methods : B7‐H3 CAR‐T and control T cells (CTRL) were co‐cultured with a positive B7‐H3 PC cell line (L3.6pl) to evaluate the cytotoxicity of engineered T cells. EVs from B7‐H3 CAR‐T and CTRL‐T cells were isolated by cell sorting followed by ultracentrifugation and phenotypically characterized by a flow cytometry method patented by our Laboratory. EVs were characterized by atomic force microscopy (AFM), western blot (WB), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The antitumour activity of B7‐H3 CAR‐T‐derived EVs was evaluated by both MTT and killing assays. Furthermore, their cargo was studied by proteomics. Results : B7‐H3 CAR‐T‐derived EVs characterization revealed that EVs displayed a globular shape and an average size of 140 nm. Furthermore, B7‐H3 CAR T‐derived EVs expressed characteristic EV markers (CD63 and Flotillin‐1) and were negative for cytochrome C and other typical contaminants (i.e., apolipoproteins). Co‐culture experiments showed that B7‐H3 CAR‐T cells induced 36% (*** p < 0.01) of killing on L3.6pl cells after 24 h. B7‐H3 CAR‐T‐derived EVs showed a time‐dependent killing activity against L3.6pl cells. In particular, the killing percentage of L3.6pl cells was 10% ( p = 0.05) and 22% (* p < 0.05) after 24‐ and 48‐h treatments with 150 µg B7‐H3 CAR‐T‐derived EVs, respectively. In line with killing assay results, B7‐H3 CAR‐T‐derived EVs significantly affected PC cell viability by MTT, compared to control. Summary/Conclusion : Our preliminary results showed a relevant antitumour activity of B7‐H3 CAR‐T‐derived EVs on PC cells in vitro, encourageing further studies to evaluate their potential use in PC therapy. Funding : This study was supported by the European Union—NextGenerationEU, under the National Recovery and Resilience Plan (NRRP), Project Title: “National Center for Gene Therapy and Drugs based on RNA Technology,” CUP: D73C22000810006.

Bacille

Ana Teixeira‐Marques 1,2 , José Pedro Sequeira 1,2 , Sara Monteiro‐Reis 1 , Rui Freitas 1,3 , Marta Dueñas 4,5,6 , Rui Henrique 1,7,8 , Carmen Jerónimo 1,8 1 Cancer Biology and Epigenetics Group, Research Center of IPO Porto Research Center/Porto Comprehensive Cancer Center (Porto.CCC); 2 Doctoral Programme in Biomedical Sciences, ICBAS School Medicine and Biomedical Sciences, University of Porto, R. Jorge de Viterbo Ferreira 228, 4050 313 Porto, Portugal; 3 Urology Clinic & Department of Urology, Portuguese Oncology Institute of Porto (IPO Porto); 4 Research Institute Hospital 12 de Octubre (Imas12), University Hospital 12 de Octubre, Av Cordoba s/n, 28041 Madrid, Spain; 5 Molecular Oncology Unit, CIEMAT (Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas), Avenida Complutense 40, 28040 Madrid, Spain; 6 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III, SPAIN; 7 Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto); 8 Department of Pathology and Molecular Immunology, ICBAS‐School of Medicine & Biomedical Sciences, University of Porto; Porto, Portugal Introduction : According to the EAU guidelines, bladder cancer patients classified into intermediate‐ or high‐risk groups are typically treated with BCG immunotherapy. However, despite this treatment, 40%–50% of patients experience tumour recurrence/progression. There are no FDA/CE‐approved biomarkers to predict BCG failure, contributing to poor outcomes and overtreatment. Small non‐coding RNAs (sncRNAs) derived from extracellular vesicles (EVs) have gained attention as potential biomarkers, as they play a key role in cell‐to‐cell communication in cancer. This study aimed to profile the EV‐sncRNAs in BCG‐treated patients to uncover candidate biomarkers that could predict BCG failure, as well as to explore the role that EV‐sncRNA targets may play in disease relapse. Methods : Urinary EVs were separated from BCG non‐failure ( n = 8) and BCG failure ( n = 7) patient samples (informed consent was obtained; IPO Porto ethical approval—CES_62/023) using differential ultracentrifugation and characterized according to the MISEV2023 guidelines. RNA was isolated, and sncRNA‐sequencing was conducted. SncRNAs were considered statistically significant when |log2(FoldChange)| ≥ 0 and p value < 0.05. KEGG and GO analyses were performed on the respective miRNAs’ targets. Results : SncRNA‐seq identified 63 miRNAs differentially expressed between the BCG non‐failure and BCG failure groups, with 5 miRNAs being significantly downregulated and 12 significantly upregulated in the BCG non‐failure group. Overall, patients who experienced BCG failure showed fewer miRNAs than the BCG non‐failure group (647 vs. 458 miRNAs, respectively), adding to the fact that these numbers were lower in progressions than in recurrences (377 and 403 miRNAs, respectively). The MAPK signalling pathway showed the highest gene ratio and count by KEGG analysis, and differences in biological processes related to small GTPase‐mediated signal transduction were found by GO analysis. Summary/Conclusion : Urinary EV‐miRNAs revealed potential as promising biomarkers for predicting BCG failure. Additionally, the lower EV‐miRNAs number in BCG failure patients, particularly in those with tumour progression, suggests that these miRNAs may have a tumour‐suppressive role and may be involved in driving disease progression. Furthermore, the regulation of MAPK pathway targets by EV‐miRNAs may be associated with BCG failure, underlying the need for a deeper understanding of these sncRNAs in BCG failure. Funding : UCIPredict‐2023/TRANSCAN3/0001/2021; FCT PhD Fellowship 2023.01232.BD.

Calcium

Yumi Kumagai, Etsuo A. Susaki, Isao Nagaoka Juntendo University, Tokyo, Japan Introduction : Extracellular vesicles (EVs), secreted in response to microbial infections, play a pivotal role in modulating immune and inflammatory responses. Sepsis, a life‐threatening condition characterized by multiple organ dysfunction due to a dysregulated systemic inflammatory response to infection, presents significant challenges in therapeutic management. Despite extensive research, effective treatment strategies remain elusive. Our previous study demonstrated that EVs derived from mouse neutrophils stimulated with LL‐37, a human cathelicidin host‐defence peptide, improved survival rates in a septic mouse model induced by caecal ligation and puncture (CLP). However, the low yield of EVs posed a limitation of EV for therapeutic use. In this study, we explored strategies to enhance EV release from neutrophils with the aim of advancing the potential for clinical application of EV‐based therapies for sepsis. Methods : EVs were isolated from the supernatant of neutrophils incubated with LL‐37 and/or the calcium ionophore A23187 using ultracentrifugation. The EVs were characterized by flow cytometry, interferometric microscopy, and western blotting. Their antibacterial efficacy was assessed by incubating them with Escherichia coli isolated from the mouse cecum. To evaluate therapeutic potential, CLP mice were intraperitoneally administered either EVs or phosphate‐buffered saline (PBS) as a control, and survival rates and body weight changes were monitored over 1 week. Results : The calcium ionophore A23187 enhanced EV secretion from LL‐37‐stimulated mouse neutrophils. These EVs (PMN‐LL‐37Ca‐EV) exhibited greater antibacterial activity compared to EVs derived from neutrophils incubated with LL‐37 alone (PMN‐LL‐37‐EV). In the CLP mouse model, administration of PMN‐LL‐37Ca‐EV resulted in a higher survival rate or a faster recovery compared to PMN‐LL‐37‐EV when an equivalent number of EVs was intraperitoneally injected. Additionally, neutrophils isolated from human donors were stimulated with LL‐37 to release EVs with antimicrobial activity. These human‐derived EVs also demonstrated sepsis‐ameliorating effects when administered to CLP mice. Summary/Conclusion : Calcium ionophore enhances the secretion of neutrophil‐derived EVs, as well as their antimicrobial activity and sepsis‐ameliorating effects. These findings provide valuable insights into the development of EV‐based therapeutic strategies for sepsis.

Car‐T

Paola Lanuti 1,2 , Francesco Guardalupi 1,2 , Giulia Corradi 1,2 , Rosalba Florio 3 , Davide Brocco 4 , Serena Veschi 3 , Domenico De Bellis 1,2 , Francesca D'Ascanio 1,2 , Anna Piro 3 , Laura De Lellis 3 , Pasquale Simeone 1,2 , Maria Concetta Cufaro 2,5 , Serena Pilato 3 , Isabella D'Amario 1,2 , Fabio Verginelli 3 , Damiana Pieragostino 2,5 , Antonella Fontana 3 , Piero Del Boccio 2,3 , Alessandro Cama 3 , Mauro Di Ianni 1,2 1 Department of Medicine and Aging Sciences, Italy; 2 Center for Advanced Studies and Technology (CAST), Italy; 3 Department of Pharmacy, Italy; 4 Department of Medical, Oral and Biotechnological Sciences, Italy; 5 Department of Innovative Technologies in Medicine and Dentistry, Italy Introduction : CAR‐T cell therapy has revolutionized cancer treatment of haematological malignancies. However, many challenges remain to be addressed, including their severe adverse effects. Extracellular vesicles (EVs) have been pointed out as new therapeutic agents for their advantages in terms of safety. Given that CAR‐T cell‐derived EVs possibly mimic the anti‐tumour activity of CAR T cells, here we studied phenotypes and functions of CD19.CAR‐T cell‐derived EVs with the final aim to investigate their therapeutic potential. Methods : EVs derived from CD19.CAR‐T cells in pre‐infusion bags ( n = 3) or circulating CD19.CAR‐T cells ( n = 3) from patients infused with CAR‐T cells were separated by cell sorting and co‐cultured with two different CD19+ cell lines (Raji and SUP‐B15), using EVs from untransduced T cells from the same donors as controls. The antitumour activity of B7H3 CAR‐T‐derived EVs was evaluated by both MTT and killing assays, using 7‐AAD incorporation. CD19.CAR‐T EV morphological (nanoparticle tracking analysis, NTA, transmission electron microscopy, TEM, and atomic force microscopy, AFM) and phenotypical analyses (flow cytometry and proteomics) were carried out. Results : CD19.CAR‐T EVs characterization revealed that EVs displayed a globular shape and an average size of 133.1 ± 65.5 nm. CD19.CAR‐T EVs expressed EV markers (CD63 and flotillin‐1), and typical EV contaminants (i.e., cytochrome C and apolipoproteins) were undetectable. CD19.CAR‐T EVs expressed higher CAR levels than their parental cells and carried a pro‐apoptotic protein cargo possibly associated with their killing abilities. Furthermore, CD19.CAR‐T cell‐derived EVs significantly affected cell viability of CD19+ cell targets in a dose‐dependent manner. Using a dose of 0.015 µg EV proteins/target cell of blood circulating CD19.CAR‐T EVs, a killing of 20.3% (±3.8) and 37.8% (±7.0) was reached when Raji and SUP‐B15 were used as targets, respectively, while the killing percentage induced by pre‐infusion bag CD19.CAR‐T EVs, was 42.0% (±12.3) and 35.8% (±13.8) for Raji and SUP‐B15, respectively. Summary/Conclusion : Overall, our data indicate that CD19.CAR‐T cell‐derived EVs may be proposed as a new CAR‐T cell product. Contributing to the direct killing of the leukaemic target, they may represent a better tolerated therapy tool that could be infused independently from CAR‐T cells. Funding : PNC‐E3‐2022‐23683269.

Caspase

Erika G. Marques de Menezes 1,2 , Scott A. Bowler 3 , Thomas A. Premeaux 3 , Cecilia M. Shikuma 4 , Mohamed Abdel‐Mohsen 5 , Lishomwa C. Ndhlovu 3,6,4 , Philip J. Norris 1,2,7 1 Vitalant Research Institute, San Francisco, CA, USA; 2 Department of Laboratory Medicine; 3 Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York City, New York, USA; 4 Department of Tropical Medicine, Weill Cornell Medicine, New York, New York, USA; 5 Northwestern University, Chicago, Illinois, USA; 6 Hawaii Center for AIDS, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA; 7 Department of Medicine, University of California, San Francisco, California, USA Introduction : We have previously demonstrated that extracellular vesicles (EVs) expressing neuroinflammatory markers in people living with HIV (PLWH) are associated with HIV‐related neurocognitive impairment, which persists despite effective suppression with antiretroviral therapy. However, the mechanistic role of EVs in contributing to neuroinflammation in PLWH remains unclear. Given that microglial and astrocyte cell injury contributes to neurological impairment, we investigated the impact of EVs from PLWH and their effects on survival of brain resident cell types involved in neuropathology. Methods : EVs were isolated by differential centrifugation from plasma samples obtained from 11 PLWH and 13 people without HIV (PWOH). The concentration and size of purified EVs were evaluated using a NanoSight instrument (Malvern). Primary human microglia and astrocytes were exposed to EVs in the presence or absence of caspase‐1, ‐10, and ‐12 inhibitors, and apoptosis and necrosis rates were quantified by staining with annexin V and propidium iodide (PI) using an Aurora spectral cytometer. Statistical analysis was performed using the Kruskal‐Wallis test and Dunn's multiple‐comparison post hoc test ( p < 0.05). Results : The levels of necrosis (annexin V‐PI+) in microglia exposed to EVs from PLWH who were aviremic or viremic were significantly increased when compared with EVs from PWOH or to unexposed microglia. Necrosis was also significantly increased after astrocyte exposure to EVs, irrespective of HIV status, when compared with unexposed astrocytes. No significant difference in early apoptosis (annexin V+PI‐) or late apoptosis (annexin V+PI+) levels between the EV treatment groups was noted; however, a significant increase in late apoptosis was observed between all EV treatment groups and untreated cells. Inhibition of caspase‐1 in EV‐exposed microglia and inhibition of caspases‐1, ‐10, and ‐12 in EV‐treated astrocytes significantly reduced EV‐induced necrosis irrespective of HIV status. Summary/Conclusion : A reduction in EV‐mediated cell death by caspase inhibitors, acting downstream of critical inflammatory pathways (TNF, Fas, calpain), highlights a potential targetable mechanism by which EVs may contribute to HIV‐associated neuroinflammation and neuronal injury in PLWH through the promotion of an inflammatory cell death in CNS resident cells. Our findings further suggest that targeting EVs holds promise for limiting apoptosis and mitigating HIV‐associated cognitive impairment even among those with viral suppression.

Changes

Presenter: Bence P. Nagy Semmelweis University, Hungary Introduction : Light exposure of embryos during assisted reproduction affects embryo quality and implantation capacity in a wavelength dependent manner. We investigated the molecular mechanism of light‐induced changes through the comparative analysis of gene expression and regulatory miRNA profile of murine embryos cultured in dark environment or exposed to white / red filtered light. miRNA sequencing was used to assess the role of embryo‐derived extracellular vesicles in the endometrium‐embryo dialogue. Methods : In vitro cultured mouse embryos (3.5 dpc) were exposed to white or red filtered light. After 24 h, the miRNA content of embryo‐derived extracellular vesicles were isolated and RNA‐sequencing was performed. Differential expression analysis and functional enrichment analysis were used for evaluating the transcriptome results. Results : Embryo‐derived extracellular vesicles wavelength‐dependently enclosed unique miRNA cargos the target genes of which play a role in embryo implantation. We have found many significantly changed miRNA within the embryo‐derived extracellular vesicles, independent from the embryonic miRNA. Within the white light treated group, miRNA targeting genes in cell signalling and extracellular matrix reorganization were downregulated. Meanwhile following the treatment with filtered red light, there were significant elevations in levels of miRNA, targeting genes, that play a role in the implantation of embyros. Furthermore there were significant changes in miRNA targeting immunological signalling pathways, namely Fc gamma signalling, DAP12 signalling and inflammatory signallization. Summary/Conclusion : Extracellular vesicles of light‐exposed embryos play a role in blastocyst‐decidua communication through the horizontal transfer of regulatory miRNAs. Our data prove that light exposure during in vitro fertilization modifies cell function that might affect the outcome of implantation. Funding : This work was supported by OTKA K 147483, TKP‐EGA10 and STIA‐KF‐17.

Culprit

Ulrike Resch 1,2 , Taras Afonyushkin 3 , Silvio Kau‐Strebinger 4 , Anna S. Ondracek 5 , Luisa M. Schmidt 2 , Marcus Krueger 2 , Christoph Binder 3 , Irene Lang 5 1 Center for Physiology and Pharmacology, Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Austria; 2 CECAD Cluster of Excellence for Aging Research, Institute for Genetics, University of Cologne, Germany; 3 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 4 Department of Biomedical Sciences and Pathobiology, Center of Pathobiology, Institute of Morphology, Vetmeduni, Vienna, Austria; 5 Department of Internal Medicine II, Cardiology, Medical University of Vienna, Vienna, Austria Introduction : Acute myocardial infarction (AMI) is a leading cause of heart failure and death. Hypoxia reperfusion leads to damage and death of microvascular cells and cardiomyocytes and the release of DAMPs. Subsequent local immune cell infiltration not only contributes to the immediate damage but also determines cardiac remodelling. Extracellular vesicles (EVs) shed during this process reflect activation and death of different cells and are also actively involved in the pathogenesis of AMI. However, potential differences of the EV protein composition at the culprit site as compared to the peripheral circulation are unknown. Characterizing the proteome of culprit‐site plasma and EVs may provide more detailed molecular insights into culprit site plaque rupture and subsequent myocardial remodelling and allow for the development of new prognostic markers and therapies. Methods : This study included a patient subset ( n = 7) of the ‘Strategic targeted temperature management’ study (STATIM, NCT01777750 ) recruited at the time of diagnosis of ST‐segment elevation myocardial infarction (STEMI). EVs were isolated from cell free citrated‐plasma aspirated from arterial culprit and peripheral sites after centrifugation at 18,000 ×  g . EV size, shape, and surface markers were analysed using NTA, TEM and flow cytometry in agreement with MISEV recommendations. Comparative proteome profiling of crude plasma, MagReSyn‐depleted plasma and EVs was realized using an EvoSep One‐30SPD‐timsTOF‐pro 2 setting operating in dia‐PASEF mode. Results : We identified 2673, 933, and 1615 proteins in EVs, crude or depleted plasma. Eight hundred sixty‐one proteins were common to all three sample types, 10 and 21 proteins were unique to crude or depleted plasma, and 1627 proteins were unique to EVs, emphasizing the valuable gain of information obtained from the plasma EV‐compartment. We found that EV, but not whole plasma proteome signatures, shows distinct site‐specific patterns. Culprit EV proteomes contained predominant neutrophil and platelet degranulation signatures. In addition, we found selective enrichment of mitochondrial proteins in culprit EVs consistent with massive cardiomyocyte death. Summary/Conclusion : We uncovered a unique acute phase EV proteome signature delineating molecular details of the STEMI culprit site that support a critical role for neutrophil activation.

Digital

Presenter: Xueming Niu The University of Queensland, Brisbane, Australia Introduction : An abnormal inflammatory microenvironment is considered a hallmark of high‐grade brain cancer. Microglia are the predominant resident immune cell population in the brain and play a key role in regulating tumour progression and response to treatment in patients. Extracellular vesicles (EVs) derived from microglia carry molecular signals that reflect the inflammatory state of the brain. In our study, we developed a single‐molecule resolution nanotechnology platform to capture microglia‐EVs from the blood of glioblastoma (GBM) patients and profile alterations in the phenotype of microglia during therapy and at recurrence. Methods : We developed a digital platform that capitalizes on a gold‐topped silicon nanopillar array integrated with superplasmonic Au–Ag alloy nanoboxes for multiplex readouts. Microglia EVs were captured on the nanopillar and detected by surface‐enhanced Raman scattering (SERS) nanoboxes targeting an inflammatory marker panel. Counting EV‐activated nanopillars generated a digital readout of single EV molecular profiles. The inflammatory biomarker panel was first validated on the platform using EVs derived from pro‐ and anti‐inflammatory murine and human microglia cells. Subsequently, we applied this platform to profile EVs in a preclinical mouse model and in GBM patients to detect tumour recurrence and track therapeutic response. Results : Based on our in vitro experiments, we demonstrated that the nanopillar chip was able to capture single microglia‐derived EVs and analyse the pro‐ or anti‐inflammatory phenotypes of microglia EVs. As a result of clinical samples, our platform also enabled longitudinal monitoring of changes in the brain's inflammatory microenvironment. We found that the upregulation of inflammatory markers in microglia EVs, which indicated more significant immune responses, correlated with therapeutic resistance and the possibility of tumour recurrence. These findings highlight our platform's potential to provide real‐time monitoring of GBM‐related inflammatory microenvironmental changes through profiling microglia‐EVs. Summary/Conclusion : Microglia‐EVs offer promising insights into the GBM microenvironment, helping us understand tumour progression. Our nanopillar platform provides a real‐time and precise strategy for monitoring tumour evolution by analysing EVs at single‐molecule resolution. This platform has the potential to become a tool in personalized patient management, allowing for the optimization of therapeutic strategies that enhance treatment outcomes.

Dissect

Presenter: Tongjian Zhao Uppsala University, Sweden Introduction : The pancreatic islets are composed of cells with distinct functions and are crucial to the pathogenesis of T1D. Islet cells can selectively and actively release EVs. However, due to the limited quantity of EVs that can be obtained from islets, currently it is only possible to perform bulk analysis on islet‐derived EVs rather than single‐EV analysis. The aim of the study is to use proximity barcoding assay (PBA) for single EV surface protein profiling to characterize the heterogeneity of human islet‐derived EVs. Methods : EVs from cultured human islets with and without cytokines (IL‐1β and IFN‐γ for 24 or 48 h) or other relevant cells were isolated by a classic ultracentrifugation procedure. NTA, TEM, immunoblotting, PBA and nanoscale flow cytometry were applied for EV characterization and quantification. Results : PBA detected distinct EV membrane profiles of EVs derived from human islets, the EndoC‐betaH1 cell line, islet stellate cells (ISC) and plasma, which highlights the possibility of using PBA to track EV origins. We observed a sub‐population of EVs that co‐expressed the combination of CD44, MUC1, and ICAM1 on the EV surface. The percentage of this population increased significantly (from 0.82% to 4.40%, n = 6, p = 0.01, paired t ‐test) after 24 h of cytokine exposure. The combination of single‐cell RNA‐seq data of islets and PBA EV data identified that CD44 and MUC1 were cell type‐specific EV markers of ductal cells. ICAM1 was a cytokine‐inducible EV marker. To further confirm this, we isolated EVs from the EndoC beta‐cell line and the ductal PANC1‐1 cell line and confirmed these markers by both PBA and nanoscale flow cytometry. Thus, this CD44, MUC1, and ICAM1 positive EV population was very likely secreted from cytokine‐stressed ductal cells. Changes of cytokine‐treated human islet EV membrane profiles were associated with faster uptake of EV by PBMC and increased secretion of pro‐inflammatory cytokines IL‐1beta, TNF alpha and IL‐6. Summary/Conclusion : The study established a method to dissect the heterogeneity of human islet‐derived EVs through membrane protein profiling and highlighted the importance of EV membrane proteins on EV uptake and downstream immune response triggering in T1D. Future study is warranted to test the feasibility of using PBA for plasma EV analysis for T1D early diagnosis. Funding : Swedish Children Diabetes Foundation

Ectopic

Presenter: Mohamed Elbeltagy University of Duisburg‐Essen, Duisburg, Germany Introduction : Mesenchymal stromal cells (MSCs) exert therapeutic effects, primarily mediated through extracellular vesicles (EVs) with immunomodulatory properties. CD73 on MSCs catalyses the conversion of extracellular ATP (eATP) into anti‐inflammatory adenosine. However, the absence of CD39 in MSCs limits this purinergic pathway. Elevated eATP, an early damage‐associated molecular pattern (DAMP), triggers severe inflammatory cascades post‐stroke. This study hypothesizes that introducing CD39 expression in immortalized MSCs (ciMSCs) will enhance the immunomodulatory efficacy of their EVs in neuroinflammatory conditions like ischaemic stroke. Methods : ciMSCs were modified via lentiviral transduction to express CD39. EVs were isolated through ultracentrifugation and characterized per MISEV guidelines, including nanoparticle tracking analysis (NTA) and BCA assay for concentration and purity. Western blot confirmed EV markers CD63, CD9, CD81, and TSG101. Image flow cytometry quantified CD39‐fused eGFP+ EVs, and ATP hydrolysis was assessed using a colourimetric assay. Immunomodulatory potential was tested via a multi‐donor mixed lymphocyte reaction (mdMLR) with mononuclear cells from 12 donors to evaluate responses with and without eATP. The therapeutic effects of CD39+ ciMSC‐EVs were assessed in a murine middle cerebral artery occlusion (MCAO) ischaemic stroke model. Neuromuscular coordination was evaluated with the tightrope test, and infarct volume and oedema were analysed using Nissl and cresyl violet staining. Results : Image flow cytometry and western blot analysis confirmed that ciMSC‐EVs carried CD63, CD81, and CD39. CD39+ ciMSC‐EVs demonstrated efficient ATP‐to‐adenosine conversion, a capability not observed in unmodified ciMSC‐EVs. In mdMLR assays, while both EV types suppressed T cell activation in the absence of eATP, CD39+ ciMSC‐EVs exhibited markedly enhanced suppression in the presence of eATP, supporting the role of adenosine pathways in modulating immune responses. In the ischaemic stroke model, administration of CD39+ ciMSC‐EVs significantly improved neuromuscular coordination, reduced infarct volume, and decreased oedema compared to EVs from unmodified ciMSCs, highlighting their superior therapeutic potential. Summary/Conclusion : CD39 expression in ciMSCs enhances the immunomodulatory and therapeutic efficacy of their EVs, especially under conditions of elevated eATP. CD39+ ciMSC‐EVs are a promising therapeutic strategy for neuroinflammatory diseases such as ischaemic stroke, with further studies underway in models like acute Graft‐versus‐Host Disease (GvHD). Funding : This project is supported by funding from the Deutsche Forschungsgemeinschaft (DFG).

Effects

Gabriela Villa Marin, Ana Claudia Torrecilhas Universidade Federal de São Paulo (UNIFESP); Instituto de Ciências Ambientais, Químicas e Farmacêuticas; Departamento de Ciências Farmacêuticas; Labouratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. Introduction : Trypanosoma cruzi is the protozoan that causes Chagas Disease (CD). The medications used to treat CD, Benznidazole (BZ) and Nifurtimox, remain to cause side effects at different phases of the disease. There is still a need for additional study into the parasite's molecular structure to develop new therapies and improved comprehension of T. cruzi's interaction mechanisms during its life cycle in both invertebrate and vertebrate hosts. Our research has previously shown that T. cruzi shed EVs during its life cycle. These particles play a critical role in host communication, interaction, and immune response regulation. Methods : The main aim of this study was to examine the effect of Dasatinib (DAS) on EV release by T. cruzi. DAS was initially developed to inhibit the dual Src/Abl tyrosine kinase, although it can also inhibit several serine/threonine kinases. A published study found that DAS suppressed the growth of parental amastigotes but not those with TcK2 depletion, indicating that it could be a promising lead for developing CD treatments that target TcK2. Trypomastigote forms were cultured at 37°C at varying time intervals (0, 15, 30 min, 1, 2, and 4 h) and concentrations of DAS, BZ, and a control (no compounds). After incubation, the cultures were centrifuged, and EVs were purified to evaluate particle the quantity and parasite viability using NTA and Cytoflex. Results : The results showed variations in the concentration of vesicles produced by parasites treated with DAS and BZ. The parasite produced a smaller amount as the concentration of the chemical increased. However, the size of EVs obtained from the parasite was not significantly different from the control. Summary/Conclusion : DAS action on TcK2 kinase, may influence parasite communication processes involving EVs, creating more prospects for the development of CD therapies. The stability of EV size under treatment situations indicates that the substances are selective for release without impacting EV structure, emphasizing the importance of additional study of these mechanisms for therapy. Funding : CAPES, CPNq e FAPESP (2020/07870‐4)

Eimeria

Joshua Seun Olajide 1,2 , Olusanya Olasehinde 3 , Cai Jianping 1 1 Chinese Academy of Agricultural Sciences, China, 2 Obafemi Awolowo University, Nigeria, 3 University of Benin, Nigeria Cai Jianping is senior co‐author Introduction : During host‐parasite interactions, extracellular vesicles (EVs) are secreted by intracellular protozoan parasites and parasite‐infected host cells. Secreted EVs usually contain proteins with important roles in disease outcome. Eimeria falciformis clinical manifestation is prominent on the 7th day post infection, coinciding with oocyst shedding and completion of parasite life cycle. This study examined plasma‐derived EVs with parasite‐proteins in E. falciformis‐infected mice. Methods : Blood samples were collected from E. falciformis‐infected mice (7 days post‐infection) and from uninfected host. EVs were isolated from the host plasma using serial centrifugation. 5 mL of the supernatant was equilibrated with PBS (40%) and EDTA. Centrifugations were followed by ultrafiltration(0.45 µm) and ultracentrifuged at 120,000 ×  g for 18 h at 4°C. The pellet was resolved on iodixanol gradient solutions. Seven gradient layers were pooled, diluted with PBS and washed for 4 h at 120,000 ×  g . EVs were characterized by TEM and NTA, and the protein composition were profiled by LC–MS/MS. Results : Identified EVs were spherical to circular in shapes. 53 and 154 proteins were identified from the infected and uninfected hosts respectively. Out of these, 31 host‐derived proteins were common to both groups. Common EVs markers were HSP70, 14‐3‐3 protein and histones. E. falciformis‐infected host plasm‐derived EVs had ras‐related protein and MHC class II as specific EVs markers. Identified parasite proteins were 69. Including synthesis, regulation of eukaryotic cellular processes, chromatin binding and stress‐response proteins. Enrichment analysis indicated that these proteins were from extracellular exosomes, vesicles, region and organelles, while response to endogenous stimulus, toxic substance and chemicals were significant biological processes of EV proteins isolated from infected host. Major molecular function were protein and enzyme binding. Antigen processing and presentation and protein processing were significant annotated genes. Protein‐protein interaction analysis showed that parasite proteins are in complex formation with overlapping genes. Summary/Conclusion : Identified parasite EV proteins are involved in parasite survival and pathogenesis while EV proteins from the host indicated oxidative stress. Nonetheless, identified proteins are important diagnostic and pathogenic molecules during coccidiosis. Funding : Key Technologies Research and Development Program (Key Technologies R&D Program) 2017YFD050040320, Innovative Special Project of Agricultural Science and Technology (Grant No. CAAS‐ASTIP2014‐LVRI‐09)

Exosome

Presenter: Chia‐Ning C. Shen Genomics Research Center, Academia Sinica, Taipei, Taiwan (Republic of China) Introduction : Exosomes derived from mesenchymal stem cells (MSCs) possess a wide range of biological functions like these cells by contributing to tissue repair. For example, exosomes released from MSCs can be used to treat by reducing inflammation and modulating extracellular matrix. Therefore, several preclinical and clinical studies have recently demonstrated the potential of MSC‐derived exosomes to be applied to treating osteoarthritis. However, limited production of exosomes has hampered their development for clinical translation. Here, a programmable all‐in‐one Panasonic automated culture machine was utilized to derive MSCs and exosomes from human induced pluripotent stem cells. Methods : Mesenchymal stem cells (iMSCs) were generated by a stepwise differentiation protocol utilizing a programmable all‐in‐one Panasonic automated culture system. Exosomes were isolated from concentrated conditioned medium and followed by size exclusion chromatography. iMSC and exosomes were characterized by immunofluorescence staining and single‐cell RNA sequencing analysis using the BD Rhapsody system. The purity of exosomes was assessed by nanoparticle tracking analysis (NTA). Results : Initial characterization revealed MSCs derived from automatic process possess multipotency. To further uncover the cellular homogeneity in MSCs derived from automated processes, FACS and single‐cell analysis using the BD Rhapsody system were performed. Automatic process derived MSCs were positive for CD29, CD44, CD73, CD90, CD105, and HLA‐ABC and negative for CD45 and HLA‐DR. To investigate whether RNA abundance is correlated between parental iMSCs and their exosomes, mRNA and small RNA sequencing were further conducted. The results suggest that exosomal RNAs indeed recapitulate the RNA abundance of their parental iMSCs. To validate whether exosomes derived from iMSCs can mediate their immune modulation activity, exosomes were further isolated from concentrated conditioned medium of iMSCs followed by size exclusion chromatography. These purified exosomes assessed by NTA and characterized by TEM were positive for CD9, CD63 and CD81. We validated exosomes isolated from iMSCs exerted similar effects on NK cells. We found exosomes derived from iMSCs can prime NK cells to produce increased levels of IFN‐γ in response to IL‐12 and IL‐18, which is just like their parent iMSCs. Summary/Conclusion : The findings of the current study elucidated the possibility of production of exosomes with superior immunomodulatory properties from iMSCs generated from iPSCs utilizing automated production methods.

Helping

Presenter: Nicolas Ansart Institut Curie, Paris, France Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed particles, which can transfer bioactive molecules to target cells, modulating their physiology. EVs are therefore the object of rising interest from scientists interested in all possible physiological or pathological questions. However, EVs are very heterogeneous and co‐secreted by cells with various other extracellular factors. Demonstration that EVs are involved in a given function thus requires several steps of isolation and analysis, for which the International Society for EVs has published guideline articles (MISEV2023, MISEV2018). Based on our long‐term expertise in EVs, and the frequent requests from colleagues for help on EV‐related questions, we created in 2020 at Institut Curie a core facility dedicated to EVs. Methods : The CurieCoretech EVs are co‐led by the head of the Cytometry Coretech (CG), the head (CT) and a senior scientist (L.M.J.) of the ‘EV, Immune responses and Cancer’ research team, and experiments are run by engineers of the facility and the team (N.A., A.Z., N.N.). It disposes of 2 EV‐specific instruments: Zetaview (ParticleMetrix), and FlowNanoAnalyzer (nanoFCM). It offers access to instruments of the CurieCoretech Cytometry: Aurora spectral and ImageStream Imaging flow cytometers (Cytek) and CytoFLEX (Beckman). It also provides access to various devices for EV isolation (differential ultracentrifugation, size exclusion chromatography) and analysis (Western blotting, MacsPlexExo). Results : The first step for any new user is a discussion with one of the senior leaders of the core facility, to define the scientific question and determine the best approaches which the facility can offer. The user will then bring their own biological samples to be trained by an engineer of the facility to perform isolation and characterization of EV preparation. The CurieCoreTech can perform these experiments for the users. The CurieCoreTech also develops protocols and pipelines for single EV analysis, in particular by one‐ or two‐colour staining for nanoFCM analysis, and for EV staining for downstream analysis of interaction with target cells. Summary/Conclusion : By providing advice on how to isolate and characterize EVs following the MISEV guidelines, and by developing novel protocols for EV analysis, the CurieCoreTech EVs aim to promote reproducible and interpretable research on EVs.

Hypoxia

Helena Branco 1,2 , Cristina P. R. Xavier 1,3,4 , Chiara Riganti 5,6 , M. Helena Vasconcelos 1,2 i3S—Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal; 2 Department of Biological Sciences, FFUP—Faculty of Pharmacy, University of Porto, Porto, Portugal; 3 UCIBIO—Applied Molecular Biosciences Unit, Toxicologic Pathology Research Laboratory, University Institute of Health Sciences (1H‐TOXRUN, IUCS‐CESPU), Gandra, Portugal; 4 Associate Laboratory i4HB—Institute for Health and Bioeconomy, University Institute of Health Sciences—CESPU, Gandra, Portugal; 5 Department of Oncology, University of Torino, Torino, Italy; 6 Interdepartmental Research Center for Molecular Biotechnology “G. Tarone”, University of Torino, Torino, Italy Introduction : Non‐small cell lung cancer (NSCLC) is the world‐leading cause of cancer‐related mortality. Despite recent breakthroughs in NSCLC treatment, the prognosis remains poor, owing to the development of resistance, including multidrug resistance (MDR). Extracellular vesicles (EVs) are key mediators of intercellular communication within the tumour microenvironment (TME). Specifically, growing evidence highlights the role of EVs in the horizontal transfer of drug resistance traits within the TME. Hypoxia and acidosis, defining features of the TME, are associated with increased EV production. Here, we aimed to study the impact of the hypoxic/acidic TME on the cargo of EVs released by MDR NSCLC cells. Methods : EVs shed by NCI‐H460/R MDR cells were isolated by differential centrifugation. Before EV isolation, cells were kept for 72 h either under normoxia and physiological pH (control), hypoxia (1% oxygen) or acidosis (pH 6.5). Hypoxia induction was confirmed by western blot (WB). EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy and WB. Protein content of EV's cargo was further assessed by mass spectrometry‐based proteomics. The most relevant differentially expressed proteins (DEPs) between control, hypoxia and acidosis cell culture conditions were validated by WB. Results : Isolated EVs exhibited the typical size range (150–170 nm) and characteristic ‘cup‐shaped’ morphology. EV‐associated protein markers (Hsp70, CD9, CD47, CD81 and Alix) were successfully detected. ApoB, cytochrome c, actinin‐4 and actin were either absent or present at minimal levels in the isolated EVs compared to their corresponding cells. A total of 101 proteins were enriched in EVs from NCI‐H460/R cells released under hypoxia, while 48 proteins were enriched under acidosis, both compared to normoxia/physiological pH. Conversely, 66 and 85 proteins were decreased under hypoxic and acidic conditions, respectively. For both conditions, PANTHER analysis revealed an enrichment in proteins involved in cell metabolism and a decline in proteins involved in JAK/STAT signalling, as well as B and T cell activation. Most relevant DEPs were validated by WB. Summary/Conclusion : Hypoxia and acidosis significantly impact the cargo of EVs shed by NSCLC MDR cells. This suggests that these EVs may contribute to immunomodulation and have an impact on immunotherapy efficacy. Future work will confirm this hypothesis.

Hypoxic

Presenter: Hargita Hegyesi, Semmelweis University, Hungary Introduction : Cardiomyocytes (CMs) comprise approximately 30% of cells in the heart and communicate with cells to maintain cardiac functions. Numerous cargoes originating from the mitochondria, cytosol, and the plasma membrane of cardiomyocytes have been identified in small and large EVs, which, once transferred to other cells, may influence their biological activity of the recipient cells. Ischaemic heart disease is associated with increased production of extracellular vesicles (EVs), and the molecular composition of EVs from hypoxic cells differs significantly from that of normoxic cells. Objective : This study investigates how hypoxia‐reoxygenation (H/R) alters EV release from CMs and assesses whether cardiac EVs influence the gene expression profile of unstressed (bystander) CMs. Methods : We cultured C57/Bl6 mouse‐derived primary neonatal cardiomyocytes. Hypoxia was induced for 4 h using Oxoid AnaeroGen paper sachets, followed by reoxygenation in an incubator for 16 h. Large EVs (lEVs) and small EVs (sEVs) were isolated via the combination of gravity driven size filtration and differential centrifugation. EV characterization was performed using flow cytometry, TEM, and nanoparticle tracking analysis (NTA). CM‐derived EV uptake was assessed by confocal microscopy and flow cytometry. The functional impact of CM‐EVs on bystander cells was analysed using the PAMM‐174ZA RT2 Profiler PCR Array (qPCR). KEGG pathway analysis was conducted using DAVID Functional Annotation Tools. Results : CM cells expressed caveolin‐3 and α‐actinin. Compared with controls, H/R‐treated cells released a significantly increased number of lEVs, as indicated by Annexin V binding, suggesting elevated lEV release following H/R treatment. H/R treatment decreased CD81+ sEV levels, with CD9 and CD107α remaining undetectable. Treatment with H/R CM EVs significantly upregulated ADRA1A, ADRA1D, AEBP1, MYH10, TNNI3, CCL11, and COL1A1 in bystander cells after 24 h in the recipient CM cells. In contrast, the expression of CREM, CCN2, TNNT2, FRZB, and MAP3K5 was reduced. The genes modulated by H/R EVs are involved in a common molecular pathway implicated in hypertrophic cardiomyopathy development. Summary/Conclusion : This study advances our understanding of the mechanisms of CM‐EV biogenesis under physiological and pathological conditions, contributing insights into potential pharmacological strategies for ischaemic heart failure. Funding : NVKP_16‐1‐201 (NKFIH), VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003 (NCLP), 2019‐2.1.7‐ERA‐NET‐2021‐00015, 2024‐1.2.5. TÉT and the EU's Horizon 2020 No. 739593.

Induced

Alisa Jokela, Kerttu Airavaara, Julia Monola, Chris S. Pridgeon, Riina Harjumäki University of Helsinki, Helsinki, Finland Introduction : Mesenchymal stromal cell (MSC)‐derived extracellular vesicles (MSC‐EVs) hold great potential for regenerative medicine. However, the quality of tissue‐derived MSC‐EVs varies with donor characteristics, and MSC cultures often face challenges, for example, in early senescence and reduced therapeutic potency. To obtain an inexhaustible source for MSC‐EVs with consistent quality, induced pluripotent stem cells (iPSCs) could be used to produce induced MSCs (iMSCs). However, a standardized protocol for a large‐scale iMSC production has not yet been established. This study aims to optimize the iMSC differentiation protocol for therapeutic EV production and explore if tissue‐derived MSC‐EVs could enhance the differentiation. We hypothesize that this optimized protocol could be used in future tissue regeneration applications. Methods : Three protocols, modified from previous studies, were explored using pathway inhibitor‐based approaches. The methods include direct differentiation and differentiation via trophoblast (TB) or neural crest cell (NCC) checkpoints. After optimization, tissue‐derived MSC‐EVs were tested at different time points. All EVs in this study were isolated by differential centrifugation and size‐exclusion chromatography. The characteristics of iMSCs and EVs were analysed using qPCR, proteomics, and nanoparticle tracking analysis. Results : Differentiation via NCCs produced iMSCs with a typical MSC gene expression profile (CD73+, CD105+, CD90+), robust proliferation, and high cell yield. Tissue‐derived MSC‐EVs did not significantly enhance differentiation processes but showed a trend of increased gene expression profiles under some conditions. Further analysis of the resultant iMSC‐EVs is ongoing. Summary/Conclusion : Based on the preliminary data, induction of iMSCs through NCCs appears to offer the most promising protocol for generating iMSCs with sufficient cell yield for EV production. This protocol will be further optimized to produce iMSCs with the most potential EVs for future tissue regeneration applications. Funding : Research Council of Finland: GeneCellNano flagship project [#337430] and Finnish Cultural Foundation [#00220283].

Linking

Presenter: Juan P. Hinestrosa Xzom, USA Introduction : Extracellular vesicles (EVs) offer immense potential in disease detection, treatment choices, and screening by reflecting disease states through biomolecules like proteins, nucleic acids, and metabolites. Although EV markers are well‐studied (per ISEV guidelines), their link to specific cancer biomarkers remains underexplored, with most research focused on nucleic acids and free proteins. Our work seeks to bridge this gap, examining the relationship between known EV markers and cancer‐related proteins. Methods : EVs were isolated via ultracentrifugation from cancer cell lines H1975, A549, AsPC‐1, and SKOV‐3. Using multiplex ELISA, we measured classic EV markers (CD63, CD9, CD81, TSG101) and 52 cancer‐associated markers, analysing correlations with cancer biomarkers while adjusting for EV particle concentration and size. Biomarkers were tested across multiple concentration levels. To confirm results, EVs were spiked into buffer and human plasma, then assessed on a microelectrode array platform, closely reflecting their native state. Results : As anticipated, EV markers (CD9, CD63, CD81) correlated strongly across cell lines and directly with particle input. Each cell line type displayed a unique EV protein profile aligned with its cancer type. Among the 52 cancer biomarkers tested, 15 were detected in H1975, 4 in AsPC‐1, 2 in A549, and 4 in SKOV‐3. Notably, the lung cancer biomarker CYFRA21‐1 was present in both lung cancer cell lines and showed correlation with CD63 and CD81, indicating its presence on the EV surface. Full correlation data will be presented between all the EV biomarkers and the cancer biomarkers detected. Following EV spiking into buffer and plasma, biomarker signals were confirmed in an environment as close as possible to their native state. Further analyses regarding EV concentration, morphology, and findings post‐lysis for cytosolic cancer biomarkers will also be shared. Summary/Conclusion : The promise of EVs for early cancer detection is closer than ever to becoming a reality. Demonstrating that EVs are directly linked to cancer biomarkers and that this information can be easily accessed brings us a step nearer to achieving reliable, near‐patient diagnostics. Here, we illustrate the specific correlations between EV markers and cancer biomarkers across cell lines, showing their feasibility for simple, direct detection. Funding : This study was sponsored by Xzom, Inc.

Mapping

Niké Guilbert 1,2 , Amélie Vander Cruyssen 1,2 , Nele De Langhe 1,2,3,4 *, Sofie Van Dorpe 1,2,5 *, Sarah Deville 1,2 , Sándor Dedeyne 1,2 , Quentin Roux 1,2 , Hannelore Denys 2,6 , Linos Vandekerckhove 3,4 , Olivier De Wever 1,2 , An Hendrix 1,2 1 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium; 2 Cancer Research Institute Ghent, Ghent, Belgium; 3 HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University, Ghent, Belgium; 4 Department of General Internal Medicine and Infectious Diseases, Ghent University Hospital, Ghent, Belgium; 5 Department of Gynecology, Ghent University Hospital, Ghent, Belgium; 6 Medical Oncology, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent, Belgium. * These authors contributed equally Introduction : Bacterial extracellular vesicles (BEV) play a pivotal role in interkingdom communication, shaping unique ecosystems that influence both physiological and pathological processes. Given their emerging significance, the study of BEV has gained increasing attention, revealing tremendous potential for clinical applications. However, the diverse nature of BEV—including variations in their biophysical and biochemical characteristics, as well as their complex cargo—poses significant challenges. We performed a systematic analysis to shed light on the global BEV research landscape and map current practices in BEV research, highlight critical knowledge gaps and offer actionable recommendations. Methods : We analysed all 845 original BEV research articles published from 2015 to 2021, encompassing a total of 3338 experiments. For each experiment, we employed a detailed checklist comprising 233 parameters related to the source, preparation protocols, and characterization methods. The extracted data is publicly accessible through the EV‐TRACK platform (https://evtrack.org/). Results : Our analysis revealed a concerning inconsistency in nomenclature, with fewer than 20% of studies utilizing the term ‘bacterial extracellular vesicle.’ Meanwhile, the variety of BEV preparation methods used is extensive, with 934 unique protocols reported. Strikingly, only 24.5% of experiments included both particle and protein characterization, and a significant one‐quarter failed to report any form of characterization. Consequently, biophysical and biochemical characteristics of BEV preparations remain largely unknown. As an initiating endeavour, we catalogue commonly explored BEV‐enriched/depleted proteins. Furthermore, the average EV‐METRIC score—a measure for quality and transparency—of BEV studies plateaued from 2018 onwards, contrasting with the exponential increased in EV‐METRIC observed in eukaryotic EV studies since the release of the MISEV2018 guidelines. Summary/Conclusion : Analysis of over 800 studies reveals that MISEV2018 best practices are underutilized in the emerging field of BEV, reflected by a low average EV‐METRIC score of 14.5%. We integrate specific experimental parameters into the EV‐TRACK knowledgebase, adapting the tool to meet specific BEV research needs. As the field of BEV research continues to expand, prioritizing transparency and reproducibility will be essential to unlock biological insights and to advance BEV‐based applications. Funding : This work was supported by UGent, FWO, Horizon Europe MSCA grant agreement No. [722148] and ERC grant agreement No. [101045156].

Mbsomes

Presenter: Mariane Shouky Institut Curie, France Introduction : Orchestrated intercellular communication is essential to maintain skin homeostasis. Extracellular vesicles (EVs) play a crucial role in intercellular crosstalk in the skin, allowing exchange of proteins, lipids and genetic material. EVs are a heterogeneous group of cell‐derived membranous structures that are released by cells into the extracellular environment as exosomes and ectosomes, which originate from the endosomal system or which are shed from the plasma membrane, respectively. The mitotic midbody bridge at the final stage of cytokinesis could be a potential source of released midbody bridge‐derived ectosomes and MBsomes. Whether proliferating primary keratinocytes release MBsomes and midbody‐bridge ectosomes that could act as a means of intercellular communication in skin homeostasis, to our knowledge, has not been investigated yet. The aim of this project is to decipher the midbody bridge as a potential source of ectosomes as well as their signalling function in the epidermal/dermal crosstalk and skin homeostasis. Methods : To study the biogenesis and the release of midbody bridge vesicles, we used electron microscopy and immunofluorescence approaches. In addition, purification of MBs and EVs from primary epidermal keratinocytes was performed by differential ultracentrifugation followed by sucrose cushion and size exclusion chromatography, respectively. The composition of MBs and EVs was investigated by proteomics and transcriptomics. In addition, to decipher MBs and EVs signalling function and potential role in skin epidermal/dermal crosstalk, migration and proliferation of recipient dermal fibroblasts were also assessed by Incucyte scratch‐wound assay and real‐time proliferation assay, respectively. Results : Our results show that proliferating epidermal skin keratinocytes release MBsomes and midbody bridge vesicles carrying cytokinetic proteins. In addition, MBsomes and EVs, released by epidermal keratinocytes, enhance migration and proliferation of dermal fibroblasts. Summary/Conclusion : This project highlights the midbody bridge as a potential source of a new subtype of large EV, MBsomes, in addition to other midbody bridge‐derived vesicles. It also shows the potential role of MBs in intercellular crosstalk in the skin. Funding : NIH National Institute of Health.

Methods

Presenter: Thomas Maslanik Cellarcus Biosciences, San Diego, California, USA Introduction : Enrichment of extracellular vesicles (EVs) is required for many studies. Screening methods, functional studies, and analysis of rare subsets of EVs all require some form of EV enrichment. However, it is important to characterize any enrichment method to understand how it impacts the composition of EVs in a given sample and validate the approach to EV enrichment for downstream applications. A rigorous analytical approach for evaluating enrichment methods is needed. Methods : Vesicle Flow Cytometry (vFC) and BCA measurements were used to characterize media from hollow‐fibre bioreactor (HFB) DiFi cell cultures pre‐ and post‐enrichment. Results : There were 8.5E9 total EVs/mL in HFB DiFi cell culture media. Fifty‐nine percent of EVs were positive for EGFR above the limit of detection (25 antibodies per vesicle). Enrichment by UF (100k MWCO) resulted in 52% recovery of total EVs with similar EGFR positivity and a significant increase in EV/protein ratio. Other pore sizes differed in total EV recovery (300 kDa MWCO – 35%, 30 kDa MWCO – 25%) and EGFR positivity (300 kDa MWCO – 67%, 30 kDa MWCO – 54%). SEC enrichment resulted in a > 90% recovery of total EVs across all fractions (41% EGFR+). Sixty‐five percent of total EVs (86% of EGFR+ EVs) were present in fractions 7 and 8. Selecting appropriate fractions also significantly increased the EV/protein ratio. Summary/Conclusion : In this study, we demonstrate workflows for enrichment method characterization. This study demonstrates a broadly applicable analytical approach to characterizing enrichment methods for a given sample. Adopting such an approach in workflows requiring EV enrichment will improve our understanding of input material in any downstream application and will enhance rigour and reproducibility of these studies. Funding : Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R44CA272098. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Müller

Presenter: Sun Young Lee University of Southern California, USA Introduction : Müller glia (MG) are the primary glial cells unique to the retina and serve as a major source of neuroprotective factors and regulators of vascular permeability. Increasing evidence indicates that diabetes adversely affects the retinal neurovascular unit (NVU), impacting its interdependent vascular, neuronal, glial, and immune cells as diabetic retinopathy (DR) progresses. MG have been shown to release small extracellular vesicles (MG‐sEVs), which play an important role in cell‐to‐cell communication. The current study aims to investigate the therapeutic potential of intraocular treatment with MG‐sEVs to protect the NVU in DR. Methods : sEVs were isolated from the conditioned cell culture media (CCM) of the primary mouse MG cells using the tangential flow filtration (TFF) method and comprehensively characterized as established in our lab. Two DR mouse models, the STZ‐induced model for type I and the db/db model for type II DR, received three weekly intravitreal injections of MG‐sEVs post‐DR induction. We assessed neuroretina thinning, retinal vascular leakage, macrophage infiltration, and leukocyte recruitment through in vivo fluorescein angiography (FA) and optical coherence tomography (OCT) and immunohistochemistry (IHC) of retinal sections using anti‐F4/80 ab and FITC‐ConA perfusion of retinal flat mounts. In addition, electroretinograms (ERGs) were conducted. Results : We confirmed DR induction in both models ( n = 10) through inner retinal thinning, increased vascular leakage (in db/db mice only), and heightened retinal macrophage and vascular leukocyte infiltration. In contrast, intraocular MG‐sEVs treatment significantly preserved retinal thickness, particularly in the inner neuronal layers, and reduced vascular leakage, macrophage infiltration, and leukocyte recruitment. Although there was a mild reduction in a‐ and b‐wave amplitudes in scotopic ERG for DR, MG‐sEV treatment did not significantly improve these ERG findings. Summary/Conclusion : MG‐secreted sEVs may have the potential to mitigate early‐stage DR by protecting NVU, which addresses the gap in early‐stage DR treatment. Despite advances in understanding DR pathophysiology, no treatments currently exist for early‐stage DR beyond screening and managing systemic risk factors. Additionally, treatments for advanced DR, such as anti‐VEGF injections and laser photocoagulation, often fail to restore vision. While these initial results are encouraging, additional studies in biological cargos of MG‐sEVs are underway. Funding : National Institutes of Health (NIH)/National Eye Institute (NEI) R21EY035425.

Natural

Frederic St‐Denis‐Bissonnette 1,2 , Karan Mediratta 2 , Andrew Stalker 1 , Gauri Muradia 1 , Michele Ardolino 2,4 , Seung‐Hwan Lee 2 , Dylan Burger 2,3 , Lisheng Wang 2,3 , Jessie R. Lavoie 1, 2 1 Health Canada, Ottawa, Canada; 2 University of Ottawa, Ottawa, Canada; 3 Ottawa Hospital Research Institute, Ottawa, Canada Introduction : Natural Killer cell‐derived extracellular vesicles (NK‐EVs) are promising cancer immunotherapeutics due to their immunomodulatory and cytotoxic effects. However, the mechanisms by which administered NK‐EVs interact with host immune cells in immunosuppressed environments like triple‐negative breast cancer (TNBC) remain unclear. This study investigates NK‐EVs' immunomodulatory effects on immune cells from TNBC patients and healthy controls using a multi‐OMIC approach (i.e., flow cytometry and single‐cell RNA sequencing—scRNA‐seq). Methods : Clinical‐grade NK‐EVs were produced from the NK92‐MI cell line using a scalable biomanufacturing process, following MISEV guidelines (published in JEV in 2023). Human peripheral blood mononuclear cells (PBMCs) obtained from TNBC patients (3 donors) and healthy controls (3 donors) were exposed to NK‐EVs in a time and dose‐dependent manner. The effects of NK‐EV treatment on immune cells were assessed using flow cytometry and scRNA‐seq. NK‐EV‐stimulated immune cells were co‐cultured with TNBC cells to monitor cytotoxicity using a live‐cell monitoring assay. NK‐EV uptake was evaluated using CFSE‐labelling (dye associated with EV membrane) and flow cytometry. Ethical approval and informed consent were obtained. Results : Preliminary assessment of healthy PBMCs by flow cytometric analysis, performed using an unsupervised data reduction algorithm (i.e., fitSNE) and a state‐of‐the‐art clustering algorithm (i.e., FlowSOM), revealed significant activation of T, NK, and myeloid cells (e.g., macrophage, monocyte, and dendritic cells) following a 4‐h NK‐EV treatment. EV uptake experiments confirmed that these immune cell subtypes were selectively associated with NK‐EV treatment, with myeloid cells showing a disproportionately higher association with NK‐EVs than other immune subtypes after a 48‐h treatment. Interestingly, NK‐EV‐stimulated immune cells exhibited increased tumour‐killing potential (72‐h treatment) over the non‐stimulated counterparts. scRNA‐seq analysis of NK‐EV‐stimulated PBMCs is ongoing to decipher activation pathways. Summary/Conclusion : This is the first multi‐OMIC study combining flow cytometry and scRNA‐seq to investigate NK‐EV‐induced immunomodulation in TNBC‐derived immune cells. Our findings align with previous research showing that NK‐EVs can educate the immune system (i.e., T cell activation and macrophage polarization), further reinforcing the therapeutic potential of NK‐EVs against cancer.

Paeonia

Presenter: Fang Ma Carrier Biomed (Suzhou) Co.,Ltd., China (People's Republic) Introduction : UV exposure‐induced photoageing accounts for over 80% of facial ageing, resulting in apparent damage to HDFs and HaCaTs. Excessive ROS is observed in these damaged cells, leading to serious oxidative damage and senescence. Plant‐derived exosomes have garnered significant interest due to their inherent bioactivities, excellent biocompatibility, low immunogenicity, and cost‐effectiveness. Paeonia lactiflora Pall (PLP), a traditional herbal with pain‐relieving, anti‐inflammatory, and anti‐oxidant properties, has been widely used in skin administration. However, the role of PLP‐derived exosomes in repairing skin photodamages and preventing photoageing has not been explored. Methods : First, exosomes from fresh PLP (FP‐Exo), dried PLP (DP‐Exo), and decocted dried PLP (DPd‐Exo) were obtained by differential centrifugation. Second, the morphology and biological functions of FP‐Exo, DP‐Exo, and DPd‐Exo were evaluated to determine the most suitable extraction method. Third, the anti‐photoageing and ROS scavenging efficacy of DP‐Exo was investigated in UV‐damaged HDFs and HaCaTs. Next, the acute and chronic photoageing of rodent models and human volunteers were created to evaluate its photodamage repairing and photoageing preventing performances. Results : TEM images and SDS‒PAGE analysis revealed that FP‐Exo and DP‐Exo maintained intact exosomal membranes, whereas DPd‐Exo exhibited noticeable membrane rupture and protein loss due to the boiling process. All exosomes showed particle sizes of about 200 nm. DP‐Exo demonstrated superior biological functions, including enhanced cell migration, ROS scavenging, and apoptosis reduction in both UV‐induced cellular models. Furthermore, DP‐Exo enhanced the expression of collagen I and reduced the secretion of MMP‐1. In vivo studies revealed that mice treated with DP‐Exo experienced substantial improvement in erythema and skin lesions, with epidermal thickness and inflammation levels comparable to those of healthy controls. Additionally, a cream formulation incorporating DP‐Exo demonstrated pronounced anti‐photoageing effects and skin‐improvement benefits in human volunteers. Summary/Conclusion : Collectively, we identify an optimal extraction method for PLP‐derived exosomes and underscores the therapeutic efficacy of DP‐Exo across cellular assays, murine models, and human trials. DP‐Exo exhibits promising anti‐photoageing effects through mechanisms including ROS scavenging, anti‐inflammatory activity, extracellular matrix remodelling, and anti‐senescence. This study shed light on the potential application of DP‐Exo in repairing skin photodamages and preventing photoageing.

Pd‐L1

Caihong Li, Miriam Zentgraf, Jan Van Deun, Katja Blume, Susanne Rößner, Andreas S. Baur Department of Dermatology Unikilinikum Erlangen, Friedrich‐Alexander‐Universität (FAU) Erlangen‐Nürnberg, Deutsches Zentrum Immuntherapie (DZI), Erlangen, Germany Introduction : In melanoma, phosphatase and tensin homolog (PTEN) loss frequently occurs and is associated with tumour progression, metastasis, and response to immunotherapy. In certain cancers, PTEN loss enhances the production of programmed death ligand‐1 (PD‐L1)‐enriched extracellular vesicles (EVs) in a PI3K‐dependent manner. Some research had shown that metastatic melanoma released high levels of EVs carrying PD‐L1, which suppressed CD8+ T cell function and mediated resistance to immune checkpoint inhibitor therapy. Hence, exploring the relationship between PTEN‐deficient melanoma and immune response through the release of EVs may offer new perspectives for melanoma treatment. Methods : Fusing PD‐L1 to a modified oestrogen receptor (mER) ligand‐binding domain generated a tamoxifen‐inducible form. The construct was expressed in melanoma cell lines with PTEN wild type, PTEN mutation, and PTEN loss. The induced mature PD‐L1 in melanoma cell lines was visualized by GFP/RFP analysis using a confocal laser scanning microscope. EVs from supernatants of PD‐L1‐overexpressing cell lines were purified using OptiPrep density gradient and size‐exclusion chromatography (SEC). EV characterization was performed through Western blot and nanoparticle tracking analysis (NTA). The levels of PD‐L1 carried by EVs were analysed by western blot. An MCSP‐CAR expression plasmid was constructed for producing CD8+ MCSP‐specific CAR T cells, targeting/killing melanoma cells. Results : In the tamoxifen‐inducible system, when the hormone‐binding domain of the mER bound to tamoxifen, the receptor was released from its inhibitory complex, enabling PD‐L1 to exert its function. We observed that after induction, PD‐L1 cleavage/maturation occurred in the perinuclear region, with the active C‐terminal part translocating to the cell membrane while the cleaved‐off N‐terminus remained around the nucleus. The level of EV containing PD‐L1 released from PTEN‐loss melanoma cells was higher than that from PTEN‐wt melanoma cells. The impact of PD‐L1‐enriched EVs produced by melanoma cells with different genotypes on CD8+ T cell‐mediated immune response will be compared. Summary/Conclusion : PTEN modulates the release of PD‐L1‐carrying EVs, thereby regulating immune evasion in melanoma.

Priming

Arantza Infante 1 , Leire Cabodevilla 1 , Natividad Alcorta‐Sevillano 1,2 , Iratxe Macías 1 , Blanca Gener 1,3 , Javier Aldazabal 4 , Esperanza González 5 , Ana Talamillo 5 , Daniela Gerovska 6 , Marcos Araúzo‐Bravo 2,6,7 , Ibai Diez 1,7,8 , Juan M. Falcón 5,7 , Clara I. Rodríguez 1 1 Biobizkaia Health Research Institute, Barakaldo, Spain; 2 University of Basque Country (UPV/EHU), Leioa, Spain; 3 Cruces University Hospital, Barakaldo, Spain; 4 University of Navarra, Pamplona, Spain; 5 Center for Cooperative Research in Biosciences (CIC bioGUNE), Derio, Spain; 6 Biogipuzkoa Health Research Institute, Donostia, Spain; 7 IKERBASQUE, Bilbao, Spain; 8 Harvard Medical School, Boston, Massachusetts, USA Introduction : Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range of clinical manifestations. The majority of cases are due to mutations in the COL1A1 or COL1A2 genes, which encode type I collagen, the main protein of bones. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cells, have been tested as an innovative therapy for OI with promising but transient outcomes. Concerning the mechanism of action of cell therapy in OI, the paracrine hypothesis has superseded the initially expected host tissue replacement by donor MSCs. In this regard, an emerging concept is gaining attention, the ability of MSCs to change their paracrine responses depending on environmental clues. Methods : In this work we have tested this hypothesis by priming MSCs with serum samples coming from either severely affected OI adults or control, age‐matched donors. Then, we have isolated, characterized and functionally tested the EVs secreted by MSCs under these conditions (OI‐priming‐EVs, CTRL‐priming‐EVs). In addition, the miRNoma of their cargo has also been analysed. For the isolation of EVs, conditioned medium from MSCs was subjected to tangential flow filtration, followed by size exclusion chromatography and ultracentrifugation. Then, isolated EVs were characterized by standard methods and EVs cargo was evaluated by miRNAseq. Results : We found a significant enrichment of miRNAs which target type I collagen in OI‐priming‐EVs, an interesting finding since the downregulation of the mutant type I collagen is being considered a possible new strategic approach for OI. Moreover, when administered in vivo in an OI mouse model, mice treated with OI‐priming‐EVs showed enhanced improvements in bone quality parameters when compared to those treated with CTRL‐priming EVs. Summary/Conclusion : These results provide evidence that the priming of MSCs with disease‐relevant signals can induce the secretion of EVs with enhanced therapeutic potential for OI. Funding : This work was supported by the Spanish Ministry of Health, Instituto de Salud Carlos III through the project “PI21/0077” (co‐funded by European Regional Development Fund; “A way to make Europe”), the Basque Government Health Department 2023333014; Gangoiti Foundation, AHUCE Foundation and the Spanish Federation of Rare Diseases (FEDER).

Probing

Raluca Ghebosu 1 , Jenifer Pendiuk Goncalves 1 , Nur Indah Fitri 1 , Dalila Iannotta 2 , Mohammad Farouq Sharifpour 3 , Elaina Coleborn 4 , Alex Loukas 3 , Fernando Souza‐Fonseca‐Guimaraes 4 , Joy Wolfram 1,2, 1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia; 2 School of Chemical Engineering, The University of Queensland, Brisbane, Queensland, Australia; 3 Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia; 4 Frazer Institute, Faculty of Medicine, The University of Queensland, Woolloongabba, Brisbane, Queensland, Australia Introduction : Recent evidence suggests that other circulating biological nanoparticles, primarily lipoproteins, can bind to extracellular vesicles (EVs) and change their biological identity. This has been demonstrated using complex techniques; however, there remains a need to rapidly and simply quantify EV‐lipoprotein binding and identify intergroup differences in EV binding to lipoproteins. Uncovering altered lipoprotein binding to certain EVs could have major implications for understanding EV biology and developing medical applications. Methods : Here we demonstrate a first‐of‐its‐kind, simple and quick proprietary assay to assess EV binding to lipoproteins. The assay (proprietary, limiting disclosure) provides sensitive readouts that can effectively predict the extent of EV binding with lipoproteins. Non‐human derived EVs were isolated by size‐exclusion chromatography, while EVs from various human cell types, including natural killer cells, osteosarcoma and breast cancer, were isolated using tangential flow filtration. All EVs were authenticated using nanoparticle tracking analysis, western blot, and cryogenic transmission electron microscopy. Results : The assay was validated with synthetic nanoparticles, small molecules, polymers, and proteins that display known interactions with lipoproteins. We then probed EV interactions to demonstrate that EVs from various human and non‐human (nematode and bacteria) sources bind to lipoproteins. Notably, EVs derived from cancerous cells displayed substantially greater binding to lipoproteins compared to EVs from cell type‐matched healthy cells. Additionally, EVs from a metastatic osteosarcoma line were found to bind to lipoproteins significantly more than those from corresponding patient‐matched non‐metastatic osteosarcoma cells. This trend was also shown in a breast cancer model where poorly metastatic breast cancer cell line derived EVs have significantly less lipoprotein binding when compared to highly metastatic breast cancer cell line EVs. Direct associations between osteosarcoma EVs and lipoproteins were also shown by cryogenic transmission electron microscopy, with a substantially greater number of interactions seen in the metastatic osteosarcoma cell line—mirroring the assay. Summary/Conclusion : Taken together, we show that the developed assay is capable of measuring EV‐lipoprotein binding in a simple, rapid, and quantitative manner and can be utilized to uncover differing levels of EV binding to lipoproteins. These findings open new avenues to probe EV biology and develop novel therapeutics and diagnostics.

Process

Presenter: Tomas Mesurado acib GmbH, Graz, Austria Introduction : Extracellular vesicle (EV)‐based advanced medicines are demonstrating promising therapeutic potential in early‐stage clinical trials, both as delivery systems and intrinsic therapeutics. However, to transition EV‐based therapies to late‐stage trials and commercialization, scalable manufacturing processes are essential. Current EV downstream processes, which involve multiple unit operations such as ultracentrifugation, filtration, and chromatography, face challenges including poor scalability, low selectivity, and inadequate recovery. Chromatographic filtration utilizing non‐woven fibres offers an attractive method to intensify the process. Methods : In this study, we evaluated the impact on EV quality if the traditional clarification steps of differential centrifugation and sterile filtration were replaced by a single chromatographic unit operation. Functionalized non‐woven fibres have been used to intensify and streamline manufacturing processes for monoclonal antibodies. Here, we tested their applicability to a typical EV production process. EV recovery and process yield were determined through nanoparticle tracking analysis, Western blotting, and bead‐based flow cytometry using different EV markers. We compared the biological activity of EVs produced in a conventional process to those produced with the intensified process. Results : Replacing two centrifugation and one filtration step with a single chromatographic filtration step, resulted in drastically reduced process times while not affecting the recovery of EVs. The impurity profile was comparable, with the chromatographically clarified material showing lower concentrations of dsDNA. The biological activity after filtration were compared to an untreated control and a significant effect on the anti‐inflammatory activity was observed. Summary/Conclusion : In this study, we developed the effective EV preparation method that enable a large quantity at high purity without decreasing the bioactivity of EVs. Furthermore, this method can also remove critical DNA impurities alongside of cellular debris in a single step. Therefore, non‐woven fibre filtration method, which overcomes all issues of current EV preparation method, must be gold standard for the therapeutic EV preparation in the future. Funding : This work was supported by Solventum Corporation.

Protein

Presenter: Nikoleta Alchus Laiferová Slovak Academy of Sciences, Slovakia Introduction : The health benefits of regular physical exercise are driven by a range of mechanisms acting in synergy. Extracellular vesicles (EVs) released into circulation during exercise are promising mediators of interorgan crosstalk, potentially involved in exercise‐induced adaptations and modulation of brain plasticity. The aim of this study is to identify the changes in protein content of EVs, isolated from cerebrospinal fluid (CSF) and serum, induced by acute intense aerobic exercise. Methods : EVs were isolated from CSF and serum of young healthy active adults (M/F 13/6; median age 25(IQR 22‐31yrs), BMI 23.2(IQR 21.7‐24.5 kg/m2) & VO2max (spiroergometry) 47(IQR 38.1‐51.2 mL/kg/min) by ExoQuick (System‐Biosciences, US). Sampling of CSF was performed (i) before and (ii) 60 min after a monitored outdoor 90 min run by atraumatic lumbar puncture. Serum was obtained at baseline (R1), immediately after (R2), and 60 min after (R3) the run (90 min, 80%HRmax). EVs were characterized by Nanopartical Tracking Analysis (Nanosight‐NS500, Malvern, UK), TEM and immunoblotting & Exo‐Check Array (CD63, CD81, GAPDH, ApoE, Albumin, GM130). Cytokine content of EVs isolated from CSF and serum was determined (RayBio array C‐2000, US). Running‐induced changes cognitive performance (MemTrax, Cogstate) were assessed. Results : Ten out of 174 cytokines were significantly downregulated in EVs from CSF after exercise (fold change range ‐0,42 to ‐1,81). Acute exercise reduced levels of two and upregulated levels of six cytokines in serum EVs (fold change R2/R1 ‐0, 40 to 1,22). Compared to baseline, two cytokines were upregulated 1 h postexercise (fold change R3/R1 0,39–0,74), while 1 h post‐exercise recovery decreased levels of four and increased levels of one cytokine (fold change R3/R2, ‐0, 95 to 0,34) in serum EVs. Summary/Conclusion : The regulated cytokines participate in the regulation of essential metabolic pathways, immune functions, vascular integrity and angiogenesis, as well as in extracellular matrix remodelling. Our results support the role of EVs as mediators of adaptive responses to exercise. Funding : APVV 20‐0466, ADDIT‐CE Horizon Europe, VEGA 2‐0076‐22, FWF‐KLI‐1122, EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project No. 09I03‐03‐V04.

Quality

Presenter: Chadaporn Attakitbancha Chulalongkorn University, Thailand Introduction : Quality by Design (QbD) represents a systematic approach to pharmaceutical development that begins with predefined objectives and emphasizes product and process understanding and strategic control. For the emerging field of therapeutic extracellular vesicles (EVs), implementing QbD principles is crucial to ensure product quality, patient safety, and optimal therapeutic outcomes. This study applies QbD methodology to develop a robust GMP manufacturing process for human mesenchymal stem/stromal cell (MSC)‐derived EVs, based on ICH guidelines (Q8‐Q11), PDA recommendations, and ISPE baseline guides. Methods : We implemented a comprehensive QbD framework incorporating iterative risk assessments throughout the product lifecycle. The process began with defining the Quality Target Product Profile (QTPP) for MSC‐derived EVs, followed by systematic identification of Critical Quality Attributes (CQAs), Critical Process Parameters (CPPs), and Critical Material Attributes (CMAs). Process Development Risk Assessment utilized Failure Mode and Effects Analysis (FMEA) to establish links between raw materials, starting cell quality, process parameters, and final product attributes. The Control Strategy was developed through risk‐based approaches and multivariate analysis, culminating in Process Performance Qualification (PPQ). Results : Our QbD implementation established a knowledge‐based manufacturing process with reduced product risk through enhanced process understanding. The analysis yielded: 1) A comprehensive QTPP defining target specifications for therapeutic EVs; 2) Risk‐prioritized CQAs, CPPs, and CMAs with established acceptance criteria, including critical attributes for starting materials and raw materials; 3) A documented Control Strategy incorporating in‐process controls and release specifications; and 4) A Continued Process Verification (CPV) program enabling continuous improvement through systematic monitoring and feedback loops. The process demonstrated consistent production of EVs meeting predefined quality attributes across multiple validation batches. Summary/Conclusion : Applying QbD principles to hMSC‐derived EV manufacturing resulted in a well‐characterized process where quality is proactively designed for each step of production, ensuring consistent product quality critical for patient safety and therapeutic efficacy. This systematic approach enhances process robustness, facilitates regulatory compliance, and establishes a framework for continuous improvement in therapeutic EV manufacturing for clinical uses. Funding : This study is supported by the National Research Council of Thailand (NRCT) and Chulalongkorn University, Thailand

Removal

Presenter: Nico Lingg Austrian Centre of Industrial Biotechnology, Graz, Austria Introduction : Advanced medicines based on extracellular vesicles (EVs) are showing promising results as therapeutic agents (both as delivery systems and intrinsic therapeutics) in several ongoing early‐stage clinical trials. To advance EV‐based medicines into late‐stage clinical trials and commercialization, scalable manufacturing processes are still needed. Currently available EV downstream processes require the combination of several unit operations, including ultracentrifugation, filtration, and/or chromatography, and suffer from poor scalability, low selectivity, and inadequate recovery. DNA in particular is a critical impurity that is challenging to remove, especially in the form of chromatin. Methods : In this study, we developed a downstream process for the purification of EVs that combines the use of functionalized non‐woven fibres in flowthrough mode with tangential flow filtration. Non‐woven fibres are a type of convective chromatography media with an open‐pore architecture, offering a promising solution to the limited surface area and mass transfer limitations encountered with conventional chromatography resins, thereby improving process productivity. EV recovery and process yield were determined through nanoparticle tracking analysis, Western blotting, and bead‐based flow cytometry using different EV markers. Removal of chromatin was determined by Picogreen assay and anti‐histone Western blot. Results : EV material that was purified using an efficient non‐woven fibre flow‐through step showed a strong decrease in histones and double stranded DNA. The concentration of EVs remained comparable in most cases. The biological activity after filtration were compared to an untreated control. A significant effect on the anti‐inflammatory activity was observed. Summary/Conclusion : Our work demonstrates that EVs produced in different cell lines can be recovered, while critical impurities such as host cell DNA and chromatin are bound to the non‐woven fibre. The material resulting from the non‐woven fibre purification could then further processed using tangential flow filtration. The combination of these unit operations allowed the removal of host cell DNA, especially in the form of chromatin, increasing product purity and safety while maintaining its biological activity. Funding : This project was funded by Austrian Research Promotion Agency FFG and Solventum Corporation.

Retinal

Presenter: Neha Sharma L V Prasad Eye Institute, India Introduction : Retinopathy of prematurity (ROP), a major ocular complication of preterm birth, is characterized by abnormal angiogenesis and formation of FVM (fibrovascular membrane) at the vitreo‐retinal junction, causing retinal detachment and blindness. Herein, we examined retinal gene profiling and extracellular vesicles, elucidating their involvement in disease pathogenesis. Methods : Retinal transcriptome analysis was performed using the microarray platform, and pathway analysis was performed using GeneSpring GX (ROP = 3, controls = 3). EVs were isolated and characterised using NTA, TEM and western blotting. Further, small RNAseq was performed in vitreous humour (ROP = 8, control = 7), and validations were done by qPCR. Results : 201 genes were significantly differentially regulated between the ROP retina and control retina (FC: ±1.5, p value < 0.05). Angiogenesis, retinal neural development, the complement pathway, macrophage activation, gliosis, and photoreceptor development were the top significant pathways. Interestingly, extracellular vesicle processes were seen among the significant ( p value = 1.429 × 10 −2 ) pathways in the retinal transcriptome. Earlier studies from our lab demonstrated the involvement of active microglia cells in ROP pathogenesis. EVs released from retinal cells are known to diffuse to vitreous humour. From our EVs small RNA sequencing data, 883 miRNAs were identified, of which 739 showed differential expression. Among these, 9 miRNAs exhibited significant upregulation, while another 9 were notably downregulated. Pathway analysis revealed an enrichment of inflammation, angiogenesis and development related pathways in the EVs of ROP subjects. Summary/Conclusion : A significant involvement of inflammation, immune activation, and angiogenesis in ROP was seen in the retinal transcriptome and further confirmed by extracellular vesicle profiling. Further functional investigation could help in delineating their involvement in exacerbation/alleviation of ROP progression. Funding : Department of Science and Technology Innovation in Science Pursuit for Inspired Research (INSPIRE), Government of India (IF190699).

Section

Presenter: Weiyin Zeng Guangzhou University of Chinese Medicine, Guangzhou, People's Republic of China Introduction : The incidence of alcoholic liver disease (ALD) is increasing in China; however, no effective natural ingredient‐based drugs have been approved for market release to date. Traditional Chinese medicine, specifically Artemisiae scopariae Herba, is a natural plant that has demonstrated significant efficacy in alleviating hangovers, revitalizing the spleen, and protecting both the intestines and liver. Chinese herbal derived extracellular vesicle‐like particles (CHM‐EVLP) are characterized by their stability, target specificity, and bioactivity, retaining the pharmacological properties of their source plants. They are readily absorbed by macrophages across various organs and serve as natural drug carriers within traditional Chinese medicine. This study investigates the anti‐hangover and hepatoprotective effects of Artemisiae scopariae Herba, leverageing the unique properties of CHM‐EVLP. Our objective was to explore the potential therapeutic effects and underlying mechanisms of action of Artemisiae scopariae Herba derived extracellular vesicle‐like particles (AH‐EVLP) in mitigating alcoholism and safeguarding the liver against alcohol‐induced damage in vivo. Methods : AH‐EVLP were isolated from dried Artemisiae scoparia e Herba juice using ultracentrifugation and characterized by TEM and nano‐flow analysis. Their proteins, RNAs, and lipids were identified using silver‐staining, agarose gel, and thin‐layer analysis. The alcohol detoxification effect was evaluated through recovery reflex and serum alcohol and acetaldehyde levels. To study hepatoprotective effects against acute alcoholic liver injury (AALI) in mice, liver injury markers, inflammation, oxidative stress, damage, and apoptosis were monitored. Potential active targets and mechanisms for AALI treatment were investigated using WB, qRT‐PCR, IHC, RNA sequencing, LC‐MS, and network pharmacology. Results : AH‐EVLP resemble mammalian extracellular nanovesicles in size and shape. Their administration alleviated toxic symptoms and liver injury in a dose‐dependent manner. They are also anticipated to reduce alcohol‐induced liver inflammation and oxidative stress‐related hepatocyte death by inhibiting the IL‐17 signalling pathway and lowering Ripk4 expression in mice with acute alcohol intoxication. Summary/Conclusion : AH‐EVLP has the potential to mitigate alcohol‐induced liver injury by reducing the inflammatory response, suggesting its development as an innovative nutraceutical or pharmacological intervention for the treatment of alcoholic liver disease (ALD).

Setting

Enrico Ragni 1 , Paola De Luca 1 , Simona Landoni 1 , Michela Taiana 1 , Giuseppe Talò 2 , Matteo Moretti 2,3,4,5 , Laura de Girolamo 1 1 IRCCS Ospedale Galeazzi—Sant'Ambrogio, Labouratorio di Biotecnologie Applicate all'Ortopedia, Italy; 2 IRCCS Ospedale Galeazzi—Sant'Ambrogio, Cell and Tissue Engineering Laboratory, Italy; 3 Ente Ospedaliero Cantonale, Labouratories for Translational Research, Switzerland; 4 Ente Ospedaliero Cantonale, Service of Orthopedics and Traumatology, Switzerland; 5 Università della Svizzera Italiana, Faculty of Biomedical Sciences, Switzerland Introduction : Therapies for cartilage regeneration may lead to incomplete or fibrous tissue formation. Fibrocartilage is poorly performant, and over time joint degeneration progresses, eventually requiring prosthesis. Mesenchymal stromal cells (MSCs) release extracellular vesicles (EVs) able to stimulate naïve cartilage that are studied in clinical trials. The pitfall of EV‐based interventions into the joint cavity is dispersion. This leads to low concentration at the lesion site, resulting in reductions of efficacy and inflation of production costs to increase EVs dose. Biological scaffolds embedding MSC‐EVs are a promising option to concentrate and release MSC‐EVs at the lesion site. This work aimed at producing an enhanced platelet‐derived fibrin gel, already used in clinical practice for cartilage repair, loaded with MSC‐EVs. Methods : Ethics Committee approval and informed consent were obtained. MSCs were isolated from waste adipose tissue, and at passage 1 after serum starvation, EVs were collected by differential centrifugation (100,000 × g , 3 h, 4°C). For microscopy, conditioned medium was 10 µM CFSE supplemented (1 h, 37°C). Particles were characterized by NTA, TEM, WB (positive markers: CD9/81, TSG101, Flotillin; negative markers: Calnexin), FC (lineage markers: CD73/90; EVs markers: CD9/63/81), ELISA, and miRNA‐arrays. MSC‐EVs were added to platelet rich plasma, and fibrin gel was obtained with CaCl 2 . MSC‐EVs’ embedment efficiency, release and incorporation in osteoarthritis (OA)‐patients’ chondrocytes and cartilage explants were monitored by SEM, FC, microfluidic‐based confocal approaches and real‐time quantitative multimodal nonlinear optics imaging. The therapeutic effect of highly‐concentrated gel‐released MSC‐EVs was tested in OA chondrocytes by qRT‐PCR. Results : A high‐efficiency incorporation protocol (> 70% input EVs) was defined, relying on a one‐step modification of standard procedures used in clinical practice for platelet‐gel preparation for cartilage lesions. MSC‐EVs were released for up to 4 weeks and taken up by OA‐chondrocytes and cartilage explants. The release from MSC‐EVs‐loaded platelet gel had synergistic anti‐inflammatory/matrix remodelling effects on chondrocytes with respect to pure MSC‐EVs or released factors from unloaded gels. Summary/Conclusion : A platelet gel loaded at high concentration with MSC‐EVs is an enhanced and easy‐to‐translate tool to foster chondrocyte homeostasis, a key requisite for cartilage repair. Funding : This project (PNRR‐MCNT2‐2023‐12377836) has been funded by the European Union—Next Generation EU—NRRP M6C2 ‐ Investment 2.1 Enhancement and strengthening of biomedical research in the NHS.

Surface

Presenter: Apurba Dev Electrical Engineering, Sweden Introduction : The elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. However, accurate quantification of their elastic modulus is challenging due to their nanoscale dimensions and their fluid‐like lipid bilayer. Methods : Here we apply the Canham‐Helfrich model to quantify the elastic properties of EVs from force spectroscopy performed by an atomic force microscope. We then apply our method to EVs derived from the human embryonic kidney (HEK293T) cell line and different families obtained by genetically altering it's tetraspanins expression. Each of the EVs were carefully analysed with over 150 of force indentation to capture their fluctuations and the data were theoretically modeled. The probability distribution functions obtained were differentiated with Jensen‐Shanon divergence. Results : We find that in contrast to previous reports the EVs behave as surface‐adhered vesicles showing a linear dependence of tether force on the tether length. The bending modulus estimated for the different EV families range from 15.7 − 33.0 kBT with large fluctuations within each family of vesicles which is consistent with inherent EV heterogeneity. Interestingly, our experiments also show large fluctuations of measured values even for a single EV when repeatedly examined under identical conditions. The data consequently show a large spread in their elastic modulus and bending modulus in the range 4‐19 mN/m and 15.7‐33 kBT, respectively, concealing any potential differences among the families of the EVs. Only the family of CD63‐knockout sample exhibited detectable differences with the wild‐type sample. Summary/Conclusion : We show that linear dependence of tether force on tether length is consistent with the theory of adhered vesicles. We suggest that the fluctuations in the individual vesicles is driven by both thermal and compositional effects, as expected for such nanoscale objects. We also show that the probability distribution function (PDF) of elastic moduli are best described by not a single Gaussian but superposition of Gaussians, thus, necessitating appropriate statistical approach. The study suggest that protein alternations might have an influence on the elastic moduli, but they are often hidden in such large fluctuations. Funding : The Swedish Research Council; The Swedish Cancer Foundation.

Tcvps23

Nadjania Saraiva de Lira Silva 1 , Ana Claudia Trocoli Torrecilhas 2 , Sergio Schenkman 3 1 Universidade Federal de São Paulo, campus São Paulo 2 Universidade Federal de São Paulo, campus Diadema 3 Universidade Federal de São Paulo, campus São Paulo Introduction : Endosomal Sorting Complex Required for Transport (ESCRT) plays a role in eukaryotic cell secretion. It consists of four multi‐subunit complexes: ESCRT‐0, I, II, and III. This complex can be found in protozoan parasites, which cause serious infectious diseases in humans. One of these is Chagas disease, which is caused by Trypanosoma cruzi infection and frequently leads to cardiac fibrosis in people. We envisioned examining whether the ESCRT complex affects extracellular vesicle (EV) secretion and the potential function of EVs in Chaga's disease pathogenesis. Methods : We created parasites by replacing one copy of the TcVPS23 gene, a key element of the ESCRT‐I machinery, with the hygromycin resistance gene using CRISPR/Cas9 technology and evaluated the resulting parasites' EV generation, infectivity, immune response, and pathogenesis. Results : TcVPS23 expression was reduced, resulting in altered surface proteins, increased soluble protein release, and decreased extracellular vesicle secretion by the parasite's infectious trypomastigote form. Mice infected with the mutated parasite lines showed reduced blood parasitemia and mast cell recruitment in the cardiovascular system during the acute phase of infection. Despite the high parasite load in heart tissue, they also showed reductions in IFN‐gamma and TGF‐β levels in the circulatory system and heart. Mice in the chronic stage showed lower levels of IL1‐β and IL‐6, fewer mast cells, lower levels of MCPI, KC, and IL6, and less cardiac fibrosis than the parental parasite line. Summary/Conclusion : These findings show that TCVPS23 of ESCRT‐I component affects the release of extracellular vesicles and soluble molecules, altering the immune response and myocardial fibrosis in the T. cruzi‐infected host. Funding : Supported by CAPES, CPNq, and FAPESP 2020/07870‐4, and 2021/12883‐0.

Testing

K. Barreiro 1 , R. Hartman 1 , I. Quintero 1 , F. Roessler 1 , P. H. Groop 2,3,4 , M. Puhka 1 1 HiPREP core, Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland; 2 Folkhälsan Research Center, Biomedicum Helsinki, Helsinki, Finland; 3 Department of Nephrology, University of Helsinki, Helsinki University Hospital, Helsinki, Finland; 4 Faculty of Medicine, Research Program for Clinical and Molecular Metabolism, University of Helsinki, Helsinki, Finland Introduction : Urinary extracellular vesicles (uEV) provide valuable non‐invasive biomarkers for urogenital diseases. However, progress in biomarker discovery and validation is slow because of lack of uEV isolation methods suitable for robust high‐throughput processing. For this purpose, we tested methods based on precipitation, ultrafiltration and immunomagnetic bead capture in comparison to ultracentrifugation. Methods were evaluated based on processing format and time, RNA or protein yield and quality and uEV sample morphological analysis. Methods : uEVs were isolated from urine samples with or without a preconcentration step. The urine donors included healthy donors or donors with type 1 diabetes with or without kidney disease. uEV samples were viewed with transmission electron microscopy, RNAs profiled with the Bioanalyzer Pico kit and proteins quantified with micro BCA or similar methods. For a deeper insight, proteins were run on SDS‐PAGE gels and EV‐enriched markers and contaminants detected by Western blotting, or samples were subjected to RNA sequencing. Results : EVs, EV‐like structures, small RNA and proteins were detected using all methods. The yield, quality and purity assessments showed small differences between the methods. EV markers, contaminants and sequencing results were at an acceptable level in comparison to ultracentrifugation. Processing time and sample format varied the most between the methods. Summary/Conclusion : All methods showed initial promise for scalable EV isolations. However, the need for sample preprocessing and output sample format impact the throughput or the selection of usable downstream techniques. Further optimization may allow a wide variety of large‐scale studies for biomarker discovery. Funding : This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115974. The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA and JDRF.

Through

Invited Speaker: Frederik J. Verweij Utrecht University, The Netherlands

Towards

Maximilian Kieckhöfer 1 , Caroline Schmidt 1 , Franziska Mitrach 1 , Christian Preußer 2 , Michaela Schulz‐Siegmund 1 1 Leipzig University, Leipzig, Germany; 2 Philips University Marburg, Marburg, Germany Introduction : In a prior study, we established an siRNA loading strategy yielding an average of 150 siRNA copies per milk extracellular vesicle (mEV) utilizing calcium phosphate nanoparticles (CaP‐NP) and dual centrifugation (DC). However, we believe this siRNA distribution is unevenly distributed across the mEV population. A homogenous siRNA loading is crucial for reliable siRNA dosing and development of new delivery systems. To distinguish between loaded and non‐loaded vesicles, we systematically investigated and compared nanoparticle tracking analysis (NTA), flow cytometry (FCM) and nano flow cytometry (nFCM). Methods : After enrichment, deploying differential centrifugation and size exclusion chromatography followed by ultracentrifugation, the mEV pellet was loaded by DC with fluorescently labelled siRNA bound to CaP‐NP. As a control, mEV‐mimicking liposomes (EVmL) were manufactured by a solvent evaporation protocol and loaded with CaP‐NP or just with siRNA. For fluorescence detection on single vesicles, we used NTA, FCM and nFCM. The loading efficiency was measured by total fluorescence with a plate reader after particle lysis. Results : NTA analysis showed big differences in fluorescence between CaP‐NP‐loaded mEVs and EVmL loaded without CaP‐NP. We detected much more siRNA‐loaded EVmL than mEVs. The fraction of fluorescent mEVs and EVmL, presumably carrying CaP‐NP, showed a higher particles size than average. With flow cytometry we confirmed a small mEV population with large particles, loaded with fluorescent siRNA. We also stained CaP‐NP with calcein to circumvent low fluorescence intensity when using siRNA. Interestingly, when measuring total siRNA loading in a plate reader after lysis, we detected much higher fluorescence in mEV and EVmL than expected by the small fraction of positive vesicles. Summary/Conclusion : Our findings show that direct measurement of siRNA loading inside single mEVs is possible but may be limited by a minimal fluorescence yield per vesicle, depending on the instrument employed. The finding that fluorescence was mainly found in large vesicles may result from the concept of encapsulating CaP‐NP in mEVs or just by the minimum fluorescence intensity of a vesicle. Finally, there is a need for higher sensitivity instruments for assessing mEV loading in future research and higher loading concentration to increase the fluorescence intensity per vesicle.

Urinary

Presenter: Haitao Sun Southern Medical University, Guangzhou, People's Republic of China Introduction : Glioma, the most aggressive malignant tumour of the central nervous system, is currently diagnosed primarily through invasive biopsy procedures, and effective early screening methods remain elusive. Brain‐derived extracellular vesicles (EVs) can traverse the blood–brain barrier into the systemic circulation and are subsequently excreted into urine via renal filtration. As these EVs carry tumour‐derived metabolites that can be efficiently enriched and detected from noninvasively collected urine, they represent promising carriers for constructing liquid biopsy biomarkers for glioma. Methods : In this study, we systematically evaluated four EV isolation techniques—kit‐based methods, the EXODUS method, ultracentrifugation, and size‐exclusion chromatography—in order to identify the optimal method for isolating urinary EVs. Subsequently, we conducted a case‐control study including 30 age‐, sex‐, and BMI‐matched healthy controls, 30 meningioma patients, and 30 patients each with WHO grade I–II and grade III–IV gliomas. Following EV isolation from urine samples, we performed untargeted metabolomic analysis using ultra‐high‐performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS) to identify differential metabolites and construct a diagnostic model. Results : The findings indicate that size‐exclusion chromatography offers significant advantages in purifying urinary EVs. Transmission electron microscopy (TEM) revealed that the isolated EVs possess a typical bilayer membrane structure with diameters ranging from 30 to 150 nm. Nanoparticle tracking analysis (NTA) demonstrated a particle concentration of 1.9 × 10 1 ⁰ particles/mL, reflecting a significantly higher enrichment compared to other methods ( p < 0.05). Furthermore, purity analysis showed that EVs isolated via size‐exclusion chromatography were over ten times purer than those obtained by alternative techniques ( p 2.0, p < 0.005), and a multi‐marker diagnostic model constructed using a random forest algorithm achieved an area under the curve (AUC) exceeding 0.80, with both sensitivity and specificity above 80%. Summary/Conclusion : This study represents the first systematic comparison of urinary EV isolation methods, revealing that size‐exclusion chromatography yields superior purity and enrichment. Our optimized approach enabled the discovery of robust glioma‐specific metabolic biomarkers, underscoring the potential of urinary EV‐based liquid biopsies for noninvasive early diagnosis and targeted treatment of glioma.

Analysis

Presenter: Alexis Wilhelm Technical University Munich, Munich, Germany Introduction : Breast cancer, one of the most prevalent cancers among women, is characterised by the expression of hormone receptors and the growth factor receptor HER2. This information is crucial for the development of personalised tumour treatment using HER2‐targeted therapies. Tumour biopsies are necessary for the histopathological diagnosis of HER2 expression by breast cancer cells, but they are subject to sampling error. Methods : In this study, we present a successful method for identifying and analysing cancer‐derived extracellular vesicles (EVs) from plasma for the detection of HER2 expression in breast cancer without the need for additional processing steps. We have optimised the detection of nano‐sized particles by flow cytometry and employed these settings to detect HER2‐expressing EVs and quantify the expression level of HER2 on these EVs. Results : In a clinical study of 115 patients with breast cancer, optimised flow cytometric analysis detected between 50 and 1.3 × 103 HER2+EVs per µL in the plasma of patients. While the numbers of HER2+EVs did not directly correlate with HER2 expression of tumour tissue, tumour size, grade, or metastasis, the integration of the data deriving from the enumeration of HER2‐positive EVs and the determination of their HER2 expression levels enabled the reliable identification of HER2‐expressing tumours, while preventing the misclassification of false positive results. Summary/Conclusion : Our findings demonstrate the viability of analysing cancer‐derived EVs from plasma by integrating the count of HER2+ EVs and the expression level of HER2 on these EVs for the diagnosis and personalised therapy of breast cancer patients. Funding : This project was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) (ZIM) to P.A.K. and O.W. B.H. was supported by funding from the School of Medicine and Health. P.K., T.B. and J.K. are supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project‐ID 210592381 ‐ SFB 1054 (TP B03; TP Z02), CRC‐TRR179.

Breaking

Invited Speaker: Claudia Günther UKER, Medizinische Klinik 1, Germany

Building

Invited Speaker: Rienk Nieuwland Amsterdam University Medical Center, The Netherlands

Calcific

Presenter: Mohammed Rabiul Hosen University of Bonn, Germany Introduction : Calcific aortic valve disease (CAVD) is one of the leading causes of cardiovascular death in the elderly population worldwide. MicroRNAs are highly dysregulated in patients with CAVD undergoing surgical aortic valve replacement (SAVR). However, the miRNA‐dependent mechanisms regulating inflammation and calcification or miRNA‐mediated cell‐cell crosstalk during the pathogenesis of CAVD remain poorly understood. We investigated the role of extracellular vesicle (EV)‐associated miR‐145‐5p, which we showed to be strongly upregulated in CAVD in mice and humans during valve calcification. Methods : Human miRNA arrays identified dysregulated miRNAs in CAVD tissue explants compared to non‐calcified HV tissue explants from patients undergoing SAVR. Echocardiographic parameters were measured in conjunction with the quantification of dysregulated miRNAs in a murine CAVD model. Integrated OMICS analyses were performed to analyse molecular miRNA signatures and their effects on signalling pathways. RNA‐seq, high‐throughput TF assays, osteogenesis, and proteomic arrays revealed that several genes, miRNAs, TFs, and proteins are critical for calcification and apoptosis involved in the pathogenesis of CAVD. Results : Among several miRNAs dysregulated in valve explants from CAVD patients, miR‐145‐5p was the most highly sex‐independently dysregulated miRNA (AUC, 0.780, p value, 0.01). MiRNA arrays utilizing CAVD samples from patients and mice showed that the expression of miR‐145‐5p is significantly upregulated and positively correlated with cardiac function based on echocardiography. In vitro experiments confirmed that miR‐145‐5p is encapsulated in EVs and transported into VIC of the AV. The results of integrated OMICs show that miR‐145‐5p is related to markers of inflammation, calcification, and apoptosis. The calcification experiments demonstrated that miR‐145‐5p regulates the ALPL gene, a hallmark of calcification in heart valve cells. EV‐mediated shuttling of miR‐145‐5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3′ untranslated region to inhibit its translation, thereby diminishing the calcification of target VIC. Summary/Conclusion : Elevated levels of pro‐calcific and pro‐apoptotic EV‐associated miR‐145‐5p contribute to the progression of CAVD via the ZEB2‐ALPL axis, which could potentially be therapeutically targeted to minimize the burden of CAVD. Funding : German Cardiac Society (DGK), Ernst und Grimmke Foundation, and Corona Foundation Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Grant No. 397484323 ‐ TRR259 ‐ Project B04

Chitosan

Mengyun Wang, Jin Wang, Shuangshuang Liu, Lingting Kong, Linli Lv, Bicheng Liu, Taotao Tang Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, China Introduction : Extracellular vesicles (EVs) hold significant potential for sepsis treatment; however, their therapeutic efficacy can be compromised by the formation of plasma protein corona (PC) upon entering the circulatory system. This study aims to characterize the composition and function of the plasma PC and to explore how chitosan oligosaccharide (COS)‐mediated electrochemical modification can inhibit PC formation around EVs, thereby enhancing their therapeutic potential for sepsis. Methods : Small EVs derived from umbilical mesenchymal stem cells (MSCs) and HEK293F cells were harvested via serum‐free 3D culture. A dynamic system simulated blood circulation for plasma PC formation, characterized by NTA, Raman spectroscopy, proteomics, and super‐resolution imaging. Additionally, COS was utilized for electrochemical modification of EVs to assess their inhibitory effect on PC formation and influence on macrophage uptake. A septic mouse model was established to evaluate the in vivo distribution and therapeutic efficacy of COS‐modified EVs. Results : Following plasma PC formation, MSC‐EVs and HEK293F‐EVs showed increased particle size and decreased zeta potential. Raman spectroscopy revealed significant changes in EV composition, characterized by increased protein peak intensities. Proteomic analysis identified 263 and 209 plasma proteins on MSC‐EVs and HEK293F‐EVs, respectively, with notable enrichment of immunoglobulins and complement proteins. Super‐resolution imaging further confirmed the presence of plasma proteins like IGHG1, C3 and APOA1 around EVs. Additionally, PC formation enhanced macrophage uptake of EVs and activated inflammatory responses. To address this issue, we modified EVs with COS, which significantly reduced PC formation. Consequently, COS modification effectively inhibited macrophage uptake and the inflammatory response triggered by the PC. IVIS imaging indicated that COS‐modified EVs had decreased distribution in the liver and spleen but enhanced uptake in the brain, heart, lungs, and kidneys of both normal and septic mice. Importantly, COS modification improved survival rates compared to unmodified MSC‐EVs and enhanced protective effects on the lungs, heart, and kidneys in sepsis. Summary/Conclusion : Plasma proteins readily form a PC on EVs, while COS modification can reduce the adsorption of immunoglobulins, complements, and others, thereby inhibiting plasma PC formation. This modification decreases EV uptake by the mononuclear phagocyte system, enhancing their biodistribution in extrahepatic organs and subsequently ameliorating multi‐organ damage in sepsis.

Clinical

Odett Kaiser 1 , Susanne Sasse 1 , Jennifer Harre 1 , Mario Gimona 2,3 , Eva Rohde 2 , Daniela Auer 2 , Tanja Schally 2 , Stefan Rund 2 , Carina Kals 2 , Christina Folie 2 , Elisabeth Bayer 2 , Hinrich Staecker 3 , Nils Kristian Prenzler 1 , Athanasia Warnecke 1 1 Hannover Medical School, Hannover, Germany; 2 Paracelsus Medical University (PMU), Salzburg, Austria; 3 Paris‐Lodron University Salzburg, Salzburg, Austria; 4 University of Kansas School of Medicine, Kansas City, Kansas, USA Introduction : Following cochlear implantation, immune response can lead to loss of residual hearing and impaired hearing performance. So far, there are no pharmacological therapies to compensate for this. A new medicinal product derived from mesenchymal stromal cells, vesicle‐enriched secretome fractions (VSF), comprises extracellular vesicles (EV), particles and soluble factors. Preclinically, VSF demonstrated immunomodulatory and/or neuroprotective effects and was evaluated for safety. Clinically, VSF has been used in a first named patient approach to demonstrate the feasibility of intracochlear application during cochlear implantation. A first‐in‐human phase I/II clinical trial named ESCRT has been submitted to the Central European system for clinical trials. Methods : In this first‐in‐human open‐label monocentric phase I/IIa clinical trial, a single dose (30 µL of 0.5–1 × 10 11 particles/mL) of VSF is applied to the inner ear during cochlear implant surgery as adjuvant treatment prior to insertion of the electrode array. Eleven patients (aged ≥ 18) with profound hearing loss receiving cochlear implantation are included. The number and severity of adverse events (AEs) are documented, and immune response, auditory nerve function, and speech perception are evaluated. A sentinel approach will be included. Study duration per subject is approximately 6 (±1) months, with the entire trial scheduled to last approximately 21 months. Results : The submission of ESCRT via the Clinical Trials Information System (CTIS) was approved in October 2024 and was initiated immediately. Safety as a primary endpoint, based on the detection and documentation of severe and serious AEs, will be recorded using a sentinel approach based on the results of the first three patients. Summary/Conclusion : The process of taking a new drug candidate from the idea to the bench to a clinical trial is challenging, time‐consuming and expensive. Many unexpected hurdles have to be overcome on the way to approval. Thus, this clinical trial of the first intracochlear approach of EV application can serve as an exemplary pathway to achieve the broader goal of developing precision medicines with the potential to improve and preserve hearing after cochlear implantation trauma. Funding : This work was funded by MED‐EL (Austria), by Deutsche Forschungsgemeinschaft (DFG; EXC 2177/1, Project ID 390895286), and by the Project “ExtraNeu” from the State of Salzburg, Austria.

Coenzyme

Rachele Agostini 1 , Mattia Tiboni 1 , Stephanie Fondi 1 , Paola Ceccaroli 1 , Emanuela Polidori 1 , Patrick Orlando 2 , Sonia Silvestri 2 , Andrea Frontini 2 , Luca Casettari 1 , Michele Guescini 1 1 University of Urbino Carlo Bo, Urbino, Italy; 2 Polytechnic University of Marche, Ancona, Italy Introduction : Coenzyme Q (CoQ) is an endogenous lipophilic quinone ubiquitous in biological membranes endowed with antioxidant and bioenergetic properties. CoQ deficiency can result from either a genetic defect or secondary deficiencies due to ageing, oxidative stress, or drug interaction with biosynthesis. In these conditions, CoQ supplementation represents a relevant strategy to mitigate CoQ deficit; however, numerous studies have demonstrated that the efficiency of dietary CoQ uptake is a limiting step. Exosomes and, more generally, extracellular vesicles (EVs), the endogenous nanocarriers that can deliver biological information between cells, were recently proposed as a new drug delivery system. EVs resemble liposomes in terms of size, shape, and structure. The ability of EVs to transport biomolecules to recipient cells has made them attractive for drug delivery purposes. Here, we developed bioinspired exosome‐mimetic nanovesicles to deliver ubiquinone (CoQ10) to different cell types. Methods : The CoQ10‐loaded nanovesicles (Q10‐eNVs) were produced using a mixture of synthetic choline‐based phospholipids, membrane proteins isolated from C2C12 myoblasts, and oxidized CoQ10. Unilamellar vesicles were obtained by extrusion through cellulose acetate membranes. Results : The Q10‐eNVs have a similar diameter to exosomes (about 110 nm), and the obtained CoQ10 packageing into Q10‐eNVs was 25 µg/mL. Human fibroblasts, HeLa, mice skeletal muscle (C2C12), and rat cardiomyocytes (H9c2) were treated with 6.25 µg/mL of Q10‐eNVs for 24 h. The obtained results showed that H9C2 ware able to take up 3 times more CoQ10 than the other cell types. Summary/Conclusion : Taken together, these studies suggest that the bioengineered nanovesicles can serve as novel exosome‐mimetics to deliver CoQ10 to cardiomyocytes effectively. Funding : This work has been funded by the European Union—NextGenerationEU—under the Italian Ministry of University and Research (MUR) National Innovation Ecosystem grant ECS00000041 ‐ VITALITY—CUP [H33C22000430006].

Collagen

Presenter: Ming‐You Shie China Medical University, Taiwan (Republic of China) Introduction : Diabetic wounds are characterized by impaired healing due to chronic inflammation, reduced angiogenesis, and insufficient collagen deposition. Extracellular vesicles (EVs), derived from adipose‐derived mesenchymal stem cells (ADSC), have shown potential in promoting wound healing through immunomodulation and tissue regeneration. This study explores the therapeutic effects of calcium silicate (CS)‐stimulated ADSC‐derived EVs (CSEV) incorporated into collagen hydrogel for diabetic wound healing. Methods : The in vitro and in vivo diabetic wound models were utilized, including a diabetic human dermal fibroblast (HDF) model and a diabetic rabbit wound model. CSEV were characterized for their physical and biological properties, and their therapeutic efficacy was assessed, particularly in improving wound healing and regulating oxidative stress. Molecular analysis revealed that CSEV contains distinct miRNA and proteins linked to angiogenesis, wound healing, and anti‐inflammatory effects, highlighting their potential for diabetic wound treatment. Results : CSEVs were characterized by their enhanced protein content, surface marker expression, and bioactive cargo, including pro‐angiogenic and anti‐inflammatory factors. In vitro studies demonstrated that CSEV‐loaded collagen significantly reduced reactive oxygen species (ROS) production and enhanced cell proliferation and migration compared to standard EV‐loaded collagen. Cytokine profiling further revealed the upregulation of anti‐inflammatory cytokines (IL‐10) and extracellular matrix components (Col I) in the CSEV‐treated groups. In vivo, histological evaluation of diabetic rabbit models treated with CSEV‐loaded collagen exhibited superior re‐epithelialization and organized collagen deposition, indicating accelerated wound closure. Summary/Conclusion : The findings of this study demonstrate that CS‐stimulated EVs (CSEV), incorporated into collagen hydrogel dressings, significantly enhance the healing of diabetic wounds. CSEV exhibit superior bioactivity, as evidenced by increased protein content, enhanced cellular uptake, and modulation of inflammatory and regenerative pathways. The sustained release of CSEVs from the collagen matrix supports long‐term therapeutic efficacy, promoting angiogenesis, reducing oxidative stress, and accelerating re‐epithelialization and collagen deposition in vivo. These results highlight the potential of CSEV‐loaded collagen dressings as a novel therapeutic strategy for improving outcomes in diabetic wound care, particularly in cases where traditional treatments are insufficient due to impaired tissue regeneration.

Crafting

Prima Dewi Sinawang 1 , Priyanka Multani 1 , Mehmet O. Ozen ,1 , Jodie Wong 2 , Demir Akin 1 , Claire Hanson 3 , Matt Larsen 3 , Liang Wang 2 , Brian T. Cunningham 4 , Manish Kohli 3 , Utkan Demirci 1 1 Stanford University, Stanford, California, USA; 2 H. Lee Moffitt Cancer Center, Tampa, Florida, USA; 3 University of Utah, Salt Lake City, Utah, USA; 4 University of Illinois at Urbana‐Champaign,  Urbana, Illinois, USA Manish Kohli and Utkan Demirci are co‐senior authors . Introduction : Extracellular vesicles (EVs) offer minimally invasive means for cancer detection and monitoring. However, the lack of standardized methods for clinical biospecimen preparation and EV isolation hampers the clinical utility of EV‐based biomarker assessments. Our study investigates the impact of clinical sample preparation and our ExoTIC device on the quality of plasma‐derived EVs in metastatic castration‐resistant prostate cancer (mCRPC) patients. Methods : We assessed sample preparation variables, including blood anti‐coagulant choice (EDTA or sodium citrate), type of plasma platelet fraction (platelet‐rich or platelet‐poor), and use of protease inhibitors. EVs were isolated using the ExoTIC device, followed by EV characterizations and biomarker analysis using nanoparticle tracking analysis, cryogenic electron microscopy, Western blot, and digital PCR. Blood was collected from all patients after obtaining written consent, adhering to an institutional IRB‐approved protocol for specimen collection. Results : We detected mCRPC‐relevant proteins ARv7 and PSMA in EVs from all plasma sample types. Our findings indicate that platelet‐poor plasma (PPP) from EDTA tubes without protease inhibitors, or sodium citrate tubes with protease inhibitors, is optimal for detecting EV‐ and biology‐associated mCRPC biomarker miR‐375. Elevated EV miR‐375 levels from PPP samples correlate with the poor therapeutic response to docetaxel chemotherapy. Summary/Conclusion : Optimal biospecimen preparation for EV analysis could enhance detection accuracy and patient management, highlighting detection of plasma EV‐associated mCRPC‐specific marker proteins (e.g., ARv7 and PSMA) and microRNA miR‐375. Funding : NIH R01EB029805 and R01CA212097; P.D.S. acknowledges support from the James D. Plummer Graduate Fellowship, EDGE Doctoral Fellowship Program, Summer First Fellowship Program, Dean's Office of the Stanford School of Engineering, Cancer Imaging & Early Detection Award, Canary‐ACED Graduate Fellowship, and Stanford Bio‐X Interdisciplinary Initiatives Program Seed Grant.

Decoding

Bhuvanesh Kumar Raju, Vikram Gujar Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, Tulsa, Oklahoma, USA Introduction : Respiratory diseases, like chronic rhinosinusitis, allergic rhinitis, and asthma, pose global health concerns, affecting over 500 million people and significantly contributing to the healthcare burden. Traditional diagnostic methods often lack sensitivity and specificity, necessitating the development of novel biomarkers for early and accurate detection of specific disease conditions. Small extracellular vesicles (sEVs), nano‐sized vesicles released by cells, have gained attention as carriers of molecular biomarkers due to their role in cell communication and presence in various biological fluids. This study aims to identify respiratory‐specific biomarkers within sEVs derived from nasal mucus, offering a non‐invasive approach to diagnosing respiratory conditions. Methods : We received deidentified nasal mucus and serum samples from healthy individuals and extracted the sEVs using precipitation and size exclusion‐based techniques. We characterized these sEVs using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and western blotting. We subsequently employed RNA sequencing to identify potential biomarkers within these vesicles. In addition, we performed single‐cell RNA sequencing for nasal tissue to achieve a high‐resolution view of cellular heterogenicity and to compare cell‐type‐specific biomarkers. We processed the sequencing data using bioinformatic tools, including alignment, normalization, and differential gene expressions, to identify potential respiratory biomarkers. Results : Our analysis showed a higher level of specific mRNA transcripts, associated with inflammation, cellular stress response, and membrane integrity, which are crucial in respiratory diseases in nasal sEVs compared to serum sEVs. The findings suggest that sEVs from nasal mucus are a rich source of respiratory‐specific biomarkers, which could be utilized to diagnose and monitor inflammation and olfactory dysfunction in respiratory conditions. Summary/Conclusion : These biomarkers offer a promising avenue for developing non‐invasive diagnostic tools that could improve patient outcomes by enabling earlier and more accurate detection of respiratory diseases. Future research should focus on validating these biomarkers in larger, disease‐specific cohorts. Funding : This study was supported by the Startup Fund—Oklahoma State University.

Deletion

Johannes Oesterreicher 1,2 , Magdalena Metzger 1 , Sergiu Dumitrescu 1 , Sivun Dmitry 3 , Peter Dungel 1 , Regina Grillari 4 , David Hercher 1 , Johannes Grillari 1,4 , Jaroslaw Jacak 1,3 , Marcin Osuchowski 1 , Wolfgang Holnthoner 1 , Johannes Zipperle 1 1 Ludwig Boltzmann Institute for Traumatology, Vienna, Austria; 2 BOKU University, Vienna, Austria; 3 University of Applied Sciences Upper Austria, Wels, Austria; 4 Evercyte GmbH, Vienna, Austria Introduction : The potential role of EVs as therapeutics has attracted major attention in the scientific community. Yet, application of EVs, particularly from a mesenchymal origin, may produce deleterious side effects such as activation of coagulation via tissue factor (TF). Possible lethal thrombotic events are currently disregarded in pre‐clinical therapeutic studies using EVs. This project aimed to generate a cell line that does not pose the risk of thrombogenicity when used to generate therapeutic EVs. Methods : We generated a TF knockout (TF KO) immortalized adipose‐derived stromal cell line using CRISPR/Cas9. Single‐cell clones were screened for loss of gene expression using RT‐PCR, western blot, and flow cytometry. EVs were enriched using tangential flow filtration and analysed via nanoparticle tracking analysis, atomic force microscopy, bead‐based flow cytometry, and dot blot. An inflammation assay using RAW 264.7 cells assessed potential changes in TF KO EV potency. Procoagulant properties of EV preparations were tested via whole blood thromboelastometry. Results : Expression analysis confirmed the successful TF knockout. Flow cytometry revealed no difference in lineage‐specific markers. The inflammation assay resulted in no altered immune modulation capacity of generated EVs. Thromboelastometric analysis revealed a significant reduction of clotting time (human: by 39%, rat: by 59%) and clot formation time (human: by 42%, rat: by 62%) for the native EV preparations; this thrombogenic effect was abrogated by the TF‐blocking antibody (HTF‐1). The analysis of TF KO‐derived EVs showed no significant modulation of the respective coagulation parameters. Summary/Conclusion : Our study shows the successful generation of a TF KO cell line for EV generation that diminishes previously reported possible lethal side effects. This work contributes to the development of safer therapeutic applications of EVs in pre‐ and clinical practice.

Delivery

Anthony Yan‐Tang Wu, Wendy Wan‐Ting Wong, Phyllis Shao‐Fang Pan, Shannon Yu‐Hsuan Yeh, Felicia Djurijanto, Jou‐Min Wang, Christian Hanson, Charles Pin‐Kuang Lai Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan (Republic of China) Introduction : Extracellular vesicles (EVs) can be categorized by size into large EVs (lEVs; > 200 nm) and small EVs (sEVs; < 200 nm), both of which transport bioactive cargo, including nucleic acids and proteins, to facilitate intercellular communication under (patho‐) physiological processes. While lumen cargo contributes to functional effects, our studies showed that EV membrane proteins affect EV organotropism in vivo. Although cells are reported to release both sEV and lEVs, simultaneous tracking and biodistribution analysis of multiple EV subpopulations remain largely unexplored. Methods : We developed PalmSORBET, a spectrally multiplexed EV reporter system composed of two bioluminescence resonance energy transfer (BRET)‐based reporters, PalmSORET and PalmGRET, to concurrently track organotropism and biodistribution of labelled PalmGRET‐lEVs and PalmSORET‐sEVs. Each BRET reporter comprises a paired bioluminescent and fluorescent protein: PalmSORET (Rluc8.6‐535SG and iRFP713), PalmGRET (nanoluciferase and GFP). By labelling EV inner membranes via a palmitoylation motif, PalmSORBET enables multimodal and multi‐resolution EV imaging through bioluminescence, BRET‐induced fluorescence, and fluorescence. To investigate the dynamics between lEVs and sEVs, we co‐administered both populations overexpressing CD13, a protein we identified to reduce the lung organotropism of liver cancer‐derived EVs, followed by the spleen organotropism change. Results : PalmSORBET successfully labelled lEV and sEV inner membranes without altering EV biophysical properties and biodistribution. Intravenous co‐administration of lEVs and sEVs showed that CD13 overexpression (CD13‐OE) confers EV spleen tropism at 0.5 and 72.5 h post‐injection. A single CD13‐OE in lEV or sEV shared a similar organotropism pattern to that of CD13‐OE in both lEV and sEV. Notably, co‐administration of lEV (wildtype) and sEV (CD13‐OE) leads to lEV distribution to the kidney and plasma when compared to co‐injected lEV (CD13‐OE) and sEV (CD13‐OE). On the other hand, spleen‐tropic lEV (CD13‐OE) did not significantly affect the biodistribution of the sEV (wildtype or CD13‐OE). Summary/Conclusion : We identified CD13 as a key EV membrane protein for spleen tropism in both lEVs and sEVs. Detailed biodistribution analysis via the PalmSORBET system first discovered the interdependent behaviour of lEV and sEV populations under altered spleen tropism, suggesting potential long‐term effects on their biodistribution and functional roles. Funding : This study was supported by the NSTC 113‐2628‐B‐001‐003, i‐MATE Program AS‐iMATE‐107‐33, IAMS 30‐08, Academia Sinica Career Development Award 109‐M04.

Detailed

Hirotaka Nishimura, Tomofumi Yamamoto, Masato Kiyoshi, Noritaka Hashii, Akiko Ishii‐Watabe National Institute of Health Sciences, Tokyo, Japan Introduction : The number of clinical trials involving EV administration has been increasing in recent years, and the day when the first EV drug is launched is drawing near. However, there is still little consensus on how to evaluate the quality and ensure the safety and efficacy of EVs in the development of pharmaceutical products. In particular, it is necessary to establish a new evaluation method to assess particle heterogeneity in EV formulations, which is not used for conventional pharmaceutical products. In this study, we propose a detailed characterization method using ion‐exchange HPLC fractionation and various analytical methods as a method to evaluate the heterogeneity of particle components in EV formulations. Methods : We used EVs derived from adipose tissue MSC and purified with TFF as a test sample. EVs were analysed using anion exchange HPLC with a multiangle light scattering detector (AEX‐MALS), and the main peaks containing particles were collected. These purified fractions and input EVs were characterized by TEM, NTA, nano‐flow cytometry, and proteomic analysis by LC‐MS/MS. Results : AEX‐MALS revealed that the particulate contents in the EVs were classified into two major groups by their charge properties. TEM observations provided precise particle structures and accurate size distribution information for each sample, but the size distribution was quite different from those measured by NTA and nano‐flow cytometry. Detailed evaluation of expressed molecules by nano‐flow cytometry revealed significant differences in RNA loading rates and expression rates of common EV markers such as CD9 and 81 among the samples, suggesting that each fraction separated by AEX depending on the charge is composed of different types of EVs. Proteomic analysis showed that one fraction after purification contained many proteins that are considered EV markers in the database, such as Exocarta and Vesiclepedia (about 70% of the top 30 proteins and 50% of the top 100 proteins), suggesting that AEX is useful for evaluation of EV‐derived proteins in EV samples. Summary/Conclusion : AEX‐MALS is useful not only for evaluating the charge heterogeneity of particles in EV formulations, but its use in purification will also contribute significantly to the manufacturing of EV products with higher purity. Funding : AMED JP22mk0101218, JP24am0521007.

Detectev

Giorgia Adamo *1 , Sabrina Picciotto 1 , Paola Gargano 1 , Angela Paterna 2 , Samuele Raccosta 2 , Estella Rao 2 , Daniele Paolo Romancino 1 , Giulio Ghersi 3 , Mauro Manno2, Monica Salamone 1 , Antonella Bongiovanni 1 1 Cell‐Tech HUB and Institute for Research and Biomedical Innovation (IRIB), National Research Council of Italy (CNR), Palermo, Italy; 2 Cell‐Tech HUB and Institute of Biophysics (IBF)—CNR, Via Ugo La Malfa, Palermo, Italy; 3 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Palermo, Italy Introduction : The therapeutic and diagnostic promise of extracellular vesicles (EVs) as bioactive nanocarriers in cell‐free therapies necessitates robust quality control, particularly in assessing bioactivity, membrane integrity, and batch consistency. Given the typical heterogeneity of EV preparations, these assessments require functional assays that provide essential quality control metrics to complement existing physicochemical methods and ensure EV preparations meet stringent quality standards. The DetectEV assay introduced here was developed to address these limitations, offering a fast, sensitive, and quantifiable evaluation of EV functionality. Methods : DetectEV is an enzymatic‐based assay that quantifies esterase activity within the EV lumen and assesses membrane integrity, presenting results in enzymatic units. Using microalgae‐derived EVs, or nanoalgosomes, as an EV‐model, we optimized assay parameters and validated its performance in detecting changes in bioactivity and integrity across different EV types (including human cell‐derived EVs), isolation methods, and storage conditions, requiring only minimal sampling for rapid analysis. Results : DetectEV demonstrated high sensitivity and specificity in quantifying esterase activity in nanoalgosomes and various human‐derived EV types. The assay effectively distinguished batch‐to‐batch variability, with its readout correlating to specific functional properties, such as antioxidant capacity in nanoalgosomes. Comparative analyses showed that DetectEV could detect differences in EV bioactivity and integrity resulting from various isolation, loading, and storage methods, outperforming conventional approaches that primarily focus on physicochemical EV characteristics. Summary/Conclusion : The DetectEV assay provides an efficient, single‐step analysis of EV functionality and membrane integrity, enabling reliable quality control for various EV applications. Its broad compatibility with diverse EV types and minimal sample requirements make it a versatile and practical tool for standardizing EV functionality assessments prior to specific EV‐based potency tests. The predictive capability of the DetectEV assay for EV functionality underscores its potential to support EV‐based therapeutic and diagnostic development. Funding : This work was supported by the VES4US and the BOW projects, funded by the European Union's Horizon 2020 research and innovation programme, under grant agreements nos. 801338 and 952183, MUR PNRR “National Center for Gene Therapy and Drugs based on RNA Technology” (Project no. CN00000041 CN3 RNA) and by the Institute for Research and Biomedical Innovation (IRIB), National Research Council of Italy of Palermo (Internal Call@IRIB2023).

Diameter

Presenter: Sayam Ghosal Semmelweis University, Hungary Introduction : Recent advancements in extracellular particle (EP) research have unveiled novel non‐vesicular extracellular particles (NVEPs), such as exomeres and supremeres, yet much remains unknown about their biogenesis and physiological roles. This study investigates the properties of NVEPs, isolated from a 167k g pellet (167k‐NVEP), and compares them to well‐characterized extracellular vesicle (EV) subtypes: small EVs (100k‐sEVs) and large EVs (14k‐lEVs) isolated from 100k g and 14k g pellets respectively. Our goal was to explore the biogenesis, molecular composition, and structural diversity of these EPs, thereby contributing to a deeper understanding of EP dynamics. Methods : We employed an integrative approach, combining an image meta‐analysis from various published studies on intraluminal vesicles (ILVs) across species and kingdoms, with extensive characterization of 167k‐NVEPs and EV subtypes. Characterization techniques included transmission electron microscopy (TEM), biochemical assays, Raman spectroscopy, lipidomic profiling via mass spectrometry, high‐resolution flow cytometry and super‐resolution microscopy. These methods enabled us to identify structural, biochemical, and molecular distinctions among the EP subtypes. Results : TEM image meta‐analysis revealed that ILVs within multivesicular bodies (MVBs) consistently measured with a mean ± SD diameter of 109.60 ± 36.77 nm. This size range closely aligns with sEVs but not lEVs. The maximum observed ILV size was 197.00 nm, showing that lEVs exceeding 200 nm in diameter may be of ectosomal origin but not derived from the exosome biogenesis pathway. 167k‐NVEPs demonstrated a significantly higher protein‐to‐lipid ratio and unique lipid composition, marked by cholesteryl ester enrichment. Raman spectroscopy revealed a similar trend of significantly high protein‐to‐lipid ratio in 167k‐NVEP compared to 100k‐sEV and super‐resolution microscopy showed strikingly stronger Lamp1 positivity. Summary/Conclusion : Our findings suggest that EPs >200 nm likely do not originate from the exosomal pathway. Elevated Lamp1 expression in 167k‐NVEPs indicates a lysosomal association, suggesting distinct functions and physiological roles from traditional EVs. Funding : EU's Horizon 2020 Research and Innovation Programme (739593), OTKA FK 147023, The Higher Education Excellence Program (FIKP) and the Therapeutic Thematic Programme (TKP2021‐EGA‐23), National Cardiovascular Laboratory Program (RRF‐2.3.121‐2022‐00003) and 2019‐2.1.7‐ERA‐NET‐2021‐00015, NVKP_16‐1‐2016‐0004 grant of the Hungarian National Research, NKFIH, VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, EKÖP‐2024‐237 and Stipendium Hungaricum Scholarship 2021.

Distinct

Inge Varik 1 , Katariina Johanna Saretok 1 , Kristine Rosenberg 1,2 , Ileana Quintero 3 , Maija Puhka 3 , Nataliia Volkova 1 , Aleksander Trošin 4 , Paolo Guazzi 5 , Agne Velthut‐Meikas 1 1 Department of Chemistry and Biotechnology, Tallinn University of Technology, Estonia; 2 Nova Vita Clinic AS, Estonia; 3 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland; 4 East‐Tallinn Central Hospital, Centre for Infertility Treatment, Estonia; 5 HansaBioMed Life Sciences Ltd, Estonia. Introduction : While several studies have isolated extracellular vesicles (EVs) from human follicular fluid (FF), research detailing the specific functions of FF EV subtypes remains limited. Understanding EV subtype‐specific roles could provide new insights into ovarian function. In this study, we isolated large (LEVs) and small EVs (SEVs) from human FF, characterized their short non‐coding RNA (ncRNA) composition, and assessed their impact on ncRNA expression in KGN cells. Methods : Ethical approval was obtained from the University of Tartu Research Ethics Committee, and written informed consent was obtained from all study participants. FF subtypes were purified by size exclusion chromatography, followed by tangential flow filtration (pore size 200 nm), and characterized according to the MISEV2023 guidelines. KGN cells were treated with 10 8 /mL LEVs or 10 9 /mL SEVs for 24 h. Small RNA‐seq libraries were prepared for EVs and EV‐treated KGN cells and sequenced with NovaSeq X. Unique molecular counts of miRNAs and piRNAs were used for differential expression (DE) analysis with DESeq2. Over‐representation analysis (ORA) was conducted with miEAA to identify gene ontology terms related to the DE genes. Results : Principal component analysis separated SEV and LEV samples along the PC1 score, indicating significant differences in their ncRNA content. We identified 46 DE miRNAs between SEVs and LEVs, with 43 upregulated in SEVs, and 3 in LEVs. Additionally, 45 DE piRNAs were detected, with 13 upregulated in SEVs and 32 in LEVs. ORA of DE miRNAs indicated enrichment in pathways related to cell cycle, TGF‐β signalling, DNA damage response, cellular senescence and oxidative stress. DE analysis between SEV‐ and LEV‐treated KGN cells identified 152 DE ncRNAs, including 84 DE miRNAs and 68 DE piRNAs. Similarly to DE EV miRNAs, ORA demonstrated the involvement of DE miRNAs in cell cycle, TGF‐ β signalling, and DNA damage response, but also SUMOylation and WNT signalling. Summary/Conclusion : This study highlights differences in short ncRNA composition and function of FF EV subtypes. Differential expression of miRNAs in SEVs and LEVs, particularly in pathways like cell cycle regulation, TGF‐β signalling, and oxidative stress, suggests SEVs and LEVs may play distinct roles in maintaining follicular health. Funding : The study was funded by the Estonian Research Council.

Efficacy

Hiroki Kaneta 1 , Tomoyuki Nakasa 2 , Yimiti Dilimulati 1 , Dan Moriwaki 1 , Shigeru Miyaki 1, 3 , Nobuo Adachi 1 1 Department of Orthopaedic Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University; 2 Department of Artificial Joints and Biomaterials, Graduate School of Biomedical and Health Sciences, Hiroshima University; 3 Medical Center for Translational and Clinical Research, Hiroshima University Hospital Introduction : Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint destruction due to inflammation. Despite advancements in pharmacotherapy, complications and inadequate disease control highlight the need for new treatments. Extracellular vesicles (EVs), including exosomes and microvesicles, play a vital role in intercellular communication, carrying proteins, miRNAs, and mRNAs that influence cell function. Plant‐derived EVs offer potential advantages like stability, cost‐effectiveness, and mass production capabilities. Ginger, known for its anti‐inflammatory effects, has demonstrated efficacy in RA by inhibiting the proliferation and migration of synovial fibroblasts. This study aimed to investigate the therapeutic potential of ginger‐derived EVs in RA treatment. Methods : Ginger‐derived EVs were isolated using ultracentrifugation and characterized for miRNA content. In vitro, the anti‐inflammatory effects were assessed on TNFα/IL‐1β‐stimulated RA synovial fibroblasts (RASFs) by measuring MMP3 and IL‐6 gene expression, cell proliferation (MTT assay), and migration (scratch assay). In vivo, collagen antibody‐induced arthritis (CAIA) was induced in DBA/1J mice, and the effect of orally administered ginger‐derived EVs was evaluated based on arthritis scores, histological assessments (HE, Safranin‐O, TRAP staining), and miRNA profiling. All animal experiments were approved by the relevant animal ethics committee. Statistical significance was set at p < 0.05. Results : Ginger‐derived EVs significantly suppressed MMP3 and IL‐6 expression, reduced RASF proliferation, and inhibited migration in vitro. In vivo, ginger‐derived EVs markedly reduced arthritis scores, decreased synovitis, inhibited cartilage degradation, and reduced osteoclast activity without adverse effects. Microarray analysis revealed that ginger‐derived EVs contained miRNAs identical to human miRNAs with reported anti‐inflammatory properties. Summary/Conclusion : This study provides the evidence of ginger‐derived EVs effectively suppressing inflammation and proliferation in RA models, highlighting their potential as a novel, cost‐effective, and scalable therapeutic approach. Our findings suggest that ginger‐derived EVs could represent a new avenue for RA treatment that minimizes adverse effects while leveraging natural anti‐inflammatory properties. Further studies are warranted to evaluate their long‐term stability and to optimize extraction methods for broader clinical application. Funding : The JCR Grant for Promoting Research for Early RA.

Enhanced

Presenter: Xuhuang Xiao Jinan University, Guangzhou, People's Republic of China Introduction : The clinical translation of plasma‐derived extracellular vesicle (pEV)—relevant achievements was challenged by the abundant proteins and non‐vesicular extracellular particles (NVEPs) leftovers during isolation. Among which, the overwhelming of low‐density lipoproteins (LDL) were the primary contaminant in EVs acquired by size‐based isolation. We have previously developed the dichotomic size‐exclusion chromatography (dSEC) that could simply and efficiently remove abundant proteins and high‐density lipoproteins from pEVs. To provide a rapid method for the clinical isolation of high‐quality pEVs, we aimed to improve dSEC to selectively remove LDL. Methods : Heparin‐conjugated agarose beads were employed to capture LDL and remove them from pEVs during dSEC isolations. The efficiency and specificity of the removal were parallelly estimated by nanoparticle tracking analysis (NTA), BCA, transmission electron microscopy (TEM), immunoblotting (IB), mass spectrometry (MS) and nanoflow cytometry (nFCM). Label‐free MS analysis was then performed to provide insights into the protein contents of the purified pEVs. Results : Heparin beads could significantly remove 62% of particles and 60% of proteins in the EV fraction acquired by dSEC through overnight incubation, resulting an elimination of ApoB (a marker of LDL) without affecting the signal of EV markers in IB. EV‐like cup‐shaped illusion could only be observed in the TEM images of purified EV isolates, but none were found in the leftover on the beads. By integrating these heparin beads into our well‐characterized dSEC columns (dSEC_HP), we could remove over 90% of NVEPs including LDL‐particles by a single elution. Label‐free MS quantification reported that compared to the original dSEC, EV isolates from dSEC_HP showed increases in all identified transmembrane and cytosolic EV protein markers listed in MISEV2023, while various apolipoproteins including ApoB‐100 were found to be significantly decreased. Such high‐quality pEV isolates were proved to be capable for immunofluorescent nFCM analysis. Summary/Conclusion : Lipoproteins in pEVs could be selectively removed or even eliminated by heparin‐conjugated agarose beads. Combining such beads with the dSEC, we have developed a simple and rapid isolation technique to acquire pEVs with low lipoprotein contaminants. Such a major‐equipment‐free method could be easily performed in clinical settings, thus could potentially facilitate the clinical translation of EV‐relevant achievements.

Exosomal

Min‐Jung Ma Kyungpook National University, Daegu, Republic of Korea Introduction : Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal disorder characterized by persistent inflammation of the digestive tract, significantly affecting patients' quality of life. IBD is also associated with psychological stress, creating a bidirectional relationship between disease progression and stress responses. Despite advances in understanding its pathogenesis, the exact aetiology remains unclear, and current diagnostic methods rely on invasive procedures such as endoscopy and biopsy, necessitating the development of alternative biomarker‐based approaches. Exosomes, known as small extracellular vesicles carrying bioactive molecules, have gained attention as potential biomarker sources for various diseases. Methods : In this study, serum‐derived exosomes were analysed to identify IBD related stress biomarkers using a mouse model. Exosomes were isolated from serum samples obtained from a mouse model induced with IBD, and exosome characterization was performed using NTA, TEM, and flow cytometry analysis. For flow cytometry, exosomes were labelled with antibodies targeting CD9, CD63, and CD81. Protein profiling of exosomes from IBD and control mice was subsequently performed using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Results : Our findings revealed distinct protein expression patterns in the exosomes of IBD mice model compared to controls. Notably, proteins associated with inflammation, gut barrier integrity, and stress pathways were significantly altered in IBD samples. These results suggest that exosome analysis could provide valuable insights into both the physiological and psychological dimensions of IBD. Summary/Conclusion : This study highlights the potential of exosome‐based biomarkers discovery in advancing the understanding of IBD pathogenesis and stress‐related mechanisms, offering new avenues for diagnosis and disease management.

Exosomes

Presenter: Serena Mastantuono University of Udine, Italy Introduction : Glioblastoma multiforme (GBM) is the most aggressive primary brain cancer characterized by an extensive intra‐tumour and inter‐patient heterogeneity. While glioma stem cells (GSCs) are considered responsible for GBM recurrences, the crosstalk between tumour cells and tumour microenvironment (TME) strongly influences tumour behaviour. Glioma associated stem cells (GASCs) are representative of the TME since they are not tumourigenic, but, in vitro, they increase the biological aggressiveness of tumour cells through the release of exosomes. Here we aimed at evaluating the efficacy of selected drugs, acting on the TME, to blunt its tumour supporting ability, taking advantage of a patient‐derived in vitro model of TME, represented by GASCs and GASC‐derived exosomes (GDEs). Methods : We selected 3 drugs: bacitracin, a5b1 integrin antibody and ciclopirox, known to inhibit migration and invasion of tumour cells. GASCs were treated first to select the maximum dose and the timing of treatments, and then to investigate a possible reduction in their activated phenotype evaluating proliferation, motility and anchorage‐independent growth, in vitro. GDEs were isolated from the collected supernatants of treated and not treated GASC, using ExoQuick‐TC precipitation solution and their concentration and particle size were measured by Nanosight. Moreover, U87 MG and GSCs were conditioned with GDEs, treated or not, with different drugs, to evaluate their internalization and their effects on proliferation, motility, anchorage‐independent growth, migration and invasion of tumour cells. Finally, GDEs miRNAs were extracted and sequenced to identify modifications in their contents, after drugs treatments. Results : Drugs were able to reduce the activated phenotype of GASCs, inhibiting cell proliferation, motility and anchorage‐independent growth. GDEs were efficiently internalized into U87 and GSC being able to inhibit their proliferation, motility, migration, invasion and anchorage‐independent growth. Moreover, we found that drugs were able to modify the GDEs cargo, upregulating some oncosoppresors miRNA such as microRNA‐100‐5p, microRNA‐21‐5p and microRNA‐34a‐5p. Summary/Conclusion : We confirmed the important supporting role, mediated by GDEs, of the TME on tumour cells behaviour. In addition, we demonstrated that the 3 selected drugs can efficiently revert its activated phenotype and change GDEs cargo reducing their tumour‐supporting function. Funding : PRIN ARREST CUP G53D23003680006

Fasciola

Christian M. Sánchez‐López 1,2 , Aránzazu González‐Arce 1 , Mari Cruz Manzaneque‐López 3 , Marta González‐Warleta 4 , Jose Antonio Castro‐Hermida 4 , Mercedes Mezo 4 , Roberta Gasparro 5 , Carla Soler 2,6 , Alba Cortés 1 , Dolores Bernal 7 , Pedro Pérez‐Bermúdez 3 , Antonio Marcilla 1,2 1 Àrea de Parasitologia, Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot (Valencia), Spain. 2 Joint Research Unit on Endocrinology, Nutrition and Clinical Dietetics, Health Research IIS La Fe‐Universitat de València, Valencia, Spain 3 Departament de Biologia Vegetal, Universitat de València, Burjassot (Valencia), Spain. 4 Labouratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, AGACAL‐Xunta de Galicia, Abegondo, A Coruña, Spain 5 Department of Biomedicine, Neurosciences, and Advanced Diagnostics (Bi.N.D), Section of Biology, Via Divisi 83, University of Palermo, Palermo, Italy. 6 Instituto de Ciencia de los Materiales, Parque Científico, Universitat de València, Burjassot, Valencia, Spain. 7 Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Burjassot, Valencia, Spain Introduction : Fasciola hepatica infection, a major concern in European dairy cattle, is known to impact milk yield and composition, although studies addressing these effects are limited. Among milk components, milk‐derived extracellular vesicles (mEVs) have garnered attention due to their roles in immune regulation, antioxidant activity or pathophysiological processes. This study explores the presence of F. hepatica EVs (FhEVs) in milk and examines how F. hepatica infection influences the abundance and proteomic content of mEVs. Methods : Milk samples were collected from dairy farms in Galicia, Spain. The presence of specific antibodies against F. hepatica infection was assessed using the MM3‐SERO ELISA test. mEVs were isolated through a combination of differential centrifugation, EDTA treatment, ultracentrifugation and size exclusion chromatography (SEC). mEVs were characterized through NTA, TEM and LC‐MS/MS and label free semi‐quantitative proteomics. Results : mEVs were successfully isolated from the milk of both infected and uninfected cows. Notably, the quantity of mEVs recovered was significantly higher in samples from infected animals compared to uninfected ones, while both mEVs preparations exhibited similar particle/protein ratios. A total of 338 proteins were identified in mEVs through LC‐MS/MS, with 46 proteins showing significantly lower levels, and 30 proteins displaying higher levels in mEVs infected animals compared to those from uninfected animals. No proteins from F. hepatica were detected. The absence of FhEVs was confirmed by ELISA, using PAb against FhEVs. Summary/Conclusion : F. hepatica infection significantly alters the abundance and proteomic composition of mEVs in dairy cattle, indicating their potential as biomarkers for infection status and milk quality. While infection affects the host response, the parasite may not shed EVs into the milk. Further research is needed to clarify the functional implications of these mEV changes on the health and productivity of infected cows. Funding : Work supported by Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Spain (Grants PID2019‐105713GB‐I00 and PID2023‐146116NB‐I00 and ERDF/EU), and Conselleria d'Educació, Cultura, Universitats i Ocupació, Generalitat Valenciana, Valencia, Spain (Grants PROMETEO/2020/071 and CIPROM/2023/54). C.M.S‐L is the recipient of a Postdoctoral fellowship funded by AEI /10.13039/501100011033.

Improved

Beatrice Crestani 1 , Noemi Torriero 1 , Chiara Maceri 1 , Valentina Moccia 1,2 , Valentina Zappulli 2 , Davide Ferraro 1 1 Department of Physics and Astronomy, University of Padova, Padova, Italy; 2 Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro, Italy. Introduction : Microfluidics and droplet microfluidics permitted significant steps forward in biomedical diagnosis, allowing high throughput analysis and improved sensitivity. Droplet microfluidics, consisting of the generation of emulsions where aqueous droplets are suspended in oil, is currently applied in ddPCR and single‐cell sequencing. Here, we propose to use droplet microfluidics for the isolation of extracellular vesicles (EVs), mediated by immunocapturing on magnetic beads. Monophasic microfluidic has been employed for this scope with a low capture efficiency, mainly due to lack of mixing. Differently, droplets introduce recirculation zones promoting mixing between beads and EVs. In this work, we optimize the hydrodynamics of microparticles within droplets in microchannels to improve the EV isolation from plasma. Methods : We analysed the behaviour of beads denser than the medium (2‐fold) under various experimental conditions (droplet speed, droplet volumes, channel diameters). We applied the best mixing conditions to optimize reaction time in a hydrogen peroxidase assay, and then to improve the EV isolation from frozen raw plasma. For the latter, experiments were carried out both by the microfluidic device and by an in‐batch protocol, varying the incubation time (1–4 h). Validation techniques were performed, including nanoparticle tracking analysis (NTA), BCA assay, and western blotting (WB), according to the MISEV‐2023 standards. Results : From the microfluidic point of view, we observe that the redispersion of beads inside droplets is more homogeneous when the droplet aspect ratio is small (length‐width ratio between 1 and 3) and when they flow slowly ( 10‐fold higher WB signal intensities for EV markers CD9, CD81, and CD29 than in‐batch. The optimized droplet size and flow rates allowed to shorten the bead incubation time, achieving similar WB signals across 2‐, 3‐, 4‐h incubations. Summary/Conclusion : This microfluidic platform improves EV isolation, addressing limitations of conventional approaches. Enhancing EV recovery from human plasma, droplet microfluidics offers a significant advance in liquid biopsy, showing a way for accessible, reproducible clinical diagnostics. Funding : This research was funded by “Next Generation EU” Missione‐4 Componente‐1, for projects no. P2022J2ZC2 and 2022YYLSJ2.

Injected

André Görgens 1 , Svetlana Pavlova 1 , Doste Mamand 1 , Daniel W. Hagey 1 , Yesid Velasquez 1 , Wenyi Zheng 1 , Guannan Zhou 1 , Risul Amin 1 , Antje M. Zickler 1 , Manuela Gustafsson 1 , Samir EL Andaloussi 1 1 Karolinska University, Sweden Introduction : The application of extracellular vesicles (EVs) for targeted delivery of drugs, proteins, and RNA to specific cell types or organs is a promising approach in molecular therapy. However, in order to achieve efficient and targeted delivery, it is crucial to improve our understanding of the fate of injected EVs in terms of tissue and cellular biodistribution. Data by us and others has demonstrated that EVs are rapidly cleared from the blood circulation to different organs following administration, with large fractions ending up in the liver and lungs within min after injection. In this study, we investigated the key steps and kinetics of this clearance process and particularly focused on studying how intravenously injected EVs would interact with circulating blood cells shortly after injection. Methods : For this study, we used thoroughly characterized EV preparations from different stable producer cell lines, and EVs were prepared with different established EV isolation methods. Following the injection of fluorescently tagged EVs into mice, we utilized high‐sensitivity multicolour imaging flow cytometry to quantify the binding of EVs to different blood cell types in the circulation and their tissue resolution in terms of target cell type in the spleen and liver. Results were validated by single‐cell RNAseq of EV‐receiving cells in mouse models and by scanning electron microscopy (EM). Results : We demonstrate that a significant proportion of injected EVs binds to blood cells within min after intravenous injection. EVs were particularly found to directly bind to erythrocytes and platelets in the circulation, which is highly relevant since those two blood cell types account for > 99% of all blood cells and have been largely neglected in this context up to now. We further show that EVs ‘hitchhike’ on both Erythrocytes and Platelets to different organs, where they are cleared by macrophages. Summary/Conclusion : In summary, we report a highly relevant yet largely overlooked mechanism that significantly contributes to clearance of EVs bound to Erythrocytes and Platelets from the blood circulation to distant organs.

Insights

Presenter: Kübra Bekar ULiege (GIGA Research Center), Liège, Belgium Introduction : Breast cancer (BC) is one of the most common cancers worldwide. While it can be treated, metastatic BC remains incurable. Prior research from our group demonstrated that endothelial cell‐derived extracellular vesicles (EVs) enriched in miR‐142‐5p, miR‐183‐5p, and miR‐222‐3p (i.e., ‘miR‐TAM’) participate in the polarization of macrophages towards an M2‐like phenotype, promoting tumour growth in a BC mouse model (Njock et al. 2022, JEV). However, the impact of miR‐TAM in metastasis remains unexplored. A crucial step before metastasis is the formation of a pre‐metastatic niche (PMN), which is notably initiated by EVs. This study aims to unravel the role of miR‐TAM in PMN formation and metastasis both in vitro and in vivo. Methods : Mouse endothelial cell‐derived EVs were isolated using a differential ultracentrifugation method and characterized through western blotting and nanoparticle tracking analysis (NTA). EV uptake was assessed in macrophages (i.e., RAW264.7) and fibroblasts (i.e., MEF; mouse embryonic fibroblasts) via confocal microscopy. Subsequently, changes in PMN‐related gene expression profiles following miR‐TAM‐enriched EV treatment of these cells were assessed by quantitative PCR. To further investigate the impact of miR‐TAM in 4T1 tumour‐bearing mice, peritumoural administration of miR‐TAM‐ or control microRNA‐enriched EVs was performed, followed by flow cytometry and immunohistochemical analysis. Results : Notably, isolated EVs were enriched for EV markers CD63, CD81, and syntenin but lacked the negative marker cytochrome C. Furthermore, endothelial EVs exhibited a major peak in the size range of small EVs (< 200 nm) and were successfully internalized by recipient cells. Interestingly, treatment with miR‐TAM‐enriched EVs upregulated the expression of pro‐tumourigenic genes Csf3, Cxcl1, Col3a1, Il‐1β, and Ccl3 in macrophages and fibroblasts, which are two major cell types in PMN formation. Strikingly, repeated peritumoural administration of miR‐TAM‐enriched EVs resulted in a significant reduction of CD4+ and CD8+ T cells in the lungs of 4T1 tumour‐bearing mice, establishing an immunosuppressive environment that favours metastatic outgrowth. Summary/Conclusion : Here, we elucidate a previously unrecognized role of miR‐TAM‐enriched endothelial EVs in advancing metastasis both in vitro and in vivo, identifying them as potential modulators of PMN formation. These preliminary findings highlight promising avenues for novel BC therapeutic strategies.

Isev2025

IOC Chairs: Eva Rohde (Austria), Eva‐Maria Krämer‐Albers (Germany) IOC Members: An Hendrix (Belgium), Andreas Moeller (China), Bo Li (China), Edit Buzas (Hungary), Johannes Grillari (Austria), Lucia Languino (USA), Mario Gimona (Austria), Sun Young Lee (USA), Wolf Holnthoner (Austria), Cristóbal Cerda‐Troncoso (Belgium), Madhusudhan Bobbili (Austria).

Lgals3Bp

Presenter: Akane Kanamori Osaka University, Osaka, Japan Introduction : The Epstein‐Barr virus (EBV) causes several poor prognostic cancers, including lymphoma. Recently, we revealed that tumour‐associated macrophages (TAM) express high levels of secretory phospholipase A2‐X (sPLA2‐X) in EBV‐positive lymphoma. This enzyme modifies the function of tumour‐derived extracellular vesicles (EVs) by hydrolysing their membrane phospholipids. The modified EVs are then incorporated by macrophages, facilitating TAM polarization and activating the cell surface GPCR, promoting tumourigenesis (Kudo et al. 2022, Cell Metabolism). Methods : To analyse the factors involved in sPLA2‐X activity, proteomic analysis was performed. sPLA2‐X activities were then analysed by LC‐MS/MS to detect degradation products. The roles of LGALS3BP were assessed using western blot and immunostaining. Results : Interestingly, patient‐derived EVs were much more susceptible to hydrolysis than those from healthy individuals despite having a similar composition of phospholipid. Proteomic analysis of the EVs identified LGALS3BP as a patient‐specific component. LGALS3BP has been reported to be abundant in the serum of several cancer patients; however, the functions were still elusive. LGALS3BP was mainly secreted by TAM, as was sPLA2‐X, and exerted as a ‘protein corona’ that binds to EV membranes rather than a constituent of them. LGALS3BP enhanced the hydrolytic activity of sPLA2‐X by directly binding to the EV surface, possibly acting as a scaffold between them. Although LGALS3BP alone increased the uptake of EVs by target cells, the synergistic activity of LGALS3BP and sPLA2‐X greatly enhanced this uptake, which depended on Galectin‐3 (LGALS3). Finally, the EVs cotreated with LGALS3BP and sPLA2‐X remarkably accelerated lymphomagenesis in vivo. Summary/Conclusion : These results suggest that LGALS3BP contributes to lymphomagenesis by promoting the hydrolysis of EV membranes through sPLA2‐X activation and enhancing EV uptake to target cells mediated by LGALS3. This results in accelerated formation of the tumour microenvironment through malignant communication between tumour cells and TAM.

Lifespan

Presenter: Alessia Brancolini Evercyte GmbH, Austria Introduction : Mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) have shown promise as safe and effective therapeutic agents, exhibiting regenerative, immunomodulatory, and anti‐inflammatory properties comparable to those attributed to parent cells. Donor heterogeneities, limited replicative life span, and alterations in cellular phenotype throughout in vitro cultivation, however, continue to be significant obstacles for adequate cell expansion required for scalable EV manufacturing. This study investigates the use of hTERT immortalized (‘telomerized’) Wharton's Jelly (WJ)‐derived MSCs as source for efficient, standardized, reliable MSC‐EVs production by comparing primary WJ‐MSCs and the secreted EVs to their telomerized counterparts. Methods : Primary WJ‐MSCs (WJ‐MSC273) were immortalised by non‐viral overexpression of the catalytic subunit of human telomerase (hTERT). hTERT immortalized cells (WJ‐MSC/TERT273) and primary cells were compared in terms of morphology, cellular doubling time, surface/senescence marker expression, and tri‐lineage differentiation ability. Primary and telomerised WJ‐MSCs‐derived EVs were enriched from xeno ‐free conditioned medium by tangential flow filtration, and characterized for morphology, size, EV‐marker expression and miRNA cargo composition. The biological activity of primary and telomerised WJ‐MSC‐derived EVs was assessed by anti‐inflammatory, anti‐fibrosis and wound healing in vitro assays. Primary and telomerized WJ‐MSCs, as well as secreted EVs were characterized for their safety profile. Results : hTERT overexpression enabled stable and extended in vitro proliferation of WJ‐MSC/TERT273 cells, while ensuring cellular morphology and doubling time comparable to their primary counterparts. Telomerized WJ‐MSCs did not show increased SA‐ß‐gal activity at late population doublings and retained the canonical surface marker expression and tri‐lineage differentiation potential of WJ‐MSC273 cells. Furthermore, the immortalization process did not impact particle release, EV characteristics or biological activities. Notably, no increase of hTERT DNA, mRNA or activity was measurable in WJ‐MSC/TERT273‐derived EVs, and telomerized WJ‐MSCs showed no in vitro tumourigenic potential. Summary/Conclusion : MSC immortalization by ectopic expression of hTERT generates cell lines capable of continuous MSC‐EV production without compromising neither key cellular and EV functionalities nor their safety profiles. This approach suggests that telomerization of human cells from single donors is a promising strategy to overcome some of the limitations of using primary MSCs as EV cell factories, while supporting stable EV production.

Magnetic

Daniela Dzubinska, Jana Frydlova, Martin Vokurka, Petr Prikryl Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Prague, Czech Republic Introduction : Extracellular vesicles (EVs) are crucial for intercellular communication, with emerging diagnostic and therapeutic potential in cardiovascular and metabolic disorders. However, EV isolation remains challenging due to their small size and the complexity of biological samples. Traditional isolation methods like density gradient ultracentrifugation (DGUC) and size‐exclusion chromatography (SEC) often require post‐isolation washing and concentration steps that risk EV sample loss and their functional protein corona or can disrupt EV integrity. We adapted and optimized the single‐pot solid‐phase‐enhanced sample preparation (SP3) method using paramagnetic bead‐assisted precipitation to simplify and accelerate EV isolation for proteomic analysis by mass spectrometry. Methods : EV proteins were enriched and lysed on beads and solubilized proteins were recaptured, trypsin digested on the same beads and UHPLC‐MS were used for detection and identification EV proteome. The SP3‐EV method applicability was tested on various biological samples, including human plasma, urine, pericardial fluid, cell culture media, and explant culture media of adipose and pancreatic tissue of cardiac/diabetic patients and prediabetic cell culture or mouse models. Ethical approval was obtained, and informed consent was received from all participants. Results : The SP3‐EV method efficiently concentrated and purified EV proteins from density gradient ultracentrifugation fractions and is capable of eliminating contaminating iodixanol so that it would not interfere with proteomic mass spectrometry. This was documented by gel electrophoresis, western blot analysis, NTA, MALDI‐TOF etc. SP3‐EV method was compared to standard DGUC approach using qualitative mass spectrometry analysis. This approach can replace the washing and concentration steps by directly preconcentrating EV proteins from diluted samples, such as DGUC iodixanol or SEC fractions. Summary/Conclusion : Our SP3‐EV approach offers a versatile, rapid, and efficient possibility of EV protein enrichment for proteomic analysis, with broad‐ranging potential for biomedical and clinical research. In combination with DGUC, SP3‐EV preserves protein corona and EV integrity and facilitates high‐purity EV isolation from complex samples, so SP3‐EV enables precise profiling of EV subpopulations, which is crucial for understanding functional heterogeneity. Its compatibility with automation and high‐throughput workflows aligns with emerging trends in clinical EV research, enhancing reproducibility and reducing hands‐on time. Funding : The project Programme EXCELES LX22NPO5104‐Funded by the European Union‐Next Generation EU.

Microrna

Presenter: Yusei Fujioka Kitasato University, Japan Introduction : Extracellular vesicles (EV) mediate cell‐to‐cell communication via transferring various molecules, such as microRNA (miRNA). We previously demonstrated that plasma‐derived EV from spontaneously hypertensive rats (SHR), an animal model of human essential hypertension, increased systemic blood pressure in normotensive Wistar Kyoto rats (WKY). In the present study, we performed a comprehensive analysis of miRNA contained in plasma‐derived EV from SHR and WKY to identify miRNA and their target genes that regulate the pathogenesis of hypertension. Methods : EV were isolated from plasma of 9‐week‐old male WKY or SHR using the size‐exclusion chromatography method. After miRNA was extracted from isolated EV, miRNA‐sequence was conducted. We then conducted mRNA expression analysis in the isolated tissues of SHR and WKY by using quantitative real‐time PCR. Results : In plasma‐derived EV from SHR, we found that three miRNAs were significantly upregulated and six miRNAs were significantly downregulated compared with those from WKY. Among the target genes of the upregulated miRNAs, the mRNA expression of several genes was downregulated in the isolated brain, heart, and kidney of SHR. Summary/Conclusion : We revealed that plasma‐derived EV from SHR had different profiles of miRNAs compared with those of WKY. We also found that the mRNA expression of the target genes of miRNAs contained in plasma‐derived EV from SHR was downregulated in the tissues regulating systemic blood pressure. Additional research may contribute to identifying novel pathological mechanisms underlying essential hypertension.

Mirnomic

Beatrice Spokeviciute 1 , Fabrizio Buffolo 1 , Maddalena Arigoni 2 , Raffaele Calogero 2 , Ilaria Barchetta 3 ,Salvatore Cocola 3 , Gisella Cavallo 3 , Maria Felice Brizzi 1 1 Department of Medical Sciences, University of Turin, Italy; 2 Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy; 3 Department of Experimental Medicine, University of Sapienza, Rome, Italy Introduction : SGLT2 inhibitors (SGLT2i) are oral medications that lower blood glucose in type 2 diabetes mellitus (T2DM). In addition to their glucose‐lowering effects, SGLT2i improve cardiovascular and renal outcomes by reducing HbA1c and blood pressure, decreasing inflammation, and improving kidney function, although the underlying mechanisms remain unclear. Extracellular vesicles (EVs) offer a non‐invasive way to monitor cellular responses to SGLT2i, as they carry bioactive molecules like miRNAs and reflect the physiological state of their origin cells. This study investigates renoprotective mechanisms of SGLT2i by analysing EVs from urine in T2DM patients treated with dapagliflozin. Methods : EVs were isolated from patient urine samples via ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and western blotting for CD63 and TSG101 markers. miRNA expression profiles were assessed via next‐generation sequencing. Differentially expressed miRNAs were annotated using miRBase and linked to target genes through miRTarBase, followed by pathway analysis with Enrichr and network clustering with Gephi. The MACSPlex Assay was performed to identify changes of EV surface proteins following treatment. Results : NTA confirmed a homogeneous EV population (100–150 nm), while Western blot verified EV identity. Sequencing identified 3827 miRNAs, with 16 miRNAs differentially expressed between baseline and 7‐month follow‐up samples. Pathway analysis linked these miRNAs to NLRP3 inflammasome regulation, platelet calcium homeostasis, and ion transport, suggesting reduced inflammation and stress responses. EV protein analysis revealed increased CD326 (EpCAM), indicating renal epithelial regeneration, and decreased CD146, associated with reduced kidney injury. Summary/Conclusion : Urine EVs from SGLT2i‐treated patients showed shifts in miRNA expression and protein markers, suggesting reduced inflammation, cellular stress, and kidney injury alongside enhanced epithelial repair. Our findings suggest that EVs may serve as potential biomarkers for monitoring therapeutic response in T2DM and provide the basis to uncover the biological process associated with SGLT2i‐induced renal protection. Funding : Supported by the University of Turin, BRIM_RILO‐19_01.

Monocyte

Presenter: Nur Azira Mohd Noor, Universiti Sains Malaysia, Malaysia Introduction : Monocytes and macrophages play pivotal roles in immune activity during inflammation associated with osteoarthritis (OA). An imbalanced polarisation of M1 and M2 macrophages has been implicated in exacerbating OA. However, the interaction of monocytes with exosomes in OA conditions remains unexplored. This study intended to investigate the immunomodulatory role of exosomes derived from synovial fluid of OA patients on monocyte differentiation and function. Methods : Exosomes were isolated from the synovial fluid of OA patients using ultracentrifugation and characterised using nanoparticle tracking analysis, scanning electron microscopy, and Western blotting. To assess the exosomal influence on monocyte biology, monocytes were cultured with exosomes prior to differentiation into M1 macrophages, M2 macrophages, and monocyte‐derived dendritic cells (MoDC). The phenotypic profiles and gene expressions of all monocyte‐derived cells were evaluated using flow cytometry and real‐time PCR, respectively. Functional validation included assessing phagocytosis activity, and the ability to promote the proliferation of naïve CD4+ T cells was determined by flow cytometry. Results : Exosomes were found in a round spherical shape with sizes ranging from 30 to 150 nm and enriched with CD9, CD63, CD81, and HSP70. The presence of exosomes in monocyte culture induced monocyte differentiation into proinflammatory profiles characterized by increased CD86 expression and decreased CD206 expression on both M1 and M2 macrophages. The expression of CD80 and HLA‐DR was increased on MoDC. Gene expression analyses revealed significant upregulation of IL1A and downregulation of CD64 and AMPK on M1 macrophages. Similarly, IL1A was significantly upregulated, while CD64 was downregulated in M2 macrophages. In MoDC, HLA‐DR, MMP7, and AMPK were upregulated. The phagocytosis activity of M2 macrophages and MoDC increased in the presence of exosomes. The proliferation of naïve CD4+ T cells was markedly impaired, indicating dampened MoDC‐naïve CD4+ T cell interaction. Summary/Conclusion : These findings suggest the immunomodulatory role of synovial fluid‐derived exosomes on monocytes, thus potentially contributing to the alteration of monocyte biology and influencing the inflammatory process in OA. Funding : Grant number: FRGS/1/2020/SKK06/USM/03/6.

Multiple

Kieran Brennan 1 , Katrine F Iversen 2 , Alfonso Blanco‐Fernández 3 , Thomas Lund 4 , Torben Plesner 2 , Margaret M Mc Gee 1 1 School of Biomolecular & Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Dublin, Ireland; 2 Department of Internal Medicine, Section of Hematology, Lillebaelt Hospital, and Institute of Regional Health Science, University Hospital of Southern Denmark, Vejle, Denmark; 3 Flow Cytometry Core Technology, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Belfield, Dublin, Ireland; 4 Department of Hematology, Odense University Hospital, Odense, Denmark Introduction : Our previously published data revealed that peripheral blood plasma (PB) EVs, isolated by differential centrifugation and density gradient ultracentrifugation, from 57 multiple myeloma (MM) patients treated with daratumumab (DARA) contain elevated CD55, CD59 and CD147 relative to healthy PB EVs ( n = 12), with elevated PD‐L1 associated with patient response to DARA (Brennan et al. 2022, PMID: 36359760). The aim of this study was to determine if a mass spectrometry could identify additional proteins altered in these MM patients and if these proteins could be combined with the proteins from our previous study in order to generate two multiple myeloma plasma EV protein signatures for 1, detecting Myeloma, and 2 monitoring patient response to Daratumumab. Methods : A shortlist of 8 additional proteins upregulated in MM patients or associated with patient response to DARA were identified by mass spectrometry and validated by flow cytometry analysis. Results : Flow cytometry analysis revealed that all 8 proteins were present in all EV samples with 3 of the 8 proteins being significantly elevated in MM PB EVs relative to healthy PB EVs, while 2 of the 8 proteins were significantly decreased. Since the original study was performed, there is now 2+ years additional follow‐up on these patients, which allowed these patients to be subdivided into long‐term responders (> 2 years), short‐term responders (6–24 months) and non‐responder (< 6 months). Three proteins were significantly higher in PB EVs of patients with a long‐term response to DARA compared to the PB EVs of non‐responders and short‐term responders. PCA analysis revealed a distinct separation between healthy PB EVs from MM PB EVs, as well as separating long‐term responders from both short‐term responders and non‐responders. Multivariate ROC curves revealed that the MM PB EV signature has an area under curve (AUC) of 0.92 (Sens: 0.86/Spec: 0.92), while a long‐term response signature has an AUC of 0.733 (Sens: 0.80/Spec: 0.91). Summary/Conclusion : In conclusion we identified two EV signatures that may have potential as a non‐invasive liquid biopsy to complement or replace invasive Bone Marrow sampling for monitoring patient response to DARA. Funding : This study was supported by Research Ireland RD&I Fellowship (23/IRDIFB/12112) and the TwinFlag consortium (HORIZON‐WIDERA‐2021‐ACCESS‐03‐01).

Neuronal

Presenter: Zhixin Ma Sun Yat‐sen University, China Introduction : Alzheimer's disease (AD) is a progressive neurodegenerative disorder that accounts for the majority of dementia cases. Synaptic loss is a key factor driving cognitive dysfunction in AD. The APOE gene is strongly associated with increased AD risk. Synapse formation relies on filamentous actin (F‐actin) polymerization signalling, but it is unclear whether APOE modulates this process in AD. Extracellular vesicles (EVs) mediate inter‐neuronal communication and may play a crucial role in AD progression. However, the impact of neuronal EVs on synapses in the AD brain remains poorly understood. Methods : WT and APP/PS1 (AD model) primary neuronee‐derived EVs were isolated by multi‐step ultracentrifugation, termed WTNEVs and APPNEVs. Nanoparticle flow cytometry, electron microscopy, and Western blot confirmed EV purity and characteristics. Proteomics and bioinformatics identified proteins differentially expressed in APPNEVs. Synapse formation and the APOE‐Rac1‐N‐WASP‐Arp2/3 signalling pathway were analysed using Western blotting and super‐resolution microscopy. Stereotaxic injections of EVs into mouse brains, followed by fluorescence staining, enabled in vivo analysis of synapse formation. Results : Compared to WTNEVs, APPNEVs were released at higher concentrations. APPNEVs reduced PSD95 and synaptophysin expression, significantly decreasing the number of synapses, particularly mature synapses, in neuronees. Proteomic analysis revealed elevated levels of APOE protein in APPNEVs, suggesting its involvement in the synaptogenesis signalling pathway. Further investigation demonstrated that APPNEVs, through APOE cargo, downregulated neuronal GTP‐Rac1 levels, attenuated N‐WASP‐mediated phosphorylation of Arp2, and inhibited Arp2/3 complex‐driven F‐actin branching, thereby impairing synapse formation. In vivo experiments confirmed that APPNEVs suppressed synapse formation and downregulated PSD95 and synaptophysin expression. Additionally, inhibiting APOE function prevented APPNEVs from downregulating Rac1‐N‐WASP‐Arp2/3 signalling or impairing synapse formation, both in vitro and in vivo. Summary/Conclusion : EVs released by APP/PS1 neuronees carry APOE to recipient healthy neuronees, where they inhibit synapse formation by downregulating Rac1‐N‐WASP‐Arp2/3‐mediated F‐actin polymerization. This disruption exacerbates synaptic deficits and accelerates AD progression. Funding : National Natural Science Foundation of China (32200638); Basic and Applied Basic Research Fund of Guangdong Province (2021A1515110512 and 2023A1515010090); Shenzhen Science and Technology Innovation Program (JCYJ20230807110316034 and JCYJ20210324134612035); Research Start‐up Fund of the Seventh Affiliated Hospital, Sun Yat‐sen University (ZSQYBRJH0021).

Overcome

Sergio Lucio‐Gallego 1 , Rocío Mato‐Basalo 1 , Lama Berjawi 1 , Nerea Lago‐Baameiro 2 , María Pardo 2 , Mónica Carrera 3 , Concha Gil 4 , Lola Gutiérrez 4 , Jesús Mateos 5 , María C. Arufe 1 , Juan A. Fafián‐Laboura 1 1 Therapy Cellular & Medicine Regenerative Group, University of A Coruña (UDC), Interdisciplinary Center for Chemistry and Biology (CICA), INIBIC—University Hospital Complex A Coruña (CHUAC), A Coruña, Spain; 2 Obesidomics Group, Endocrinology area, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain; 3 Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain; 4 Proteomics Facility‐Complutense University and Scientific Park Foundation of Madrid, Madrid, Spain; 5 Clinic Pharmacology group (FarmaCHUS). Health Research Center of Santiago de Compostela (IDIS). University Hospital Complex of Santiago de Compostela (CHUS). Santiago de Compostela, Spain Introduction : Cells secretome is composed of small extracellular vesicles (sEV) among other secreted molecules such as interleukins, cytokines or chemokines that can modulate the cellular microenvironment. Senescent cells, characterized by being less proliferative, acquire a specific secretome known as SASP (senescence associated secretory phenotype). This SASP leads the cellular microenvironment to a more pro‐inflammatory one and induces senescence in the neighbouring cells in a paracrine and autocrine way. With time, senescent cells accumulate in the tissues, triggering age‐related diseases. In this study, we focused on paracrine senescence transmission mediated by sEV. Our objective is to avoid the transmission of senescence by modulating the secretion of the sEV. Methods : We reduced the levels of RAB27A, a protein implicated in sEV biogenesis, in human mesenchymal stem cells using CRISPR/Cas9 technology. Isolated sEV by ultracentrifugation from senescent and non‐senescent cells were used to treat control cells and RAB27A KO cells. Treated cells were analysed by proliferation and β‐galactosidase assays to assure the senescent phenotype. We also performed a shotgun proteomic study in the recipient cells, identifying interesting proteins. Results : The reduction in RAB27A protein accomplished a reduction in the senescence phenotype of recipient cells. The proteomic analysis shows upregulated ORAI1 and COG4 and downregulated LAPTM4A, proteins associated with the endomembrane system, in RAB27A KO cells treated with senescent sEV. Summary/Conclusion : We developed a model of paracrine senescent transmission in which RAB27A inhibition prevents the spreading of senescence. We further see by the proteomic study that proteins related to the endomembrane system, such as ORAI1, LAPM4A and COG4, could have a role in the process of paracrine senescence transmission and may be possible targets for the development of drugs against SASP and senescence. Funding : Xunta de Galicia, grant number ED481D‐2021‐020, Ministerio de Ciencia e Innovación (RYC2021‐032567‐I), funded by MCIN/AEI/10.13039/501100011033 and from the European Union «NextGenerationEU»/PRTR», InTalent program from UDC‐Inditex for the research grant and Project PI23/01347, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union. PI20/00497, funded by Instituto de Salud Carlos III (ISCIII) and co‐funded by the European Union. Xunta de Galicia (ED431F 2023/30). Transference Project financed by FINIBIC (RESISFERRO and ENDOPROT, respectively).

Particle

Presenter: Roman I. I. Koning Leiden University Medical Center, Leiden, The Netherlands Introduction : One of the key characteristics of extracellular vesicles (EVs) is the presence of a lipid bilayer. Visualization of the lipid bilayer allows positive identification of EVs, distinguishing EVs from non‐EV particles. Cryo‐electron microscopy (cryo‐EM) is able to directly visualize lipid bilayers. Additionally, cryo‐EM can detect non‐EV particles, such as lipoproteins, lipid droplets and protein aggregates. The use of cryo‐EM is, however, limited by low throughput and complex image analysis. Therefore, we developed a workflow combining automated cryo‐EM image acquisition and supervised machine learning (sML)‐assisted detection of EVs and other particles, enabling reproducible sample imaging and comprehensive particle analysis. Methods : EVs were purified from MDA‐MB‐231 cells using a combination of centrifugation, ultrafiltration and size exclusion chromatography. Samples were prepared for cryo‐EM by plunge‐freezing and imaged using a cryo‐electron microscope on a direct electron detector. Automated imaging was performed using EPU software, which enabled reproducible acquisition. sML and size measurements were performed using a custom web‐interface, Keras, TensorFlow, FIJI and the Hough circle transform. Results : Automated cryo‐EM imaging combined with manual particle classification and size analysis of 200 images of purified EVs from MDA‐MB‐231 cells revealed several particle classes with distinct morphologies and sizes. Notably, ∼25% of the particles lacked a lipid bilayer, indicating they were not EVs. Cryo‐EM imaging and sML particle analysis of an additional 2000‐image dataset identified two particle classes, of which less than half were EVs. The remaining particles consisted of small amorphous structures, resembling exomeres. Additional advanced sML modelling enabled automatic detection of the lipid bilayer, as well as lipoprotein particles from blood plasma. Summary/Conclusion : Automated cryo‐EM imaging, combined with sML particle detection, enables reproducible data collection and objective particle classification, quantification and (size) analysis of EVs. By specifically detecting EVs by their lipid bilayer, it was shown that a significant part of particles present in purified EV samples are non‐bilayer containing contaminants. The combination of cryo‐EM and sML‐assisted detection of both the lipid bilayer and particles in general, classification and analysis provides a powerful approach to explore the prevalence and detailed structures of EVs and contaminants in purified EV samples.

Physical

Ilaria Bellini 1 , Bradley J Whitehead 2 , Claudia Chiovoloni 1 , Silvia Rondón 1 , Agostina Pietrantoni 3 , Anna Kashkanova 4 , Federica Rinaldi 5 , Maria Gioia Fabiano 5 , Eleonora D'Intino 5 , Ilaria Rago 6 , Luca Cecchini 6 , Stefano D'Amelio 1 , Peter Nejsum 2 and Serena Cavallero 1 Serena Cavallero and Peter Nejsum are co‐senior authors 1 Department of Public health and infectious diseases, Sapienza University of Rome, Italy. 2 Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus, Denmark. 3 Core facilities, Istituto Superiore di Sanità, Rome, Italy. 4 Max Planck Institute for the Science of Light, Erlangen, Germany. 5 Department of Drug Chemistry and Technology, Sapienza University of Rome, Rome, Italy. 6 Department of Physics and IFNF, Sapienza University of Rome, Italy. Introduction : Helminth extracellular vesicles (EVs) in parasitic diseases represent a growing area of interest. However, challenges in parasite EVs characterization hamper the understanding of their properties. This research investigated the physical and functional features of Anisakis EVs, a zoonotic nematode known to cause allergic reactions and gastrointestinal inflammation in humans, with a potential link to cancer. EVs were isolated using size exclusion chromatography (SEC) and characterized using orthogonal optical approaches: Nanoparticle Tracking Analysis (NTA), Interferometric NTA (iNTA)‐ applied to helminth EVs for the first time‐ Dynamic Light Scattering (DLS); Atomic, Scanning and Transmission Electron Microscopy (AFM, SEM, TEM, CRYOTEM). Biological effects were evaluated on human peripheral blood mononuclear cells (PBMCs) and complement activation assays. Methods : 1000 Anisakis third stage larvae (L3) were collected from fish and cultured in RPMI with 1% of pen/strep, media were collected daily. EVs were analysed for size, concentration, inner water content, zeta potential, and protein amount. Proteinase K treatment was used to explore the influence of furry coat on EV properties. Functional assay used human serum treated with increasing concentrations of Anisakis and HEK293 EVs followed by ELISA to quantify levels of C5a. Human PBMCs were treated with Anisakis EVs and analysed for TNF production. Results : Anisakis EVs showed 10 10 particles/mL concentration and an average diameter of 150 nm. Following “shaving” treatments, DLS showed a mild reduction in size, suggesting the presence of a furry coat, confirmed by microscopy. iNTA revealed that 5–20% of EVs' internal content was non‐aqueous, suggesting the presence of a bioactive cargo. Anisakis but not human EVs dose dependently activated the complement pathway resulting in production of the anaphylatoxin C5a. Furthermore, Anisakis EVs induced production of TNF by human PBMCs in a mechanism that was augmented by the presence of human serum. Summary/Conclusion : Anisakis EVs revealed biological properties able to expand the knowledge in host‐parasite dynamics. Additionally, the exploration of the parasitic EVs procedures are essential for progress, particularly regarding their applications in in vitro and in vivo models and clinical trials.

Pi3K/Rab

Shuo Wang, Yosuke Tanaka The University of Tokyo, Japan Introduction : Sonic hedgehog (SHH)‐containing large exosomes are a new type of large EVs. They may be equivalent to ‘ART‐EVs’ that contain SHH and Rab18 proteins (Coulter et al., Cell Rep 2018); and the hMSC‐derived ones relevant for post‐myocardial infarction recovery (Mackie et al., Circ Res 2012). Here we show that PI3K/Rab signalling facilitates SHH‐containing large exosome release from hMSCs. In our previous study of polydactyly mice (Wang et al., Dev Cell 2022), we detected an FGFR/PI3K signalling dependency of a posterior‐to‐anterior SHH protein gradient formation in developing limb buds. Regional regulation of SHH‐containing large exosome release may contribute as an underlying mechanism, of which rate was significantly augmented by FGFR/PI3K signalling in the peripheral layer. In the present study, we have investigated precise molecular and cellular mechanisms of PI3K‐induced large exosome release. Methods : We stimulated human MSCs and mouse fibroblasts with PI3K agonists and Rab18‐modulating compounds and/or constructs. Then we measured the changes in the large exosome releasing cascade by immunocytochemistry, protein biochemistry/immunoblotting, NTA, TEM, and live imaging. In addition, we investigated the physiological relevance of the large exosomes using HUVEC cell assay for in vitro angiogenesis. Results : PI3K agonists significantly stimulated hMSCs to release large exosomes of 0.6–1.5 um large, bounded by lipid bilayers, and highly enriched by SHH protein. The EVs from stimulated hMSCs showed 4 folds higher angiogenic capacities on HUVEC cells than control EVs. Rab18 GTPase may act as a downstream target of PI3K signalling that critically facilitates the membrane trafficking of large exosome components. We show evidence that PI3K signalling increases the GDP‐bound form of Rab18 GTPase, which was preferentially accumulated in the cell centre according to the microtubule‐mediated intracellular trafficking. SHH‐containing large exosomes were then directly released from this perinuclear endosomal compartment, probably by the function of Rab18‐GDP effector proteins. Summary/Conclusion : This study describes a PI3K/Rab‐mediated regulation mechanism of SHH‐containing large exosome production. It will provide cell biological insights on how the noncanonical secretion drives large exosome release, which will be of high clinical values for regenerative medicine.

Podocyte

Karen Lahme 1 , Wiebke Sachs 1 , Desiree Loreth 1 , Stephanie Zielinski 1 , Johannes Brand 1 , Kristin Surmann 2 , Uwe Völker 2 , Thorsten Wiech 3 , Tobias N. Meyer 4 , Lars Fester 5 , Catherine Meyer‐Schwesinger 1 1 Institute of Cellular and Integrative Physiology, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 2 Interfaculty Institute for Genetics and Functional Genomics, Greifswald, Germany; 3 Institute of Pathology, Nephropathology Section, University Medical Center Hamburg‐Eppendorf, Germany; 4 Nephrology, Asklepios Klinikum Barmbek, Hamburg, Germany; 5 Institute of Neuroanatomy, Medical Faculty, University Bonn, Bonn, Germany Introduction : Membranous nephropathy (MN) is an autoimmune glomerulonephritis of kidney podocytes caused by circulating autoantibodies directed against podocyte foot process proteins. The morphologic hallmark of MN is glomerular antigen‐autoantibody deposition. The underlying mechanisms and clinical significance of glomerular aggregate formation and release in MN are unknown. This study analysed podocyte‐derived urinary extracellular vesicle (EV) formation in human nephrotic patients and experimental murine MN and identified exophers as the pathobiological correlate of glomerular urinary space antigen/autoantibody aggregates. Methods : EVs were isolated from mouse and human urine by differential ultracentrifugation, followed by ultrafiltration (human urine) or by dialysis (mouse urine). EVs were quantified by nanoparticle tracking analysis, characterized by electron microscopy, ImageStream, and immunoblotting. The proteostatic content of human urinary EVs was further investigated by mass spectrometry. Immunohistology from human nephrotic patient biopsies was performed. Results : We identified podocyte exophers as biological correlates of glomerular urinary space aggregates in MN. These large, stalked vesicles are formed by translocating THSD7A and bound autoantibodies from the subepithelial to the urinary side of podocyte plasma membranes. Exophers in MN patients contain antigen/autoantibody complexes and disease‐associated proteins like complement. Exopher‐release enhanced by proteotoxic stress reduces the glomerular aggregate burden in experimental MN. Monitoring urinary exopher abundance and autoantibodies is essential for assessing immunologic activity in MN, especially in the setting of negative serum autoantibody titres. The proteostatic content of podocyte‐derived urinary EVs changes disease‐dependently, reflecting the disease‐associated proteostatic situation of podocytes. Summary/Conclusion : Exopher‐genesis is a protective pathomechanism in MN. Tracking of the autoantibody from the serum to the urinary exopher‐bound state assesses the podocytes’ propensity to deal with the disease initiating autoantibodies. This will provide a non‐invasive diagnostic tool with prognostic potential for clinical diagnostics of MN.

Proteome

Presenter: Rose W. Gatheru Kemri‐Wellcome Trust Research Programme, Kilifi, Kenya Introduction : Globally, the post‐discharge mortality rate observed in children under 5 years poses a great health crisis, especially in low‐ and middle‐income countries. There is clear evidence from emerging data that severe acute malnutrition (SAM) in children under 5 years contributes towards this burden. Hospitalized children with malnutrition are at eight times higher risk of death within 1‐month of discharge than well‐nourished children. Currently, the biological mechanisms underlying the cause of death, which could inform novel interventions to minimize the risk of death in this group of children, are not well understood. Traditional biomarkers and risk factors such as whole plasma and clinical anthropometric measures have low and unspecific predictive value. Recently, studies have revealed that a proportion of the plasma proteome is encapsulated in extracellular vesicles whose content is used in specific inter‐cellular signalling pathways and might not be adequately accessible to trypsin digestion during processing of samples for mass spectrometry analysis. We aim to identify novel proteomic biomarkers in EVs that are associated with post‐discharge mortality in severely malnourished children that will inform therapeutic interventions of post‐discharge mortality. Methods : This will be accomplished by complementary approach analysis targeting EV‐associated proteins and cytokines and whole plasma using samples obtained with informed consent at discharge from sick, undernourished children who were admitted to a hospital and followed up post‐discharge for 12 months. Quantifying EV‐associated proteins, as well as those in whole plasma, may increase our understanding of the risk of early death post‐discharge and prognostic biomarkers. Results : We hypothesize a model of ineffective innate and adaptive immune responses contributed to inadequate clearance of infections, hence inflammation. Glucose utilization is thus prioritized to the activated immune cells at the expense of the growth of the recovering infants. Hence, this inflammation‐induced component of starvation may contribute to organ dysfunction, multi‐organ failure and death. Summary/Conclusion : This is the first study proposing identification of novel biomarkers associated with post‐discharge mortality through characterization EV fractions. The potential biomarkers identified will aid in the management of SAM children that will include specific interventions to reduce inflammation in addition to antimicrobial and nutritional interventions.

Refining

Presenter: Daniele Reverberi IRCCS Ospedale Policlinico San Martino, Genoa, Italy Introduction : Extracellular vesicles (EVs) are membrane‐bound particles released by cells, playing a key role in intercellular communication and serving as promising biomarkers for various diseases. The ability to isolate and characterize specific EV subpopulations, particularly those circulating in plasma/serum, holds great potential for enhancing biomarker precision and developing targeted therapies. Cancer‐derived EVs often express unique surface markers, enabling their distinction from EVs of other cellular origin. Thus, the accurate sorting of tumour EVs could provide valuable insights into cancer progression, metastasis, and treatment response. Methods : We developed a robust method for isolating and sorting CD9‐positive plasma EVs as a proof‐of‐concept for broader EV subpopulation analyses. Plasma EVs were isolated using sucrose cushion ultracentrifugation, balancing purity and yield. After a bulk EV characterization, an unconventional flow cytometry strategy based on fluorescence threshold triggering was initially applied to assess the percentage of CD9‐positive EVs and subsequently to sort them. CD9 is one of the most highly expressed tetraspanins by plasma‐derived EVs and was selected here as a proof‐of‐concept antigen to describe our standardized sorting protocol. Results : To determine the minimal prerequisites for EV detection and sorting, instrument setup in terms of sheath and sample pressure, as well as particle concentration, were thoroughly considered. The effect on event rates per second, side light scattering, and fluorescent signals obtained during the analysis of different EV numbers was assessed using various nozzles, demonstrating that particle concentration can significantly affect their accurate quantification and characterization. Analyzing an excessive number of EVs leads to the coincident detection of nano‐sized vesicles, resulting in an unwanted swarming effect. Moreover, we demonstrated how the removal of unbound reagents (dyes, antibodies), by size exclusion chromatography, after EV staining profoundly impacts the percentage of CD9‐positive EVs and consequently affects their sorting efficiency in terms of event rate/second and volume of sorted material. Summary/Conclusion : The advancements here proposed could enable high‐purity isolation of EV subpopulations, facilitating their use in downstream applications, such as the identification of cancer‐specific biomarkers or development of EV‐based targeted therapies. Funding : This work was supported by Worldwide Cancer Research under Grant (Reference 24‐0042).

Response

Lara Bieler 1,2 , Rodolphe Poupardin 3 , Fabian Blessing 1,2 , Eva Grasmann 4 , Daniela Auer 4 , Stefan Rund 4 , Carina Kals 4 , Christina Folie 4 , Elisabeth Bayer 4 , Eva Rohde 6,7 , Mario Gimona 4,6,7 , Sebastien Couillard‐Despres 1,2 1 Institute of Experimental Neuroregeneration, Paracelsus Medical University, Salzburg, Austria; 2 Austrian Cluster for Tissue Regeneration, Salzburg, Austria; 3 Institute of Experimental and Clinical Cell Therapy, Paracelsus Medical University, Salzburg, Austria; 4 GMP Laboratory Paracelsus Medical University, Salzburg, Austria; 5 Department of Transfusion Medicine, University Hospital Salzburg, Salzburg, Austria; 6 Research Program for Nanovesicular Therapies, Paracelsus Medical University Salzburg, Salzburg, Austria; 7 Ludwig Boltzman Institute for Nanovesicular Precision Medicine, Salzburg, Austria Introduction : At present, no effective therapies are available for traumatic spinal cord injury (SCI). Following the initial trauma, persistent inflammation exacerbates neuronal and functional losses. Our recent findings highlight the potential of acute treatment with MSC‐derived extracellular vesicles (MSC‐EVs) in mitigating inflammation and its consequential damages after SCI. However, with numerous protocols available for EV preparation from MSC secretomes, we sought to identify the most efficacious method for generating EV‐preparations capable of reducing the pro‐inflammatory polarization of microglia. Methods : We assessed the potential and biological activity of EV fractions produced with several protocols involving various pore sizes for tangential flow filtration (TFF), in optional combination with size exclusion chromatography, or ultrafiltration, and supplementation of the culture medium with human platelet lysate. EV fractions were applied in vitro on LPS‐treated BV‐2 microglia. Thereafter, cells and medium were harvested for analyses. Results : We observed that all EV fractions reduced the production of nitric oxide by LPS‐treated BV‐2 microglia comparably, regardless of the preparation protocol used. Nevertheless, EVs enriched via small pore size (100 kDa) TFF led to higher anti‐inflammatory activity based on PCR analyses of pro‐inflammatory cytokine expression. We conducted a transcriptomic analysis comparing samples obtained from BV2 microglia which were activated with LPS and treated with the most and least active EV preparation or left untreated, which are currently being analysed. Elucidating the specific signalling pathways activated by potent anti‐inflammatory EV‐fractions, will provide valuable insights to further refine EV preparation protocols and identify molecular targets for combating inflammatory conditions. Summary/Conclusion : Elucidating the specific signalling pathways activated by potent anti‐inflammatory EV‐fractions, will provide valuable insights to further refine EV preparation protocols and identify molecular targets for combating inflammatory conditions.

Scalable

Stephen Lenzini, Jae Jung, Madeline Cramer, Elie Zakhem, Jon A. Rowley RoosterBio, Inc., Frederick, Maryland, USA Introduction : Scalable GMP‐compatible production of MSC‐EVs requires bioreactor expansion and downstream processing (DSP) for purification. We have published MSC‐EV bioreactor processes from 3‐50L with DSP operations including clarification, tangential flow filtration (TFF), chromatography, and formulation. Total cumulative yields of MSC‐EVs are often < 10%, representing a major challenge by limiting lot sizes and increasing cost of goods. Recently, we discovered a novel reagent (Agent V, patent pending) for addition during cell culture or to harvested conditioned medium (CM) that streamLines MSC‐EV DSP by greatly increasing yield. Our goal in this study was to determine the Agent V concentration in bioreactor‐harvested conditioned medium (CM) that maximizes post‐DSP yield while maintaining quality attributes. Methods : Human bone marrow MSC‐EVs were produced in a 3L bioreactor (Eppendorf) using RoosterCollect‐EV for a 5‐day collection. Agent V was added to harvested CM at 0.1×, 1× and 5× for at least 15 min and MSC‐EV yield though clarification filters (Sartorius) was quantified by NTA (Particle Metrix). The optimal Agent V dose (1X) was chosen to treat remaining CM. MSC‐EV yield and purity (particles/mg protein) for each additional unit operation (TFF, Repligen; chromatography, Cytiva) were measured +/‐ Agent V treatment. Purified MSC‐EVs were tested for MSC‐EV identity (CD63/CD9/CD81 via ProteinSimple Jess) and function (CD73 activity). Results : Treating CM with Agent V led to a dose‐dependent yield improvement through a 5 µm clarification filter. Without Agent V, pressure in the filter rises, leading to filter fouling and particle loss. With Agent V, pressure rise during filtration is minimized. Treated and untreated CM were processed further through each DSP unit operation, demonstrating that treatment leads to significant cumulative yield improvements (∼5% vs. ∼50%, a 10‐fold increase). Treatment and filtration steps did not affect particle size distribution. Furthermore, MSC‐EV markers (ProteinSimple Jess) and MSC‐EV potency (CD73 activity) in the purified EVs from the Agent V treated group were maintained. Summary/Conclusion : In conclusion, Agent V is a novel, GMP compatible reagent that simplifies and streamLines EV purification by increasing MSC‐EV DSP yield 10‐fold while maintaining their quality attributes.

Skeletal

Presenter: Hagit Shoyhet Technion‐IIT, Israel Introduction : Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by insulin resistance and impaired glucose homeostasis, resulting in systemic complications. In T2D, disrupted intercellular signalling exacerbates inflammation and metabolic dysregulation, driving disease progression. Current therapies primarily address symptom management rather than underlying cellular communication deficits. Recent studies suggest that extracellular vesicles (EVs) are instrumental in modulating these cellular interactions, as they carry molecular cargo that influences metabolic and inflammatory pathways. Tissue engineering, particularly of skeletal muscle, offers a promising approach to utilizing EVs for therapeutic purposes in T2D. Our previous work showed that engineered muscle tissue overexpressing GLUT4 positively impacts diabetic phenotypes in Diet‐Induced Obesity (DIO) mice, improving blood glucose levels, insulin response, and cytokine profiles. Here, we explore the role of EVs derived from engineered constructs in mediating these systemic effects. Methods : EVs were isolated from the conditioned medium of 3D‐engineered skeletal muscle tissue overexpressing GLUT4 using an affinity column kit. The EVs were characterized via DLS, AFM and cryo‐TEM. EV functionality was tested using in vitro glucose uptake assay and in vivo experiments, including the glucose tolerance test and histology. We also analysed their protein and small RNA content via proteomics and MiR‐seq, respectively. Results : In vitro assays indicate that EVs isolated from GLUT4 overexpressing 3D skeletal muscle tissue increase glucose uptake in WT myotubes compared to EVs isolated from WT 3D skeletal muscle tissue. In vivo experiments on diabetic mice indicated that the GLUT4 OE myotubes EVs improve response in the glucose tolerance test (GTT). MiR‐seq and proteomic analyses suggested a regulatory pattern affecting various metabolic pathways, such as IGF‐1 and insulin signalling through the delivery of IGF‐1 and downregulation of specific MiRs, additionally confirmed by in vitro molecular assays. Summary/Conclusion : Myogenic EVs derived from GLUT4 OE skeletal muscle constructs can improve glucose metabolism of WT muscle in vitro and of DIO mice in vivo. The EVs' unique cargo can help explain their mechanism of action and the in vivo effect of a small implanted construct. Our work suggests that tissue engineering can be used to affect the host metabolic systems, reaching beyond the small‐sized implanted tissue.

Specific

Presenter: Qianbei Li Southern Medical University, Guangzhou, People's Republic of China Introduction : Bacterial outer membrane vesicles (OMVs) are nano‐sized structures derived from the outer membrane of Gram‐negative bacteria. These vesicles have emerged as key players in host‐pathogen interactions, but their potential as biomarkers remains largely unexplored due to the difficulty of identifying them in complex biological samples. Methods : We used the polymyxin B‐fluorescein probe (PmbF) to label bacterial OMVs in blood samples and analysed them using nano‐flow cytometry. In a mouse model of pneumonia, we collected serum samples at various time points post‐infection to monitor changes in PmbF+ EVs. To confirm the origin of these EVs, we utilized an engineered mCherry‐expressing E. coli strain. Additionally, we collected clinical samples and compared the performance of PmbF+ EVs with C‐reactive protein (CRP) and procalcitonin (PCT) in diagnosing bacterial infections and differentiating them from viral or mycoplasma infections. Finally, we conducted co‐culture experiments to assess the effects of OMVs on macrophages. Results : In the mouse model of pneumonia, we observed a significant increase in serum PmbF+ EVs as early as 6 h post‐infection, preceding positive blood cultures. In clinical samples, PmbF+ EVs outperformed CRP and PCT in diagnosing bacterial infections and distinguishing them from viral or mycoplasma infections. Co‐culture experiments revealed that OMVs triggered macrophage apoptosis by inducing mitochondrial dysfunction and reactive oxygen species (ROS) production. Summary/Conclusion : Our findings highlight the potential of circulating OMVs as promising biomarkers and mediators of bacterial infections. By using the PmbF probe and nano‐flow cytometry, we can efficiently detect and quantify these OMVs, providing a new tool for the early diagnosis and monitoring of bacterial infections. Furthermore, the role of OMVs in host immune responses offers new insights into the pathogenesis of bacterial infections. Funding : This work was supported by the National Science Fund for Distinguished Young Scholars (82025024); the Key project of the National Natural Science Foundation of China (82230080); the National Natural Science Foundation of China (82302593 and 82272438); Guangdong Natural Science Fund for Distinguished Young Scholars (2023B1515020058); the Natural Science Foundation of Guangdong Province (2023A1515012512); the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2022J001).

Syntenin

Barnabas Irmer 1,2 , Allegra Angenendt 1,2 , Luc Camoin 3 , Stéphane Audebert 3 , Christiane Geyer 1 , Mirjam Gerwing 1 , Hanna Spiessbach 1 , Mira Hebel 1 , Émilie Baudelet 3 , Darius Wlochowitz 4 , Uwe Hansen 1 , Annalen Bleckmann 1,2 , Pascale Zimmermann 3,5 , Kerstin Menck 1,2,3,4 1 University of Muenster, Germany, 2 University Hospital Muenster, Germany, 3 Centre de Recherche en Cancérologie de Marseille, France, 4 University Medical Center Goettingen, Germany, 5 K. U. Leuven, Belgium Introduction : Despite knowing that cancer‐derived small extracellular vesicles (sEVs) support tumour growth, the specific roles of different sEV subpopulations remain unclear. Syntenin has been identified as one of the main regulators of endosomal‐derived sEVs. Our study investigates how syntenin affects the protein cargo and tumour‐supporting function of sEVs in breast cancer. Methods : Small EVs were isolated by differential ultracentrifugation from syntenin‐deficient murine 4T1 and human MCF‐7 breast cancer cells and comprehensively characterized based on the MISEV guidelines using NTA, electron microscopy and immunoblots. Syntenin knockout (KO) and corresponding wild‐type (WT) sEVs from 4T1 cells were further characterized by mass spectrometry for their proteomic cargo, and differentially expressed proteins were validated by immunoblotting. The influence of Syntenin silencing on the adhesive capacity of breast cancer sEVs was tested in vivo by biodistribution analysis in syngeneic mice and in vitro by cell and sEV adhesion assays. Results : We detected 178 down‐regulated proteins on sEVs upon Syntenin KO. Pathway enrichment analysis suggested a strong enrichment of adhesion‐related processes. Accordingly, sEVs from Syntenin‐deficient 4T1 and MCF‐7 cells showed a reduced expression of several focal adhesion and cell‐cell junction proteins. In line, Syntenin silencing reduced the fibronectin‐binding capacity of sEVs from both cell lines, which was mediated by sEV‐associated Integrin alpha‐V/beta‐3 (αVβ3). Compared to sEVs from WT cells, Syntenin KO sEVs showed decreased tropism towards the fibronectin‐rich liver microenvironment in vivo, provided less adhesive support for 4T1 cells and thereby failed to induce cancer cell migration, which appeared to be independent of EV uptake. Summary/Conclusion : Syntenin regulates the proteomic cargo and concomitantly the adhesive, organotropic and pro‐migratory properties of sEVs in breast cancer. Funding : Else Kroener‐Fresenius‐Foundation (project 2019_A162), Deutsche Forschungsgemeinschaft (DFG, project ME 4573/1‐1) and the Heidenreich‐von‐Siebold programme of the University Medical Center Goettingen.

Systemic

Presenter: Tang‐Long Shen National Taiwan University, Taiwan (Republic of China) Introduction : Viroids are a type of plant pathogens solely consisted of naked, circular single‐stranded RNAs that do not encode any protein. Citrus exocortis viroid (CEVd) enables infecting not only citrus but also many Solanaceae spp. Neverthelss, the pathogenesis and the systemic symptomatic mechanistic nature of CEVd are largely unknown. Methods : To clarify whether CEVd enables transmission through tomato‐derived extracellular vesicles (tEVs), we isolated tEVs from stems or leaves of tomato plants at six wpi or mock. Results : After measurement of the isolated tEV particle concentration, protein, and RNA amounts, as well as examination by transmission electron microscopy (TEM), the CEVd RNA genome was detected in the CEVd‐infected tEVs but not in the mock, implicating that CEVd might be able to transmit in the host plant via tEVs. Additionally, tEVs derived from CEVd‐infected leaves bear several catabolic and defense pathway‐associated proteins, including catalase, subtilisin‐like protease, and pathogenesis‐related (PR) proteins at a relatively high expression level compared to the mock counterpart. Furthermore, to speculate the pathogenicity of the CEVd‐contained tEVs, we re‐inoculated the CEVd‐infected tEVs on tomatoes and subsequently found significant decreases in the biomass and the leaf area at 4 wpi compared to the mock control. Summary/Conclusion : Our study provides first and novel insight for EV‐mediated transmission of CEVd within infected tomatoes attributed to the systemic symptom development during CEVd pathogenesis. Funding : NSTC 112‐2313‐B‐002‐024‐MY3

Targeted

Farzaneh Malekian, Alireza Shamsian, Van Manh Hung Le, Biljana Gigic, Christoph Kahlert Department of General, Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg, Germany Introduction : Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer‐related deaths worldwide. Its tumours contain heterogeneous cell populations, complicating treatment. Among these, cancer stem‐like cells (CSLCs) contribute significantly to tumour progression, chemoresistance, and metastasis. CSLCs, characterized by markers such as CD133, are challenging therapeutic targets. Developing strategies to selectively target and eliminate CSLCs is essential for improved treatment outcomes. Extracellular vesicles (EVs), natural vehicles for intercellular communication, have emerged as promising carriers for targeted therapy due to their ability to deliver therapeutic agents and their potential for modification for targeted delivery. Methods : In this study, EVs have been isolated from HEK293T cells that were modified to present CD133 antibodies on their surface. EV isolation and characterization followed MISEV 2023 guidelines to ensure purity and quality. EVs were decorated with CD133 antibodies using a chemistry‐based technique and loaded with the cytotoxic agent 5‐fluorouracil (5‐Flourouracil (5‐FU) and Doxorubicin) via electroporation, achieving an efficient cargo‐loading process. The success of EV decoration was assessed using a MACSQuant Analyzer 10. To evaluate targeting specificity, CRC cell lines were genetically modified using CRISPR/Cas9 to create two distinct target cell populations: CD133‐overexpressing cells (DLD1 cell line) and CD133 knockout cells (SW620 cell line). The targeting ability of the modified EVs was assessed by comparing their binding and uptake in these cell populations with their wild‐type counterparts. Results : The strategies for extracellular vesicle (EV) decoration and cargo loading achieved approximately 80% efficiency, with target specificity enhanced twofold compared to unmodified EVs. Uptake assays were conducted on wild‐type DLD1 and SW620 cells, with further analysis planned for engineered cell lines. The cytotoxic effects of modified EVs will be evaluated across various colorectal cancer (CRC) cell lines, alongside an investigation of their mechanisms of action. Summary/Conclusion : Primary results from our study suggest that CD133‐modified EVs can target CSLCs, delivering therapeutic cargo effectively in vitro and potentially in vivo. However, further in vitro and in vivo studies are needed to validate and expand these findings. This approach holds promise for targeting chemoresistant populations within CRC tumours, advancing the potential for more precise and effective therapies. Funding : This study was supported by the DAAD Scholarship.

Tracking

Anna Lischnig 1 , Yusuf C. Erdoğan 1 , Wolfgang F. Graier 1,3 , Roland Malli 2,3* 1 Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; 2 CF Bioimaging, Center of Medical Research, Medical University of Graz, Graz, Austria; 3 BioTechMed Graz, Graz, Austria Introduction : The tetraspanin CD63 is commonly associated with extracellular vesicles (EV) and plays an important role in EV biogenesis and secretion. CD63 is being used for the real‐time visualization of multivesicular body (MVP) and plasma membrane (PM) fusion events. The available reporters for CD63 secretion rely on pH‐sensitive fluorescent proteins (FPs), which show an intensity increase during MVB fusion due to pH change. However, current CD63‐based reporters have limited dynamic range and background from CD63‐FP accumulation at the PM, posing a challenge to detect fast, transient exocytosis events. Therefore, advanced strategies are essential to expand and refine the toolkit for EV secretion visualization. Methods : We fused a genetically encoded single‐FP‐based Ca 2+ biosensor, GreenT‐EC.c, to the first extracellular loop of CD63 as an alternative to only pH‐sensitive FPs. The new design also consists of protease‐cutting sites flanking the biosensor to lower the PM background signal and a red reference FP fused to the intracellular C terminus of CD63 to normalize the readout. Upon protein structure validation in silico, we tested the new reporter in mammalian cell lines (HeLa S3, EA.hy926). We characterized the sensor dynamics and the visualization of CD63 secretion events via live cell imaging. Results : We demonstrate that the CD63‐fused calcium biosensor emits strong green fluorescence upon exocytosis, as elevated extracellular Ca 2 ⁺ and neutral pH enhance calcium binding to the sensor. This increase in green fluorescence, without elevation in red fluorescence, enhances the GFP/RFP ratio, allowing clear visualization of secretion events. Protease treatment reduces background GFP signal at the PM, improving the dynamic range and enabling precise detection of transient exocytosis events. Overall, the protease‐mediated removal of externalized FP significantly enhances signal quality. Summary/Conclusion : Our findings demonstrate that Ca 2 + biosensors can effectively be used to visualize secretion events in live‐cell imaging. We expect that with this new reporter system, EV secretion can be monitored in high spatial‐temporal resolution, thus being very suitable for live cell imaging applications.

Transfer

Presenter: Maide Zehra Gencali Friedrich‐Alexander Universität Erlangen‐Nürnberg, Germany Introduction : HEK WT cells express the proteases ADAM10 and ADAM17, whereas HEK dKO cells lack these proteases. Located on the cell membrane, ADAM10/17 can shed the inactive form of the cell surface metalloproteinase Meprin β. This study aims to demonstrate the proteolytic activity of ADAM10/17 in HEK WT cells and show that this activity can be transferred through EVs to HEK dKO cells. Additionally, we investigate the release of ADAM10/17 on EV in dependency of bacterial OMV from THP‐1 macrophage cells. Methods : EVs generated from HEK WT cells were transfered to HEK dKO cells that had been transfected with Meprin β. The shed Meprin ß was assessed by Western Blot. Meprin ß shedding from EVs was assessed on EV derived from HEK WT cells expressing Meprin ß. The proteolytic activity of ADAM10/17 present on EVs was additionally measured with a quenched fluorescent peptide substrate. Furthermore, THP‐1 cells were stimulated with Gram‐negative bacteria derived OMV or OMV with reduced LPS surface expression (clear coli). We then characterized THP‐1 cell released EV for ADAM10/17 content. Results : Our results show that HEK WT cells transfected with Meprin β exhibit significant shedding of Meprin β due to the activity of ADAM10/17. This shedding also occurs on EVs derived from such Merpin ß expressing HEK WT cells and when EVs containing ADAM10/17 are added to HEK dKO cells. Notably, ADAM10 is preferentially released onto HEK WT cell derived EVs. To investigate the release of ADAM10 and 17 in a more meaningful setting, we stimulated THP‐1 cells with OMV and determined the release pattern of both proteases onto EV. Summary/Conclusion : Using ADAM10/17 deficient cells, we can now show that ADAM10 proteolytic activity is present on EV and can be transferred to other cells. This basic finding opens new avenues for mechanistic studies capitalizing on this finding. IT will be of interest to investigate the ratio between ADAM10 and 17 released on EV in different physiological and pathological conditions and how transfer of these proteases to distant cells will change cell behaviour and properties.

Tribbles

Wen‐Wei Chang 1 , Yu‐Hao Huang 1 , Yen‐Min Huang 2 , Hsueh‐Te Lee 3 1 Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan; 2 School of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan; 3 Institute of Anatomy & Cell Biology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan Introduction : Extracellular vesicles (EVs) are key mediators in cancer progression, largely due to the cargo they carry. CD276, an immune checkpoint molecule, plays a significant role in cancer behaviour, contributing to immune evasion, proliferation, and therapy resistance. Tribbles pseudokinase 3 (TRIB3) is a pseudokinase with a scaffolding function, and its role in regulating the EV loading of CD276 in oral squamous cell carcinoma (OSCC) remains unclear. This study aims to investigate the function of TRIB3 in the EV loading process of CD276 and its potential as a biomarker in OSCC. Methods : The Cancer Genome Atlas (TCGA) datasets were used to assess correlations between TRIB3, CD276, and ESCRT‐associated proteins ALIX/TSG101. EVs were collected from OSCC cells by ultrafiltration. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot. A CD276‐EGFP cDNA plasmid was used for studying the intracellular trafficking of CD276 and its escape from lysosomal degradation. Plasma EVs from OSCC patients and healthy donors were obtained to analyse CD276 levels by western blot and ELISA. Results : Analysis of TCGA datasets revealed a positive correlation between TRIB3 or CD276 expression and ALIX/TSG101. TRIB3 knockdown did not significantly affect EV size or particle concentration, but reduced TSG101, ALIX, and CD9 protein levels in EVs, with no change in whole cell lysate levels. Structural modelling suggested interactions between TRIB3 and ESCRT proteins, indicating TRIB3's role in regulating EV cargo loading. TRIB3‐silenced OSCC cells exhibited reduced CD276 on cell membranes and in EVs, despite unchanged total CD276 levels, indicating TRIB3's involvement in intracellular trafficking. Transfection with CD276‐EGFP showed CD276 escaping lysosomal degradation, suggesting that TRIB3 might prevent lysosomal fusion. Plasma EVs from OSCC patients showed detectable CD276 in three out of four samples, with the highest levels in a recurrent case, while a relatively low level of CD276 was detected in healthy donors. Summary/Conclusion : TRIB3 regulates CD276 loading onto EVs and prevents its lysosomal degradation in OSCC cells. These findings suggest that TRIB3 is a promising therapeutic target and CD276 in EVs could serve as a potential biomarker for OSCC diagnosis, especially in recurrent cases. Funding : National Science and Technology Council in Taiwan.

Tumoural

Bianca C. Pachane 1,2 , Pedro H. T. Bottaro 1 , Aline M. Machado 1 , Cynthia A. de Castro 3 , Gabriela Guerra 1 , Larissa T. Gozzer 1,4 , Marina M. Grigoli 5 , Artur D. Zutião 5 , Angelina M. Fuzer 5 , Marcia R. Cominetti 5 , Wanessa F. Altei 2,6 , Heloisa S. Selistre‐de‐Araujo 1 1 Biochemistry and Molecular Biology Laboratory, Department of Physiological Sciences, Universidade Federal de São Carlos—UFSCar, São Carlos, SP, Brazil; 2 Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, SP, Brazil; 3 Pathology and Biocompatibility Laboratory, Department of Morphology and Pathology, Universidade Federal de São Carlos—UFSCar, São Carlos, SP, Brazil; 4 Universidade de Aveiro, Aveiro, Portugal; 5 Department of Gerontology, Universidade Federal de São Carlos—UFSCar, São Carlos, SP, Brazil; 6 Radiation Oncology Department, Barretos Cancer Hospital, Barretos, SP, Brazil Introduction : The highly metastatic triple‐negative breast cancer (TNBC) relies on its tumour microenvironment (TME) to maintain phenotypic heterogeneity and progression. Extracellular vesicles from hypoxic TNBC were previously shown to aid tumoural invasion, but their function in the tumour microenvironment is still unclear. In this study, we sought to understand the effect of TNBC‐derived hypoxic EVs (EVh) in the TME in vitro. Methods : EVh were separated from hypoxic (1% O 2 ) MDA‐MB‐231 conditioned media using ultracentrifugation and filtration, and characterized following MISEV2023 guidelines. Using direct, indirect and multicellular circulating co‐culture (MC‐CC) models, we observed how EVh interfered with tumoural and endothelial cells, fibroblasts, monocytes and macrophages. Results : EVh promoted monocyte differentiation to M2‐like macrophages and inhibited macrophage‐derived phagocytosis in endothelial and tumoural cells. The protection of endothelial, tumoural and stromal cellular integrity by EVh increased pro‐tumoural and pro‐angiogenic signalling, collagen matrix synthesis and potential differentiation to cancer‐associated fibroblasts. The multicellular circulating co‐culture method was corroborated by other co‐culture protocols, leading to an adequate system with potential for investigating other tumour‐related processes, including circulating tumour cells and metastasis. Summary/Conclusion : Overall, our results suggest that tumoural EVs from hypoxic cells are essential for maintaining a cohesive TME and contribute to understanding tumour basic biology. Funding : This work was funded by the São Paulo Research Foundation through grants 2019/05149‐9, 2021/01983‐4, and 2022/04146‐9; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), code 001; and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Unbiased

Presenter: Nathalie Nevo Institut Curie Research Center, INSERM, France Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed particles released by most cell types. EVs are able to transfer signals and cargoes between donor and recipient cells and are thus instrumental in intercellular communication. However, EVs released by a single donor cell source are increasingly recognized as extremely heterogeneous in terms of size, intracellular origin, and cargo composition. EVs are routinely characterized by the presence of given proteins in bulk preparations. However, analysing large numbers of EVs at the single‐vesicle level is the only way to truly decipher their heterogeneity. Methods : Here, we developed a reliable pipeline of single EV analysis by an EV‐dedicated flow cytometer (Flow NanoAnalyzer), which detects all particles and measures their size down to 55 nm in diameter without the need for immobilization or fluorescent labels. The necessity to remove unbound antibody and determine the optimal antibody concentration was demonstrated using EVs devoid of the analysed markers as negative controls. The pipeline was then developed to reliably quantify the proportion of EVs bearing none or any combination of two markers and their size. Results : We thus observed, depending on the cell source (MDA‐MB‐231 versus HeLa), around 15% versus 20% of EVs bearing none of the CD9 and CD63 tetraspanins, often used to define EVs, 37% versus 23% of EVs bearing both CD9 and CD63, and either a similar proportion of single CD9+ and CD63+ EVs (MDA‐MB‐231) or 3 times more CD9+ than CD63+ EVs (HeLa). We also observed CD29 (ITGB1) as a protein present as frequently on EVs as the tetraspanins (MDA‐MB‐231). Other surface markers, by contrast, were present in less than 20% of EVs, and mostly of relatively large size. Finally, we confirmed that treatment of cells with homosalate, a drug previously proposed to increase the release of plasma membrane‐derived ectosomes (Grisard et al. JEV 2022 11, no. 7: e12242), increased the proportion of CD9+/CD63− EVs (likely ectosomes) to the detriment of CD63+/CD9‐ EVs (likely exosomes), and we identified two other drugs with the reverse effect. Summary/Conclusion : Overall, nano‐flow cytometry implemented with the proper negative controls and validated antibodies allows us to reliably quantify proportions of EV subpopulations suggested by bulk analyses of EV markers.

Vivazome

Min Chen 1 , Baani Bagga 1 , Smriti Krishna 2,3 , Ella Johnston 2 , Johannes Weber 2 , Xenia Sango 2 , David Reutens 1 , David Haylock 2,3 1 Centre for Advanced Imaging, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia; 2 VivaZome Therapeutics Pty Ltd, Jenny Graves Building, La Trobe University, Bundoora, Victoria 3083, Australia; 3 La Trobe University, School of Molecular Science, Kingsbury Drive, Bundoora, Victoria, 3083, Australia Introduction : Inflammatory mechanisms exert a profound influence on the outcome of traumatic brain injury (TBI). Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have been shown to modulate the inflammatory response and improve functional recovery in preclinical TBI models (J Mol Neurosci 2020; 70(5): 677–688). VivaZome's proprietary cells (VZT‐PC) share many of the characteristics of MSCs including cell surface markers, immunomodulatory properties and differentiation potential (Eur J Cell Biol 2020; 99(7): 151123). VZT‐PC‐conditioned culture medium has been shown to accelerate wound healing, reduce inflammation and increase angiogenesis (NPJ Regen Med 2020;5(1):24.). EVs derived from VZT‐PCs contain several microRNAs known to modulate inflammation and have anti‐inflammatory activity in a cell‐based assay. Based on these properties, this study evaluated the treatment effect of VZT‐PC‐derived EVs in a preclinical TBI model. Methods : Adult C57 mice were subjected to a cortical contusion by compressing the cortex to a depth of 1.0 mm at a velocity of 3.5 m/s and 400 ms duration with a bevelled tip 3 mm in diameter (TBI‐0310). Mice were randomLy allocated to treatment groups: 1. sham injury + vehicle, 2. TBI + vehicle, or 3. TBI + EVs (2.19 × 1010 particles, iv, post‐injury). Rotarod trials, open field tests, elevated plus maze, and Barnes maze were performed by investigators blinded to the group to assess functional recovery. Results : Fall latency of rotarod trials was significantly increased in EV‐treated TBI mice compared to vehicle‐treated TBI mice, indicating that EV treatment effectively improved the recovery of grip strength, balance, and motor coordination after TBI. Velocity and distance travelled during the open field test were comparable across the three treatment groups, suggesting that locomotor function was not affected by TBI and EV treatment. In the elevated plus maze test, exosome treatment enhanced exploratory behaviours in TBI mice compared to vehicle, as evidenced by the increased duration in the open arms. Barnes maze results showed that TBI caused impairment in spatial learning, which was not reversed by EV treatment. Summary/Conclusion : VZT‐PC‐derived EVs improved motor function and increased exploratory behaviour. The accessibility and high proliferative potential of GFs make them an attractive source of EVs for treating TBI. Funding : Cooperative Research Centres Projects (CRC‐P) Grants.

V‐Disk

Presenter: Ehsan Mahmodiarjmand Hahn‐Schickard, Freiburg, Germany Introduction : The clinical application of extracellular vesicle (EV) research, such as in liquid biopsies, is limited by the lack of reproducible, automated, and affordable EV purification methods. An ideal method would standardize the entire workflow from blood collection and plasma separation to EV purification for reliable and comparable results. Our microfluidic cartridge (V‐Disk) aims to achieve this. The current version of V‐Disk processes 1 mL of pretreated plasma, and the next version will automate plasma separation from 3 mL of blood, completing the purification workflow. Methods : Using disposable centrifugal microfluidic cartridges manufactured by injection molding, we automate a multimodal EV purification process consisting of cation exchange chromatography (CEX), two filtration steps to remove particles > 800 nm and < 20 nm and concentrate the EVs, and a final treatment with a mixed‐mode chromatography (MMC) resin to remove remaining protein contaminants. Results : After establishing the layout and manufacturing processes of the V‐Disk, we conducted an initial study comparing it with size‐exclusion chromatography (SEC), using three melanoma patient samples. ELISA results of EV surface markers reveal that the V‐Disk isolates exhibit a higher EV yield than those obtained via SEC. Measuring proteins, HDLs, and (V)LDLs, we demonstrate that the V‐Disk can remove impurities from plasma samples to achieve purity comparable to that of SEC. Nanoparticle‐tracking analysis reveals that isolated particles' size distribution is around 100 nm. Summary/Conclusion : Starting from CEX‐treated plasma, the V‐Disk version already demonstrates significant potential, competing with SEC in terms of EV purity and yield. In the future, the V‐Disk will offer a fully automated, standardized label‐free isolation of EVs from whole blood in a highly reproducible manner in a small point‐of‐care compatible device. Funding : Financial support by the Federal Ministry of Education and Research, Germany within the project KI‐VesD‐2 (No. 16LW0339) is gratefully acknowledged.

Achieving

Presenter: Gang Han Tianjin Medical University, Tianjin, People's Republic of China Introduction : Exosomes are promising drug delivery vehicles due to their low immunogenicity and favourable safety profile. Efficiently loading proteins or nucleic acids onto exosomes is critical for their successful application. Our team previously identified peptide‐ and DNA aptamer‐based anchoring molecules that mediate efficient loading to exosomes without modifying the source cells. However, the limited affinity of these molecules restricts their loading capacity, highlighting the need for higher‐affinity alternatives. Methods : We employed phage display technology to screen for nanobodies with high affinity for exosomes. The best candidates were validated using single‐molecule imaging and flow cytometry. We systematically analysed the size and quantity of proteins that could be loaded by the selected nanobody. Results : Through screening and validation, we identified the nanobody Nexo3 as having the strongest affinity for exosomes from various sources. Nexo3 facilitated the efficient loading of a range of molecules, including mCherry, Cre, double‐stranded RNA‐binding domain (DRBD)/siRNA, IgG antibodies, and CRISPR/Cas9‐gRNA (RNP). It successfully delivered DRBD/siRNA, Cre, and Cas9‐gRNA to target cells, resulting in effective interference with GAPDH mRNA and editing of reporter genes. Importantly, Nexo3 was able to bind to exosomes in both cell culture supernatants and the bloodstream of mice, leverageing the liver tropism of circulating exosomes for targeted delivery of mCherry and DRBD/siRNA to hepatic cells. Summary/Conclusion : Nexo3 exhibits strong affinity for exosomes, enabling the efficient loading and delivery of high molecular weight proteins. Additionally, Nexo3 can modify circulating exosomes, facilitating targeted protein delivery to liver cells, thereby enhancing the potential of exosomes as effective drug delivery systems.

Advancing

Presenter: Irina Nazarenko University of Freiburg, Freiburg, Germany Introduction : Extracellular vesicles (EVs) hold great promise for liquid biopsy applications. However, challenges in high‐purity EV isolation, precise quantification, and molecular characterization hinder clinical translation. We address these limitations by introducing an integrated suite of novel methodologies: (1) Superose 6‐based size exclusion chromatography (SEC) for high‐resolution EV isolation, (2) PHoNUPS, an advanced software for standardized EV size and concentration analysis, and (3) protein interaction coupling assay (PICO) for absolute EV biomarker quantification at the single‐vesicle level. Methods : We developed and validated a Superose 6 FPLC‐based SEC method to obtain highly pure small EVs (sEVs) from biofluids. To improve NTA and DLS data analysis, we created PHoNUPS, an open‐source R‐based software enabling standardized, high‐throughput EV sizing and concentration analysis across multiple platforms, ensuring broad accessibility. Finally, we optimized the PICO assay, enabling absolute single‐vesicle quantification of EV biomarkers in a reagent tube without requiring specialized infrastructure. Results : Superose 6‐based SEC enabled precise separation of EV‐rich fractions from biofluids, significantly reducing lipoprotein contamination and facilitating easy detection of disease‐related EVs, including tumour‐derived vesicles. PHoNUPS efficiently processed NTA and DLS data, delivering automated, standardized particle size distribution analysis, enhancing reproducibility across platforms. The PICO assay provided absolute EV biomarker quantification with high specificity and reproducibility. This integrative suite was evaluated on cell models and clinical samples. Summary/Conclusion : Our integrated suite provides a transformative approach to EV research, overcoming key challenges in isolation, quantification, and biomarker detection. The advantages include high‐purity EV isolation, standardized size and concentration analysis, and a breakthrough single‐vesicle biomarker quantification method that requires no specialized equipment or costly infrastructure. These innovations pave the way for next‐generation EV‐based liquid biopsy diagnostics and precision medicine applications. Funding : Supported by the German Federal Ministry of Education and Research (EV‐Surf 13GW0605F, Nanodiag 03ZU1208CA, Next‐GenNTA KK518401 ) and the European Union HORIZON‐EIC‐2021‐TRANSITIONOPEN‐01 (Nexus 101058200).

Assessing

Šárka Hrachovinová 1 , Stanislava Sladeček 1 , Janka Rakytová 1 , Martina Hýžďalová 2 , Nikol Straková 2 , Michal Felsinger 3 , Markéta Bednaříková 4 , Jitka Hausnerová 5 , Daniel Pinkas 6 , Václav Pustka 7 , Zbyněk Zdráhal 7 , Vít Weinberger 3 , Vendula Pospíchalová 1 1 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; 2 Department of Pharmacology and Toxicology, Veterinary Research Institute, Brno, Czech Republic; 3 Department of Obstetrics and Gynecology, University Hospital Brno and Medical Faculty, Masaryk University, Brno, Czech Republic; 4 Department of Internal Medicine—Hematology and Oncology, University Hospital Brno and Medical Faculty, Masaryk University, Brno, Czech Republic; 5 Department of Pathology, University Hospital Brno and Medical Faculty, Masaryk University, Brno, Czech Republic; 6 Cryo‐EM and Tomography Core Facility, Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 7 Proteomics Core facility, Central European Institute of Technology, Masaryk University, Brno, Czech Republic Introduction : Malignant ascites is a common feature in advanced ovarian cancer, particularly high‐grade serous carcinoma (HGSC) of the ovaries, fallopian tubes, and peritoneum. Ascitic fluid, often collected during cytoreductive surgery, contains abundant components from the tumour microenvironment, including extracellular vesicles (EVs). Isolating EVs from ascites via size exclusion chromatography (SEC) may improve diagnostic accuracy and offer prognostic insights by enabling detailed EV profiling. This approach provides a valuable, accessible resource for translational cancer research. Methods : EVs were isolated from five malignant ascites samples using two SEC column types: IZON qEV1 70 nm (IZON) and Immunostep EVs SEC 70 nm (Immunostep). Parameters evaluated included sample volume requirements, separation times, and EV recovery efficiency. EV fractions were analysed using Western blotting, cryo‐electron microscopy (cryo‐EM), multi‐angle dynamic light scattering (MADLS), and proteomics to assess purity, size distribution, and molecular profiles. Results : Results indicated that EVs isolated with the Immunostep column exhibited higher levels of lipoprotein contamination, as confirmed through proteomic analysis. Proteomic profiling revealed distinctive protein enrichment patterns between the two columns: IZON‐isolated EVs predominantly showed MISEV2023 protein categories 1 and 2, while category 3 proteins were more abundant in Immunostep isolates. Categories 4 and 5 showed differential enrichment across both columns, suggesting isolation of distinct EV subtypes. Size distribution analysis demonstrated that IZON columns isolated larger EVs, while Immunostep columns captured smaller particles. Notably, both columns preserved the EV corona, ensuring robust isolation of intact EVs across a broad size range. Summary/Conclusion : Optimizing EV isolation from malignant ascites could improve HGSC diagnostics and prognostics. This study outlines the unique strengths of IZON and Immunostep columns, helping researchers choose the best option for their needs. StreamLined EV isolation may boost the reliability and impact of biomarker research. Funding : This work was supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102)—funded by the European Union—Next Generation EU. CIISB, Instruct‐CZ Centre of Instruct‐ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127, is gratefully acknowledged for the financial support of the measurements at the CEITEC Proteomics Core Facility.

Automated

Presenter: Jason Dixon STEMCELL Technologies, Vancouver, Canada Introduction : Traditional extracellular vesicle (EV) isolation methods such as differential ultracentrifugation (UC) are time consuming, difficult to implement in clinical settings, and cannot be automated. We have recently developed immunomagnetic methods (EasySep) to isolate EVs using tetraspanin surface markers. We have now fully automated these methods using RoboSep‐S (up to 4 samples) and RoboSep‐16 (up to 16 samples) platforms, allowing for the scalable isolation of EVs for sample volumes of 0.5 to 8.0 mL. Methods : EVs from human plasma were labelled directly with a tetraspanin (CD9, CD63, and/or CD81)‐specific antibody cocktail. The labelled EVs were bound to magnetic particles and separated from unwanted EVs using a specialized magnet. Automated isolation using RoboSep‐S or RoboSep‐16 was performed as follows: guided by the on‐screen prompts, plasma, reagents, tips, tubes, and wash solutions were loaded onto the instrument; the instrument automatically labelled EVs with antibody cocktails and particles and transferred the sample to the magnets on the instrument deck for magnetic separations. EVs isolated manually or via the automated platforms were analysed by western blot to assess EV recovery and albumin contamination. Results : The fully automated RoboSep‐16 pan‐EV protocols recovered 89%–98% EVs ( n = 6) while the RoboSep‐S protocols recovered 93%–111% ( n = 6) EVs compared to the manual separation using EasySep magnets. The automated platforms consistently demonstrated lower albumin contamination compared to manual isolation. Both platforms were able to isolate EVs in less than 60 min. Summary/Conclusion : EasySep allows for fast and easy immunomagnetic enrichment of EVs. Fully automated EV isolation on RoboSep‐S and RoboSep‐16 platforms further reduces impurities and user variability, offering scalable EV isolation solutions for labouratories using EVs as biomarkers of disease.

Bacterial

Camila Leiva‐Sabadini 1 , Paula Saavedra 1 , Pablo Berrios 1 , Estefanía Tarifeño‐Saldivia 2 , Christina M.A.P. Schuh 3 , Sebastian Aguayo 1 1 Pontificia Universidad Católica de Chile, 2 University of Concepción, Chile, 3 Universidad del Desarrollo, Chile. Introduction : Streptococcus mutans is the main bacteria in the development and progression of dental caries, a disease that impacts billions of people globally. Recent research has highlighted important mechanobiological changes in tooth collagen as a result of glycation, yet no studies have researched whether these alterations influence the production of bacterial extracellular vesicles (bEVs) by S. mutans biofilms and promote disease. Thus, this study aimed to conduct a multimodal analysis of oral biofilm‐derived S. mutans bEVs formed on both native and glycated type‐I collagen substrates. Methods : Coverslips were coated with type‐I collagen and experimental glycation was obtained through incubation with methylglyoxal (MGO) and monitored via autofluorescence quantification for 96 h. Subsequently, S. mutans UA 159 biofilms were grown for 24 h under aerobic conditions and at 37 C on collagen‐coated substrates and bEVs from the biofilms were isolated with a combination of ultrafiltration (Amicon) and ultracentrifugation. All bEVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy, fluorescence microscopy with lypophilic dye staining, atomic force microscopy, and mass‐spectrometry‐based proteomics. Protein enrichment, abundance, and differential expression were analysed across the different bEV groups. Results : NTA and microscopy analysis demonstrated that bEVs isolated from biofilms growing on both types of collagen surfaces were smaller in size than their counterparts isolated previously from planktonic cultures. No differences in particle yield and protein concentration were found between bEVs from the native collagen and the glycated‐collagen adhered biofilms. However, proteomic analysis demonstrated different cargo signatures among groups and a decreased protein diversity for bEVs isolated from glycated collagen‐bound biofilms, as well as the differential expression of several virulence‐associated proteins in the bEV cargo. Summary/Conclusion : Collagen‐glycation by MGO induces relevant morphological and protein cargo alterations in S. mutans bEVs that have potential biological and clinical impact on cariogenic biofilm promotion, and may be an important driver for disease development in the oral cavity. Funding : ANID FONDECYT 1220804

Cellulose

K. Yoshida, A. Yokoi, Y. Nagao, M. Kitagawa, E. Asano‐Inami, H. Kajiyama Nagoya University, Japan Introduction : Extracellular vesicles (EVs) are promising non‐invasive biomarkers. We developed a novel and simple EV isolation device, the cellulose nanofibre (CNF)‐based EV sheet, designed to capture EVs in micro‐volume bio‐fluids. This study aimed to investigate the utility of the EV sheet for identifying ovarian cancer biomarkers. Methods : We collected EVs from micro‐volume ascites on multiple organ surfaces, including the liver, pelvic peritoneum, tumour, and fallopian tube, using the EV sheet during surgery. Additionally, EVs were collected from pre‐, intra‐, and postoperative body fluids (serum and urine) using the EV sheet. Small RNA sequencing was performed on 429 EV samples from ovarian cancer and benign tumour patients, followed by qRT‐PCR validation. Results : miRNA profiles of ascites‐EVs differed based on sampling locations. Compared to normal tissues, eight miRNAs were highly expressed in ovarian cancer ( p < 0.05), with these miRNAs also showing high expression on the tumour surface. Four well‐defined miRNAs (miR‐200a‐3p, miR‐200c‐3p, miR‐425‐5p, and miR‐429) were selected as candidate targets. These four candidate miRNAs were validated by qRT‐PCR to be highly expressed on the tumour surface and in pre‐ or intraoperative body fluids in ovarian cancer. Postoperatively, urinary miR‐200a‐3p and serum miR‐429 levels were downregulated. In addition, candidate EV‐miRNAs were more highly expressed on the fallopian tubes than on tumour surfaces in some cases, facilitating location‐based differential expression comparisons. Summary/Conclusion : EV‐miRNAs captured by CNF‐EV sheets could serve as novel biomarkers, potentially leading to new clinical strategies for ovarian cancer diagnosis, staging, and treatment efficacy.

Classical

Chen Wang 1 , Tobias Tertel 2 , Yiqiao Zhang 1 , Yanis Mouloud 2 , Xiaolong Liu 1 , Matthias Mack 3 , Bernd Giebel 2 , Dirk M. Hermann 1 1 Department of Neurology, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany; 2 Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany; 3 Department of Nephrology, University Hospital Regensburg, Regensburg, Germany Introduction : An appropriate understanding of the modes and sites of action is essential for the successful clinical translation of mesenchymal stromal cell‐derived small extracellular vesicles (MSC‐sEVs). Our previous studies demonstrated that MSC‐sEVs fail to synergistically provide neuroprotection when neutrophils are depleted in a mouse ischaemic stroke model. In addition to neutrophils, monocytes/macrophages may also act as key cellular targets of MSC‐sEVs, considering the close interaction between neutrophils and monocytes/macrophages and the pronounced response of monocytes/macrophages to MSC‐sEVs. Herein, we explored the effects of MSC‐sEVs in the presence and absence of monocytes/macrophages in our mouse ischaemic stroke model to identify the specific monocyte/macrophage subpopulation responsible for MSC‐sEV‐induced neuroprotection. Methods : Male C57BL/6J mice were subjected to transient middle cerebral artery occlusion (MCAO). Following reperfusion, either vehicle or MSC‐sEVs (equivalent to 2 × 10 6 MSCs), were administered intravenously. Prior to MCAO and sEV treatment, peripheral monocytes/macrophages—or, more specifically, classical Ly6C + high monocytes, were depleted using clodronate liposomes (50 mg/kg) or anti‐CCR2 antibodies (MC‐21; 20 µg), respectively. Neurological deficits and ischaemic brain injury were evaluated over 3 days post‐MCAO. Results : MSC‐sEVs consistently induced neuroprotection, reducing neurological deficits, brain infarct size, and brain oedema in MCAO mice treated with PBS‐loaded control liposomes or isotype antibodies in both study sets. Administration of clodronate liposomes induced total peripheral monocyte/macrophage depletion, which ensued with neuroprotection similar to that of MSC‐sEV treatment. Yet, when MSC‐sEVs were administered to monocyte/macrophage‐depleted MCAO mice, the neuroprotection provided by MSC‐sEVs was abolished, resulting in worsened stroke outcomes. MC‐21 antibody‐induced selective Ly6C + high monocyte depletion provided milder neuroprotection than total monocyte/macrophage depletion, reducing neurological deficits only. When MSC‐sEVs were administered to Ly6C + high monocyte‐depleted MCAO mice, neurological deficits were again worsened, and infarct size increased. Summary/Conclusion : Classical Ly6C + high monocytes are key mediators of MSC‐sEV‐induced neuroprotection in ischaemic stroke.

Combining

Rodríguez de Lope, Miriam M. 1,2 ; Sánchez‐Pajares, Ibone R. 1,2 ; Herranz, Estela 1,2 ; López‐Vázquez, Cristina M. 3 ; González‐Moro, Ainara 1,2 ; Rivera‐Tenorio, Alan 1,2 ; González‐Sanz Carlos 4,5 , Soledad Sacristán 4,5 , Chicano‐Gálvez, Eduardo 3 ; de la Cuesta, Fernando 1,2 . 1 Department of Pharmacology, School of Medicine, Universidad Autónoma de Madrid, Spain. 2 Instituto de Investigación Hospital Universitario La Paz (IdiPaz), Madrid, Spain. 3 IMIBIC Mass Spectrometry and Molecular Imaging Unit, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Reina Sofia University Hospital, University of Cordoba (UCO), Córdoba, Spain. 4 Centro de Biotecnología y Genómica de Plantas (CBGP, UPM‐INIA/CSIC), Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Madrid, Spain. 5 Departamento de Biotecnología‐Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid (UPM), Madrid, Spain. Introduction : Although the field of Plant EVs is experimenting an exponential growth, rigorous characterization complying with MISEV guidelines has not been yet implemented due to the lack of bona fide reference markers. The ISEV Plant Task Force is working on this standardisation, among other goals, and will benefit of deeper and broad proteomic dataset mining. Methods : In this work, we have paved the way for the standardization of genuine Plant EV markers, providing the most profound proteomic data so far from apoplastic washing fluid‐derived EVs, a sample enriched in genuine extracellular vesicles from plant tissue, as opposed to methods involving tissue disruption; of two reference plant species: Arabidopsis thaliana (Arath‐EVs) and Brassica oleracea (Braol‐EVs). Additionally, we have conducted an exhaustive analysis of the proteomic data available so far from genuine EVs from any plant species, evaluating current potential markers, together with those found in our proteomic analyses. Next, we analysed the presence of orthologues and their degree of conservation throughout plant taxa, as well as in 2 reference species from the animal kingdom: human and mouse. Their degree of conservation was compared with that of current EV markers used in animal‐derived EVs: CD63, CD81 and CD9. Results : Our results provide evidence supporting the potential use of the following families as Plant EV markers: fasciclin‐like arabinogalactan proteins (FLAs), patellins, germin‐like proteins, aquaporins, heat shock 70 proteins, 14‐3‐3 like proteins, Rab proteins, and vacuolar‐type ATPase complex subunits. Among these, 3 were found to be plant‐specific: FLAs, patellins and germin‐like proteins. FLA proteins, in particular FLA1, FLA2 and FLA8, were recurrently identified in previous EV datasets from different plant sources showing great abundance. Summary/Conclusion : This work contributes to addressing more rigor and defining the utility of the Plant EV markers proposed so far, also providing novel potential markers to the field. Besides, FLAs arise as a novel family of plant‐specific GPI‐anchored proteins with great potential as Plant EV markers, since they have orthologues across the different plant subdivisions and display a similar phylogenetic distribution in plants to that of currently used markers in animals. Funding : Comunidad de Madrid 2023‐5AIND‐28938 and Ministerio de Ciencia e Innovación (PID2021‐126274OB‐I00; CNS2022‐135368).

Comparing

Shan Wang, Qiubai Li Union hospital, Tongji medical college, Huazhong University of Science and Technology, China Introduction : Nanoparticles possess protein coronas that significantly affect their biological properties and in vivo behaviour. Extracellular vesicles(EVs) and liposomes are, respectively, natural and synthetic nanoparticles that resemble in size, morphology and structure. This systematic review comparatively analyses EVs and liposomes from the protein corona perspective, aiming to leverage insights from liposome corona research to enhance future exploration in the emerging field of EV corona research. Methods : A comprehensive search was conducted using Web of Science, PubMed and Scopus to identify articles studying protein coronas of EVs and liposomes from 2010 to October 2024. Queries were derived from several related published articles and the MeSH Database. A total of 37 studies were included based on the PRISMA guidelines. Results : Primary formation mechanisms of liposome corona—the Vroman effect and cooperative adsorption—partially apply to EVs, with cooperative adsorption observed in EV corona but the Vroman effect needing further validation. EVs can acquire corona proteins like ApoE and fibronectin intracellularly, showing a distinct difference in formation compared to liposome corona. Several studies identified immunoglobulins, fibrinogen, complement factors and apolipoproteins as universal corona components of EVs and liposomes, though their relative abundance differs. Albumin was consistently observed as a major component of liposome corona; however, studies reported conflicting findings on its presence in the EV corona. Biological significance of EV and liposome coronas were mainly assessed in the context of drug delivery regarding cellular uptake, circulation, and biodistribution, whereas EV corona also demonstrated potential as disease biomarkers for rheumatoid arthritis or ovarian cancer, as well as therapeutic applications in promoting tissue regeneration and wound healing. Furthermore, methods for isolating and characterizing protein coronas of EVs and liposomes were convergent. Yet, state‐of‐the‐art techniques for characterizing protein conformation, spatial arrangement, and affinity kinetics in EV corona require further exploration. Summary/Conclusion : EV corona introduces a paradigm shift in EV research but is still undergoing early development. Integrating expertise from liposome corona research with the inherent strengths of EVs as natural nanoparticles may exert synergy effects, paving the way for new advancements in EV‐based nanomedicine for diagnosis, drug delivery, and therapeutics. Funding : the National Key Research and Development Program of China (No. 2021YFA1101500)

Curvature

Presenter: Veronika Kralj‐Iglic University of Ljubljana, Slovenia Introduction : A better understanding of basic physicochemical mechanisms of formation and stability of extracellular vesicles (EVs) is highly warranted in the development of methods based on EVs. Micro‐sized membrane‐enclosed vesicles without internal structure have been theoretically described by the area‐difference elasticity model which considers the membrane as an elastic laterally isotropic continuum whereas due to the small size of the EV, its membrane is sensitive to the shape of the membrane building blocks. Methods : EV membrane was theoretically described using ensemble statistical physics methods. The membrane is represented by a surface divided into patches, each containing a very large number of constituents (inclusions) deriving from both closely coupled membrane layers. Free energy of a single inclusion takes into account curvature mismatch and in the case of anisotropic inclusions also the in‐plane orientation of the inclusion. The equilibrium shape of the membrane is obtained by minimization of the free energy obtained by summing up the contributions of all the inclusions. Minimization is performed by numerical solutions of Lagange‐Euler differential equations and Monte Carlo simulations. In the experimental part EVs were isolated from blood and tomato homogenate by differential centrifugation and observed by Scanning Electron Microscopy. Results : Based on agreement of theoretical and experimental results we propose a model in which general properties of highly curved membranes are taken into account. In narrow necks and tubular parts, the inclusions undergo orientational ordering which stabilizes the shape. The model describes a variety of EV shapes and their possible transformations. Some of them (cuboid, discoid, dumbbell, and starfish) were imaged by SEM in isolates from blood and tomato homogenate. Summary/Conclusion : The model explains the stability of nano‐sized membrane‐enclosed vesicles without internal structure which could not be explained by the area‐difference elasticity model relevant for mildly curved (micro‐sized or larger) structures. The results indicate that specific chemical composition is not a prerequisite for EV stability or its shape. The key parameter is the intrinsic shape of the inclusions, which reflects the properties of groups of molecules and their interactions. Funding : Slovenian Research Agency: J3‐3066, P3‐0388, P2/0132, P2‐0232, J2‐4427, J2‐4447, EU Horizon 2020 MSC Staff Exchange: FarmEVs 101131175.

Defective

Bushra Saleem, Shah Bahrullah Shah, Anna Roth, Elke Winterhager, Tienush Rassaf, Ulrike Hendgen‐Cotta University Hospital Essen, Germany Introduction : Heart failure (HF) following reperfused myocardial infarction (repAMI) remains a major clinical challenge, with mitochondrial dysfunction playing a central role in disease progression. Although timely reperfusion is the optimal treatment for acute myocardial infarction (AMI), the risk of HF persists due to unresolved cellular damage. Mitochondria, vital for cellular energy production, are severely affected during repAMI and their impaired clearance could exacerbate cardiac injury. While cardiac tissue‐resident macrophages are essential for removing dysfunctional mitochondria via extracellular vesicles (EVs), the mechanisms behind accumulation of damaged mitochondria remain poorly understood. This study investigates how defective communication of macrophage‐cardiomyocyte together with mitochondrial debris clearence machinery contributes to mitochondrial accumulation, autophagy blockade, and adverse cardiac remodelling in repAMI. Methods : Using electron microscopy, we identified damaged mitochondria in left ventricular biopsies from HF patients and detected defective mitochondria within EVs in the process of secretion by cardiomyocytes as well as in the cardiac interstitium. Results : In a mouse model of repAMI, we employed immunofluorescence and confocal microscopy to track the dynamics of cardiac‐resident macrophages and their longitudinal depletion, revealing a corresponding accumulation of dysfunctional mitochondria over time. Additionally, we explored the presence of molecular pathways regulating mitochondrial secretion and degradation, uncovering disruptions in proteostasis and metabolic fitness. Summary/Conclusion : Our findings from the HF patients biopsies and immunofluorescence in murine repAMI model suggest that impaired macrophage‐mediated clearance of damaged mitochondria via EVs leads to mitochondrial accumulation and left ventricular dysfunction. These results underscore the critical role of macrophage‐cardiomyocyte crosstalk across the basal lamina in maintaining cardiac function and suggest that targeting this signalling axis could offer novel therapeutic strategies to prevent heart failure post‐reperfusion. Future studies will focus on identifying specific molecular mediators of this mitochondrial debris removal to inform potential interventions.

Designing

Presenter: Kapadia Devarshi Lonza, USA Introduction : Extracellular vesicles (EVs), including exosomes, are being studied extensively and have emerged as a promising novel technology for the delivery of complex therapeutic payloads such as nucleic acids or proteins. EVs are naturally occurring within the body and have several potential advantages over other delivery vehicles such as LNPs or viral vectors, such as their ability to evade the immune system response and therefore support repeat dosing, as well as their capacity to access biological barriers and deliver a wide range of cargo to specific target tissue types given their tropism profiles. These attributes make EVs favourable candidates to engineer as drug delivery systems. Methods : Here, we discuss a plug‐and‐play platform approach to express biomolecules using engineered EVs. Therapeutic cargo may be loaded onto or within EVs via identified scaffold proteins using novel plasmid design. This concept was explored in the development of therapeutics such as exoSTING, exoASO‐STAT6 and exoIL‐12, which have completed Phase 1 clinical studies within oncology applications. Alternatively, small molecules may be linked to EVs using a proprietary linker technology. A dedicated cell line for EV production was also generated, which resulted in a clone capable of high particle titer, integrity, and purity. Results : Results are to be discussed. Summary/Conclusion : These technologies will be made available to the wider scientific community under a research license, or via Lonza's CDMO service offerings.

Detection

Vladislav Semak 1 , Manuel Hessenberger 2 , Marwa Mostageer 1 , Tanja Eichhorn 1 , Gerald J. Obermair 2 , Viktoria Weber 1 1 University for Continuing Education Krems, Krems, Austria; 2 Karl Landsteiner University of Health Sciences, Krems, Austria Introduction : Fluorescently labelled antibodies for detecting cell surface epitopes may fail to detect these epitopes on extracellular vesicles (EVs). These limitations are related to the small size and low number of antigens on EVs, as well as to self‐aggregation and batch‐to‐batch variations in the fluorochrome‐to‐protein ratio of the conjugated antibodies. We hypothesise that nanobodies (single‐domain antibodies) can improve the detection of epitopes on EVs due to their smaller molecular size and consistent labelling. Here, we present the initial results of our study, demonstrating the use of fluorescently labelled anti‐CD9 nanobodies for the detection of EVs. Methods : Anti‐CD9‐nanobodies fused with green fluorescent protein (GFP) were recombinantly expressed in E. coli and HPLC purified. Aliquots of 100 µL platelet‐derived EVs (protein conc. 1 µg/mL) stained with anti‐CD9‐GFP‐nanobody, anti‐CD41‐PC7‐antibody, and/or AnnexinV‐APC were analysed using a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA) and nanoparticle tracking analysis ZetaView TWIN NTA (Particle Metrix, Inning am Ammersee, Germany). Results : Anti‐CD9‐GFP‐nanobodies were successfully expressed and purified. Flow cytometry confirmed CD9 expression on AnxV+ CD41+ platelet‐derived EVs, as detected using the anti‐CD9‐GFP‐nanobody vs. anti‐CD33‐GFP‐nanobody as control. Nanoparticle tracking analysis (NTA) of platelet‐derived EVs stained in parallel with anti‐CD9‐GFP‐nanobody and CellMask green (CMG) in particle‐free PBS yielded 20% CD9+ particles and 57% CMG+ particles. Summary/Conclusion : We show that nanobodies are suitable for EV detection by flow cytometry and NTA. We will further optimise the flow cytometric and NTA protocols for detecting CD9+ EVs and compare anti‐CD9‐GFP‐nanobody to commercially available fluorescently labelled CD9‐antibodies. Furthermore, we will screen existing nanobody sequences for additional targets, such as more specific EV markers and test additional fluorescent proteins (e.g., mCherry). Funding : This research was funded by the Technology Fund of Lower Austria (grant number WST3‐F‐5030664/032‐2022).

Different

Ana Flores‐Chova 1 , Olga Martinez‐Arroyo 1 , Sarah Tassinari 2 , Marta Méndez 1 , Laia Garcia‐Ferran 1 , Josep Redón 1,3 , María José Forner 1,4,5 , Benedetta Bussolati 2 , Ana Ortega 1,6 , Raquel Cortes 1 1 Cardiometabolic and Renal Risk Research Group, INCLIVA Biomedical Research Institute, Valencia, Spain; 2 Department of Molecular Biotechnology and Health Science, University of Torino, Torino, Italy; 3 CIBER of Physiopathology of Obesity and Nutrition (CIBEROBN), Institute of Health Carlos III, Minister of Health, Madrid, Spain; 4 Internal Medicine, Hospital Clínico Universitario, Valencia, Spain; 5 Faculty of Medicine, Department of Medicine, University of Valencia, Valencia, Spain; 6 CIBER of Cardiovascular Diseases (CIBERCV), Institute of Health Carlos III, Minister of Health, Madrid, Spain Ana Flores‐Chova and Olga Martinez‐Arroyo are co‐first authors. Ana Ortega and Raquel Cortes are co‐senior authors . Introduction : The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, the effect of biofluid or disease on their profile remains under‐investigated. This study aimed to characterize the tetraspanin profile in several human biofluids and the effect of kidney diseases. Methods : We characterized this tetraspanin profile in plasma and urine EVs from 76 lupus patients (22 with lupus nephritis, LN), 18 patients with diabetic nephropathy (DN), and 20 healthy controls using flow cytometry (MACSPlex Kit). Additionally, single EV surface protein characterization was performed using SP‐IRIS (Exoview platform). Written informed consent was obtained, and the study was approved by the Clinical Research Committee of the University Clinic Hospital of Valencia (ref: 2021/062). Results : In plasma, CD9 showed the highest mean fluorescence intensity (MFI) across all groups; however, CD63 and CD9 had the highest MFI in urine. According to kidney disease, LN plasma samples showed increased CD81 and decreased CD9, while DN plasma had elevated CD9. In urine, LN patients had increased CD9 and CD81 but decreased CD63, whereas DN showed decreased CD9. Single‐vesicle analysis revealed no differences in particle sizes across biofluids or patient groups, but distinct tetraspanin colocalization patterns were noted. In urine, > 65% of CD63+ vesicles in LN were captured by CD63; in plasma, CD81+ and CD63+ vesicles were largely captured by CD81, especially in kidney disease groups. CD81 captured the highest proportions of triple‐ and double‐positive vesicles in both urine and plasma, with the highest levels in LN patients. Summary/Conclusion : Biofluid and kidney disease have an effect on the EV‐tetraspanin profile. This finding establishes the importance of the tetraspanin characterization before EVs isolation methodologies based on these membrane markers. In addition, further studies are necessary to analyse in more depth what origin‐cell condition tetraspanin profile in kidney disease. Funding : Carlos III Health Institute: PI18/01405 and PI21/00249 and PI23/01179, I+D+I Projects; FI20/00096 and FI22/00032, PFIS grants and Miguel Servet Contract CP22/00069; CNS2022‐136175, Ayudas consolidación Investigadora, funded by MCIN/AEI/10.13039/501100011033 and Unión Europea «Next Generation EU»/PRTR». IJC2020‐045308‐I, Juan de la Cierva Incorporación, funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future” and co‐funded with European Funds for Regional Development (FEDER).

Discovery

Manju Babu, Deepthi Ann Thomas, Anaekshi Gogoi, Arnab Datta Laboratory of Translational Neuroscience, Division of Neuroscience, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore 575018, Karnataka, India Introduction : Ischaemic stroke (IS) remains a leading cause of death and disability globally, traditionally viewed as a neuronee‐focused disorder. However, the evolving concept of the neurovascular unit, which encompasses neuronees, glial cells, and endothelial components, has broadened our understanding. Recent studies highlight the role of extracellular vesicles (EVs) as key modulators of intercellular communication, carrying proteins, lipids, and nucleic acids. Notably, EVs have the ability to cross the blood‐brain barrier, positioning them as potential biomarkers for IS. Methods : To uncover the cell‐specific role of EVs during IS, we isolated EVs from neuronees (N 2 a), astrocytes (C8‐D1A), and endothelial cells (bEnd.3) exposed to oxygen‐glucose deprivation (OGD) conditions, mimicking stroke‐induced stress. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide and lactate dehydrogenase assays were employed to determine the optimal OGD time point. EVs were isolated using polyethylene glycol‐based precipitation, adhering to MISEV 2023 guidelines. The characterization of EVs was conducted through nanoparticle tracking analysis, western blot, and transmission electron microscopy. Proteomic profiling of EVs was performed via mass spectrometry, followed by Bayesian data analysis to generate a ranked list of IS‐related EV protein biomarkers. Results : EVs were successfully isolated following 4 h of OGD, a time point validated by metabolic assays showing no significant drop in viability and no significant increase in the cytotoxicity across all cell types. Comprehensive characterization revealed distinct size distributions, protein markers, and structural features in EVs from control and OGD conditions. Proteomic analysis identified both cell‐specific and shared EV proteins under ischaemic stress, revealing distinct differential expression patterns within each cell type and between the different cell types. Bayesian analysis integrated these findings with existing datasets, producing a prioritized list of potential EV protein‐based biomarkers for IS. Summary/Conclusion : This study demonstrates that EVs released from neuronees, astrocytes, and endothelial cells under OGD conditions contain distinct proteomic signatures. These findings provide valuable insights into the role of EVs in IS pathology and highlight their potential as biomarkers for early diagnosis and therapeutic intervention. Funding : DST‐SERB (SRG/2021/001357), YU/SG/169‐2024, ICMR (BMI/11(67)/2022).

Education

Presenter: Valentina R. Minciacchi Universitätsmedizin Mainz, Germany Introduction : Within the bone marrow microenvironment (BMM), leukaemia cells have been shown to hijack some of the intercellular communication routes usually employed by benign cells of the haematopoietic system. This results in the establishment of an environment permissive to leukaemia growth and protection from the action of drugs. A major adverse communication route of leukaemia cells is the release of extracellular vesicles (EVs) that are capable of reprogramming the BMM. Highly motile and aggressive tumour cells are known to shed large oncosomes (LO). These have been shown to condition the tumour microenvironment and contribute to disease progression. Given the high deformability of leukaemia cells, this study therefore focused on the role of LO in the communication between leukaemia cells and the BMM. Methods : Leukaemia mouse models; differential and density gradient centrifugation; flow cytometry analysis and sorting; bulk RNAseq; fluorescent microscope imaging. Results : We show that LO isolated from mice with BCR‐ABL1+ B‐cell acute lymphoblastic leukaemia (B‐ALL) alter the phenotype of target primary mesenchymal stromal cells (MSC). Treatment of MSC with purified B‐ALL‐derived LO results in an increased expression of peroxisome proliferator‐activated receptor gamma (PPAR‐g) and fatty acid‐binding protein 4 (FABP4), which are indicators of increased adipocyte differentiation potential. Consistently, these MSCs show an augmented ability to differentiate into adipocytes following stimulation. Furthermore, LO‐treated MSC specifically support the growth of BCR‐ABL1+ BA/F3 B‐LL cells but do not increase proliferation of the empty vector control cells. Differentiation of LO‐treated MSC into adipocytes not only increases their ability to sustain leukaemia cell growth but also further stimulates the colony forming potential of leukaemia cells. Finally, transcriptome analysis together with targeted secretome profiling indicate that conditioned by B‐ALL‐derived LO contributes to the recently identified role of ECM remodelling and growth factor availability in B‐ALL progression. Summary/Conclusion : This study shows that LO released by B‐ALL cells contributes to the establishment of a tumour‐permissive environment which supports leukaemia progression. Funding : DFG grant MI 2669/2‐1.

Effective

Ji Hoon Ha 1 , Sung Eun Kim 1 , Jin Young Kim 1 , Jin Mo Kim 1 , Sang‐Geun Han 1 , Yeon Gyun Jung 2 , Yoon Soo Cho 2† 1 Kolmar Korea, Korea, Republic of. 2 Hanllim university, Hangang Sacred Heart Hospital, Korea, Republic of. Introduction : This study includes various strategies and results for exosome penetration into the skin. Unlike blood vessels, the skin is composed of a hard stratum corneum, the outermost layer of the skin, making it difficult for exosomes to pass through the skin barrier and reach the cell layer. Although exosomes are known to be very small, about 100 nm in size, and can pass through the gap of the skin barrier. However, exosomes are not affected by gravity because of it's size, so they cannot move spontaneously under the skin, and are greatly affected by the concentration gradient. Methods : We adjusted three methods to properly insert exosomes into the skin. Surface engineering of exosomes Hybridization of exosomes with soy lecithin by extrusion and surface modification with cations and anions. Loading support control Increasing the skin adhesion of polymers in formulations containing exosomes and evaluating the delivery ability of exosomes Opening the skin barrier with negative pressure Applying exosome formulations to octopus suction cup‐mimicking patches using biomimetic technology and evaluating them. Results : More exosomes were delivered when the surface of the exosome was positive rather than negative, and when the polymer in the polymer support solution containing exosomes increased adhesion to the skin, the concentration of exosomes increased as they moved to the skin surface, passing through the skin. In particular, when the skin barrier was relaxed by negative pressure, the space in the stratum corneum opened, accelerating the movement of exosomes. It showed excellent effects on improving wrinkles and moisturizing the skin of women aged 40‐60s. Summary/Conclusion : Exosomes have various effects on the skin, such as wrinkle improvement, whitening, and moisturizing. However, due to the structural limitations of the skin barrier and the side effects caused by the small size of exosomes, they are difficult to pass through. The various strategies presented in this study will be important indicators for increasing the efficacy of exosomes in the skin. Funding : “This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (grant number : HP23C0061)”.

Efficient

Presenter: Carlotta Schudy University Medical Center Hamburg‐Eppendorf, Hamburg, Germany Introduction : Prostate cancer (PCa) is the most prevalent malignant tumour among men. Despite early detection, diagnosis and treatment, PCa remains among the most frequent causes of cancer‐related deaths. Extracellular vesicles (EVs), with their cargo that reflects the characteristics of their parent cells, offer a potential means of communication with other cells and present various opportunities for therapeutic and diagnostic applications. A primary aim of this study is to identify an efficient, time‐saving method for exosome isolation, as the current gold‐standard method, ultracentrifugation, is time‐intensive, unsuitable for small volumes, and risks damaging exosomes. Additionally, this study explores the potential role of exosomes in the prognosis of PCa. Methods : Hormone sensitive (LNCaP) and hormone‐resistant LNCaP sublines were used. EVs were isolated by ultracentrifugation, precipitation, and a combined method. Nanoparticle tracking analysis assessed EV size and concentration, and Western blotting and PKH67 staining characterized CD9/CD63 expression and exosome uptake, respectively. LC‐MS/MS compared EV proteomics to parent cells, with validation through patient‐derived tissue slice cultures. Informed consents were obtained from all patients. Results : Our findings indicate that the combined concentration‐precipitation method achieves the highest EV yield and concentration. EVs were characterized by identifying specific markers, and confocal microscopy confirmed robust cellular uptake of PKH67‐labelled exosomes. Proteomic analysis revealed significant differences between EVs and their parent cells, with distinct proteomic profiles noted between EVs from LNCaP cells and their hormone‐resistant sublines. Several pathways were uniquely enriched in EVs. These results were further validated using EVs isolated from patient‐derived tumour slice cultures. Summary/Conclusion : Our study highlights the effectiveness of a newly established isolation method using combined concentration‐precipitation approach, revealing unique cargo and functional differences between EVs and their parental cells. These distinctions suggest that EVs may play key roles in prostate cancer progression. Ongoing mechanistic studies are now focused on uncovering how EVs influence the response to anti‐hormone therapy in PCa.

Enhancing

Sahba Mobini, Luna Pérez Troncoso, María Ujué González Instituto de Micro y Nanotecnología, IMN‐CNM, CSIC (CEI UAM+CSIC), Madrid, Spain Introduction : Stem cell transplantation shows promise for neurological conditions, but challenges like low cell survival and immune rejection limit its success. Extracellular vesicles (EV), key players in stem cell paracrine signalling, are emerging as an alternative treatment, though they struggle with low yield and variable cargo. Electrical stimulation (ES) has recently been suggested to boost stem cell regeneration capacity. We hypothesize and demonstrate that low voltage/low frequency ES could enhance the therapeutic potential of the mesenchymal stem cell (MSC) secretome, but the influence of ES parameters on MSC EV biogenesis and cargo remains unexplored. Methods : We examined how frequency in voltage‐controlled pulsed ES affects EV production and their therapeutic content. EV from immortalized MSC lines were analysed under different ES conditions. Human adipose‐derived (ASC/TERT1) and bone marrow‐derived MSCs (BM‐MSC/TERT1) were cultured in a custom electro‐bioreactor and subjected to 1‐h daily ES for 3 days at 25 mV/mm, using 1 and 500 Hz frequencies with a 25% duty cycle. ES‐treated EVs (ES‐EV) from these regimes were then tested on MSCs and differentiated neurones to assess uptake and regenerative effects. Results : EV were isolated using ultracentrifugation and characterized following ISEV guidelines, through nanoparticle tracking analysis and mass spectrometry‐based proteomics. We observed significant differences in yield and cargo of ES‐EV depending on the specific ES regimes and cell sources, up to 2 folds increase. our proteomics results show that ES‐EV enriched with distinct proteins compared control EV. Our bioassays confirmed that the effect of ES‐EV on cell proliferation in MSCs are more pronounced compared to control EV. Summary/Conclusion : These preliminary results suggest that MSC‐derived EV are manipulable through ES and that ES parameters could potentially control EV properties. ES has the potential to be used as an effective tool for enrichment of EV from stem cells for regenerative medicine. Future experiments are required for defining mechanisms and fine‐teuning. Funding : This study was supported by the Agencia Estatal de Investigación (AEI/Spain): CNS2023‐144736 funded by MCIN/AEI/10.13039/501100011033 and Unión Europea «Next Generation EU»/PRTR; PID2021‐128611OB‐I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe; “JAE Intro ICU” REF: IQM‐27. CSIC: I‐ILINK22025 funded by CSIC (I‐LINK2022); Connexion Nanomedicina CSIC

Epimedium

Presenter: Xiuping Cai The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, China (People's Republic) Introduction : Postmenopausal osteoporosis (PMOP) is a common and multiple systemic bone disease. Epimedium brevicornu Maxim (EP), the sovereign herb used for the treatment of PMOP among traditional Chinese medicines, has active ingredients and mechanisms that are still unclear. Herein, Epimedium brevicornu Maxim‐derived extracellular vesicle‐like particles (EP‐EVLPs) were naturally isolated from fresh EP leaves and strongly demonstrated the potential targeted treatment of EP‐EVLPs for PMOP. Methods : EP‐EVLP was isolated from fresh Epimedium brevicornu Maxim leaves by differential ultracentrifugation. They were characterized and identified by transmission electron microscopy (TEM), flow nano analyser, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), agarose gel electrophoresis, thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC). In vivo experiments were conducted to explore the bone‐targeting, anti‐osteoporosis activity and biocompatibility of EV‐EVLP. In vitro, alizarin red staining (ARS) and qRT‐PCR were used to detect osteogenic differentiation of hBMSCs and MC3T3‐E1. In addition, Human Umbilical Vein Endothelial Cells (HUVECs) activity and apoptosis were evaluated by flow cytometry and CCK‐8, while angiogenesis of HUVECs was verified by wound healing assay, Transwell migration assay and tube formation assay. Results : Epimedium brevicornu Maxim‐derived extracellular vesicle‐like particles (EP‐EVLPs) were naturally isolated from fresh EP leaves and strongly demonstrated the potential targeted treatment of EP‐EVLPs for PMOP. EP‐EVLPs had a cup membrane structure and contained a variety of proteins, small‐molecule compounds, lipids, and nucleic acids. In vivo EP‐EVLPs were able to accumulate and act on bone tissue to exert an anti‐osteoporotic effect without toxicity. It was interesting that the results of metabolomics and network pharmacology analyses combined with cellular experiments revealed that the anti‐osteoporotic effect of EP‐EVLPs was achieved by promoting angiogenesis and then improving the bone microenvironment in HUVECs instead of promoting osteogenic differentiation. Summary/Conclusion : In summary, our study showed that EP‐EVLPs may be the effective components of EP with the bone‐targeting activity and anti‐osteoporosis effect, which may represent a new clinically promising nano platform for the prevention and treatment of PMOP.

Evolution

Presenter: Irene Dirignani Istituto Ortopedico Rizzoli, Bologna, Italy Introduction : Ewing sarcoma (EWS), a paediatric bone and soft‐tissue cancer, is characterized by a high level of intra‐tumour heterogeneity and metastatic potential. Extracellular vesicles (EVs) released by tumour cells play a crucial role in tumour progression, metastasis and immune system suppression. MicroRNAs (miRNAs) are small non‐coding RNAs that can be packaged into EVs and influence various cellular processes. This study aims to investigate the expression of 179 miRNAs within EVs in the plasma of Ewing sarcoma patients (with informed consent) before and after neoadjuvant chemotherapy. Methods : EVs were isolated from 1 mL of frozen plasma sample from 3 healthy donors and 4 Ewing sarcoma patients pre‐ and post‐therapy using Izon qEVs single 35 nm columns. EV‐miRNAs were extracted using the Qiagen miRNeasy Kit and retrotranscribed with the Qiagen miRCURY LNA RT Kit. During reverse transcription, RNA spike‐in templates were added to monitor performance. Real‐time PCR was performed with the Qiagen miRCURY LNA Focus PCR Panel Serum/Plasma. Data analysis was performed with the Qiagen proprietary software GeneGlobe. Results : Comparative analysis revealed significant differential miRNAs expression between Ewing sarcoma patients and HDs with a fold regulation >2. Compared to HDs, 34 miRNAs were upregulated in patients before therapy, while 11 were downregulated. Similarly, 40 miRNAs were upregulated after therapy, and 19 were downregulated. Interestingly, only 2 miRNAs were differentially expressed (upregulated) in post‐therapy compared to the pre‐therapy samples (miR‐122‐5p, miR‐885‐5p). Of particular interest, miR‐223‐3p was consistently upregulated in Ewing sarcoma patients compared to healthy donors. Analysis of target pathways is underway to elucidate the potential functional implications of these differentially expressed (DE) miRNAs in Ewing sarcoma. Summary/Conclusion : We identified different DE‐miRNAs in EVs isolated from the plasma of healthy donors and Ewing sarcoma patients, before and after neoadjuvant chemotherapy. Among them, mir‐223‐3p is of interest due to its role in suppressing the immune system and inflammation. Its effect is being investigated in macrophages, which are the main immune population that infiltrate this overall cold tumour. Further cases (2 HDs and 5 patients) are being analysed and will be ready for the congress date. Funding : This research has received funding from AIRC under IG 2019—ID. 22805—P.I. Katia Scotlandi.

Ev‐Zone

Rodolphe Poupardin 1 , Martin Wolf 1,2 , Nicole Maeding 1 , Gregor Fuhrmann 3 , Dirk Strunk 1,2 1 Cell Therapy Institute, Paracelsus Medical University, Salzburg, Austria; 2 Austrian Red Cross Research, Vienna, Austria 3 Department of Biology, Friedrich‐Alexander‐University Erlangen‐Nürnberg, Erlangen, Germany Introduction : The rapidly expanding extracellular vesicle (EV) research field necessitates rigorous reporting standards and efficient tools for navigating the growing body of information. Our EV‐Zone platform (www.ev‐zone.org) addresses these challenges through two innovative applications: EV‐Checklist and EV‐PMC Search. Methods : We developed EV‐Checklist, a digital tool designed to facilitate rapid standardized reporting on EV studies during manuscript preparation. Our second tool, EV‐PMC Search, provides an EV‐focused comprehensive searchable database of constantly updated open‐access publications. We are integrating Large Language Models (LLMs) into EV‐PMC Search to enhance its capabilities for complex, context‐aware literature exploration. Results : EV‐Checklist quickly guides researchers through a comprehensive checklist covering nomenclature, source, isolation, characterization, and functional studies of EVs, compliable in under 30 min. It generates a concise two‐page PDF‐formatted table for easy integration into manuscripts and stores the information in a searchable database. The checklist is adaptable to evolving research needs, as demonstrated by the MiBlood EV checklist variant. EV‐PMC Search offers advanced search functionalities, including Boolean and regular expressions, enabling efficient exploration of full‐text or specific sections within over 23,500 open‐access EV publications. To enhance its capabilities, we are integrating Large Language Models (LLMs) into EV‐PMC Search, allowing for more nuanced and context‐aware literature exploration. This AI‐powered feature will enable researchers to perform complex semantic searches, extract relevant information from papers, and generate summaries of multiple publications. Summary/Conclusion : The open access EV‐Zone suite of tools streamLines the reporting and discovery process in EV research. EV‐Checklist complements existing registries with unmet time‐saving efficiency, and the AI‐augmented EV‐PMC Search revolutionizes literature exploration, enabling researchers to navigate the rapidly growing EV knowledge base with unparalleled precision.

Exploring

Presenter: Marie Auquière UCLouvain, Louvain‐la‐Neuve, Belgium Introduction : Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system. Various resident and peripheral immune cells are involved in MS, including microglia. Microglia can adopt different phenotypes, thus playing a dual role: ‘neuroprotective microglia’ (M2) slow MS progression, while ‘neurotoxic microglia’ (M1) favour MS progression. Extracellular vesicles (EV) have received considerable attention due to their intrinsic therapeutic properties and their use as drug delivery systems. We aim to develop a new nanomedicine‐based therapy to decrease neuroinflammation in MS by combining a bioactive lipid and the intrinsic properties of EV. Our approach is to select EV, either from resting or polarized microglia, based on their impact on microglial cell activation, and to associate them with the prostanoid 15‐deoxy‐∆‐12,14‐prostaglandin J2 (15d‐PGJ2) to dampen microglial activation. Methods : EV were isolated from resting (M0) and polarized (M1 and M2) microglia cells (BV2) and were characterized by NTA (size and concentration), NanoFCM (markers) and TEM (morphology). We compared the effect of these EV (10,000 EV/cell) on mRNA cytokine expression in microglia cells (BV2) and on primary rat mixed glial cells (MGC). Then, the bioactive lipid 15d‐PGJ2, selected for its anti‐inflammatory effect, was loaded in EV (by incubation at 37°C). The impact of 15d‐PGJ2‐loaded EV on pro‐ and anti‐inflammatory cytokines was evaluated in BV2 cells and in MGC by measuring the mRNA expression of inflammatory markers. Results : EV‐M0, EV‐M1, or EV‐M2 have no significant effect on the expression of inflammatory markers in activated BV2‐cells. EV‐M1 increased the mRNA expression of pro‐inflammatory markers in activated MGC. The 15d‐PGJ2‐loaded EV (using EV‐M0) significantly decreased mRNA expression of pro‐inflammatory markers in activated microglia. Summary/Conclusion : We successfully isolated and characterized EV from resting and polarized microglia. However, none of the tested EV significantly impacted the expression of pro‐ or anti‐inflammatory markers in microglial cells. The bioactive lipid 15d‐PGJ2 encapsulated in EV shows promise for its anti‐inflammatory effects on microglial cells and warrants further investigation.

Filaggrin

Presenter: Adrian Kobiela University of Gdańsk, Poland Introduction : Atopic dermatitis (AD) is a highly prevalent inflammatory skin disease, where insufficiency in the late epidermal protein filaggrin is frequently observed in the lesional skin of patients, with direct and indirect impact on the skin barrier quality and function. Using filaggrin knockdown keratinocytes generated by shRNA interference (shFLG), we have previously observed that filaggrin insufficiency greatly impacts cellular compartments related to exosomes/small extracellular vesicles (sEVs), but the significance of this is unknown. Methods : ShC and shFLG keratinocytes were grown in DMEM, and sEVs were isolated from keratinocyte‐conditioned media by ultracentrifugation protocol. We used mass spectrometry to detect proteins in the isolated fractions, the Alpha Fold2 tool for protein binding modelling, and functional uptake assays (with prior PKH67 labelling). Dendritic cell (DC) models were generated by monocyte differentiation in IL‐4/GM‐CSF, and LPS (a maturing factor) was added when needed; IL‐10 was used to generate tolerogenic DCs. Results : We established that filaggrin insufficiency in keratinocytes results in changes in the content of sEV‐associated proteins, and specifically, increased association of anti‐adhesive proteins (tenascin C; TNC and matrillin‐2; MATL2) with sEVs. At the functional level, such an increased abundance of TNC and MATN2 resulted in a reduction in shFLG‐sEV uptake by all DC subsets studied. We identified that these proteins belong to the fibronectin 1 network and can interact with fibronectin 1 (FN1), likely competing with sEVs adhesion to the cell surface. Accordingly, the rescue experiments with excess recombinant human rhFN1 demonstrated an increase in sEV uptake for shFLG, but not for shC‐derived sEVs. Summary/Conclusion : An increase in the anti‐adhesive proteins within the sEV corona resulting from filaggrin insufficiency results in changes in the propensity of keratinocyte‐released sEVs to undergo cellular uptake by dendritic cells. This change may affect the ability of keratinocytes to transfer antigens and innate signals and, as such, contribute to less effective pathogen clearance in AD. Funding : POIR.04.04.00‐00‐21FA/16‐00 project, carried out within the First TEAM program of the Foundation for Polish Science, co‐financed by the European Union under the European Regional Development Fund (awarded to DGO). UK Medical Research Council, NIHR Oxford Biomedical Research Centre and NIHR Clinical Research Network (GSO).

Glycan‐

Presenter: Quan Zhou The University of Queensland, Australia Introduction : Lung cancer is the leading cause of cancer‐related deaths worldwide, largely due to late‐stage diagnosis that limits treatment options. Current screening methods like low‐dose CT lack accuracy in distinguishing early‐stage malignant from benign nodules, highlighting the need for more precise, cost‐effective tools. Additionally, identifying patients likely to benefit from immunotherapy is crucial due to variable responses and possible side effects. Reliable biomarkers could guide immunotherapy decisions, improving outcomes and personalizing care. Extracellular vesicles (EVs), as valuable components in liquid biopsy, show promise for lung cancer screening and assessing immunotherapy response. Methods : We developed a multiplex microfluidic platform that integrates alternating current electrohydrodynamics and surface‐enhanced Raman scattering (SERS) nanoparticle barcodes to capture and analyse EVs for lung cancer screening and immunotherapy prediction. For screening, a specific protein and glycan panel was identified through an isogeneic cancer cell line model and subsequently applied to the SERS microfluidic platform in a clinical cohort, distinguishing between benign and malignant nodules. For immunotherapy prediction, bioinformatic analyses identified glycosylation‐associated genes enriched in lung cancer, correlating with immune infiltration. Knockdown experiments in lung cancer cell lines allowed for lectin microarray analysis of glycan alterations in EVs, with functional assays elucidating their immunomodulatory roles. The glycan signature was validated on the SERS microfluidic platform in lung cancer patients with variable immunotherapy outcomes. Results : The SERS microfluidic platform demonstrated high specificity and sensitivity in distinguishing early‐stage malignant lung nodules from benign nodules using both protein and glycan EV panels, with AUC values above 0.85. Distinct EV glycan profiles between late‐stage and early‐stage lung cancer patients indicated progressive alterations in glycosylation associated with tumour progression and immune evasion. Furthermore, alternations in EV glycan profiles were identified in knockdown experiments, which were found to correlate with outcomes in lung cancer patients receiving immunotherapy by the SERS microfluidic platform. Summary/Conclusion : This SERS microfluidic platform offers a robust, minimally invasive approach for lung cancer screening and immunotherapy response prediction. By leveraging EV‐based protein and glycan panels, this platform provides an accurate and efficient alternative to traditional methods, addressing limitations of current screening techniques and advancing personalized immunotherapy strategies in lung cancer.

Hnrnpa2B1

Ilona Barbara Csordás 1,2 , Tamás Visnovitz 3 , Christophe Badie 4 , Katalin Lumniczky 1 1 Unit of Radiation Medicine, Department of Radiobiology and Radiohygiene, National Public Health Centre, Budapest, Hungary; 2 Doctoral School of Pathological Sciences, Semmelweis University; Budapest, Hungary; 3 Semmelweis University, Faculty of Medicine, Department of Genetics, Cell‐ and Immunobiology; Budapest, Hungary; 4 Centre for Radiation, Chemical and Environmental Hazards, UK Health Security Agency, Chilton, Didcot, UK Introduction : Bone marrow (BM) is the site of haematopoiesis, where proper function relies on interactions between BM stem and stromal cells. Extracellular vesicles (EVs) play a crucial role in this interaction by delivering bioactive cargo, such as microRNAs (miRNAs). The EV‐miRNA cargo is determined by the status of the donor cell; thus, cellular stressors, such as ionizing radiation (IR), modify its composition. In vivo, we demonstrated that IR‐modified EVs transfer radiation‐induced damage to non‐irradiated cells, with miRNAs serving as important regulators. To better understand the role of EVs in the BM microenvironment, we investigated BM‐ and EV‐miRNA cargo and EV‐miRNA loading by RNA‐binding proteins (RBPS) after IR exposure. Methods : Nine‐ to twelve‐week‐old male CBA‐mice were irradiated, and their BM was collected 24 h after IR. BM‐EVs were isolated with the precipitation method and EV markers were detected with western blot (WB). EV and BM miRNA profiles were analysed with NCoutner and/or qPCR, then clustered into BM‐IR response‐relevant pathways: leukaemia/senescence/inflammation, and a sequence motif search was also conducted. Based on identified miRNA motifs, RBPs were selected for concentration (WB) and cellular localization (confocal microscopy) analysis. Predicted miRNA‐RBP interactions were validated in vivo by immunoprecipitation. Results : Seventy‐one percent of the EV‐miRNAs associated with leukaemia/senescence/inflammation were linked to three RBPs: Anxa2, hnRNPQ and hnNRPA2b1. Among them, hnRNPQ and hnRNPA2b1 expression altered upon IR. hnRNPA2b1 exhibited upregulation in the BM but downregulation in BM‐EVs; the miRNAs carrying recognition motifs of hnRNPA2b1 mirrored the quantitative changes of their binding partner in EVs. We established the specific binding of miRNAs to hnRNPA2B1 through their binding motifs, as miRNAs lacking these motifs did not co‐precipitate with the protein. Summary/Conclusion : The miRNA content of BM‐EVs following IR exposure results from a selective sorting and packageing mechanism. One observed mechanism through which IR influences this process is by deregulating RBPs, particularly hnRNPA2b1. Selectively packaged EV‐miRNAs could be involved in the development of late radiation effects, such as leukaemia development in the BM. Funding : Funding: This research was supported by the European Union's Euratom research and training programme 2014–2018 CONCERT under the grant agreement No. 662287 and the Euratom research and training programme 2021–2025 PIANOFORTE under the grant agreement No. 101061037.

Improving

Presenter: Kuan‐Der Lee Taichung Veterans General Hospital, Taichung, Taiwan (Republic of China) Introduction : Recent advances in mesenchymal stem cells (MSC)‐secreted extracellular vesicles (EVs) provide another innovative treatment option for various diseases. Hypoxia significantly affects many properties of MSCs, including cell viability, proliferation, differentiation, and cell migration. Changes in cell culture conditions can affect the release of different exosomes. Therefore, this study explores the effect and therapeutic effect of hypoxia on clinical‐grade mesenchymal stem cell and exosomes. Methods : hUC‐MSC‐EVs were isolated and expanded using animal‐free/serum‐free medium from Wharton's jelly from healthy human umbilical cord. EVs isolated from normoxic (21% O 2 , 5% CO 2 ) or hypoxic (2% O 2 , 5% CO 2 ) cultured MSCs were sorted and concentrated by membrane‐based tangential flow filtration. We evaluate the size and distribution of nanoparticles through nanoparticle tracking analysis (NTA) and evaluate morphology through transmission electron microscopy. Immunophenotyping of two EV populations was assayed by flow cytometry (Miltenyi Biotec, Germany). Results : hUC‐MSC‐EVs were enriched by hypoxia in mean expression values for CD9 (EV‐NormO 2 89.12% ± 4.04%; EV‐2% O 2 96.76% ± 1.02%), CD63 (EV‐NormO 2 97.63% ± 1.49%; EV‐2% O 2 99.04% ± 1.23%), CD81 (EV‐NormO 2 84.88% ± 19.77%; EV‐2% O 2 94.75% ± 4.31%), and particles concentration (EV‐NormO 2 6.41 × 10 8 ± 3.47 × 10 8 /10 5 cells/mL; EV‐2% O 2 1.75 × 10 9 ± 3.34 × 10 8 /10 5 cells/mL). hUC‐MSC‐EVs cultured in hypoxia expressed much higher levels of the surface markers CD49e than those cultured in normoxia. MicroRNA‐seq showed EVs had high expression of miR‐151‐3p and miR‐21‐5p. Herein, we will present these hUC‐MSCs have demonstrated clinical improvement in children with HIE. Summary/Conclusion : hUC‐MSCs cultured under hypoxic conditions secrete EVs enriched in the extracellular matrix protein CD49e and miRNAs and may improve children with HIE. Funding : This study was supported by the Intramural research grant from Taichung Veterans General Hospital, Taichung, Taiwan.

Inclusion

Presenter: Tina Vida Plavec Jožef Stefan Institute, Ljubljana, Slovenia Introduction : Lactococcus cremoris is a gram‐positive bacterium and represents a well‐known system for recombinant protein expression. Gram‐positive bacterial extracellular vesicles (EVs) have recently gained more attention and have been hypothesized to have a possible physiological role. The bacteria producing recombinant proteins could be used as cell factories for the incorporation of functional and bioactive proteins into the EVs. In this study, we analysed the effect of the expression of different recombinant proteins in L. cremoris on the formation of EVs and their protein content. Methods : EVs were isolated following a standard protocol using an ultracentrifuge at 130,000 × g for 2 h, followed by removal of the supernatant and collection of the EVs. EVs were characterised with transmission electron microscopy (TEM), flow cytometry, polydispersity index measurement and proteomic analysis. Results : We successfully isolated EVs from recombinant L. cremoris , and characterized them by TEM and flow cytometry, which revealed differences in both the quantity and heterogeneity of the secreted EVs, depending on the recombinant protein that was expressed in L. cremoris . The size of the isolated EVs was within the expected nano‐scale range, mostly 50–200 nm. The presence and quantity of individual recombinant proteins inside the vesicle were confirmed by proteomic analysis. Summary/Conclusion : We have shown that recombinant L. cremoris can be used to successfully produce EVs. The recombinant proteins expressed by L. cremoris were detected in the EVs‐containing ultracentrifuged pellets. Our study therefore highlights the potential of using L. cremoris for the production of EVs and for loading them with recombinant proteins. Funding : ARRS N3‐0184 Small protein blockers of IL‐23/IL‐17 axis as intestinal inflammation inhibitors secreted by probiotic bacteria. ARRS P4‐0127 Pharmaceutical biotechnology: Science for health.

Influence

Presenter: Lucas F. B. Nogueira University of São Paulo, Brazil Introduction : Extracellular vesicles (EVs) produced by Aspergillus fumigatus play a pivotal role in fungal communication and host‐pathogen interactions, especially during pulmonary infections. These vesicles acquire a protein corona when exposed to biological fluids, which significantly alters their surface properties and biological behaviour. Understanding the impact of this protein corona on EV functions, particularly in interactions with immune and fungal cells, is critical for elucidating their role in pathogenesis. Methods : EVs were isolated from Aspergillus fumigatus cultures and subjected to a high ionic strength solution followed by ultracentrifugation to remove the protein corona. Nanoparticle tracking analysis (NTA) was used to assess the size distribution of the EVs before and after protein corona removal. Scanning electron microscopy (SEM) was employed to observe changes in morphology, and zeta potential measurements were taken to evaluate surface charge alterations. Protein content and composition were analysed through BCA assay and SDS‐PAGE. Functional assays were conducted to examine the uptake of EVs by macrophages and their interaction with fungal cells. Results : NTA revealed that the hydrodynamic diameter of EVs was initially around 130 nm, but after removal of the protein corona, a new distribution appeared, with vesicles measuring greater than 200 nm. SEM images confirmed that EV morphology was largely maintained, though subtle shape changes were observed. The zeta potential shifted from ‐12.4 mV to ‐2.3 mV after corona removal, indicating substantial changes in surface protein composition. Protein quantification and SDS‐PAGE analysis showed significant differences between native and corona‐free EVs. Functionally, EVs without their protein corona exhibited reduced uptake by macrophages, suggesting the corona's importance in immune recognition. In contrast, corona‐free EVs displayed altered interactions with fungal cells, potentially influencing biofilm formation and fungal virulence. Summary/Conclusion : The protein corona significantly modulates the biological activity of Aspergillus fumigatus EVs, influencing both immune recognition and fungal interactions. Its removal resulted in altered EV size, surface charge, and reduced macrophage uptake, while enhancing interactions with fungal cells. These findings highlight the role of the protein corona in immune evasion and fungal pathogenesis, offering potential avenues for developing therapeutic strategies targeting these processes in Aspergillus fumigatus infections. Funding : This work was supported by FAPESP (2024/00403‐2).

Isolation

Victoria O. Shender 1 , Polina V. Shnaider 1 , Arseniy Lashkin 1 , Alexandra Shirikova Zlenko 3 , Olga I. Aleshikova 2 , Elena Khomyakova 3 1 Lopukhin Federal Research and Clinical Center of Physical‐Chemical Medicine of FMBA, Moscow, Russian Federation; 2 National Medical Scientific Centre of Obstetrics, Gynaecology and Perinatal Medicine named after V.I. Kulakov, Moscow, Russian Federation; Exosome Analytics, Paris, France Introduction : EVs, being very stable and abundant in body fluids, hold great promise in diagnostics. Clinical applications require simple, efficient and cost‐effective approaches for EV isolation. Ultracentrifugation, the gold standard for EV isolation with close to 100% yield under properly selected centrifugation conditions, is not suitable for clinical applications because it is labour‐intensive, requires expensive equipment and does not allow for simultaneous isolation of EVs from large numbers of clinical samples. Ultrafiltration is not suitable for EV isolation from cell cultures or body fluids with high protein/lipoprotein content, such as cell cultures supplied with FBS, serum, plasma, ascites and in most cases from urine of patients with advanced cancer. Polymer‐based kits and SEC columns are expensive and both have important drawbacks. Express kits require an additional purification the viscous polymer, and SEC columns are unable to purify EVs from most lipoproteins. Thus, new approaches to EV isolation are needed. Methods : Based on the theoretical approach we have developed in Livshits et al., doi: 10.1038/srep21447, we adapted the sEV common ultracentrifugation protocol to a conventional benchtop centrifuge. According to our calculations, sEV isolation takes 3–4.5 h depending on the RCF (20,000 × g –30,000 × g ) and the size of the tube. Experimental evidence for the efficiency of isolation at 25,000 × g on a benchtop centrifuge was demonstrated using SKOV‐3 cell culture, serum of healthy people, and ascites and urine from ovarian cancer patients. EV size distribution was analysed by NTA. The expression of CD9, CD81, and CD117 membrane proteins on sEVs isolated by 25,000 × g centrifugation on a benchtop centrifuge and conventional isolation methods were analysed by bead‐based ELISA and bead‐based FACS. Results : We compared the sEV isolation yield by centrifugation at 25,000 × g on a benchtop centrifuge with that of 100,000 × g ultracentrifugation and 100 kDa ultrafiltration. Our experimental data proved that sEV isolation on benchtop centrifuge has a yield similar to or even better than 100,000 × g ultracentrifugation and 100 kDa ultrafiltration. Summary/Conclusion : sEVs isolation on benchtop centrifuge is high yield and cost‐effective method that can be used in research and clinics.

Kras/Lkb1

Presenter: Cristina Borzi IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy Introduction : KRAS/LKB1 co‐mutated non‐small cell lung cancers (KL NSCLCs) show a very aggressive clinical behaviour. They present a peculiar tumour microenvironment, with pro‐inflammatory features. The role of extracellular vesicles (EVs) in tumour‐stroma communication has been extensively demonstrated, as well as their contribution to inflammation, immunosuppression and tumour dissemination. Fibroblasts are the major component of the stroma. They are characterized by phenotypic and functional heterogeneity; however, the molecular mechanisms responsible for the modulation of their phenotype are largely unknown. Here, we investigated how EVs released by KL NSCLCs could affect fibroblast phenotype reshaping. Methods : EVs were isolated by ultracentrifuge from conditioned medium of the KRASWT/LKB1WT H1299 NSCLC cell line (WT‐EVs) and the KRASG12C/LKB1DEL isogenic clone (KL‐EVs). EVs were characterized by NTA, TEM, and EVs‐associated markers (flow cytometry – FC, western blot). Normal lung fibroblasts (CCD‐19Lu) and immortalized cancer‐associated fibroblasts (CAF154hTERT) were used for functional studies. EVs uptake was evaluated by FC and confocal microscopy. Fibroblast phenotype was assessed by gene expression analysis and ELISA. Patient‐Derived Xenografts (PDXs) were established by subcutaneous injection of WT and KL NSCLC tumour samples in SCID mice. The cBioPortal open‐source tool was used for in silico analyses. Results : KRAS/LKB1 co‐mutation significantly increased EVs secretion by H1299. However, KL‐EVs had lower protein content than WT‐EVs. EVs size distribution was not affected by the mutational status. In vitro data showed that uptake of EVs by fibroblasts is comparable between WT‐ and KL‐EVs, with 100% of EV+ CCD‐19Lu at 4 h of EVs exposure. We revealed that KL‐EVs are able to induce an inflammatory phenotype in normal and immortalized cancer associated fibroblast by upregulating the expression and secretion of IL‐6. KL‐EV long‐term exposure modulates other immune‐related genes, including CCL2, CXCL1, CXCL3, and IL‐8. The increase of IL‐6 expression level in KL compared to WT tumour samples was confirmed in vivo in PDXs and in silico analyses (CPTAC series). Summary/Conclusion : KRAS/LKB1 co‐mutation impacts EVs release and phenotype. Our results support the hypothesis of an active role of EVs in switching fibroblasts toward an inflammatory‐like subtype in KL NSCLC. Further studies are needed to fully elucidate the molecular mechanisms behind this modulation. Funding : Italian Association for Cancer Research (AIRC)

Lipidomic

Presenter: Abhay Mishra All India Institute of Medical Sciences, New Delhi, India Introduction : Breast cancer remains a leading cause of female mortality, primarily due to delayed detection. Current diagnostic methods lack specificity for early‐stage identification, necessitating novel approaches. Small extracellular vesicles (sEVs) present in plasma exhibit lipidomic signatures with potential as biomarkers. Fourier‐transform infrared (FTIR) spectroscopy provides a label‐free method for analysing these lipid profiles. Methods : Plasma samples from breast cancer patients (BrCa, n = 74) and healthy controls (HC, n = 30) were used for sEV isolation. Characterization employed transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting for markers (CD9, CD81, CD63, TSG101). FTIR spectra of sEVs were analysed to identify lipid and other biomolecular changes, employing spectral ratio calculations (e.g., I3015/I2929). Principal component analysis (PCA) and receiver operating characteristic (ROC) curve analyses assessed the diagnostic potential. Results : Significant lipid alterations were identified in BrCa sEVs, including elevated I3015/I2929 and I2960/I2929 ratios. PCA differentiated BrCa from HC with 94.5% explained variance, supported by ROC analysis where I2960/I2929 achieved 100% sensitivity and specificity (AUC = 1.000). Additionally, spectral markers of proteins, nucleic acids, and carbohydrates revealed distinct biomolecular signatures in BrCa sEVs compared to controls. Summary/Conclusion : This study demonstrates that FTIR spectroscopy of sEVs can discern breast cancer‐associated lipidomic changes with high precision. Spectroscopic lipid‐to‐lipid ratios, particularly I2960/I2929, emerged as robust markers for early detection. These findings highlight FTIR‐based sEV profiling as a promising non‐invasive diagnostic tool, paving the way for enhanced clinical outcomes through early breast cancer detection. Funding : This work was supported by the Department of Bio‐technology, India, through JRF funding‐ DBT/2021‐22/THSTI/1601.

Lysosomal

Presenter: Reena Kartha University of Minnesota, Minneapolis, Minnesota, USA Introduction : Lysosomal diseases occur due to genetic defects in enzymes or cofactors required to hydrolyse cellular waste products. The ensuing lysosomal dysfunction leads to accumulation of toxic substrates within the cells, which can trigger a multitude of cellular anomalies, including oxidative stress, mitochondrial dysfunction, inflammation, and so forth. Gaucher disease (GD) is one such disease caused by defects in the glucocerebrosidase enzyme encoded by the GBA1 gene. Over 300 different mutations have been identified, each affecting the enzyme activity to varying extents. This leads to glycosphingolipid accumulation triggering multisystem symptoms including anaemia, thrombocytopenia, hepatosplenomegaly and bone issues. Notably, mutations in the GBA1 gene have emerged as a major risk factor for Parkinson's disease, although the exact mechanism underlying this is not clear. Lysosomes are crucial in extracellular vesicle (EV) biogenesis and can influence its cargo by participating in the regulation of cell sorting mechanisms. Alterations in lysosomal function, especially in the context of diseases, can therefore directly impact the characteristics of EVs, highlighting their potential as biomarkers for these diseases. Methods : Patient‐derived GD fibroblasts harbouring either mild (N370S/84GG) or severe (L444P/S364T) variants were compared against a normal human fibroblast line (NHDF). These were characterized as mild or severe based on the percentage of glucocerebrosidase enzyme activity. EVs were isolated from three independent passages of each cell line using the ultracentrifugation method. The collected vesicles were analysed using the ZetaView next generation nanoparticle tracking analyser, to determine EV concentration and size distribution. Results : We observed significant differences in EV sizes between normal and GD cells. Specifically, both GD cells showed lower frequency of EV between 100 and 200 nm size compared to normal cells. However, only milder GD cells showed higher concentrations of EVs above 200 nm. Notably, EV size did not vary between different passages of normal cell lines that served as a control. Summary/Conclusion : Our initial findings indicate EV size and potentially EV cargo to be altered by lysosomal dysfunction and to be dependent on the severity of the defect. We are further investigating the EV biogenesis pathway genes in cells and examining the alterations in the EV cargo. Funding : Department research funds.

Measuring

Panagiotis Papoutsoglou 1 , Alain Joliot 2 , Louise Merle 2 , Lorena Martin‐Jaular 2,3 , Anna Almeida 1 , Clotilde Théry 2,3 , Antonin Morillon 1 1 ncRNA, Epigenetic and Genome Fluidity, CNRS UMR3244, Sorbonne University, PSL University, Institut Curie, Centre de Recherche, Paris, France; 2 INSERM U932, Institut Curie Centre de Recherche, PSL Research University, Paris, France; 3 Institut Curie Centre de Recherche, CurieCoreTech Extracellular Vesicles, Paris, France Introduction : Extracellular vesicles (EVs) contain heterogeneous nucleic acid content. Their role as transporters of biomolecules is well‐established. However, there is a lack of knowledge regarding the dynamics of EV RNA delivery and whether a specific transcript can preserve and exert its functionality during its intercellular travel. Here, we aim to analyse the EV RNA content of donor triple negative breast cancer (TNBC) cells, and the dynamics of its transfer into recipient monocytes. The ultimate goal is to identify functional long RNAs, transferred via EVs, that impact immune cell physiology. Methods : To study EV‐mediated cargo transfer from donor (MDA‐MB‐231) to recipient (THP‐1) cells, we first utilize a split nanoluciferase‐based (Split NanoLuc) technology to decipher the timing of EV cargo cytosolic release. We constructed donor cells expressing high‐affinity small subunit (HiBiT) tagged to a lipid‐binding domain for targeted localization to the EV inner membrane. Recipient cells expressing the large subunit (LgBiT) were also established. Small EVs were purified from cell‐conditioned medium using size exclusion chromatography (SEC) (qEV2 column, 35 nm) and used for time course uptake experiments, whereby EV cytosolic release was estimated by luciferase assays in live cells. Inhibition of EV uptake was performed by pre‐treating recipient cells with an endocytosis inhibitor. EV RNA staining was performed using SYTO RNASelect green fluorescent cell stain, and the efficiency of staining was evaluated by nano‐flow cytometry. Results : Purified MDA‐MB‐231 HiBiT‐EVs were positive for CD63, CD9, syntenin‐1, Alix and Hsc‐70 and enriched for HiBiT, compared to parental cells. The presence of HiBiT in EVs was also verified by increased NanoLuc activity in the presence of exogenous LgBiT and under mild lysis conditions. Cytosolic release into THP‐1 was observed after 1 h and peaked after 24 h of incubation with EVs. An endocytosis inhibitor greatly reduced EV uptake. Fluorescently stained EV RNA was detected in approximately 55% of isolated EVs and used for complementary EV uptake experiments. Summary/Conclusion : In summary, we demonstrate that the timing of EV uptake/cytosolic release can be efficiently assessed using Split NanoLuc technology. Moreover, TNBC EVs are taken up by monocytes mainly via endocytosis. Funding : Institut National du Cancer (INCA), grant numbers PLBIO21‐100 and PLBIO22‐222.

Mechanism

Presenter: Marc Liébana CIC bioGUNE, Derio, Spain Introduction : Nowadays more personalized and efficient cancer treatment approaches are pursued. Nanoencapsulation systems allow decreasing drug dosages and better control of the dose‐response. Extracellular vesicles (EVs) are membranous vesicles released by virtually all cells and represent a plausible biocompatible option for drug encapsulation. Mesenchymal stem cells (MSCs) represent a promising EV source due to their low immunogenicity and tumour homing ability, among other properties. Artificial systems are used with this aim, but the immunocompatibility and specificity problems that they usually exhibit lead to the exploration of alternative systems as MSC‐EVs. Another challenge for nanotherapeutic agents is to reach their targets, needing to trespass physiological barriers to deliver their effect in most of the cases. The objective of this work is the study of the use of EVs as pharmacological nanovehicles in cancer. For this purpose, biological barriers have been evaluated due to their relevance in the clinical context, and different cell lines, including representative cells from specific malignancies, have been tested for their capacity to capture MSC‐EVs. Methods : UC‐MSCs were used as EV producer cells. DiO labelling of MSCs was assessed by fluorescence microscopy and flow cytometry at periodic timepoints. EV isolation was conducted by differential ultracentrifugation. MSC‐EVs potential to cross biological barriers was evaluated in transwell co‐culture models. EVs effect on the biological barrier was assessed in terms of cytotoxicity (LDH assay) and barrier permeability (LY). Finally, MSC‐EV capture by malignant and healthy cell lines was assessed via fluorescence microscopy and flow cytometry. Results : MSCs can be labelled with the lipophilic dye Vybrant DiO for multiple days, and their derived EVs retain this dye. These EVs can be tracked across a transwell model, and they can pass through biological barriers with no significant effect on the barriers themselves. Moreover, MSC‐derived EVs display homing properties towards recipient cell lines. Summary/Conclusion : DiO‐EVs derived from MSCs could pass biological barriers and become differentially captured by distinct cell lines, thus offering a promising opportunity for their use as anticancer drug‐containing nano‐vehicles. Funding : Spanish Ministry of Universities—FPU21/05332 MCIN & ERDF/FEDER—PID2021‐125104OB‐I00 EU's Horizon Twinning programme ‐ 101079264 (EVCA) EU's Horizon Staying Healthy Programme ‐ 101095679 (halt‐RONIN)

Menstrual

Raminta Vaiciuleviciute 1 , Kieran Brennan 2 , Ilona Uzieliene 1 , Jolita Pachaleva 1 , Zaneta Kasilovskiene 3 , Lina Piesiniene 4 , Daiva Bironaite 1 , Margaret Mc Gee 2 , Eiva Bernotiene 1,5 1 Innovative Medicine Centre, Lithuania, 2 University College Dublin, Ireland, 3 Maxmeda, Lithuania, 4 Nanodiagnostika, Lithuania, 5 VilniusTech, Lithuania Introduction : Several biomolecules have been previously associated with unexplained infertility (uIF) in blood and uterine samples, immune cells and their secreted factors, endometrial tissue, menstrual blood, serum, and stromal cells, however they do not comprehensively represent different uIF endotypes and their isolation and detection involves invasive diagnostic methods and lacks precision. This ex‐vivo study was performed to examine the potential of menstrual blood extracellular vesicles (EVs) as source of non‐invasive biomarkers of uIF. Methods : Menstrual blood was collected on cycle day 2 from 9 fertile volunteers and 8 women with uIF. Menstrual blood serum (MBS) EVs were isolated from fertile and uIF female menstrual blood serum using Iodixanol Density Gradient Centrifugation and quantified by flow cytometry. EVs were characterized according to MISEV2023 guidelines: EV isolation was confirmed by Transmission Electron Microscopy; CD9, CD63, CD81 and CD147 EV markers were detected by flow cytometry, intracellular EV marker TSG101 and contamination markers albumin, APOA1, APOB and APOE were analysed by Western blot. Proteome analysis was performed using mass spectrometry, and data was analysed using bioinformatic tools. Results : Comprehensive proteomic analysis of MBS EVs and EV‐depleted MBS showed significant changes in the uIF proteome, mostly affecting cell adhesion, immune response, apoptosis, response to oxidative stress and lipid metabolism. These processes were previously linked to pathologies of the female reproductive system but never investigated in uIF and were used to stratify patients into distinct molecular endotypes. AUC analysis was used to determine the optimum set of biomarkers for each of the uIF molecular endotypes. Summary/Conclusion : This demonstrates MBS EVs carry key molecular cargo with potential to identify valuable information about alterations in the endometrium that may be associated with female infertility pathogenesis. Overall this study provides new insights into uIF that could facilitate personalised treatment approaches.

Messenger

Alice Boussaroque 1 , Samuel Holland 1 , Zongyu Gao 1 , Serena Qiao 1 , Mary Woodward 2 and Ronelle Roth 1 1 University of Oxford, Oxford, UK. 2 Vrije Universiteit Amsterdam, The Netherlands Introduction : The importance of extracellular vesicles (EVs) in modulating beneficial plant‐fungal associations such as the arbuscular mycorrhizal (AM) symbiosis remain largely unknown. Conversely, in plant pathogen interactions, EVs mediate cross‐kingdom communication by transporting defence‐related molecular cargoes such as mRNA, stress related proteins and small RNAs (sRNA) that attenuate microbial virulence. AM symbiosis is an ancient mutualistic interaction that co‐evolved with land plants around 450MYA and today persists in over 70% of terrestrial plants. Plants benefit nutritionally from the symbiosis by obtaining essential minerals such as phosphate from the fungus. In return plants provide the fungus with organic carbon. As a fatty acid auxotroph the fungus relies entirely on host lipids to complete its life cycle. Symbiotic exchange occurs in highly branched, short‐lived fungal arbuscules that form in roots and is underpinned by an intimately regulated plant‐fungal dialogue. Initial evidence that EVs might function in AM symbiosis came from transmission electron microscopy that showed EVs accumulate in the shared apoplast between arbuscule and host membranes. Here we present compelling evidence that EV encapsulated host sRNAs modulate symbiotic function. Methods : Using differential ultracentrifugation, EVs were purified from apoplastic wash fluids from AM fungal colonised and mock inoculated roots followed by LC‐MS/MS and RNAseq analyses. The Roth lab uses a combination of reverse genetics, molecular, biochemical and imaging approaches to analyse EV cargo and gene expression and to determine their role in AM symbiosis. Results : Protein and small RNA plant cargo are enriched in EVs compared to whole cell lysate. Nuclease protection assays show host sRNAs are encapsulated within EVs. Temporal analysis of sRNA target gene expression coincide with mature arbuscules and the expression of the EV biomarker Tetraspanin8. sRNAs targets include fungal genes that regulate fungal lipid utilisation. Summary/Conclusion : Our results provide evidence for plant‐to‐fungal cross‐kingdom RNA interference during AM symbiosis, likely mediated by EVs. We observed attenuation of sRNA target genes corresponding to lipid utilisation that coincide with arbuscule maturation. This points to a dynamic and highly regulated role for an EV‐encapsulated sRNA in modulating symbiotic function by rationing lipid utilisation to curb AM fungal growth. Funding : Royal Society URF. BBSRC DTP. Diamond Light Source

Metabolic

C. Guder 1 , L. Englert 2,3 , F. Gasparin 2,3 , E. Dorado 4 , M. A. Pleitez, B. Wollenberg 1 , Z. Takats 3,5 , V. Ntziachristos 2,3 , M. N. Theodoraki 1,6 V. Ntziachristos and M. N. Theodoraki are co‐senior authors. 1 Department of ENT, TUM University Clinic, Germany, 2 Institute of Biological and Medical Imaging, Helmholtz Zentrum München, Germany, 3 Chair of Biological Imaging at the Central Institute for Translational Cancer Research (TranslaTUM), School of Medicine and Health, Technical University of Munich, Germany, 4 Department of Metabolism, Digestion and Reproduction, Imperial College London, United Kingdom, 5 Department of Immunomedicine, University of Regensburg, Germany, 6 Department of ENT, head and neck, University of Ulm, Germany Introduction : Small extracellular vesicles (sEV) are emitted by all cells into the surrounding extracellular environment, facilitating communication between cells. Tumour‐derived‐EVs (TEX) are frequently found in the plasma and saliva of cancer patients. There are already known mechanisms of TEX weakening the anti‐tumour immune response mediated by proteins (e.g. PDL‐1 and CD44v3) and by RNA (e.g. miRNA). Studies additionally indicate a connection between TEX and changes in metabolism. Nevertheless, the exact impact of TEX on the tumour microenvironment remains unknown. This study investigates the interaction between TEX and protein, carbohydrate, and lipid metabolism of recipient cells. Methods : sEVs were isolated from the plasma of head and neck squamous cell carcinoma (HNSCC) patients by size exclusion chromatography. The isolated TEX were characterized by nanoparticle‐tracking‐analysis, western blot (CD63, CD9, TSG101 and CD81) and transmission electron microscopy. To investigate the interaction between sEV and protein, carbohydrate, and lipid metabolism, different cell types (e.g. UD‐SCC‐5, HUVEC) were treated with TEX over different time periods. Label‐free mid‐infrared optoacoustic microscopy (MiROM) and multiphoton microscopy were used to detect changes in metabolism. Additionally, metabolic changes were validated by immune assays and ‐omics analyses. Results : Label‐free metabolic imaging indicates early changes in lipid metabolism, but not in metabolism related to proteins and carbohydrates, after treatment of UD‐SSC‐5 with TEX. For HUVEC cells no significant differences in the metabolism after co‐incubation with TEX of HNSCC patients were detected. Further immune assays and ‐omics results support these observations. Changes in proteins were visible at later time points. Summary/Conclusion : The results of this study support the hypothesis of the connection between TEX and changes in metabolism. While the metabolites of nonmalignant HUVEC cells remained unaltered after treatment with TEX of HNSCC patients, the lipid metabolism of HNSCC cells was altered.

Microglia

Miguel Quiralte Pulido, Isabela Albuja Ron, Irene Bertolini Molecular and Oncogenesis Program, The Wistar Institute, Philadelphia Abstract unavailable

Micrornas

Presenter: Kazuki Takahashi Tokyo Medical University, Japan Introduction : Abdominal aortic aneurysm (AAA) is a life‐threatening disease due to the risk of aortic rupture. The risk factors of AAA have been identified, including ageing, male sex, hypertension, dyslipidemia, and smoking, and it shares a risk factor with atherosclerosis. Although diabetes mellitus (DM) is one of the main risk factors for cardiovascular disease, epidemiological and basic studies have described its potential protective role on the prevalence and incidence of AAA. Multiple pathological processes and molecules contribute to AAA formation, including degradation of the extracellular matrix (ECM), inflammation, apoptosis of vascular smooth muscle cells (SMC), matrix metalloprotease 2, 9, and cytokines. On the other hand, increased ECM accumulation, enhancement of TGF‐β signalling, accumulation of collagen IV, and advanced glycation end‐products have been reported as protective mechanisms of AAA in DM. However, there is no research on the mechanism of AAA formation in DM and the microRNA (miRNA) of extracellular vesicles (EVs) for AAA formation in DM. This study focused on miRNA of the serum EVs to elucidate the mechanism of AAA formation in DM. Methods : We compared mRNA in EVs from human serum between AAA with DM and non‐AAA with DM patients (controls). We collected serum samples from AAA with DM (N = 20) and non‐AAA with DM (N = 10). The EVs were isolated using ultracentrifugation according to MISEV 2023 guidelines. MiRNA was analysed using the next‐generation sequence. The microRNA in EVs with significant changes were subjected to pathway analysis using miRPath v.4. Results : The significant up‐regulation is 12 miRNAs, and a down‐regulation of 12 miRNAs was detected from AAA patients compared to controls (Fold change > |1.5|, p < 0.05). The most significant microRNA was miR‐4524a‐5p. The KEGG pathway enrichment analysis of miR‐4524a‐5p indicated the TGF‐β pathway. Furthermore, eleven of the up‐regulated 12 miRNAs were also assumed to regulate this pathway. Summary/Conclusion : The miRNA of serum EVs may regulate the TGF‐β pathway in AAA patients with DM. The activity of TGF‐β is associated with aortic aneurysm formation via SMC apoptosis, ECM degradation, and inflammatory cell infiltration.

Molecular

Miriam Sandanusova 1,2 , Gabriela Ambrozova 2 , Eva Pechackova 3 , Veronika Skockova 2 , Lukas Kubala 1,2,4 , Kristyna Turkova 2 1 Faculty of Science, Masaryk University, Czechia 2 Institute of Biophysics of the CAS, Czechia 3 Center for Regenerative Medicine, University for Continuing Education Krems, Austria 4 International Clinical Research Center, St. Anne's University Hospital Brno, Czechia Introduction : Membrane vesicles (MVs) produced by probiotic bacteria Lactobacillus rhamnosus CCM7091 hold significant potential in modulating immune responses. However, their precise molecular effects on immune cells are not fully understood. Here, we aimed to isolate and comprehensively characterize L. rhamnosus‐derived MVs and examine their immunomodulatory properties, focusing on the molecular basis of the interaction. Understanding these interactions could provide critical insights into host‐microbe communication and support the development of new therapeutic strategies for immune modulation. Methods : The MVs were isolated from the late‐stationary growth phase of the maternal culture by multiple centrifugation, filtration, and ultracentrifugation steps, followed by purification on a sucrose cushion. Subsequently, the MVs were characterized (according to MISEV2023 guidelines) by MADLS, NTA, Cryo‐EM, and western blotting. To assess their functional capacity, we evaluated the MVs’ effects in vitro on RAW264.7 macrophage cell line. Additionally, to explore the molecular mechanisms underlying L. rhamnosus MVs’ immunomodulatory effects, we used HEK293 cells overexpressing various pattern recognition receptors. To study the involvement of the inflammasome pathway in the interaction, we used primary murine bone marrow‐derived macrophages (BMDMs) and detected the production of Caspase 1, IL‐1beta, and IL‐18. Results : Our findings revealed that L. rhamnosus‐MVs elicited a robust immune response in macrophages, as shown by increased production of TNFα, IL‐6, IL‐10, and reactive species of nitrogen. At the molecular level, we observed that the L. rhamnosus‐MVs induce TLR2 signalling pathways. Moreover, the MVs treatment of BMDM showed that the MVs may interfere also with the inflammasome signalling. Summary/Conclusion : In summary, our results indicate that MVs derived from L. rhamnosus CCM7091 activate TLR2 and inflammasome pathways in macrophages, significantly enhancing cytokine and inflammasome‐associated responses. These results highlight their potential role as potential modulators of innate immune pathways. Funding : Supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104)—Funded by the European Union—Next Generation EU.

Msc‐Evs

Presenter: Luis Pedro Bernardi Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Introduction : Mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) have shown promising results for treating ischaemic stroke. Their immunomodulatory properties may influence microglial responses and local inflammation following ischaemia. However, a comprehensive investigation of MSC‐EVs' effects on microglia is lacking. Therefore, this systematic review and meta‐analysis aimed to assess whether MSC‐EVs treatment alters microglial responses in in vivo or in vitro ischaemic stroke models. Methods : In accordance with the PRISMA 2020 Statement, we searched PubMed, Web of Science, and EMBASE up to October 2023 for studies assessing microglial activation, morphology, autophagy, phagocytosis, or viability responses to MSC‐EVs treatment in in vivo or in vitro ischaemic stroke models. We calculated standardized mean differences and confidence intervals using an inverse‐variance weighted random‐effects model. Heterogeneity was estimated using the I 2 statistic, and the risk of bias was assessed with an adapted version of the SYRCLE's questionnaire. Results : The search identified 297 studies, and 27 met the inclusion criteria. In in vivo ischaemic stroke models, MSC‐EVs reduced the number (SMD = −1.45 [95% CI = −2.19, −0.71]; adjusted (adj.) p value < 0.001), surface area (SMD = −1.10 [95% CI = −2.10, −0.11]; adj. p value = 0.03), and fluorescence intensity (SMD = −1.25 [95% CI = −2.09, −0.4]; adj. p value = 0.005) of Iba1+ cells, as well as the number of Iba1+ cells co‐expressing pro‐inflammatory markers (SMD = −2.13 [95% CI = −2.96, −1.30]; adj. p value < 0.001). Conversely, MSC‐EVs increased the number of Iba1+ cells co‐expressing anti‐inflammatory markers (SMD = 1.79 [95% CI = 1.06, 2.52]; adj. p value < 0.001). In in vitro models, our results revealed decreased TNF‐α (SMD = −6.16 [95% CI = −8.95, −3.37]; adj. p value < 0.001), IL‐1β (SMD = −3.45 [95% CI = −5.57, −1.32]; adj. p value = 0.002), and IL‐6 (SMD = −2.95 [95% CI = −4.50, −1.40]; adj. p value < 0.001) concentrations in the culture medium. Summary/Conclusion : Our meta‐analysis showed that MSC‐EVs modulate microglial responses to ischaemia, decreasing pro‐inflammatory and increasing anti‐inflammatory responses. These findings highlight the potential of microglia as therapeutic targets in MSC‐EVs‐based treatments for ischaemic stroke. Funding : This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Nanoscale

Sena Yaman 1 , Ugur Aygun 2 , Ugur Parlatan 2 , and Gozde Durmus 1 1 Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California, USA; 2 Department of Radiology, Canary Center at Stanford, Stanford University, Stanford, California, USA Introduction : Extracellular vesicles (EVs) positive for epithelial cell adhesion molecule (EpCAM) hold great potential as predictive and prognostic tools for epithelium‐derived tumours. Here, we captured EpCAM+ EVs on nanometre‐sized magnetic beads and imaged them with single‐particle sensitivity using custom‐built Interferometric Scattering Microscopy (iSCAT). This approach requires < 1 µL of sample, making it highly suitable for applications with limited sample availability. Methods : EVs were isolated from MC38 Y01 (a hybrid of a murine colon adenocarcinoma) cell culture media. After 48 h of incubation in DMEM supplemented with 10% exosome‐depleted FBS, 4 mL of the culture media was collected, and EVs ranging from 30 to 200 nm were isolated using a microfluidic filtration chip. First, 50 nm streptavidin magnetic beads (Miltenyi Biotec) were functionalized with biotinylated mouse CD326 (EpCAM) monoclonal antibody (Invitrogen). Second, the antibody‐coated beads were incubated with 50 µL of isolated EVs for 1 h. For imaging, we designed a custom iSCAT setup that uses a dielectric thin film on the substrate to enhance contrast by providing a background reference field. This setup enables single‐particle visualization by capturing interference between scattered light from particles and the reference field. The resulting interferometric images reveal particle volume and shape, allowing real‐time tracking of particle dynamics. We performed interferometric imaging and nanoparticle tracking analysis (NTA) for magnetic beads, antibody‐coated magnetic beads, and magnetic bead‐antibody‐EV complexes. Results : Our setup enabled label‐free imaging of EpCAM+ EVs on magnetic beads, differentiating between bare magnetic beads, antibody‐functionalized beads, and bead‐EVEpCAM complexes. Interferometric images provided detailed size and morphology information, with quantitative analysis showing size increases and shape circularity changes upon EV binding. NTA yielded mean diameters of 58.9 nm for magnetic beads, 63.3 nm for antibody‐coated magnetic beads, and 107.7 nm for bead‐EV complexes. In the case of the bead‐EV complexes, three peaks were observed, confirming the conjugation and elongation of the particles compared to antibody‐coated magnetic beads. Summary/Conclusion : Here, we developed a rapid, cost‐effective, and easy‐to‐use method to capture, detect, and quantitatively analyse EpCAM+ EVs released from a cell line using a low volume of sample. With additional modifications, this protocol could be adapted for detecting other EV subtypes directly from plasma. Funding : UA, MSCA‐101066038.

Naturally

Presenter: Pauline S. Ley Institute for Experimental and Clinical Cell Therapy, Austria Introduction : Extracellular vesicle (EV)‐based biomarker discovery can be more sensitive than from conventional liquid biopsies. Here we studied nano‐vesicle reference material for theranostic EV‐biomarker discovery for Parkinson's disease (PD). EV isolation methods, like tangential flow filtration (TFF) and size exclusion chromatography (SEC), showed limitations concerning time and resource consumptions. We therefore explored cell extrusion as efficient method to create artificial cell derived vesicles (CDVs) as reference material. We investigated whether CDVs are comparable to naturally produced EVs derived from the same cell lines. Methods : We used fluorescent neuroblastoma cells SH‐SY5Y expressing GFP‐coupled alpha‐synuclein (a‐Syn), a key protein for PD development and progression. Two control cell lines HEK‐CD63‐mNEON and HEK‐WT were included for comparison. Vesicles were enriched by TFF and cell extrusion. We determined EV quantity and size by tunable resistive pulse sensing (TRPS), and EV quality by tetraspanin and a‐Syn expression in dot blots and, fluorescence nano‐tracking analysis (fNTA). CDVs and EVs were imaged by cryo‐transmission electron microscopy (cryoTEM). Results : We found that CDVs were comparable to EVs in size with a mode diameter of 88±11 nm and 93±16 nm, respectively. We obtained mean 1E11 CDVs and 1E10 EVs per two million starting cells. Both expressed a‐Syn and tetraspanins CD9/63/81 but CDVs were mean 10‐fold less fluorescent than corresponding EVs. Bilayer membrane‐coated EVs and CDVs of appropriate size were readily detected by cryoTEM in a frequency corresponding to input concentration. Summary/Conclusion : Testing additional extrusion parameters will be necessary to improve reference nano‐particle production.

Nicotiana

Presenter: Mahmoud Eldahshoury Leeds Beckett University, UK Introduction : Extracellular vesicles (EVs) are promising drug delivery nanocarriers, though research is mostly focused on costly, hard‐to‐scale mammalian models. This study investigates small EVs from nicotiana benthamiana (Nb), a plant model used in biopharma, as a scalable, cost‐effective, and biocompatible alternative. Plant EVs allow for easy genetic manipulation, potentially enabling transgenic plants to produce disease‐targeted EVs, offering an affordable, ethical drug delivery approach. Methods : Nb EVs were isolated via ultracentrifugation and iodixanol gradient following MISEV guidelines. EV size and morphology were assessed by nanoparticle tracking analysis (NTA) and electron microscopy, while proteomics and transcriptomics characterized EV content and functions. Cytotoxicity assays (MTT, LIVE/DEAD) tested interactions with SH‐SY5Y neuroblastoma cells, and tetraspanin8‐GFP‐labelled EV uptake was analysed by flow cytometry and FlowSight. Loading efficiency was confirmed using Exofect kits and Tx‐red‐tagged siRNA, and BACE1‐targeting siRNA‐loaded EVs’ delivery efficiency was evaluated by fluorescence microscopy, qPCR, and flow cytometry. Results : NTA and electron microscopy revealed an enrichment of vesicles under 200 nm, with negative staining confirming the presence of characteristic cup‐shaped vesicles. Western blot and proteomic analyses confirmed the presence of a CD63 protein homologue (tetraspanin‐8) and the absence of the SYP121 protein (associated with a distinct EV subpopulation in plants). Proteomic analysis further revealed a notable abundance of homologues to mammalian EV markers, indicating significant cross‐kingdom similarities in EV composition. In vitro, Nb EVs were non‐cytotoxic and were efficiently internalized by SH‐SY5Y cells. When loaded with BACE1‐targeting siRNA, Nb EVs significantly reduced BACE1 mRNA and protein levels compared to controls (free siRNA and unloaded EVs). These results highlight Nb‐derived EVs as efficient, low‐immunogenic, and cost‐effective carriers for RNA‐based therapeutics. Summary/Conclusion : This study provides compelling evidence for the potential of nicotiana benthamiana EVs as a drug delivery platform. The findings demonstrate that Nb EVs are not only readily taken up by mammalian cells but also effectively deliver functional cargo in vitro. These attributes underscore their promise as a scalable and affordable alternative to conventional EV‐based delivery systems, with particular relevance to RNA‐based therapeutics. Funding : This study was funded by Leeds Beckett University.

Omega‐3

Liam Barry‐Carroll 1 , Ivan Marniquet 1 , Claire Bruzaud 1 , Juliette Dupont 1 , Flavie Crespo 1 , Charlotte Madore 1 , Sophie Layé 1 , Jean‐Christophe Delpech 1 1 University Bordeaux, INRAE, Bordeaux INP, NutriNeuro, UMR 1286, F‐33000, Bordeaux, France—Bordeaux (France) Introduction : A hallmark of the Western diet is a marked decrease in omega‐3 polyunsaturated fatty‐acids (n‐3 PUFA) consumption. This is a cause for concern, as reduced n‐3 PUFA consumption during development may cause long‐lasting cognitive abnormalities. Evidence from murine studies suggests the functions of microglial cells (brain immune cells) and neuronees are impacted in n‐3 PUFA deficient animals, resulting in cognitive changes; however, the exact mechanisms remain unclear. Therefore, we propose to study how reduced dietary n‐3 PUFAs can impact the content of brain‐derived EVs (BDEVs) and their resultant functions in recipient cells. Methods : Male and female C57BL/6 mice were subjected to either a control diet (balanced) or a diet low in n‐3 PUFAs (deficient) from gestation until 3 months old. BDEVs were subsequently enriched from whole‐brain homogenate samples using a protocol of differential centrifugation and density‐based sucrose gradient. BDEVs were assessed by nanoscale flow cytometry, western immunoblot, TEM, and proteomic analysis. The functional impact of BDEVs was tested in vitro using BV2 microglial cells and human iNeurones treated with BDEVs using an Incucyte microscope. Results : Deficiency in n‐3 PUFAs was associated with morphometric, synaptic, and proteomic changes in the brain. Specifically, the expression of proteins relating to EV biogenesis and release was altered in n‐3 PUFA deficient brains. Changes in the proteome were also reflected at the EV level in n‐3 PUFA deficient animals in a sex‐dependent manner, with a notably increased expression of proteins involved in cellular metabolism in males and synuclein processing and neurodegeneration in females. At the functional level, microglial phagocytosis was increased when subjected to deficient BDEVs compared to balanced BDEVs, while neurite length was reduced when iNeurones were treated with deficient BDEVs. Interestingly, only BDEVs from n‐3 PUFA balanced animals showed pro‐mitogenic capabilities, resulting in an increased percentage of Ki67+ microglia. Summary/Conclusion : The results here reveal the capacity of specific nutrients to alter brain EV content and function, shedding light on novel mechanisms involved in the nutritional modulation of cognitive abilities. Funding : Nouvelle‐Aquitaine

Placental

Presenter: Isabel Graf University Medical Center of Hamburg‐Eppendorf, Germany Introduction : During pregnancy maternal (immune) cells are transferred to the developing foetus via the placenta. There is growing evidence that these cells, referred to as maternal microchimeric cells (MMc), can reduce the offspring's susceptibility to diseases later in life, as reflected, for example, by a lower risk for early life infections. However, there is limited understanding of how MMc overcome the placental barrier and which factors regulate their transfer. This study aims to reveal the mechanisms of transplacental MMc trafficking by analysing placenta‐specific extracellular vesicles (EVs) in maternal serum in normally progressing pregnancies and pregnancies affected by maternal infections. Methods : EVs from third‐trimester serum samples of healthy and SARS‐CoV‐2‐infected pregnant women ( n = 60) were isolated via differential ultracentrifugation. Successful EV isolation was validated by nanoparticle tracking analysis, western blot analysis and transmission electron microscopy. Subsequently placental EVs were enriched by fluorescence activated vesicle sorting, and their proteome was analysed by Imaging Flow Cytometry and Mass Spectrometry. The EVs’ proteome was then correlated with the MMc frequencies detected in the newborns’ cord blood. Additionally, in order to evaluate the uptake of placental EVs by maternal immune cells, cell culture experiments were performed. Lastly, C57BL/6J (CD45.2, H‐2Db) and Balb/c CD45.1 (CD45.1, H‐2Dd) mice were mated allowing the identification of MMc in the foetus with subsequent validation of the identified proteins potentially driving MMc trafficking by qPCR. Results : Following maternal SARS‐CoV‐2 infection, MMc transfer is significantly decreased. Paralleling decreased MMc transfer rates, the placenta comprises an altered EV messaging with increased EV secretion and an altered protein cargo after infection. This includes the significant downregulation of PSME1 (proteasome activator complex subunit 1), a component of the immunoproteasome, and upregulation of CACYBP (Calcyclin binding protein) after infection. Placental EVs are taken up by maternal immune cells. In a murine model we proof that the proteins which are significantly upregulated in placental EVs, are also significantly upregulated in those maternal cells which crossed the placenta (MMc). Summary/Conclusion : Our data suggest that placental EV messaging plays a significant role for the recruitment of MMc to the foetus. These findings could potentially lead to the development of new strategies for improving foetal health.

Polybrene

Andrea De Luna 1 , Alexander Otahal 1 , Karina Kramer 1 , Markus Neubauer 1 , Stefan Nehrer 1 1 Department for Health Sciences, Medicine and Research, Center for Regenerative Medicine, University for Continuing Education Krems, Krems, Austria Introduction : As a result of the ageing population, osteoarthritis (OA) became the fifteenth‐highest leading cause of disability, affecting over 7% of the global population. OA is not only a disease affecting joints but also has a major impact on the social life of patients. Current treatment options only lead to alleviation of the symptoms, including pain and inflammation, but not to regeneration of the cartilage and therefore restoration of its mechanical function. Extracellular vesicles (EVs), especially derived from mesenchymal stem cells, represent cell‐free and less restrictive tools for cartilage regeneration compared to conventional cell‐based therapies. It has been shown that only a small percentage of EVs are being taken up by the recipient cell. Therefore, methods to enhance EV uptake and therefore maximize their regenerative effect have to be established. Polybrene (hexadimethrin bromide) is a polycationic polymer that has been used to enhance transduction efficiency of enveloped viral particles via surface charge neutralization. The aim of this study was to determine whether application of polybrene enhances uptake efficiency of HFP‐MSC‐EVs into OA chondrocytes. Methods : Primary MSCs were isolated from Hoffa's fat pad of knee replacement patients. HFP‐MSC‐EVs were generated in a vertical wheel bioreactor, were enriched via ultrafiltration and were characterised via nanoparticle tracking analysis in scatter and fluorescence mode for marker protein co‐localisation. Patient‐derived osteoarthritic chondrocytes were treated with different concentrations of polybrene, and its influence on cellular metabolic activity was assessed via XTT assay. To analyse the effect of polybrene on the uptake efficiency of EVs into cells, OA chondrocytes were treated with calcein AM labelled HFP‐MSC‐EVs in the presence or absence of polybrene. Confocal microscopy was performed to determine the ratio of internalized HFP‐MSC‐EVs. Results : Lower concentrations of polybrene did not influence metabolic activity of OA chondrocytes. Supplementation of culture media with polybrene in combination with adding HFP‐MSC‐EVs leads to a four times higher EV uptake rate into OA chondrocytes. Summary/Conclusion : Polybrene has the potential to enhance EV uptake into OA chondrocytes without influencing cells´ metabolic activity. This approach could be used to further boost the regenerative properties of EVs to treat OA. Funding : This study was supported by the Gesellschaft für Forschungsförderung NÖ, grant number LSC18‐014.

Polygonum

Presenter: Qi Xiu Southern Medical University, Guangzhou, People's Republic of China Introduction : The therapeutic potential of herbs has been extensively studied, primarily focusing on small molecule metabolites. However, research on cross‐kingdom interactions between plants and humans, particularly involving plant nucleic acids, remains limited. Emerging evidence suggests that plant‐derived nanoparticles play a crucial role in regulating human health and diseases through interkingdom crosstalk. In this study, we demonstrate for the first time that polygonum cuspidatum‐derived nanoparticles (PDNs) effectively scavenge reactive oxygen species (ROS) and reverse oxidative stress in HUVECs by transferring incorporated miRNAs, thereby activating the Nrf2‐Keap1 pathway. Methods : Firstly, polygonum cuspidatum‐derived nanoparticles (PDNs) were isolated from the plant's juice, with other disruptive components removed. The physical characteristics of PDNs were analysed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS), confirming their nano‐sized bilayer membrane structure. Secondly, PDNs were incubated with various skin‐derived cell lines to assess their biosafety, pro‐migration, and anti‐oxidation properties. Subsequently, PDNs were applied to the wound areas of C57/6J mice with 10 mm 2 deep second‐degree burns. Thirdly, multi‐omics analysis and experimental validation were performed to elucidate the specific mechanisms involved. Results : First, PDNs are slightly negatively charged spherical structures with a diameter of 150 nm, capable of stable preservation at 4°C, −20°C, and −80°C for over a month. Second, in vitro experiments showed that PDNs have significant antioxidant effects, promote the migration of vascular endothelial and keratinized epithelial cells, and induce M0 macrophages to differentiate into anti‐inflammatory M2 macrophages. In vivo, PDNs significantly accelerated wound healing by reversing oxidative stress and promoting angiogenesis. Thirdly, seven miRNAs from PDNs were identified as targets of human Keap1 mRNA, leading to the activation of the Nrf2‐Keap1 antioxidant pathway. Summary/Conclusion : PDNs, enriched with plant‐derived biomacromolecules, can enter the human body and effectively modulate human cell signalling pathways through cross‐kingdom interactions. Specifically, PDNs activate the Nrf 2‐Keap1 pathway by delivering multiple miRNAs to HUVECs, enhancing their antioxidant capacity and promoting rapid healing at the wound site. Funding : This work was supported by grants from the National Science Fund for Distinguished Young Scholars (82025024), National Natural Science Foundation project of China (82472387), and the Guangzhou Municipal Science and Technology Program (SL2024B03J00976).

Potential

Presenter: Thi Nhu Ngoc Van CNRS/ALCEN, France Introduction : Salivary extracellular vesicles (sEVs) have recently emerged as biomarkers for detection and monitoring of various diseases. Compared to whole saliva, sEVs are loaded with specific cargos such as protein, nucleic acids, lipid and metabolites, whose concentration might reflect pathological or physiological states. In our previous work (1), we demonstrated that miRNAs are consistently enriched in sEVs independent from the used isolation methods. Additionally, we highlighted, in another work (2), that specifically quantifying salivary miRNAs using RT‐qPCR is highly challenging. We addressed these challenges by establishing methodological recommendations to reduce data inconsistency and enhance data interpretation accuracy (2). This study aims to decipher further the profile of miRNAs enriched in sEVs compared to that of whole saliva. First, new RT‐qPCR assays were developed employing stem‐loop priming strategy, displaying highly specific detection of individual miRNA targets. Second, miRNA expression profiles of sEV‐ and whole‐saliva‐derived miRNAs were analysed by newly developed assays. Lastly, a comparative analysis between stem‐loop based (newly developed) and polyA (commercially available) based RT‐qPCR was conducted on sEV‐ and whole‐saliva‐derived miRNA samples. Methods : Saliva samples were collected from healthy individuals followed by sEV isolation using co‐precipiation method. Small RNA extraction and RT‐qPCR analysis were performed in accordance with our previously optimized protocol (2). Results : Our data showed that newly developed RT‐qPCR assays display highly specific detection of individual miRNA targets, allowing distinction between two highly homologous miRNA molecules whose sequences are differed by only one nucleotide. Comparative analysis on sEV‐ and whole‐saliva‐derive miRNAs reveals method dependent profiles of both miRNA populations. Specifically, the polyT‐based approach exhibited broader detection of target miRNAs with a pan specific tendency, whereas the Stem‐Loop based approach reports on specific quantification of individual miRNA targets. Summary/Conclusion : These findings underscore the importance of taking into account the assay performance and its specificity into data interpretation process in order to reduce study‐dependent data in miRNA research. By enhancing the reproducibility and specificity of salivary miRNA quantification method, this study pave the way to clinical application of sEV‐associated miRNAs as reliable diagnostic and prognostic biomarkers. Funding : Agence Nationale de la Recherche. ANR T‐ERC_STG2 278927 ReGuCel. Dr Marc Van Der Hofstadt

Precision

Presenter: Anna Kashkanova Max Planck Institute for the Science of Light, Erlangen, Germany Introduction : Characterization of the size, material properties and concentration of particles in liquid suspensions is in high demand, for example, for the analysis of extracellular vesicles (EVs) in body fluids. However, existing methods are often limited in sensitivity and cannot handle a high degree of polydispersion. Methods : We present interferometric nanoparticle tracking analysis (iNTA), which combines nanoparticle tracking analysis (NTA) with interferometric scattering (iSCAT) microscopy to achieve higher sensitivity than traditional NTA instruments. We show that iNTA can measure the size and refractive index of individual nanoparticles with unprecedented precision. Furthermore, we demonstrate that the concentrations of different subpopulations in polydispersions can be determined from the trajectory numbers. Results : We benchmarked iNTA with synthetic nanoparticles and showed that gold nanoparticles as small as 10 nm can be measured. Afterwards we show that iNTA can extract the size and internal structure of liposomes and EVs. In addition, we demonstrate that iNTA can differentiate EVs from lipoproteins in blood plasma‐derived samples, which is not possible with conventional NTA. Finally, we show that iNTA can differentiate viruses from EVs in SARS‐CoV2 infected cell supernatants. Summary/Conclusion : We have shown that iNTA is a useful tool not only for characterization of particle size and refractive index, but also for quantitative assessment of particle concentration, even when sizes overlap. This is very useful for the quantification of EVs where co‐isolates are often present in the samples. Funding : This study was supported by the Max Planck Society, Bundesministerium für Bildung und Forschung, Alexander von Humboldt Foundation, Christiane Nüsslein‐Volhard Foundation.

Probiotic

Agnieszka Razim 1,2 , Anna M. Schmid 1 , Magdalena E. Paschal 1 , Tamara Weinmayer 1 , Michael Thaler 1 , Paweł Migdał 2 , Magdalena E. Skalska 3 , Mattia Morandi 4 , Catherine Daniel 5 , Dagmar Srutkova 6 , Martin Schwarzer 6 , Ursula Wiedermann 1 , Sabina Górska 2 , Irma Schabussova 1 1 Medical University of Vienna, Austria; 2 Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Poland; 3 Jagiellonian University, Poland; 4 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Czech Republic; 5 Institut Pasteur de Lille, France; 6 Institute of Microbiology of the Czech Academy of Sciences, Czech Republic Introduction : The immunomodulatory potential of probiotics is well known. Not long ago, we learnt that probiotic bacteria produce extracellular vesicles (EVs), the role of which is not yet fully understood. However, since EVs are non‐living and non‐replicating, they offer many advantages over live bacteria in clinical applications. Therefore, EVs can be used in immunocompromised individuals and appear to be ideal for intranasal administration. Methods : We isolate postbiotics in the form of EVs from the probiotic Lactiplantibacillus plantarum NCIMB8826 (LpEVs). We purify these EVs with size exclusion chromatography and characterized them with respect to their physical characteristics (size, zeta potential, and stability studies), their composition (proteins, lipids, DNA, and RNA) and their immunomodulatory properties in vitro and in vivo. We use SDS‐PAGE, Bioanalyzer, proteomics, and lipidomics to analyse the cargo. In in vitro studies we analysed receptor recognition and cytokine induction using epithelial and immune cell lines. Results : We used multiple methods to show that L. plantarum produces significant amounts of EVs with a size of about 70–80 nm in the MRS medium. High doses of LpEVs significantly reduced the number of recruited eosinophils in the lungs of allergic mice. Moreover, these LpEVs are resistant to long‐term storage, physiological pH, different salt concentrations and detergents (found in the digestive tract), making them perfect for intranasal or oral administration. However, EVs produced in MRS differed from those produced in MRS + 0.25% bile in size and protein content. Summary/Conclusion : We have shown that LpEVs retain some immunomodulatory properties of the live strain, but at the same time their characteristics are highly dependent on the conditions under which they are produced. These data suggest that the bacterium actively responds to changing environmental conditions. The immunomodulatory potential of the ‘bile‐stressed’ EVs is currently being investigated. Funding : This research was funded by HORIZON‐MSCA‐2021‐PF (project no. 101066450), START2022 by the Foundation for Polish Science, SONATA (2023/51/D/NZ7/02220) financed by the National Science Centre Poland and the Danube Allergy Research Cluster, mobility OEAD WTZ CZ 07/2023 and PL 03/2022 and FWF P 34867. We acknowledge CEITEC/Brno—Nanobiotechnology of CIISB, Instruct‐CZ Centre, supported by MEYS CR (LM2023042)) and European Regional Development Fund‐Project “UP CIISB” (No. CZ.02.1.01/0.0/0.0/18_046/0015974).

Profiling

Miks Priedols 1 , Edgars Dauss 2 , Reinis Vangravs 2 , Dārta Pūpola 2,3 , Rihards Mikilps‐Mikglebs 2,3 , Arnis Kiršners 2,4 , Ilmārs Stonāns 2 , Artūrs Ābols 1 , Alvils Krams 2,3 1 Latvian Biomedical Research and Study Centre, Riga, Latvia; 2 University of Latvia, Institute of Clinical and Preventive Medicine, Riga, Latvia; 3 Riga East University Hospital, Riga, Latvia; 4 Riga Technical University, Institute of Information Technology, Riga,, Latvia Introduction : Lung cancer remains the leading cause of cancer‐related mortality globally, with early detection crucial for improving patient outcomes. Despite advancements in diagnostics, peripheral lung cancers are often identified at advanced stages due to non‐specific symptoms and the absence of effective screening programs. Extracellular vesicle (EV)‐associated microRNAs (miRNAs) represent a promising biomarker for minimally invasive diagnostics, reflecting the molecular landscape of lung tumours. We evaluated the diagnostic utility of EV‐associated miRNA profiles from bronchoalveolar lavage fluid (BALF) for detecting peripheral lung cancer. Methods : EVs were isolated from BALF using differential ultracentrifugation. RNA isolation and cDNA synthesis was performed using commercially available kits. Seven hundred fifty‐four miRNAs profile analysis was performed on 10 malignant cancer and 10 benign tumour patient BALF samples using the TaqMan Advanced miRNA assay panel. The most promising miRNA targets identified were subsequently validated in an additional cohort of 50 patients with peripheral lung lesions. Results : On average, 232 target genes were detected in the malignant cancer patient group, compared to 209 in the benign group. Notably, hsa‐let‐7a‐5p was detected approximately 22 times higher in the malignant cancer group compared to the benign group, while hsa‐miR‐576‐3p was lower by approximately 27 times. Summary/Conclusion : Our findings underscore the potential of BALF‐derived EV‐associated miRNAs as minimally invasive biomarkers for enhancing early peripheral lung cancer detection. Funding : This study was supported by the Latvian Council of Science, Project No: lzp‐2022/1‐0410

Prolonged

Presenter: Masahito Nakazaki Sapporo Medical University, Sapporo, Hokkaido, Japan Introduction : In previous studies, we showed that three intravenous infusions of bone marrow mesenchymal stem cell‐derived small extracellular vesicles (MSC‐sEVs) over three days starting one week post‐spinal cord injury (SCI), improved motor recovery and growth in young adult rats, whereas a single equivalent dose did not. Tracing DiR‐labelled MSC‐sEVs indicated uptake by M2 macrophages at the lesion, with excess cleared by the kidneys within 24 h. These findings suggest that extending MSC‐sEV exposure through prolonged, continuous delivery may improve their therapeutic efficacy by sustaining interaction with target cells at the lesion site. In this study, we evaluated the therapeutic efficacy of continuous intravenous infusion of MSC‐sEVs in an SCI rat model. Methods : Severe contusive SCI (T9) rats were randomized into eight treatment groups based on administration method (daily or continuous), duration of treatment (3 or 6 days) and treatment type (human MSC‐sEVs or PBS control) starting on Day 7 post‐SCI. Continuous infusion was administered via osmotic pumps connected to an intravenous catheter, while daily injections were delivered through the tail vein. Open field locomotor recovery was assessed weekly until day 70 post‐SCI using the Basso–Beattie–Bresnahan (BBB) scoring protocol, and body growth was evaluated by weekly body size measurements. For mechanistic analysis microRNA sequencing was performed, and data were analysed using Ingenuity Pathway Analysis (IPA) software. Results : We found that continuous delivery of MSC‐sEVs over three days accelerated the onset of recovery compared to daily injections, and continuous infusion over six days significantly enhanced recovery compared to daily injections with the same total dosage. The timing of recovery in growth trajectories was comparable across all MSC‐sEV treatment conditions. Mechanistic analyses revealed that numerous miRNAs associated with fibrosis pathways were highly enriched in MSC‐sEVs compared to control sEVs. Summary/Conclusion : These results suggest that prolonged continuous exposure to MSC‐sEVs is essential for maximizing specific therapeutic benefits, particularly in enhancing functional motor recovery in SCI. Furthermore, at least part of the therapeutic effect of MSC‐sEVs may be mediated by microRNA changes related to fibrosis pathways.

Proteomic

Ivona Pavkova 1 , Jana Klimentova 1 , Adela Cermakova 1 , Jitka Zakova 1 , Dominik Pinkas 2 , Jaroslav Hanus 3 1 University of Defence, Military Faculty of Medicine, Czech Republic, 2 Electron Microscopy Core Facility, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic, 3 University of Chemistry and Technology, Prague, Czech Republic Introduction : Extracellular vesicles (EVs) are composed of various biomolecules through which they participate in cellular crosstalk. During infection, their effect might be bidirectional. They can either promote or inhibit the infection progress. In this study we focused on the proteomic analysis of fractions enriched in EVs produced by macrophages infected with intracellular bacterium Francisela tularensis, the causative agent of severe disease tularaemia. Despite the intensive research, the knowledge of molecular mechanisms of pathogenesis are incomplete, and represents the major challenge to the development of an effective vaccine. The release and role of EVs in relation to this pathogen has not been reported so far, and is expected to play a key role in host‐pathogen interaction. Methods : Mouse bone marrow‐derived macrophages (ca. 9x107cells) were infected with virulent strain of F. tularensis subsp. holarctica for 10 h and the cell culture supernatant was enriched for extracellular vesicles (EVs) using differential centrifugation. The presence of EVs was confirmed by TEM, NTA and immunoblot analysis. Further, proteomic analysis of EV containing fractions were performed using LC/MS/MS followed by analysis using the Metascape platform. For comparison, samples from non‐infected cells were processed in the same way. Results : In response to infection, the macrophages were found to release EVs with mode diameter 77.1 ± 3.8 nm. The presence of EVs was further successfully validated by TEM and immunodetection of EV markers CD9, CD81 and TSG101 On the other hand, almost no vesicles could be detected in control samples prepared from non‐infected cells. The MS analysis reveals the presence of about 1270 proteins, many of them are involved in cellular processes in response to infection indicating the potential role of EVs in tularaemia pathogenesis. In addition to host cell proteins, several bacterial proteins were identified as well, most of them with known relation to virulence. Summary/Conclusion : This study shows the production of EVs from cells infected with F. tularensis together with a dataset of their protein cargos, providing a preliminary view of their potential function in pathogenesis of tularaemia. Funding : The work was supported by the Ministry of Defence of the Czech Republic (project no: DZRO‐FVZ22‐ZHN II).

Proximity

Presenter: Kyung‐Min Kim Hanyang University, Seoul, Republic of Korea Introduction : We propose an innovative approach for universal labelling of cellular membrane and EP surfaces in indirect and direct manners. Our key idea is centred on cyclic ascorbate peroxidase 2 with a membrane‐accumulating peptide tag (cAPEX2P), which demonstrates strong membrane association and exceptional proximity labelling efficiency while retaining its inherent peroxidase activity. Methods : We designed and characterized split intein‐mediated self‐splicing cyclic APEX2 variants in vitro. Then, mammalian cellular membrane labelling of modified APEX2 was performed with biotinylated substrate and evaluated using western blotting and confocal fluorescence microscopy using biotin‐streptavidin interaction. To identify protein candidates related to APEX2‐mediated universal labelling, protein lysates were analysed using LC‐MS/MS. After that, we isolated EPs secreted from APEX2‐labelled cells and visualized labelling signal with super‐resolution microscopy using fluorescent APEX2 substrate. Also, we optimized the conditions for proximity labelling, confirming with super‐resolution microscopy. Finally, we applied our universal labelling methods to other EPs derived from bacteria and plants, and the labelling efficiency was analysed using flow cytometry. Results : We measured the peroxidase activity of cAPEX2, and the negative effect from structural alteration was not observed. Then, we optimized the universal labelling process of biotinylated substrate for mammalian cells. We could visualize the universal labelling signal, and the signal was co‐localized with plasma membrane staining. With our labelling method, we could perform spatiotemporal imaging of plasma membrane proteins and their translocation. We isolated EPs from universally labelled cells and visualized them using super resolution microscopy. LC‐MS/MS data revealed that labelled proteins were enriched and categorized as focal adhesion, semaphorin receptor families, and extracellular matrix proteins. While the EPs cannot be labelled directly with metabolic glycan labelling, our method could be applied to direct universal EP labelling to pre‐isolated mammalian, lactobacillus and lily EPs. Engineered APEX2 showed increased labelling efficiency up to 10 times. Summary/Conclusion : Our results revealed that cyclic cAPEX2P can easily access cellular or EP membranes, maintaining its function and labelling EPs universally in indirect and direct ways. Without any genetic modification, cAPEX2P can be applied to labelling mammalian, bacterial, and plant‐derived EPs for labelling and visualizing. Our method is promising for understanding the biodistribution of donor EPs and its fate.

Recurrent

Presenter: Ashok K. Shetty Texas A&M University College of Medicine, USA Introduction : Alzheimer's disease (AD), a progressive neurodegenerative condition, results in significant cognitive and mood dysfunction. Neuroinflammation is one of the vital factors contributing to AD progression. Intranasal (IN) administrations of extracellular vesicles produced by human induced pluripotent stem cell‐derived neural stem cells (i.e., hiPSC‐NSC‐EVs) have been shown to reduce neuroinflammation by inducing transcriptomic changes in activated microglia and reactive astrocytes and curtailing neuroinflammatory signalling cascades in 5xFAD mice, a model of early‐onset AD (Madhu et al., Journal of Extracellular Vesicles, 2024). Methods : Using the 5xFAD mice, this study investigated the efficacy of hiPSC‐NSC‐EV treatments at 3 months of age (two 30 billion intranasal doses separated by a week) versus monthly intranasal treatments at 3–7 months of age (30 billion EVs/month) for maintaining better cognitive and mood function at 8 months of age. Treatments at 3 months represent intervention at an early stage of AD, whereas treatments at 3–7 months reflect regular interventions in the early and mid‐phases of AD. Results : 5xFAD mice receiving two intranasal doses of hiPSC‐NSC‐EVs at 3 months of age displayed cognitive and mood impairments at 8 months. In contrast, 5xFAD mice receiving monthly doses of hiPSC‐NSC‐EVs at 3–7 months of age maintained better cognitive and mood function, evidenced by their ability to (1) discern minor changes in their environment in an object location test, (2) encode similar but not identical experiences in a non‐overlapping fashion in a pattern separation test, and (3) prefer sweet water in a sucrose preference test. 5xFAD mice receiving monthly doses of vehicle treatment were impaired in such cognitive tasks and displayed anhedonia. Analyses of brain tissues revealed that AD mice receiving monthly doses of EVs exhibited reductions in astrocyte hypertrophy, microglial clusters, microglia presenting inflammasomes, amyloid‐beta plaques, and the concentrations of oxidative stress markers and proinflammatory cytokines in the hippocampus. Summary/Conclusion : Intranasal hiPSC‐NSC‐EV treatments only during the early stage of AD are insufficient for maintaining better brain function in the advanced AD stage. However, intermittent intranasal hiPSC‐NSC‐EV administrations are efficacious for restraining neuroinflammation and maintaining better cognitive and mood function for extended periods. Funding : Supported by a grant from the National Institutes for Aging (1RF1AG074256‐01A1 to A.K.S.)

Revealing

Gabriella Dobra 1,2* , Edina Gyukity‐Sebestyen 1,2* , Matyas Bukva 1,2 , Timea Boroczky 1,2,3 , Regina Csordas 4 , Zoltan Szabo 4 , Almos Klekner 5 and Krisztina Buzas 1,2 1 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, H 6726 Szeged, Hungary; 2 Department of Immunology, Albert Szent‐Gyorgyi Medical School, Faculty of Science and Informatics, University of Szeged, H‐6720 Szeged, Hungary 3 Doctoral School of Interdisciplinary Medicine, Albert Szent‐Gyorgyi Medical School, University of Szeged, H‐6720 Szeged, Hungary 4 Department of Medical Chemistry, Albert Szent‐Gyorgyi Medical School, University of Szeged, H‐6720 Szeged, Hungary; 5 Department of Neurosurgery, Faculty of Medicine, University of Debrecen, H‐4032 Debrecen, Hungary; * Equally contributed to this work Introduction : Current diagnostic methods for brain tumours, such as computed tomography, MRI, and tissue biopsy, have limitations that highlight the need for less invasive testing, including molecular analysis of body fluids. Small extracellular vesicles (sEVs) are promising targets for liquid biopsy in brain tumours due to their specific properties. By analyzing the protein content of sEVs, tumour markers may be identified; however, isolation and proteomic analysis methods for sEVs need refinement. Our goal is to advance the quantitative and qualitative proteomic analysis of sEVs, enabling effective differentiation between patients with different tumour types. Our method development was focused on selecting an optimal EV isolation technique and creating a robust database. Methods : In our study, we analysed plasma samples from 9 Glioblastoma and 9 Meningioma patients. sEVs were isolated using differential ultracentrifugation (dUC) and size exclusion chromatography (SEC), and protein coronas obtained with 1.5 M NaCl digestion (EVcorona). Regarding sEV characterization, morphology was measured with transmission electron microscopy (Tecnai G2 20 X‐Twin), size distribution with nanoparticle tracking analysis (NS300), and sEV markers with flow cytometry (Cytoflex S). Proteomic analysis was performed using liquid chromatography‐mass spectrometry (ACQUITY M‐Class UPLC, Exploris 240). Patients were clustered based on proteomes from plasma, dUC and SEC isolates, and EVcorona; while protein intensities were compared using Welch's test. Results : We established a vesicle proteome database with 719 identified proteins: 553 in plasma, 614 in dUC samples, 681 in SEC‐3 isolates, 637 in SEC‐4, and 584 in SEC‐5 isolates. The SEC‐3 isolate contained the highest number of identified proteins (681). Abundant serum proteins not associated with sEVs, such as albumin, decreased progressively from plasma to dUC and SEC samples. Vesicle marker concentrations were the highest in the SEC‐3 fraction, while plasma markers fell below detection thresholds. Clustering and Welch's test showed optimal separation of patient groups based on SEC‐3. Summary/Conclusion : Our results indicate that the third SEC fraction is best suited for comparative plasma EV proteomics. Based on the developed method, the two patient groups are distinguishable, though identifying tumour markers requires a larger sample size and the inclusion of healthy controls. Funding : TKP‐EGA‐09; OTKA‐K143255; MEC_R 149857; MEC_R 149846;

Ribosomal

Jiangang Ren Wuhan University, China Introduction : Venous malformations (VMs), predominantly arising from activating mutations of TIE2 in endothelial cells (ECs), are characterized by dilated and tortuous vessels with a paucity of perivascular cells (PCs). The mechanisms of interaction between mutant ECs and PCs remain largely elusive. Here, we aim to investigate the characteristics of extracellular vesicles (EVs) from VM ECs, especially the microRNAs (miRNAs) carried and their roles in crosstalk between ECs and PCs in VM pathogenesis. Methods : miRNA profiles of human umbilical vein endothelial cells overexpressing TIE2L914F (L914F cells) and TIE2WT [wildtype (WT) cells], along with their EVs, were analysed by RNA sequencing. In vitro studies using umbilical cord stem cells (UCSCs) were done to perform functional assays of VM EVs and their enriched miRNA‐4432 (miR‐4432). miRNA pulldown and RNA interference techniques were used to identify the sorting regulator of miR‐4432 into VM EVs. Results : RNA secretion was upregulated in L914F EVs vs. WT EVs. miRNA sequencing revealed a distinct profile of L914F EVs vs. L914F cells, WT cells, and WT EVs, identifying miR‐4432 as being preferentially encapsulated in EVs from L914F cells. Functional assays demonstrated that VM EVs and EV‐carried miR‐4432 inhibited the differentiation, adhesion, and proliferation of UCSCs. Furthermore, ribosomal protein L36 (RPL36) was identified as an RNA‐binding protein and sorting regulator of miR‐4432 during the EV secretion process in L914F cells. Summary/Conclusion : This study, for the first time, identified an interaction between VM ECs and PCs via EVs, and offers valuable data on the miRNA profiles of VM ECs and normal ECs, along with their EVs. Our findings suggest that the RPL36‐mediated selective loading of miR‐4432 into EVs may contribute to the aberrant PC coverage in VMs, providing novel insights into VM pathogenesis and potential treatment strategies. Funding : the National Natural Science Foundation of China (81870361, 81922038, 81600385, 82101036)

Searching

Klára Hánělová 1 , Jan Balvan 1 , Monika Kratochvílová 2 , Martina Raudenská 2 , Jiří Navrátil 1 , Tomáš Vičar 2 ’⁵, Michal Masařík 1 ’ 3 ’⁴ 1 Faculty of Medicine, Department of Pathological Physiology, Masaryk University, Brno, Czech Republic; 2 Faculty of Medicine, Department of Physiology, Masaryk University, Brno, Czech Republic; 3 BIOCEV, First Faculty of Medicine, Charles University, Prague, Czech Republic; 4 International Clinical Research Centre (ICRC), St. Anne's University Hospital, Brno, Czech Republic; 5 Faculty of Electrical Engineering and Communication, Department of Biomedical Engineering, VUT, Brno, Czech Republic Introduction : Various populations of small extracellular vesicles (sEVs) are widely heterogeneous, even within a single cell type. These populations are marked by different surface molecules, such as EpCAM, CD63, or phosphatidylserine (PS). This implies that EV subpopulations may carry diverse proteomes, which could be essential for their wide range of biological functions. These variations allow sEVs to be involved in processes, such as the immune response, disease progression, and fibroblast activation into CAFs. This work aimed to explore, how distinct surface markers shape the functional roles of sEVs in the context of head and neck cancer (HNSCC). Methods : EVs were isolated from conditioned media (obtained from HNSCC cell line FaDu) that were subjected to differential centrifugation, filtration, and concentration and EVs were isolated using affinity‐based methods targeting EpCAM, CD63, and PS on EV surface. Then, isolated EVs were characterised using NTA or DLS, cryo‐EM, TEM, WB for EV surface markers, and LC‐MS for their proteomic composition. Furthermore, the effect of various groups of EVs on recipient cells was assessed using WB for CAF activation, LC‐MS, and measurement of mitochondrial energetics in recipient cells. Results : So far, we have been mainly focused on the first subgroup, PS‐positive sEVs, as PS is of high importance in the tumour microenvironment. By inhibition of autophagy, specifically lysosomal degradation, we observed the accumulation of proteins in PS‐EVs as an alternative pathway for autophagic degradation. This led us to the assumption that isolated EVs are of endosomal origin rather than microvesicles. Accumulation of lysosomal membrane marker, LAMP1, and decrease in the plasma membrane marker, basigin, in PS‐EVs after bafilomycin treatment supported our previous finding. Summary/Conclusion : Our results indicate that the clinical use of autophagy modulators in cancer must be taken cautiously, since they also affect the PS‐EVs content. We also observed that PS‐EVs derived from cancer cells were taken up by normal fibroblasts, activating them into CAFs. Thus, molecules carried in PS‐EVs can be critical for cancer development, progression, and potential treatment resistance. Further research will be focused on other aforementioned subgroups of EVs. Funding : This study was supported by the Ministry of Health, Czech Rep. NU22‐03–00202 Internal grant MUNI/LF‐SUp/1405/2024

Secretion

H. Seda Vatansever 1,2 , Hilal Kabadayı Ensarioğlu 1 , Aslınur Aktas 1 , Burak Kutlu Aydın 1 , Nadire Kıyak 2 , Mehmet Vatansever 2 1 Manisa Celal Bayar University, Turkey; 2 Near East University, Turkey Introduction : Microvesicular endosomes (MVEs) are characterized by containing intraluminal vesicles (ILVs), which can be controlled by multiple mechanisms. Exosome biogenesis is controlled by the ESCRT (endosomal sorting complex for transport)‐dependent and independent pathways. Rab31 plays a role in an ESCRT‐independent pathway. High intracellular levels of Rab31 are directed into the MVEs to form ILVs, which are then secreted as exosomes. Hypoxia modulates the secretion, composition, and function of exosomes, especially in various cancer. While hypoxia increased exosome release by upregulating Rab27a and reducing Rab7; however, the secretion of Rab31 by hypoxia remains unclear. Recent studies have demonstrated that the therapeutic benefits of MSCs are dependent on their extracellular vesicles (EVs). MSCs‐derived EVs can induce different immunosuppressive effects and contribute to the immunological tolerance. In our study, we investigated the detection of Rab31 during exosome secretion in adipogenic mesenchymal stem cells (AMSCs) in 2D and 3D culture conditions under normoxic and hypoxic environments. Methods : AMSCs were obtained from the ATCC culture collection. They were cultured with 10% exososme‐free FBS, 1% pen‐strepromycin, 1% L‐glutamine included in DMEM. For the 2D condition, cells were cultured in a routine culture plate; for 3D, cells were cultured in Matrigel. A hypoxic condition was performed in a chamber containing 5% CO 2 , 5% O 2 , 90% N 2 . All cells were cultured for 48 h, and after fixation of cells with 4% paraformaldehyde, the presence of Rab31, Rab7, CD9, and CD63 proteins was evaluated using an indirect immunorexidase technique. Results : Negative or very weak CD9 immunoreactivity was detected in both 2D and 3D hypoxic and normoxic conditions. While immunoreactivity of CD63 was moderate in 2D hypoxic and normoxic conditions, this immunoreactivity was strong and similar in both 3D hypoxic and normoxic conditions. Rab31 immunoreactivities were higher than Rab7 in all conditions. In addition, Rab31 immunoreactivity was higher in the 3D hypoxic condition than in the normoxic condition. Summary/Conclusion : In our results demonstrated that exosome secretion from AMSCs was affected by culture conditions. Especially hypoxic conditions trigger secretion of Rab31, and co‐localization of CD63 and Rab31 under hypoxic conditions pointed out that exosome biogenesis is controlled by Rab31 in AMSC cells under hypoxic conditions. Funding : This work was supported by the Research Fund of Near East University (Project number: SAG‐2023‐1‐026).

Selective

Presenter: Bradley Whitehead Aarhus University, Aarhus, Denmark Introduction : Trichuris trichiura , infects over 500 million people. T. suis , a whipworm of pigs provides a large mammal model of human infection with Trichuris spp. T. suis ova have been assessed in clinical trials for the treatment of inflammatory diseases, including ulcerative colitis, Crohn's disease, multiple sclerosis and allergic rhinitis. T. suis excretory/secretory (ES) products potently inhibit inflammatory responses in vitro; however, the role of extracellular vesicles (EVs) remains unknown. Methods : EVs were isolated from adult and L1 T. suis ESP and characterised using NTA, AFM and LC‐MS/MS. EVs and supernatants (SN) were subjected to miRNA sequencing to identify and quantify miRNAs enriched in EVs. miRNAs were aligned to human miRNAs to identify orthologues of known miRNAs. Immune effects of T. suis were assessed in LPS stimulated THP‐1 cells. Functional effects of T. suis miRNAs were determined by transfection of SW480 colon cancer cells followed by proteomic analysis at 48 and 72 h. Results : T. suis adults and L1 produce EVs as assessed by nanoparticle tracking analysis and AFM to confirm the presence of vesicular particles enriched from ESP. EV isolation was further confirmed by proteomic analysis. EVs did not inhibit TNF production by LPS stimulated THP‐1 cells, whereas ES and EV depleted fractions did. Analysis of T. suis secretory miRNA profiles revealed selective packageing of miRNAs in EV and SN and also by life stage. Tsu‐miR‐22‐3p and tsu‐miR‐29‐3p were enriched in EVs and their homologous miRNAs are well‐described tumour suppressor miRNAs. Therefore, the functionality of these miRNAs was tested in SW480 colon cancer cells. Proteomics at 48 and 72 h identified specific downregulation of proteins that are experimentally validated targets of hsa‐miR‐22‐3p and hsa‐miR‐29b‐3p and include well‐known tumour suppressors and are associated with poor outcomes in colon cancer. Summary/Conclusion : This work shows that T. suis produces EVs but unlike that observed for A. suum , they do not contribute to the immune effects of T. suis ES. However, T. suis EVs are enriched in miRNAs that are functional mimetics of human tumour suppressor miRNAs, suggesting a role for EVs beyond host‐immune modulation.

Serotonin

Presenter: Tugba Semerci SevimLi Eskisehir Osmangazi University, Turkey Introduction : Exosomes are extracellular vesicles that enable communication between cells. The most valuable molecules in the cargo they carry are miRNAs. We have shown the importance of one of these miRNAs, miR‐127‐5p, in chondrogenesis and cancer in our previous studies. The central and peripheral nervous systems produce serotonin (5‐HT), a neurotransmitter, while enterochromaffin cells in the intestines produce it as a hormone. Serotonin performs its functions by interacting with a large number of receptors connected to various signalling pathways. In this study, we aimed to see how human adipose tissue‐derived mesenchymal stem cell exosomes incubated with serotonin had an effect on miR‐127 and IL‐10. Methods : We first performed MTT analysis for serotonin. We then incubated mesenchymal stem cells with this dose. Exosome isolation and characterization were performed. We examined the expression of miR‐127‐5p and IL‐10 genes using qPCR analysis. We also performed an ELISA test for IL‐10. Results : Serotonin increased the expression of miR‐127‐5p and IL‐10 in mesenchymal stem cell exosomes. The IL‐10 level was higher in the exosomes. Summary/Conclusion : This study suggests that 5‐HT may be positive as it enhances the miR‐127‐5p and IL‐10 effect on mesenchymal stem cell exosomes.

Stripping

András I Försönits 1 , Eszter Á Tóth 1,2,3 , Tünde Bárkai 1 , Tamás Visnovitz 1 , Xabier Osteikoetxea 1,4 , Edit I Buzás 1,4,5 1 Department of Genetics, Cell‐ and Immunobiology, Semmelweis University, Hungary, 2 The Buda Hospital of the Hospitaller Order of Saint John of God, Hungary, 3 Department of Rheumatology and Clinical Immunology, Semmelweis University, Hungary, 4 HCEMM SU Extracellular Vesicles Research Group, Hungary, 5 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Hungary Introduction : Our group was among the pioneers in demonstrating that a biomolecular corona forms around extracellular vesicles (EVs) in biofluids. However, its impact on the accessibility of EV surface molecules remains unclear. Methods : This study aimed to evaluate whether the EV corona interferes with the immunodetection of EV surface molecules. Various approaches were applied for removing the EV corona to enhance immunodetection of EVs. We constructed an artificial EV corona using EVs derived from HEK293T‐PalmGFP cells and Cy5‐labelled human plasma proteins. The EVs were exposed to EDTA, Tween‐20, and beta‐mercaptoethanol and subjected to size exclusion chromatography and high ionic strength washes (NaCl, LiCl, and KCl). The effectiveness of corona removal was assessed by measuring the co‐localization rate of plasma proteins with EVs. We evaluated fluorescent annexin‐V binding and annexin‐V‐based affinity capture of EVs, both with and without high ionic strength washing. Additionally, EVs derived from serum‐containing and serum‐free conditioned medium of THP‐1 cells were measured with a MACSPlex kit to assess this effect. Results : We found clear evidence of EV corona stripping following high ionic strength washes. These washes significantly reduced the co‐localization of plasma proteins with EV membranes ( p < 0.0001, Mann–Whitney U test), indicating substantial corona removal. Notably, annexin‐V binding ( p < 0.01, paired t ‐test) and annexin‐V affinity capture ( p < 0.05, paired t ‐test) both increased for THP‐1 and blood plasma‐derived EVs after high ionic strength washing. The improved detection was specific to EVs derived from serum‐containing media, with no effect observed in serum‐free conditions. Enhanced immunodetection was noted for 10 out of 37 measured EV markers in plasma‐derived EVs following high ionic strength washing. Summary/Conclusion : Our findings suggest that stripping the protein corona of EVs with high ionic strength solutions can enhance immunodetection of certain EV markers, providing a potential method to improve the accuracy of EV identification. Additionally, corona stripping could enable the re‐decoration of EV surfaces with bioactive molecules, potentially enhancing their therapeutic efficacy. Funding : This research was funded by NVKP_16‐1‐2016‐0004, Hungarian National Research, Development and Innovation Office (NKFIH), VEKOP‐2.3.2‐162016‐00002, VEKOP‐2.3.3‐15‐2017‐00016, Therapeutic Thematic Programme TKP2021‐EGA‐23, RRF‐2.3.121‐2022‐00003 (National Cardiovascular Laboratory Program) 2019‐2.1.7‐ERA‐NET‐2021‐00015, EU's Horizon 2020 Research and Innovation Programme, grant agreement No. 739593.

Synthetic

Presenter: Seong Jun Park Ewha Womans University Mokdong Hospital, Seoul, Republic of Korea Introduction : Since the complexity and heterogeneity of natural extracellular vesicles (nEVs) present challenges for their use in diagnostics and therapeutics, we have previously developed synthetic EVs (sEVs) using artificial intelligence (AI) and a microfluidic mass production platform. The basic requirements for referencing the nEVs are comparable size, low polydispersity index (PDI), reliable tropism and biocompatibility. For the validation of our sEVs as reference nanoparticles of nEVs, we have conducted the physicochemical characterization and bioequivalence tests of sEVs in both cellular and animal models, demonstrating the potential of sEVs in standardizing EV‐based biomedical applications. Methods : The sEVs were characterized following MISEV 2023 guidelines, including particle size, zeta potential and encapsulation efficiency, using nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), flow cytometry, and transmission electron microscopy (TEM). Cellular uptake was assessed in HaCaT cells, and in vivo bioequivalence was evaluated in mice using nEVs from human salivary gland‐derived mesenchymal stem cells for comparison. Results : The DLS measurements confirmed particle sizes below 150 nm with a PDI under 0.1, indicating precise and uniform production. Encapsulation efficiency exceeded 95%, and zeta potential was around 50 mV. Cellular uptake assays in HaCaT cells showed higher uptake of sEVs than that of nEVs. While sEVs exhibited slightly higher cytotoxicity at low concentrations than nEVs, they were comparable at higher concentrations. In vivo mice studies demonstrated minimal hepatic and renal toxicity, similar to nEVs. Summary/Conclusion : Our findings suggest that sEVs can serve as the reliable reference particles for the EV research and applications, supporting their role in diagnostics and therapeutics. Their homogeneous mass production and customizable compositions will be able to offer a path toward more standardized and effective EV‐based biomedical solutions. Funding : This work was supported by the National Research Foundation of Korea (NRF) and the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea Government (Ministry of Science and ICT) (RS‐2024‐00432946), the NRF grant funded by the Korea government (MSIT) (No. 2021R1A2C3011254), and the Technology Development Program funded by the Ministry of SMEs and Startups (MSS, Korea) (RS‐2024‐00506850).

Targeting

Presenter: Somi Park Korea University, Seoul, Republic of Korea Introduction : Senescent cells are those that have permanently stopped dividing but remain metabolically active. Over time, the accumulation of senescent cells secretes pro‐inflammatory factors known as senescence‐associated secretory phenotype (SASP). These factors can induce senescent phenotype in nearby healthy cells, eventually contributing to age‐related diseases. The most commonly used strategy to eliminate senescent cells involves inducing apoptosis; however, this approach faces challenges with specificity. To address the limitations, we employed senescent cell‐specific receptors on extracellular vesicles (EVs) as carriers for efficient drug delivery. Here, we present a novel EV‐based system to selectively target senescent cells. Methods : Senescent cells were confirmed via SA‐β gal and rt‐pcr. EVs were isolated using serial centrifugation, tangential flow filtration and ultracentrifugation following the MISEV2023 guidelines. The function of the engineered EVs targeting senescent cells was assessed using FACS and IVIS. Results : We induced senescent cells with H 2 O 2 , UV, replicative culture, and observed a high expression of pro‐inflammatory cytokines compared to young cells before use. We also observed that nearly 50% of EVs were ligand‐positive through nano FACS. We then incubated the engineered EVs with senescent cells and confirmed that the uptake of engineered EVs was significantly higher in senescent cells than in young cells by measuring mean fluorescence intensity change. Furthermore, the function of the engineered EV was also performed in vivo using young and old mice and showed its effectiveness in targeting senescent cells through IVIS. Summary/Conclusion : We propose that the engineered EV could effectively target senescent cells and consequently can be used therapeutically to alleviate senescent cells.

Topically

Presenter: Robin L. Smith Exotropin, LLC, New York, New York, USA Introduction : More than 31 million Americans have some form of eczema that ranges from mild to severe. The standard of care includes emollients and topical steroid treatments combined with bandages and oral therapeutics. Many cases are recurrent, complicated with chronic inflammation and infection, and remain persistent, raising the need for well‐tolerated and efficacious treatments. Methods : Our data suggest that bioactive extracellular vesicles from human adipose stromal cells can be tuned, purified, and delivered to the skin via topical emulsions to effectively reprogram skin cells to reduce the symptoms associated with eczema. We have developed a patented scalable bioprocess to engineer pro‐healing EVs specifically tuned to target inflammatory skin diseases and enhance healing processes. By controlling the cellular environment in a closed‐system bioreactor, we can stimulate production and release of EVs that are biologically active and configured with a distinct miRNA cargo. Results : We demonstrate that our proprietary engineered EVs, EXO‐3, contain a unique activity profile that reduces inflammatory cytokines and stimulates collagen production, as well as activates keratinocyte and fibroblast proliferation. Research has shown that in the context of eczema, decreased levels of specific miRNAs, like miR‐29b and miR‐143, are often observed in lesional skin, which contributes to the disrupted skin barrier function and increased inflammation characteristic of the disease. Low expression of these miRNAs can exacerbate symptoms. We have identified a proprietary EXO‐3 miRNA cargo signature which includes miR‐29b and miR‐143. Replenishing decreased levels of protective miRNAs via exosomal cargo potentially offers a new treatment strategy for eczema. Under informed consent, we evaluated our EXO‐3 exosomes formulated in emulsion compositions and topically applied to afflicted skin. Our tuned EXO‐3 exosomes show a dramatic reduction of eczema symptoms, including discomfort, redness, scaling, and itchiness. Summary/Conclusion : EXO‐3 emulsion compositions are personal care formulations that are registered with the FDA under the Modernization of Cosmetics Regulation Act of 2022. EXO‐3 products were initially used solely to improve the condition and appearance of the skin. These findings were unexpected; they are supportive of engineered exosomes having targeted activity and usefulness as miRNA replacement therapies that alleviate symptoms and provide overall skin health improvement. Further exploration is warranted. Funding : This study was supported by Exotropin, LLC.

Tradeoffs

Presenter: Julie Y. Chen HORIBA Scientific, Piscataway, New Jersey, USA Introduction : Nanoparticle tracking analysis (NTA) is a high‐resolution technique for analysing extracellular particle (EP) size and concentration. Unlike dynamic light scattering (DLS), which offers bulk measurements, NTA tracks individual particle movements, making it ideal for polydisperse samples like extracellular vesicles (EVs). However, longer recording times are often required to capture sufficient data before particles leave the field of view. This study examines how reduced measurement durations impact the trade‐off between data acquisition speed and result precision. Methods : To assess the impact of shortened NTA measurements, we systematically varied the measurement duration by adjusting the number of videos and frames using NIST traceable standards (100, 150, and 400 nm polystyrene latex standards and with an EV sample). Each standard was diluted 400,000×, 100,000×, and 10,000× with 1 mM sodium dodecyl sulphate (SDS) solution. EV was diluted with phosphate‐buffered saline (PBS). Five replicates of each sample were measured per setting listed in Table 1. Video duration and number of frames were reduced incrementally to examine the threshold at which measurement precision and repeatability begin to degrade. Multiple lasers with individually adjusted laser power settings and camera gains remain constant. The particle size distributions (Dn10, Dn50, and Dn90) were compared with the supplied Certificate of Analysis, and cross‐verified with TEM; number particle concentrations were compared to fraction (1%) solids. Results : Our results reveal that shorter measurement durations can substantially decrease the accuracy of particle size distribution data. As video length decreases, under‐sampling becomes more likely, causing increased variability and decreased precision in the measured size distributions. We observed that particle count and size distribution stabilizations improve with longer measurement durations, although diminishing returns occur beyond certain thresholds. Additionally, shorter videos also led to discrepancies in data reliability, with size distributions deviating from those obtained using longer measurement durations. Summary/Conclusion : In summary, while reducing measurement duration in NTA can expedite analysis time, it compromises accuracy. Our findings indicate that shorter measurement times increase uncertainties in results, highlighting the necessity for carefully optimized NTA protocols that balance experimental speed with accuracy. This trade‐off analysis provides valuable guidelines for researchers looking to implement rapid NTA without compromising the integrity of particle size measurements.

Treatment

Presenter: Yali Yu Huazhong University of Science and Technology, China (People's Republic) Introduction : Lupus nephritis (LN) is the most common and severe refractory complication of systemic lupus erythematosus (SLE). There is an urgent clinical need for targeted and effective therapeutic strategies to repair the damaged nephron and prevent the progression of renal fibrosis. While extracellular vesicles derived from mesenchymal stem cells (MSC‐EVs) have achieved great focus as a cell‐free therapy for LN, their rapid in vivo metabolism and suboptimal target site enrichment present significant limitations. Given the high expression of CD38 in diverse immune cells and senescent cells in LN pathogenesis, we hypothesized that modification of MSC‐EVs with CD38‐targeted polypeptides could target CD38‐high immune cells and senescent cells and repair the inflammatory injured kidney. Methods : EVs were isolated from the human umbilical cord MSCs utilizing differential centrifugation and characterized following MISEV 2023 guidelines. CD38‐targeting peptides were conjugated to EV membranes via PEG‐2000 (CD38‐EVs). MRL/lpr mice, developing LN at 15 weeks, served as the experimental model. Biodistribution was assessed through immunofluorescence analysis of organ sections. LN mice received EVs or CD38‐EVs treatments at 48‐h intervals for 2 weeks. Renal function and autoantibody profiles were quantified by enzyme‐linked immunosorbent assay. Histological analyses evaluated renal inflammatory infiltration and fibrosis. Gene expression patterns associated with cellular senescence were characterized through immunofluorescence staining. Results : Our findings showed that CD38‐EVs more efficiently targeted the kidney and predominantly accumulated in renal tubular structures. Both EV and CD38‐EV treatments showed an amelioration of renal function and autoantibody levels, and a reduction of inflammatory infiltration and interstitial fibrosis. These therapeutic effects correlated with reversed stress‐induced senescence in renal tubular cells. Notably, the CD38‐EV cohort exhibited significantly superior outcomes across all parameters compared to the EV group. Summary/Conclusion : We have developed novel kidney‐targeting MSC‐EVs that demonstrate therapeutic efficacy in LN, potentially operating through the reversal of tubular cell stress‐induced senescence. While further mechanistic studies are warranted, this approach represents a promising therapeutic strategy for LN management. Funding : This research was supported by the National Key Research and Development Program of China (No. 2021YFA1101500).

Umbilical

Patrícia Freire, Sílvia C. Rodrigues, Renato Cardoso, Cláudia Oliveira, Tânia Lourenço, Andreia M. Silva, Joana Simões Correia Exogenus Therapeutics, Cantanhede, Portugal Introduction : Umbilical cord blood cells (UCBC) have been successfully used in the clinic for over 20 years. Their foetal origin and naïve status render these cells as interesting sources of extracellular vesicles (EVs) with enhanced pro‐regenerative properties. Herein, we describe how Exogenus Therapeutics developed an innovative product based on UCBC‐derived EVs (Exo‐101) and demonstrate its immunomodulatory and regenerative potential for different therapeutic uses. Methods : UCBCs were obtained from certified public/private banks and cultured under hypoxia (0.5% oxygen) for 18 h. Secreted EVs were isolated by ultrafiltration and chromatography, following a workflow developed for scalability and transfer to GMP‐compliant facilities. Exo‐101 critical quality attributes were defined upon characterization of particles, proteins, lipids and RNA content. Exo‐101's safety was evaluated in rats by repeated intravenous injection of different doses up to 12 weeks. Exo‐101's efficacy was determined using in vivo models of skin and lung injury. Exo‐101's mechanism of action (MOA) was tested in in vitro models of differentiation of macrophages cell line and peripheral blood‐derived human primary lymphocytes. Results : No major alterations in blood biochemical parameters, neither in morphology of clearance organs was detected upon repeated injection of high doses of Exo‐101 in rats, indicating systemic tolerability. Functionally, Exo‐101 significantly reduced epidermal thickening in an in vivo model of imiquimod‐induced psoriasis, and accelerated closure and re‐epithelialization of skin excision wounds in diabetic and healthy animal models. In a bleomycin‐induced lung fibrosis mice model, Exo‐101 delayed disease progression, improved animal well‐being and decreased collagen levels in the lung. In vitro studies support in vivo findings indicating that Exo‐101 has immunomodulatory functions, inducing the polarization of macrophages into the pro‐regenerative M2 phenotype, while attenuating M1 macrophages pro‐inflammatory phenotype. Exo‐101 also promoted the differentiation of Treg cells, diminishing the percentage of effector T cells in culture. Summary/Conclusion : By leverageing UCBCs as a powerful EVs source, we developed Exo‐101 as an innovative biological drug for advanced regenerative therapeutics. Tolerability and bioactivity in vivo, combined with a multifactorial MOA, suggest that it may become an effective therapy in different disease contexts. Funding : This work was co‐funded by Centro 2020 ‐ Regional Operational Program, Portugal 2020 and the European Union through FEDER.

Universal

Presenter: Takuya Kubo Kyoto Prefectural University, Kyoto, Japan Introduction : Previously, we developed a spongy‐like porous polymer consisting of poly(ethylene‐coglycidyl methacrylate) with continuous macropores that allowed efficient in situ reaction between the epoxy groups and proteins of interest. Immobilization of Protein A on spongy monolith enabled high‐yield collection of immunoglobulin G (IgG) from cell culture supernatant even at high flow rate. Additionally, this material can be moulded into any shape, enabling handling from tiny amounts of biological samples to large amounts of cell culture supernatant. In present studies, new spongy monoliths (SPMs) as separation media were developed for the separation of an extracellular vesicle, an exosome and a corona virus, SARS‐CoV‐2. Methods : In former case, the specific lectins were immobilized onto a SPM and the selective separation of exosomes were successfully achieved. Furthermore, the SPM by hybridization with TiO2 nanoparticles effectively provided the concentration of small‐extracellular vesicles (SEVs) due to the interaction between phosphate in exosomes and TiO2. In case of the virus separation, an antibody was modified onto a SPM for the selective adsorption of a spike protein on SARS‐CoV‐2. Results : In case of lectin immobilized SPMs, the separated exosomes included the different type proteins according to the proteome analysis. When we utilized TiO 2 containing SPMs, the yield of exosome was more than 100 times compared to the typical method, ultracentrifugation. This method could be applied to clinical samples, and efficient collecting of SEVs from 100 µL of plasma samples was achieved. We expect that our TiO2‐SPM will be a universal platform for collecting and purifying SEVs and significantly contribute to drug discovery research and biomarker exploration using SEVs. An anti‐spike protein (AS) based SPM (AS‐SPM) showed selective adsorption toward the spike protein in the protein mixture, and selective adsorption of SARS‐CoV‐2 was also confirmed. In contrast, the BSA‐SPM did not show adsorption of SARS‐CoV‐2 and adsorption of the rotavirus toward the AS‐SPM. Summary/Conclusion : According to these applications, newly developed SPMs can be used for the rapid and effective separation of the bio‐related targets, such as proteins, glycoproteins, extracellular vesicles, viruses, and cells.

Unlocking

Daniela Macedo 1,2,3 , Teresa Lage 1 , Lara Pierantoni 1 , Sara Abalde‐Cela 1 , Lorena Diéguez 1 , Ana Preto 2,3 , Carlos Honrado 1 1 International Iberian Nanotechnology Laboratory (INL), Braga, Portugal; 2 Centre of Molecular and Environmental Biology (CBMA), University of Minho, Braga, Portugal; 3 Institute of Science and Innovation for Bio‐Sustainability (IB‐S), University of Minho, Guimarães, Portugal Introduction : Colorectal cancer (CRC) exhibits high incidence and mortality rates, often due to late‐stage detection and limited treatment options. Changes in gut microbiota, chiefly an imbalance in short‐chain fatty acids (SCFAs), have been linked to CRC development, since SCFAs induce cell cycle arrest, lysosomal membrane permeabilization, and apoptosis. Concurrently, extracellular vesicles (EVs) are emerging as promising non‐invasive biomarkers for cancer diagnosis, prognosis, and monitoring. However, while SCFAs influence CRC cells, their effects on EV production and phenotype remain unknown. Methods : EV isolation methods, that is, ultracentrifugation (UC), size‐exclusion chromatography (SEC), ultrafiltration and an EV aggregation agent (ExoGAG), were compared. Particle size and concentration (NTA), co‐precipitated protein content (BCA), and double‐stranded DNA (dsDNA) levels (Qubit) were assessed. SCFAs were administered at IC50 concentrations, in typical normobiosis proportions (60% acetate, 25% propionate, 15% butyrate), to CRC cells to evaluate the impact on EV production and phenotype. Particle size, EV yield, protein content, dsDNA levels and integrity, and EV‐DNA analysis for microsatellite instability (MSI) using droplet digital PCR were assessed post‐treatment. Results : A 16 h UC yielded the highest particle counts but resulted in greater protein contamination compared to SEC and the optimized two‐step UC protocol. SCFA treatment increased EV production by ∼2× and significantly elevated EV‐DNA, while maintaining co‐precipitated protein levels. Notably, MSI status was assessed for the first time on CRC‐derived EVs and was shown to be detectable in EV‐DNA from all cell lines, reflecting the parent cells’ profiles, even with low dsDNA levels and after SCFA treatment. Summary/Conclusion : An optimized EV isolation protocol was developed, yielding high counts with minimal contamination. SCFA treatment significantly enhanced EV production and dsDNA content without altering co‐precipitated protein levels. Moreover, for the first time, MSI was detected in EV‐DNA, highlighting the potential of EVs as non‐invasive biomarkers for CRC, supporting their role in diagnostic and therapeutic applications. Funding : Health From Portugal (C630926586‐00465198), through the NextGenerationEU Fund.

Unpicking

Rawan Maani, Melissa Lacey, Lewis Quayle, Nick Peake Sheffield Hallam University, UK Introduction : Bowel cancer is the 3rd most common cancer and 2nd most common cause of cancer‐related morbidity worldwide. Risk factors for developing bowel cancer include obesity and diet, with increasing evidence that disease development and progression is linked to disruption to the gut microbiome. Our previous work has shown that EVs can directly target gut‐resident bacteria such as e. coli, altering behaviours including growth and biofilm formation. The aim in this work was to better understand the molecular mechanisms linking EVs to the gut microbiome. Methods : EVs were isolated from the isogenic bowel cancer cell lines SW480 and SW620 cultured in AD‐1000 bioreactors, which were fully characterised to the MISEV2023 guidelines using western blotting for positive (CD63, CD9) and negative (GM130) markers, DELFIA ELISA (CD9, CD63, CD81), nano‐flow cytometry and transmission electron microscopy. Enzyme targeting was performed using proteinase k, trypsin and PNGase to assess surface mediators of EV‐bacterial interaction. Metal ion content was assessed using commercial kits and depleted using chelators. Involvement of RNA and proteins in phenotypic changes assess using RNAse and proteinase k treatment respectively. E. coli strains MG1655 and 11G5 were treated with EVs before processing for RNAseq, and bioinformatic analysis was undertaken using BLAST alignment of putative bowel‐cancer associated EV‐miRNA with reference sequences for bacterial strains linked to bowel cancer. Results : EVs from bowel cancer cells significantly impact on growth and biofilm formation in a manner dependent on disease stage and bacterial strain. Targeting EV surface proteins with proteinase k did attenuate these effects whereas PNGase did not, however EVs lysed with detergent still had a significant affect implying a role for both direct interaction and EV cargo in altering bacterial behaviour. RNAseq showed significant upregulation of genes linked to flagella function (eg FliA), and significant down‐regulation of genes associated with zinc uptake (eg zinT, znuA, znuC). EV zinc levels were observed to be disease stage‐specific, and several miRNAs were identified with potential to target across a range of disease‐linked strains. Summary/Conclusion : EVs have potential to widely target the gut microbiome through both direct binding and indirect mechanisms, with possible involvement of both miRNA and metal ion cargo.

Unveiling

Manuti Virginia 1 , Saponaro Anna Alessia 1,2 , Murgo Emanuele 1 , Savino Giulia 2 , Marasco Maria Greta Pia 2 , Villani Rosanna 3 , Serviddio Gaetano 3 , Colangelo Tommaso 1,2 1 Cancer Cell Signalling, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy; 2 Department of Medical and Surgical Sciences, University of Foggia, Italy; 3 C.U.R.E. (University Center for Liver Disease Research and Treatment), Liver Unit, Department of Medical and Surgical Sciences, University of Foggia, Italy Introduction : Non‐alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease, characterized by excessive hepatic fat accumulation. The disease can progress from simple hepatic steatosis to non‐alcoholic steatohepatitis (NASH), fibrosis, and, in severe cases, liver cirrhosis and cancer. Understanding the molecular mechanisms underlying this progression is crucial for developing targeted therapies. Extracellular vesicles (EVs), carrying miRNAs, play critical roles in intercellular communication, impacting metabolic and inflammatory pathways. This study seeks to uncover the role of EV‐derived miRNAs in NAFLD progression. Methods : EVs were isolated from hepatocyte cell lines with induced steatosis via fatty acid exposure and characterized using nanoparticle tracking analysis and Western blotting. miRNA sequencing was performed to identify differentially expressed miRNAs in EVs derived from steatotic versus control hepatocytes. Select miRNAs of interest were validated using digital PCR. Functional assays were conducted by treating healthy hepatocyte cultures with miRNA‐enriched EVs to evaluate their impact on lipid accumulation, insulin signalling pathways and inflammatory markers (ELISA). Results : Key miRNAs were identified in NAFLD‐derived EVs, notably those influencing lipid accumulation and inflammatory response. Functional studies demonstrated that these EV miRNAs regulate pathways critical to NAFLD pathogenesis, highlighting their potential as therapeutic targets. miRNA sequencing revealed a distinct miRNA expression profile in EVs derived from steatotic hepatocytes compared to controls such as miR‐122 and miR‐34a. These miRNAs are known to influence lipid storage and insulin resistance. Furthermore, when hepatocytes were treated with EVs containing steatosis‐associated miRNAs, there was an increase in intracellular lipid accumulation. In addition, Western blot analysis of insulin signalling pathway markers showed a decrease in phosphorylation of key proteins, such as Akt, in hepatocytes treated with miRNA‐enriched EVs. Finally, ELISA results revealed elevated levels of inflammatory cytokines (e.g., IL‐6 and TNF‐alpha) in hepatocytes exposed to steatosis‐associated EVs. Summary/Conclusion : In the context of NAFLD, EV‐derived miRNAs may play a significant role in modulating pathways involved in lipid metabolism, insulin resistance and inflammation—three key aspects of the disease pathology. Our findings reveal EV‐miRNAs as modulators of metabolic dysregulation and inflammation in NAFLD, underscoring their promise as biomarkers and therapeutic targets.

Utilizing

Gréta Lilla Bányai 1 , András Merényi 2 , András Szabó 1 , Afrodité Németh 1 , Anikó Gaál 3 , Csaba Pongor 1 , Tamás Garay 1,2 1 Pázmány Péter Catholic University, Hungary; 2 Semmelweis University Hungary; 3 Research Centre for Natural Sciences, Hungary Introduction : Liquid biopsies, including the analysis of circulating tumour cells, cell‐free DNA, and extracellular vesicles (EVs), offer promising potential for early and detailed diagnosis for cancer patients. EVs, might have a distinguished potential to provide insight into a patient's physiological and pathological state as they are secreted by all cell types. Microfluidic techniques enable characterization of dissolved particles based not only on their size distribution but accounting also for their mass, density and shape. Methods : Blood samples were obtained from patients with prostate, pancreatic, and colorectal cancer. For each sample, the interval from blood draw to plasma isolation and hemoglobin levels were recorded. The microsomal fraction of plasma was isolated using size‐exclusion chromatography (SEC), and this fraction was subsequently characterized for particle size, size distribution, and concentration using nanoparticle tracking analysis (NTA). Total protein content was quantified using the Qubit protein assay, while lipid content was assessed using the SPV assay. Atto‐488 NHS ester‐stained samples were introduced into a microfluidic channel, where the distribution of protein‐bound green fluorescent signals was assessed across the channel diameter by capturing consecutive fluorescence microscopy images. Fluorescence intensity was quantified by fitting an asymmetric sigmoid function to both sides of each distribution curve, followed by determining the amplitude and growth rate of the averaged function. Results : Our experiments did not demonstrate a significant correlation between hemoglobin levels and the time elapsed prior to sample processing. Additionally, lipid and protein concentrations lacked specificity across patient groups. Although nanoparticle tracking analysis (NTA) yielded uniform results among these groups, microfluidic analysis revealed distinct variations in the growth rates of fluorescence intensity curves as samples passed through the microfluidic channel. These differences enable robust differentiation among patients with unique oncological profiles. Summary/Conclusion : This indicates that microfluidic techniques can offer a more comprehensive assessment of the extracellular vesicle (EV) fraction by leverageing diffusion characteristics alongside particle size distributions. Furthermore, the ability to differentiate between cancer patient samples using a robust, cost‐effective, and easily performed technique holds significant promise for clinical applications. Funding : This research was supported by the National Research, Development and Innovation Office through the grant TKP2021‐EGA‐42.

Versatile

Barbara Krönigsberger 1,2,3 , Sivun Dmitry 1,2,3 , Madhusudhan Reddy Bobbili 1,4,3 , Johannes Grillari 1,4,3 , Jaroslaw Jacak 1,2,4,3 1 Ludwig Boltzmann Institute for Traumatology, The Research Centre in Cooperation with AUVA, Vienna, Austria; 2 Department of Medical Engineering and Applied Social Sciences, University of Applied Sciences Upper Austria, Linz, Austria; 3 Department of Biotechnology, Institute of Molecular Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria; 4 Austrian Cluster for Tissue Regeneration, Vienna, Austria Introduction : Efficient and standardized EV internalization remains a challenge. To address this, we aim to develop a versatile strategy for EV modification to target specific cellular uptake pathways. Methods : We employed the biotin‐streptavidin system to modify eGFP‐tagged EVs derived from HEK293T cells. The first step involved the incorporation of cholesterol‐lipids into the EV membrane. A polyethylene glycol (PEG) linker of approximately 2.45 nm length conjugated cholesterol to biotin, forming a cholesterol‐PEG‐biotin (CPB) construct. For functionalization, we added Alexa Fluor 647‐labelled streptavidin (SA) to the sample, creating a tight bond to the biotinylated EVs. To optimize the labelling of unpurified EVs, we used atomic force microscopy (AFM) and single molecule fluorescence microscopy (SMFM), enabling precise characterization of surface modifications at a single‐molecule level. Building on this approach, we plan to introduce a biotinylated ligand, such as transferrin (Tf), to coat the EV surface as a test system. This strategy is intended to exploit the Tf‐transferrin receptor (TfR) pathway for targeted uptake of small EVs. Linking EVs to Tf enables specific binding to TfR, facilitating cellular internalization via the canonical clathrin‐dependent uptake mechanism. Results : Using SMFM, we confirmed the incorporation of CPB into the EV membrane and the subsequent binding of streptavidin (SA). Through dual‐colour 2D imaging, we observed that approximately 80% of the eGFP‐positive EVs also carried the CPB‐streptavidin construct, with free SA accounting for about 35% of the total detected SA in the solution. Summary/Conclusion : The proposed EV's surface modification protocol (cholesterol‐biotin‐streptavidin) is a versatile tool for wide‐range customization of EVs. Targeted delivery into cells via canonical clathrin‐dependent pathways using modified EVs may represent the first standardized method for cellular internalization. Funding : This project is supported by Interreg AT‐CZ NanoPrecMed ATCZ00052.

Activation

Presenter: Yang Lu The University of Texas, Austin, Texas, USA Introduction : Cancer immunotherapy has been successful in treating some cancer patients, but it still fails in most cases. Despite novel potential tumour neoantigens being discovered, cancer is in general an internal disorder lacking potent antigens to elicit strong anti‐tumour immune responses compared with foreign antigens. In this study, we tested a new strategy using tumour‐targeting small extracellular vesicles (sEVs) to redirect host preexisting T cell‐mediated non‐cancer immunity toward cancer cells through targeted delivery of relevant antigens to tumours. Methods : To develop a tumour‐targeting sEV platform, we designed a cell membrane‐anchoring approach to display a single chain variable fragment (scFv) antibody (derived from anti‐HER2 trastuzumab) on sEV surface. For proof of concept, we used HER2‐targeting sEVs for delivering ovalbumin (OVA)‐derived peptide SIINFEKL to targeted tumours in OT‐1 transgenic mice. We developed a method to load SIINFEKL peptides on the sEVs in an MHC‐I‐dependent manner. Results : We confirmed a targeting role of the scFv on the sEVs in facilitating receptor‐mediated endocytosis of the sEVs through binding to the HER2 overexpressed on targeted tumour cells. Treatment of OT‐1 T cells with SIINFEKL‐loaded sEVs activated OT‐1 T cells, shown by a marked increase in the percentage of CD69‐positive T cells, and stimulated proliferation of OT‐1 T cells, shown by a proliferation assay of T cells labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Co‐culture of OT‐1 T cells with HER2‐overexpressing tumour cells induced massive apoptosis of the tumour cells only after treatment with the cells with SIINFEKL‐loaded HER2‐targeting sEVs. In our studies carried out in mice, we found that intraperitoneal administration of SIINFEKL‐loaded HER2‐targeting sEVs also activated CD8+ T cells in OT‐1 mice. Compared with the findings from the OT‐1 mice treated with the HER2‐targeting sEVs without loading with SIINFEKL or treated with SIINFEKL‐loaded untargeted sEVs, treatment of the mice with SIINFEKL‐loaded, HER2‐targeting sEV strongly inhibited growth of HER2‐overexpressing tumours implanted in OT‐1 mice. Summary/Conclusion : Our findings support our ongoing and future work that pre‐existing T cell‐mediated immunity, such as anti‐viral immunity acquired through natural infection or vaccination, can be recalled and redirected to targeted tumours by tumour‐targeting sEVs loaded with MHC‐I‐compatible peptides. Funding : This study was supported by the CPRIT (RP200271) and BCRF (23‐051).

Advantages

Presenter: Mai Hazekawa Fukuoka University, Japan Introduction : The final goal of our study is to develop an siRNA therapeutic system for the cancer metastases using autologous serum‐derived small EVs as a cargo. Recently, some kinds of nanoparticles have been featured as a useful tool for siRNA or mRNA delivery. However, the differences between them have not been clarified. The purpose of this study is to show the advantages and disadvantages of small EVs derived from serum compared with other types of nanoparticles, for example, lipid nanoparticles and micelles. Methods : The small EVs isolated from mouse serum were used. To identify it, the expression of surface markers (CD9, CD63, CD81) was confirmed by Western blotting. Lipid nanoparticles and polymeric micelles were used as the comparison groups. Particle size and surface charge were evaluated using a DLS (dynamic light scattering) instrument. For cancer cells, the cellular uptake, uptake rate, and uptake mechanism of each formulation were evaluated using flow cytometry and confocal fluorescence microscopy. For the therapeutic efficacy assessment of siRNA, a WST‐8 assay was performed. Results : The particles of small EVs obtained in this study were approximately 100 nm in size and had a neutral charge. For cellular uptake, all formulations showed an improvement in uptake efficiency, with an intracellular introduction rate of 100%. However, differences were observed in the intensity and speed of cellular uptake. Particles composed of lipid membranes, including small EVs, showed earlier cellular uptake, whereas micelles demonstrated a slower uptake. Lipid nanoparticles showed the highest intensity of cellular uptake, but no correlation was observed between therapeutic efficacy and uptake intensity. Additionally, while micelles were more heavily influenced by endocytosis, small EVs were found to utilize various uptake pathways. All formulations exhibited siRNA efficacy, demonstrating enhanced therapeutic effects compared to siRNA alone. Summary/Conclusion : This study clarified the usefulness of serum‐derived small EVs as siRNA carriers and their advantages over conventional technologies. While all particles possess utility as drug delivery systems, differences in uptake speed, anticipated half‐life, and uptake pathways suggest the importance of accurately understanding each advantage and limitation. By optimizing these carriers based on the target disease, it is anticipated that ideal nucleic acid therapeutics can be realized.

Alteration

Shuzhen Chen 1 , Smara Sigdel 1 , Gideon Udoh 1 , Annie Chen 1 , Brandon Yu 1 , Rakan Albalawy 2 , Jinju Wang 1 1 Department of Biomedical Sciences; 2 Department of Internal Medicine, Marshall University, Huntington, WV 25755, USA Introduction : Vascular dementia (VD) is a chronic cerebrovascular syndrome caused by various factors, with cerebral vascular damage being one of the key pathological features. Circulating extracellular vesicles (cEVs) are emerging as novel players in neurodegenerative diseases, but the roles and implications of cEVs in hypertension‐related VD are largely unknown. In this study, we studied the cEV miR profiles/signatures in hypertensive transgenic mice. Methods : The cognitive functions of mice were assessed. CEVs were isolated from hypertensive transgenic and wild‐type mice (denoted as HPN‐cEVs and WT‐cEVs). The total RNAs, including small RNAs, were extracted from each cEV sample. RNA quality and integrity were determined using the Agilent 2100 Bioanalyzer. The libraries were prepared with the QIAseq miRNA Library Kit (Qiagen) and sequenced using an Illumina NGS system. Data analysis was performed using the RNA‐seq Analysis Portal. Results : Our data showed that the middle‐aged hypertensive transgenic mice showed impaired learning capability and short‐term memory. They had remarkably reduced cerebral microvascular density. We also found that the cells reside along the brain's blood vessels, exhibiting senescence‐like characteristics, in the hypertensive mouse brain, suggesting cerebrovascular dysfunction under hypertensive conditions, which could precede VD. According to our preliminary study of RNA‐seq, 74 miRs had a fold change less than −2 or larger than 2. Among those 74, 23 miRs had a fold change less than −4 or larger than 4 combined with an FDR p value less than 0.05. Some of those altered miRs, such as miR‐133a‐3p, miR‐196a‐5p, and miR‐296‐5p, are highly related to vascular repair and neuronee functions in the brain. Summary/Conclusion : Taken together, our data suggests the possible involvement of cEVs in hypertension‐related VD. It will shed light on establishing new strategies that interfere with the onset and progression of hypertension‐related VD and provide a new perspective for alleviating and delaying VD in individuals with hypertension. Funding : American Heart Association (AHA)‐Career Development Award (935826), AHA‐Transformational Project Award (24TPA1291189), and the National Institute of General Medical Sciences (U54GM104942).

Analytical

Stephen Lenzini, Madeline Cramer, Elie Zakhem, Jon A. Rowley RoosterBio, Inc., USA Introduction : Scalable MSC‐EV manufacturing processes are required to meet lot sizes for clinical applications. Understanding differences among MSC tissue types and donors and establishing consistent production processes are critical to overcoming scalability challenges. We used analytical tools to understand MSC‐EV quality attributes from different tissue sources when using a consistent media system across multiple production platforms. Methods : Human MSCs from 3 bone marrow (BM) donors, 2 umbilical cord (UC) donors and 1 adipose tissue (AD) donor (RoosterBio sourced) were expanded in RoosterNourish‐XF in different scalable production platforms (stirred‐tank, vertical‐wheel, spinner flask and 2D T‐flask). EVs were collected in the defined media RoosterCollect‐EV for 2 to 5 days. Particle number and size were evaluated by NTA. Total RNA content was measured via Bioanalyzer. EV marker expression was evaluated by capillary western blot, and CD73 activity was measured as a potency indicator. Fold changes in various miRNA targets were measured by qPCR. Results : Cells in all 3D bioreactor platforms produced significantly more particles than in 2D (4–5‐fold greater). Across 3D conditions, particle count increased from Day 2 to 5, with BM and UC donors demonstrating comparable maximum particle production (∼1E10/mL) while AD had less particle production (∼5E9/mL). Differences in particle size were observed with median sizes for UC 173 nm, BM 158 nm, and AD 143 nm. For all groups, protein content increased, from Day 2 to 5 as particle count increased showing a strong correlation. Total RNA content decreased from Day 2 to 5 across all 3D platforms, while RNA content remained stable in 2D. EV identity markers (CD63, CD81, CD9) and MSC‐EV identity marker CD73 were confirmed. CD73 expression and activity were demonstrated in all conditions, but the overall magnitude and trend over collection days varied between donors. Summary/Conclusion : Overall, this comprehensive study evaluated MSC‐EV quality attributes among multiple scalable production platforms using different donors from different tissue sources in a standardized cell source and media system. While EV quality attributes were consistent for each donor across production platforms, some quality attributes (particle count and CD73 activity) were donor dependent. This work helps establish a foundation for selecting clinical manufacturing processes for EV production.

Anticancer

Pisitpong Jarurachadanon 1 , Wittaya Panvongsa 1 , Intiporn Mingsakul 1 , Boon‐ek Yingyongnarongkul 2 , Suradej Hongeng 1 , Arthit Chairoungdua 1 1 Mahidol University, Thailand; 2 Ramkhamhaeng University, Thailand Introduction : Atypical teratoid/rhabdoid tumour (AT/RT) is an aggressive paediatric brain tumour with poor treatment outcomes, highlighting the need for alternative therapies. Previously, we isolated nanovesicles from Boesenbergia rotunda (fingerroot) and demonstrated their anticancer activity against colorectal cancer cells. However, the effects and mechanisms of fingerroot exosome‐like nanovesicles (FELNs) in AT/RT remain unclear. Methods : FELNs were isolated from fingerroot juice via ultracentrifugation and purified using a qEV size‐exclusion chromatography (SEC) column. Their size and morphology were characterized by DLS and TEM. Cytotoxicity was assessed in BT12 and BT16 AT/RT cell lines in 2D and 3D cultures, while migration and invasion were evaluated via transwell assays. Proteomic analysis was performed using mass spectrometry. Results : The isolated FELNs were round‐shaped, membrane‐bound vesicles with an average size of 100 nm. Metabolite profiling of FELNs identified the presence of pinostrobin, pinocembrin, and panduratin A. FELNs were taken up by BT12 and BT16 cells and exhibited dose‐ and time‐dependent cytotoxicity through apoptosis induction. Interestingly, human brain endothelial cells (hCMEC/D3) demonstrated lower sensitivity to FELNs. Notably, at non‐toxic concentrations, FELNs significantly suppressed BT12 and BT16 cell migration and invasion. Proteomic analysis further revealed that FELNs disrupted signalling pathways associated with AT/RT development, including the Wnt/β‐catenin signalling pathway. Treatment with FELNs suppressed β‐catenin protein levels and its target gene, cyclin D1. Additionally, FELNs reduced phospho‐GSK‐3β (Ser9), suggesting that FELNs promote β‐catenin degradation through a GSK‐3β‐dependent mechanism. Summary/Conclusion : Collectively, our results indicate that FELNs exhibit anticancer activity in AT/RT cells by inducing apoptosis and suppressing cancer cell migration and invasion. Mechanistically, the anticancer effects in AT/RT cells are partly mediated through the GSK‐3β‐dependent β‐catenin degradation pathway. Furthermore, FELNs demonstrated lower cytotoxicity in normal brain endothelial cells, highlighting their potential as novel therapeutic agents for brain tumour treatment. Funding : Mahidol University (Basic Research Fund: fiscal years 2022 to A.C.) and National Research Council of Thailand (NRCT) and Mahidol University (N42A660523 to A.C.).

Apoplastic

Ani Barbulova 1 , Immacolata Fiume 1 , Veronika Kralj‐Iglic 2 , Ales Iglic 2 , Anna Romolo 2 , Maneea Moubarak 3 , Gabriella Pocsfalvi 1 1 National Research Council of Italy, Institute of Biosciences and BioResources, Italy, 2 University of Ljubljana, Slovenia, 3 Damanhour University, Egypt Introduction : In vitro plant cell cultures are increasingly exploited for extracellular vesicles (EVs) farming due to their higher reproducibility, scalability and bio‐sustainability on long term scale compared to mammalian systems. Generally, the nanoparticles are isolated from the conditioned culture medium (CCM) and are considered as EVs since they are secreted in the medium during the cells life cycle. Recent studies report a relatively low yield of plant EVs from CCM. Plant cells grown in the CCM could be considered as an alternative source of apoplastic vesicles (AVs), generally isolated from plant tissues or organs applying vacuum infiltration and differential centrifugation. We have established a novel protocol that employs cell wall degrading enzymes to release AVs from in vitro grown cells. These AVs together with the EVs isolated from the CCM of the same suspension culture could open new perspectives to study plant EVs from in vitro cultures. Methods : CCM and cells from suspension cultures of tomato in the log phase of their growth were used for EVs and AVs isolation using differential centrifugation. Isolation of AVs was performed using a mix of cell degrading enzymes. Treatment conditions and enzyme composition were optimized. Obtained AVs and EVs were characterized for morphology, particle concentration, size distribution and protein profile, using cryotem, videodrop and SDS‐PAGE. Results : We optimized a method for AVs isolation from cells grown in suspension cultures of tomato using cell wall degrading enzymes. The morphology, size and size distribution of the obtained AVs were similar to that of EVs derived from the CCM of the same suspension culture, showing doble membrane layer and size of 50‐250 nm diameter. The yield and the protein content of the AVs were significantly higher than the CCM‐derived EVs. Summary/Conclusion : The developed enzymatic method for release and isolation of AVs from cells in suspension culture after treatment with cell wall degrading enzymes could be a step towards an increased EVs yields. This could further boost their exploitation for agricultural, biotechnological and one health applications. Funding : This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Staff Exchange project “FarmEVs,” grant agreement N. 101131175.

Artificial

Presenter: Wen‐Yu Lien Bionet Therapeutics, Taipei, Taiwan (Republic of China) Introduction : Osteoarthritis (OA) is a common degenerative joint disease characterized by cartilage degradation, joint inflammation, and chronic pain, leading to significant functional limitations. With an ageing population and increasing healthcare burdens, new treatment approaches are urgently needed. Exosomes, extracellular vesicles carrying bioactive molecules such as miRNAs, have emerged as potential therapeutic tools for OA due to their ability to modulate gene expression and mediate intercellular communication. ExoVia, an exosome product derived from umbilical cord mesenchymal stem cells (UCMSCs) with a defined miRNA profile, offers a promising therapeutic approach for OA. This study investigates ExoVia's miRNAs to regulate key OA‐related genes through AI‐driven deep learning models. Methods : The miRNA profile of ExoVia exosomes has been compiled and cross‐referenced with miRBase for functional annotation. OA‐related genes and pathways were identified by mining public databases such as KEGG, Reactome, and GeneCards. AI‐driven deep learning models, integrating tools like TargetScan, miRDB, and DIANA‐microT, were used to predict miRNA‐mRNA interactions. We applied CNN, RNN, and Transformer architectures, optimizing the models through feature selection and hyperparameter tuning. Predicted interactions were prioritized, and biological validation was conducted in OA chondrocyte cultures to assess changes in gene expression. Results : Our analysis identified a comprehensive miRNA profile from ExoVia, revealing several miRNAs with strong potential to regulate key genes involved in OA, such as those related to cartilage degradation and inflammation. AI‐driven models successfully predicted high‐confidence miRNA‐mRNA interactions, highlighting several miRNAs with significant regulatory potential for OA‐related genes. Additionally, in our in vitro OA model using chondrocyte cultures, treatment with ExoVia exosomes led to a significant downregulation of OA‐associated inflammatory markers. These findings confirm the therapeutic potential of ExoVia's miRNAs in modulating gene expression and reducing inflammation, further supporting its promise as a novel OA therapy. Summary/Conclusion : This study demonstrates the therapeutic potential of the ExoVia exosome product in OA, driven by miRNA regulation. AI models successfully identified key miRNA‐mRNA interactions, and in vitro validation confirmed ExoVia's ability to reduce inflammation in OA. These findings support ExoVia as a promising therapeutic approach for OA and highlight the value of combining exosome‐based therapies with AI to accelerate the development of treatments for degenerative diseases.

Assessment

Presenter: Maria Cristina Bravo University of Vermont, USA Introduction : Severe thermal injury is associated with local and systemic responses, including the release of EVs from damaged or stressed cells and coagulopathy. Here we report on procoagulant potential and EVs in plasma from thermally injured individuals. Methods : Patients ( n = 28) with varying burn severity (total body surface area, %TBSA: 39% ± 18%) were enrolled prospectively in an IRB approved study upon admission. Blood was collected, platelet poor plasma prepared within 30 min, and aliquots frozen at −80°C. For all analyses plasma was thawed in the presence of corn trypsin inhibitor to prevent contact pathway activation. Endogenous thrombin generation (TG) potential was assessed using the standardized calibrated automated thrombogram protocol, reagents (MP‐phospholipid only), and instruments (Stago); parameters of lag time and peak thrombin were extracted. To isolate EVs, freshly thawed plasma was centrifuged at 1500 × g for 15 min and 0.5 mL of supernate plasma was loaded onto an Izon Gen2 8.5 mL 70 nm agarose gel column and gravity eluted with HBS. EV isolates were defined by a purified collection volume of 2.9–4.9 mL after sample loading. Plasma concentrations of functional tissue factor (TF) and surfaces expressing phosphatidylserine (PS) were established using kinetic assays measuring the capacity of isolates to support extrinsic tenase and prothrombinase assembly and function, respectively. Quantification was based on standard curves using titrations of commercial sources of TF or PS‐bearing vesicles. Total EV (TotEV) and Intermediate sized EV (100–500 nm, IntEV) concentrations were determined in EV isolates using an Izon qNano Gold instrument. Data are presented as mean ± SD; measures were compared using Pearson correlations (significance: p ≤ 0.05). Results : 27 of 28 individuals had endogenous TG, with an average lag time of 16.8 ± 7.1 min and peak thrombin of 124 ± 66 nM. Samples had a TotEV concentration of 1015 ± 1074 × 10 9 /mL, with approximately 20% being IntEV (148 ± 176 × 10 9 /mL). Functional assays measured 60.9 ± 64.0 fM TF and 669 ± 539 nM PS in collected samples. TBSA (%) correlated with TF ( r = 0.454) and IntEV ( r = 0.409) levels but not with TG parameters or PS levels. TF correlated with IntEV ( r = 0.507) and strongly with PS ( r = 0.722). Summary/Conclusion : Thermal burn severity was associated with increased levels of both TF‐bearing vesicles and EVs of intermediate size, but not endogenous TG potential.

Atr‐Ftir

Alexander Popoff 1 , Cristian‐Tudor Matea 2,3 , Patrick Zimmerebner 2,3 , Andreas Marl 2,3 , Mario Gimona 3,4,5 , Eva Rohde 3,5,6 , Nicole Meisner‐Kober 2,3 , Daniela Schuster 1 1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Paracelsus Medical University, Salzburg, Austria; 2 Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Salzburg, Austria; 3 Ludwig Boltzmann Institute for Nanovesicular Precision Medicine, Salzburg, Austria; 4 Research Program “Nanovesicular Therapies,” Paracelsus Medical University, Salzburg, Austria; 5 GMP Unit, Paracelsus Medical University, Salzburg, Austria; 6 Department of Transfusion Medicine, University Hospital, Salzburger Landeskliniken GesmbH (SALK) and Paracelsus Medical University, Salzburg, Austria Introduction : Extracellular vesicles (EVs) harbour great therapeutic potential either by their intrinsic properties or as vehicles for drug delivery. Therefore, isolation of EVs from their biological sources is a crucial aspect. Among commonly known isolation techniques like ultracentrifugation and preparative size exclusion chromatography (SEC), tangential flow filtration (TFF) offers great potential for upscaling but is time‐consuming and requires a process control like analytical SEC. Here we show that attenuated total reflection Fourier transform infrared spectroscopy (ATR‐FTIR) can serve as a simple and fast method for the quality control of EVs. Methods : For the analysis we collected different milk‐derived EV samples during various TFF stages and determined the respective purity by analytical SEC, followed by acquisition of ATR‐FTIR spectra. This sample set we divided into a training and into a test set and created multivariate analysis models to predict the SEC purity. Accuracy and precision of the models were analysed with the test set. Results : While the training sets showed coefficients of determination (R 2 ) above 0.95, the test sets had R 2 values greater than 0.75 and errors of prediction below 10%. In addition, weighting of spectral variables indicated that wavenumbers of lipid, phosphate and carbohydrate stretching are associated with higher SEC purity. In contrast, protein‐associated wavenumbers, like for instance the Amide I and Amide II bands at 1660 cm −1 and 1550 cm −1 are clear indicators of lower SEC purity. Summary/Conclusion : Here we demonstrate that ATR‐FTIR can serve as process control during the TFF process, for example, which requires several buffer exchange steps. Due to its simple procedure, the low sample amount required and time efficiency, this technique can be a promising supplement for industrial scale EV isolation. In addition, multivariate analysis revealed spectral areas that can indicate molecule classes that can further lead to the discovery of purity markers. Funding : The project was partly funded as part of EV‐TT (WISS 2025 and EFRE/IWB 20102‐F1900731‐KZP).

Biomarkers

Presenter: Larissa Jank Johns Hopkins University, Baltimore, Maryland, USA Introduction : Multiple sclerosis (MS) is a chronic immune‐mediated demyelinating disease of the central nervous system (CNS). While therapies exist for the peripheral inflammation seen in relapsing MS (RMS), progressive MS (PMS) presents challenges due to ongoing glial activation and neurodegeneration. There are no effective treatments for PMS, partly due to a lack of biomarkers for CNS pathogenic processes in MS. Circulating EVs originating from the CNS show promise in providing these biomarkers. Methods : EVs were isolated from the plasma of 292 MS patients (195 PMS, 97 RMS), using polymer‐precipitation followed by immunoprecipitation of astrocyte‐enriched EVs ([AEVs] GLAST+) and neuronal‐enriched EVs ([NEVs] L1CAM+). We measured complement components in AEVs and synaptopodin in NEVs. Outcomes included Expanded Disability Status Scale (EDSS), the tablet‐based MS performance test (zMSPT), and clinical MRIs. Longitudinal data was collected for up to 3 years (ongoing). Biomarker associations with outcomes were analysed using linear mixed models and logistic regression models adjusted for age, sex, race, disease duration, subtype, EV isolation batch (and individual total follow‐up time). Analyses of retinal layer thickness (OCT) scans are ongoing. Results : In AEVs, C3 and C3b levels were associated with worse clinical outcomes cross‐sectionally (age‐adjusted EDSS [gARMSS]: C3 β = 3.75, p = 0.044 and zMSPT in RMS C3 β = −1.20, p = 0.028; C3b β = −0.233, p = 0.009) and longitudinally predicted faster brain atrophy in PMS (C3 in the thalamus β = −0.538, p = 0.035 and C3b in cerebral white matter β = −0.043, p = 0.015). C5 and C5a levels were cross‐sectionally associated with worse clinical outcomes (gARMSS: C5a β = 0.235, p = 0.05; zMSPT C5 β = −0.142, p = 0.016, C5a β = −0.115, p = 0.007) and MRI measures (higher WM lesion volume, C5 β = 0.667, p = 0.0007; C5a β = 0.336, p = 0.007 in RMS) but longitudinally predicted EDSS improvement (C5 OR = 2.74, p = 0.031; C5a OR = 2.61, p = 0.038). In NEVs, higher synaptopodin levels longitudinally predicted slower progression clinically (EDSS progression, OR = 0.42, p = 0.021) and on MRI (total brain atrophy, β = 0.669, p = 0.042). Summary/Conclusion : The EV biomarkers were associated with outcomes in unique patterns, suggesting they represent distinct disease processes: synaptopodin loss is likely linked to synaptic pathology, C5/C5a to acute inflammation, and C3/C3b to chronic compartmentalized inflammation. In the future, these biomarkers may serve as valuable tools for monitoring MS progression and enhance our understanding of underlying mechanisms. Funding : This work is funded by Genentech (P.A.C., P.B.), the NMSS (L.J., B.D., K.C.F., P.B.), and the NIH (E.M.M., P.A.C.).

Biomimetic

Presenter: Xiaomei Liang Southern Medical University, Guangzhou, People's Republic of China Introduction : Intrauterine adhesion (IUA) is one of the main causes of female infertility. Extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSCs) rich in multiple factors and miRNAs related to tissue regeneration, which are the main effective paracrine components of MSCs to treat IUA. However, the poor secretion and bioactivity of EVs have been obstacles to their widespread use in clinical practice. Here, we developed human chorionic villi (CV)‐derived nanofibres and then constructed the CV‐nanofibre bionic culture system for MSCs. The mechanisms of the culture system were explored in maintaining the stemness of MSCs over a long period and enhancing EV secretion and bioactivity. Finally, we formed EVs‐ECM aggregate for treatment of rat IUA model and explored its efficacy and mechanisms to provide experimental evidence for IUA clinical therapy. Methods : CV tissue was collected and separated from human placenta. CV‐nanofibre was prepared through decellularization and processing. The bionic culture system for MSCs based on CV‐nanofibre was constructed to collect high‐concentration EVs enrichment supernatant. EVs enrichment degree was detected by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot. Finally, EVs‐ECM aggregate was formed for treatment of rat IUA model. Results : We reconstructed CV‐ECM into CV‐nanofibre, which retained the ECM protein component completely and was rich in proteins of enhancing EV formation, release and transport. We constructed the bionic culture system for MSCs based on CV‐nanofibre, cultured in which MSCs stemness maintained for up to 30 days and EVs production efficiency was enhanced greatly. These EVs meeting international standards contained proteins of promoting tissue regeneration. Finally, we formed EVs‐ECM aggregate. Experimental results showed that EVs‐ECM could effectively repair endometrium, treat IUA model and support healthy live birth. Summary/Conclusion : In summary, we constructed the CV‐nanofibre biomimetic culture system to achieve large‐sale culture of MSCs and enhance EV secretion and bioactivity. EVs obtained by this system were encapsulated with CV‐nanofibres to form EVs‐ECM aggregate, which can effectively promote endometrium regeneration toward supporting foetal development and live births and overcomes the main obstacle preventing EV. Funding : This work was supported by the National Natural Science Foundation of China (82371725 and 32301204).

Biosensors

Eylul Gulsen Yilmaz 1,2 , Fatih Inci 1,2 , Yeşeren Saylan 3† 1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey; 3 Hacettepe University, Department of Chemistry, Ankara, Turkey Introduction : Early detection of breast cancer improves therapy and results, however 3D in vitro models are difficult to use to investigate biophysical influences on cancer cells. The microfluidic chip we show isolates extracellular vesicles (EVs) from MCF7 breast cancer cells, with an emphasis on EVs as cancer progression indicators. We will conduct 3D cell cultures with an extracellular matrix to investigate the impact of EVs on MCF‐12A normal breast cells and their function in alterations to the tumour microenvironment. For more accurate, up‐to‐the‐minute diagnoses, optical biosensors will be integrated with EV‐imprinted nanoparticles to enhance detection. This technique improves our knowledge of cancer biology and allows for more accurate non‐invasive cancer detection. Methods : The microfluidic chip were prepared in order to mimic continuous flow in dynamic cell culture. We cultured MCF‐7 and MCF‐12A. Isolated EVs have been examined by Western blotting, NTA, and SEM after they were extracted from MCF‐7 cells by ultrafiltration. EV‐imprinted nanoparticles were synthesized using methacrylic acid and micro‐emulsion polymerization, and their size and morphology were analysed using a Nano Zetasizer and TEM. Results : EVs extracted from a collagen‐coated microfluidic chip exhibited a size of 129.2 ± 4.5 nm and a concentration of 6.49 × 10 9 ± 3.56 × 10 8 particles/mL, as validated by NTA. SEM confirmed the elimination of bigger particles, isolating those within the 50–150 nm range, which exhibited a spherical morphology. Western Blot verified the existence of tetraspanin proteins CD63 and CD81. EV‐imprinted nanoparticles had a size of 62.78 nm with low polydispersity (PdI = 0.116), signifying uniformity in size and shape, corroborated by TEM analysis. The nanoparticles were uniform, spherical, and consistent with the size distribution obtained from zeta size analysis. Summary/Conclusion : This research seeks to create a molecularly imprinted optical biosensor for the sensitive, selective, and real‐time detection of EVs. This innovative method entails the extraction of MCF7 EVs from 3D cell cultures utilizing microfluidic chips incorporated with an extracellular matrix. The gathered EVs will be examined to ascertain their effects on MCF‐12A, yielding insights into alterations in the cancer microenvironment and the cancerization process. Funding : All authors acknowledge the support from The Scientific and Technological Research Council of Türkiye (TÜBİTAK) (Project Numbers:121C226).

Biotherapy

Serena Cecchetti 1 , Cristina Federici 1 , Rossella Canese 1 , Egidio Iorio 1 , Veronica Huber 2 , Maria Elena Pisanu 1 , Mattea Chirico 1 , Elisabetta Iessi 1 , Serena Camerini 1 , Marialuisa Casella 1 , Andrea Matteucci 1 , Daniele Macchia 1 , Massimo Spada 1 , Ann Zeuner 1 , Luana Lugini 1 1 Istituto Superiore di Sanità, Italy, 2 Istituto Nazionale dei Tumori, Rome, Italy Introduction : Extracellular vesicles of natural killer cells (NKEV) exert an antitumour effect towards haematopoietic and solid tumours and have an immune‐modulating effect, suggesting a promising role in immunotherapy. In this study, we demonstrated a network by mass spectrometry analysis between healthy donor‐derived NKEV protein cargo and antitumour effects in vivo against human B cell lymphoma. Methods : We isolated NKEV, either exosomes or microvesicles, from in vitro amplified healthy NK cells by differential centrifugation and then characterized them by NanoSight, immunoelectron microscopy, western blot and mass spectrometry (Proteome Xchange, PXD014894). NKEV cytotoxic effect against B cell lymphoma was studied by cytometry analyses, SCID mice xenograft, in vivo MRI/MRS measurements, ex vivo MRS analyses and immunohistochemistry. Results : Human healthy NKEV, both exosomes and microvesicles, have a significant and direct cytotoxic effect against human B cell lymphoma in in vitro and in vivo conditions. We observed a remarkable NKEV cytotoxic effect, mainly by apoptosis, on B cell lymphoma in vitro when exosomes and microvesicles were administered simultaneously. In vivo results showed metabolic alterations in SCID mice xenografts after NKEV treatment, associated with a significant reduction of tumour growth (64%). In the in vivo 1 H MR spectra we found a significant increase in the tumour lipid/lactate and in taurine signals, both considered as apoptosis markers. Ex vivo lymphoma metabolomics revealed a significant increase in the fatty acid (FA) pool and a decrease in unsaturated and monounsaturated FA in treated groups, as compared to the control one, thus suggesting an alteration of tumour homeostasis. Immunohistochemistry analyses confirmed the reduction of B‐cell lymphoma proliferation rate, as well as the induction of apoptosis following the NKEV treatment. Summary/Conclusion : This study underscores the importance of NKEV as a novel biological acellular tool for B‐cell lymphoma treatment, probably having a greater effect on combined treatment regimens. These nanovesicles have an extraordinary potential in innovative cancer immunotherapy, representing a safe and efficient tool naturally circulating in healthy individuals and ready to maintain the immune homeostasis and therefore a good organism healthy state. Funding : This study was supported by AIRC IG (Project IG 29148) 2023, Italy, to AZ and Ministero della Salute, Italy, WFR GR‐2011‐02351400 to LL.

Cerebellar

Pasquale D'Acunzo 1,2 , Yalan Zhang 3 , Leonard K. Kaczmarek 3 , Efrat Levy 1,2 1 Nathan S. Kline Institute for Psychiatric Research, NY, USA; 2 NYU Grossman School of Medicine, NY, USA; 3 Yale University School of Medicine, CT, USA Introduction : Kv3 voltage‐gated potassium channels mediate the repolarization of fast‐firing neuronees, including Purkinje cells. Dominant negative mutations in the Kv3.3 subunit trigger uncontrolled duration and frequency of action potentials and cause spinocerebellar ataxia type 13 (SCA13), a neurodegenerative disorder characterized by Purkinje cell loss and ataxia of gait. We have previously shown that Purkinje neuronees in the cerebellum of a mouse model of SCA13 bearing the disease‐causing mutation Kv3.3‐G592R are characterized by several endocytic abnormalities, including lysosome impairments, accumulation of undigested inclusions, and a higher number of multivesicular bodies. These abnormalities are associated with higher secretion of exosomes, without affecting ectosomes. We hypothesized that in Kv3.3‐G592R mice the concomitant presence of ion imbalance caused by the lower activation of Kv3 channels and downstream mitophagy block caused by endolysosomal abnormalities leads to alterations in mitochondria that reverberate to changes in mitovesicles, the mitochondria‐derived extracellular vesicles (EVs). Therefore, we investigated changes in mitochondria and mitovesicles in the brain of male and female Kv3.3‐G592R and Kv3.3 knockout (KO) mice, as compared with wild‐type (WT) littermate controls. Methods : EVs were isolated from cerebella and right hemibrains of Kv3.3‐G592R, Kv3.3‐KO, and WT mice using a high‐resolution iodixanol gradient and analysed by transmission electron microscopy (TEM), NTA, total protein content, and Western blotting. Mitochondria were studied in the left hemibrains and cerebella by Western blotting and TEM. Results : The number of mitovesicles in the cerebellar extracellular space was inversely correlated with the Kv3 channel activity: lowest in WT, higher in Kv3.3‐G592R (impaired activity), and highest in Kv3.3‐KO (no activity) mice. Mitochondrial fragmentation markers (phospho‐Drp1 Ser616, Ser637) followed the same trends, consistent with a higher production and secretion of mitovesicles. Under TEM, Kv3.3 channels were found to be enriched at the contact sites between the plasma membrane and mitochondria, both in WT and Kv3.3‐G592R mice, suggesting a direct regulation of the mitochondrial ion metabolism mediated by Kv3.3. Summary/Conclusion : Mitovesicles are a previously unidentified player altered in SCA13 cerebella. Mechanistically, we describe a novel pathway in vivo linking mitochondria and mitovesicle homeostasis to plasma membrane depolarization, shedding new light on the still obscure biology that regulates mitovesicle secretion in the brain. Funding : NIA ( AG086510 ).

Checkpoint

L. Steiner 1,2,4 , L. Teeuwen 1,2,4 , A. Offens 1,2 , G. Güçluler Akpinar 1,2 , J. Kuipers 1,2 , D. Martinez‐Martinez 1,2 , J. Mazouin 1,2 , B. Chambers 3 , S. Gabrielsson 1,2 1 Division of Immunology and Allergy, Department of Medicine, Karolinska Institutet, Solna, Stockholm, Sweden; 2 Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden; 3 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 4 These authors contributed equally to the work . Introduction : Extracellular vesicles (EVs) from dendritic cells have been tested in humans as cancer immunotherapy, but the treatment needs improvement to induce strong T cell immunity. In mice, EVs secreted by bone marrow derived dendritic cells (BMDCs) induce an antigen‐specific immune response and significantly delay tumour growth. Our previous work showed that BMDC EVs combined with checkpoint blockade therapy (anti‐PD1 or anti‐PD‐L1) synerize. As the BMDC EVs contain both PD1 and PD‐L1, we speculated that EVs from PD1 or PD‐L1 knock out (KO) mice would induce stronger anti‐tumour responses than WT EVs. Methods : EVs from were prepared from BMDCs by Tangential flow filtration followed by differential ultracentrifugation and characterized by nanoparticle tracking analysis, bead‐based flow cytometry and EM. Mice were immunized intravenously with WT, PD1 KO and PD‐L1 KO BMDC EVs twice, and immune responses were detected on day 21 by ELISA, ELISPOT and flow cytometry. Results : All EV‐injected groups showed an increased and similar number of antigen‐specific CD8 T cells in the spleen. However, following ex vivo restimulation of splenocytes, OVA specific CD8 T cells from both PD1 and PDL‐1 KO EV injected mice produced more IFN‐g than the WT. In contrast, when EVs were injected as tumour therapy in a mouse melanoma model, WT EVs performed better than the two KO in delaying tumour growth. All the EV treatments induced a potent anti‐tumour response by recruiting several immune cell types at the tumour sites, but the absence of checkpoint molecules on the EVs dampened the antigen specific response. This contrasts with earlier findings in tumour‐derived EVs, where tumour‐derived PD‐L1 on EVs inhibit immune responses to the tumour. Summary/Conclusion : We confirmed that EVs loaded with antigen are potent immune stimulators and promising tools for immunotherapy. Additionally, the checkpoint molecules present on theses EVs may play a functional role in immune activation and are important in eliciting a strong anti‐tumour response. Our results also highlight the need to further investigate the PD1/PD‐L1 axis in cancer therapy. Funding : The study was funded by the Swedish Medical Research Council, the Swedish Cancer Society, the Cancer Research Foundations of Radiumhemmet, and the Karolinska Institutet's KID grant.

Cloudberry

Feby Wijaya Pratiwi, 1,†* Keerthanaa Balasubramanian Shanthi, 1 Florence Naillat, 1 Ramila Mammadova, 1 Marjaana Sarpola, 1 Si‐Han Wu, 3* Marko Suokas, 4 Soile Jokipii‐Lukkari, 4 Susanna Kaisto, 1 Anatoliy Samoylenko, 1 Henrikki Liimatainen, 5 Caglar Elbuken, 6,2 Seppo Juhani Vainio 1,2* 1 Laboratory of Developmental Biology, Disease Networks Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland; 2 Kvantum Institute, University of Oulu, Finland; 3 Graduate Institute of Nanomedicine and Medical Engineering, Taipei Medical University, Taiwan; 4 Ecology and Genetics Department, University of Oulu, Finland; 5 Fiber and Particle Engineering Research Unit, University of Oulu, Finland; 6 Micro‐/Nanofluidics and Biosensor Research Group, University of Oulu, Finland Feby Wijaya Pratiwi and Keerthanaa Balasubramanian Shanthi are equal first coauthors and Florence Naillat and Ramila Mammadova are equal second coauthors . Introduction : The phrase ‘A man is what he eats’ has taken on new significance as research reveals that food provides nutrients and bioactive information that can influence mammalian genes. This information is transmitted by plant‐derived nanovesicles (PDNVs). Although PDNVs have demonstrated therapeutic potential, their effects on ageing populations remain largely unexplored. The non‐invasive nature of oral delivery is particularly appealing for this demographic, though the digestive tract poses challenges for effective drug absorption. Cloudberries (Rubus chamaemorus), which are rich in bioactive polyphenols and demonstrate high stability in the digestive tract, provide a promising source of PDNVs for oral delivery vehicles for the elderly. Methods : PDNVs from cloudberries (CNV) were isolated using differential and ultracentrifugation methods, and various properties were analysed, including protein content, particle concentration, size distribution, morphology, zeta potential, miRNA content, and antioxidant properties. Their stability in the gastrointestinal environment was evaluated using simulated digestive conditions, followed by analyses of particle concentration, morphology, and surface potential. In vitro experiments with Caco‐2 cells were used to assess cytotoxicity, cell proliferation, and CNVs uptake and transport. Additionally, in vivo imaging analysed CNVs distribution in young and aged mice after oral administration, with tissue samples and blood examined using immunohistochemistry and flow cytometry for CNVs localization and immune profiling. Results : This study highlights the unique properties and biomolecular content of CNVs and their potential for mass production. Our results show that CNVs remain stable during gastrointestinal digestion while preserving their bioactivity and properties in vitro. Additionally, they exhibit favourable biodistribution, extended retention in the GI tract, efficient cellular uptake, successful intestinal barrier translocation, entry into the bloodstream, and low immunogenicity in both young and elderly mice. Summary/Conclusion : CNVs that can withstand gastrointestinal digestion and exhibit favourable biodistribution, efficient cellular uptake, effective intestinal barrier crossing, successful bloodstream transfer, and low immunogenicity are promising candidates for stable oral delivery vehicles in both young and elderly mice. Future research should concentrate on elucidating the specific mechanisms of CNV interactions with target cells, optimizing delivery methods, and validating therapeutic efficacy in relevant disease models. Funding : This work was supported by the Kvantum Institute BioEVEngine project (University of Oulu).

Colorectal

Presenter: Subbaya Subramanian University of Minnesota, Minneapolis, Minnesota, USA Introduction : Colorectal cancer (CRC) is the third leading cause of cancer‐related deaths, and most patients are resistant to immune checkpoint inhibitors due to the immunosuppressive microenvironment. Understanding resistance mechanisms to ICIs could lead to novel combinatorial intervention strategies that make patients eligible for ICI treatment. Our previous studies have identified resistance mechanisms where extracellular vesicles (EVs) containing miR‐424 as cargo can lead to the downregulation of costimulatory CD28 on T cells and CD80/86 on dendritic cells (DCs). Furthermore, we have demonstrated that tumour EVs (TEVs) are captured and processed by DCs and are presented as antigens to T cells. The consequence of miR‐424 expression within CRC TEVs and the potential alterations in antigenic cargo within TEVs remain unexplored. In this study, we sought to understand how the expression of miR‐424 in CRC cells alters the antigenic cargo within CRC TEVs. Methods : TEVs from CT26 cells with or without miR‐424 were isolated using ultracentrifugation methods. The EVs were characterized for size and frequency using nanoparticle tracking analysis. EV tetraspanins, CD63, CD81, and Alix, were characterized using western blotting. We employed a label‐free quantitative proteomic method using mass spectrometry to characterize TEVs isolated from CT26‐miR‐424i (antimiR inhibition), CT26‐miR‐424‐KO (CRISPR/Cas9), and CT26‐miR‐424‐control cells and compared proteomic cargo to TEVs derived from CT26‐WT cells. We used FragPipe followed by data‐independent analysis using neural networks (DIA‐NN) to determine signal intensities of proteins throughout TEV samples and MSstats for statistical comparisons. Results : We observed significant differences between TEVs derived from CT26‐WT and CT26‐miR‐424KO cells. Proteins of interest enriched in TEVs derived from CT26 cells lacking miR‐424 include Tsta3, P4hb, Cdk2, and Etf1. Additionally, we saw the proteins P4hb, Ube3a, and Glb1 significantly enriched in CT26‐miR‐424KO TEVs compared to CT26‐miR‐424i TEVs. We intend to study further the antigenic potential and immune regulatory role of the protein cargos in the TEVs. Summary/Conclusion : The proteins identified in TEVs have been implicated in modulating cellular signalling. Future studies will focus on quantitative assessments of these proteins as potential tumour antigens and understanding their possible role in immune regulation using immunopeptidomics. Funding : This study was funded by the Mezin Koats Colorectal Cancer Research Funds and the Minnesota Colorectal Cancer Research Foundation.

Comparison

Presenter: Misba Khan University of Turku, Turku, Finland Introduction : Urinary extracellular vesicles (EVs) are promising source of biomarkers for non‐invasive disease detection. However, their isolation is challenged by the high abundance of non‐EV proteins like uromodulin/Tamm‐Horsfall protein (THP), which can compromise the purity and recovery of EVs. Additionally, the low concentration and heterogeneity of urinary EVs complicate standardization across different isolation methods. Optimizing pre‐isolation concentration methods and THP removal strategies is crucial to enhance EV yield, purity, and reproducibility for reliable biomarker discovery. Methods : Healthy urine samples were pooled and concentrated using 4 different Vivaspin Turbo centrifugal filters‐ with molecular weight cutoffs of 10, 30, 50, and 100 kDa. Tetraspanin and THP expression levels were analysed using a time‐resolved fluorescence‐based sandwich immunoassays (TRFIA), in which biotinylated tetraspanins were immobilized on streptavidin coated microtiter wells. The captured proteins were detected using europium doped nanoparticles conjugated with tetraspanins or THP‐specific antibody. THP was removed using NaCl treatment and filtration, both before and after the isolation of EVs. EVs were then isolated from the 10 kDa filtered urine fractions using size exclusion chromatography (SEC). Results : Among four filtration methods, urine fraction concentrated using10 and 30 kDa filters resulted in higher uEVs yields, as confirmed by tetraspanin and THP expression measured by TRFIA. Notably, the 10 kDa filter led to a reduction in THP content in the concentrated urine fractions. Comparison of THP removal strategies indicated that salt treatment was more effective than filtration. These findings were further validated in the SEC‐isolated EV fractions. Summary/Conclusion : This study introduces a comparison of four different filtration methods for concentrating urine samples and two different methods of removing THP from the urine and SEC isolated EV fractions. The results highlight the advantages of using 10 kDa filtration in combination with salt treatment for improved EV yield and purity. However, to get better yield‐driven protocol, other EV isolation methods will be included for comparison in our future experiments. Funding : This research is funded by a grant received from the Southwest Finland Cancer Society.

Converging

Plenary Presenter: Nicole Meisner‐Kober Ludwig Boltzmann Institute for Nanovesicular Precision Medicine, Vienna, Austria

Delivering

Seyedmohammad Moosavizadeh 1,2 , Jiemin Wang 1,2 , Manon Jammes 1,2 , Anastasia Walsh 4,5 , Ellen Donohoe 1,2 , Aoife Canning 1,2 , Abbas Tabasi 1,2 , Trung Bach 1,2 , Thomas Robinson 3 , Richard Moakes 3 , Aideen E Ryan 1,2,4,5 , Liam Grover* 3 , Thomas Ritter* 1,2 1 Regenerative Medicine Institute (REMEDI), School of Medicine, College of Medicine, Nursing and Health Sciences, University of Galway, Galway, Ireland; 2 CÚRAM, SFI Research Centre for Medical Devices, University of Galway, Galway, Ireland; 3 Healthcare Technologies Institute (HTI), School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom; 4 Discipline of Pharmacology and Therapeutics, School of Medicine, College of Medicine, Nursing and Health Sciences, University of Galway, Galway, Ireland; 5 Lambe Institute for Translational Research, School of Medicine, College of Medicine, Nursing and Health Sciences, University of Galway, Galway, Ireland Introduction : MSC‐EVs demonstrate immunomodulation and tissue regeneration properties while addressing the challenges associated with cell therapy. Licensing MSCs with pro‐ and/or anti‐inflammatory cytokines enhances their therapeutic effects. Delivering MSC‐EVs to the target site in a controlled manner may enhance MSC‐EVs therapeutic efficacy. Gellan gum fluid gel (GGFG) is a biocompatible delivery system which can extend the ocular retention time and overcome the obstacles of commercial eye drops due to its shear‐dependent viscoelasticity and phase transition properties. The aim of this study was to investigate the MSC‐EVs delivery to the ocular surface and evaluate their therapeutic effects. Methods : Naïve‐ and cytokine‐licensed‐MSC‐EVs were isolated by size exclusion chromatography and characterized extensively following MISEV guidelines. MSC‐EVs were investigated for their immunomodulation and tissue regeneration effects on macrophage polarization and in human corneal epithelial (HCEpi) and keratocyte (HCK) wound healing assays, respectively. GGFG was synthesized by the shear field technique and characterized for its viscosity, viscoelasticity, transparency, and biocompatibility. MSC‐EVs cumulative release from GGFG was examined over 6 hr incubation. Results : MSC‐EVs had spherical bilayer morphology with a mode size of 80 nm. The MSC‐EVs were positive for CD9, CD63, and CD81, and TSG101 markers were analysed by flow cytometry and western blotting. Both MSC‐EVs can polarize pro‐inflammatory macrophages to the anti‐inflammatory phenotype by upregulating CD206 and CD163. Furthermore, licensed MSC‐EVs were more effective at upregulating CD206 across various concentrations. The licensed MSC‐EVs better‐promoted cell migration and wound healing in HCEpi and HCK scratch models analysed by an Olympus microscope. Transparent GGFG acts as solids at rest but as fluids under force. GGFG didn't show any toxicity on the human corneal epithelial and keratocyte cells or on the bovine eye ex‐vivo model. The GGFG released 20% of the MSC‐EVs after 6 h and demonstrated a controlled profile. Summary/Conclusion : Together, these results evidenced that cytokine licensed MSC‐EVs have enhanced immunomodulation and tissue regeneration activities in 2D in vitro corneal models. In addition, the GGFG demonstrates a promising system for the MSC‐EVs ocular delivery in a biocompatible and controlled manner in 2D in vitro and ex vivo models, necessitating further investigation of the therapeutic efficacy on in vitro and in vivo corneal injury models.

Denervated

Presenter: Maria Vittoria Giraudo Umeå University, Umeå, Sweden Introduction : Peripheral nerve injuries lead to muscle loss due to the disruption of neural input. If reinnervation does not occur, muscle fibres become atrophic, significantly hindering functional recovery. Our study aims to investigate extracellular vesicles (EVs) from denervated muscles to understand their role and molecular content in response to nerve injury. Methods : Denervation of the rat gastrocnemius, tibialis, and soleus muscles was induced through sciatic nerve transection. Muscle samples were collected and weighed at 1 day and 2 weeks post‐injury. EVs were isolated from these tissues using enzymatic digestion and ultracentrifugation and characterized via western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Proteome profiling was also performed. Results : Initial proteome profiling revealed distinct differences in the protein composition of EVs from control and denervated tissues, as well as between samples collected at 1 day and 2 weeks post‐injury. These findings highlight potential injury‐related changes in the protein content of EVs that may correlate with the tissue's state of denervation, with some protein changes potentially involved in atrophic reprogramming and axonal regeneration. Further studies are underway to explore the mRNA content and biological functionality of these EVs. Summary/Conclusion : This study suggests that EVs derived from denervated muscle tissue could serve as biomarkers for the physiological state of muscles following nerve injury and may provide insights into new therapeutic approaches to promote muscle repair. Detailed analysis of their mRNA and functional profiles is expected to deepen our understanding of muscle responses to nerve injury and identify potential targets for treatment.

Developing

Presenter: Munzur Lacin Erasmus Medical Center, Rotterdam, The Netherlands Introduction : The treatment of glioblastoma (GB), the most aggressive brain tumour, remains a significant clinical challenge. Despite extensive treatment, tumour recurrence is inevitable, leading to a median survival of only 10 to 13 months. The tumour's ability to evade therapy is driven by inherent mechanisms like genetic instability and immune evasion. By engineering extracellular vesicles (EVs) as a new delivery vehicle, with the potential to load multiple therapeutic cargos at once, we aim to advance targeted therapy for GB cells, addressing limitations of conventional drug delivery. Methods : To engineer therapeutic EVs, we employed a method to encapsulate full‐length therapeutic DNA derived from adenoviral vectors within EVs, resulting in hybrid particles termed ‘Adenosomes.’ To target GB cells, we developed novel Adenosome variants carrying DNA encoding kinase‐inhibiting peptides (KIPs). We hypothesized that this approach will enable highly efficient delivery of gene therapy to GB cells, leading to targeted disruption of aberrant kinase signalling pathways and subsequent cell death. Results : We initially pre‐screened several KIP peptides (Myr‐KIPs) on GB cell cultures to identify the most effective variants. This screening included Myr‐KIPs targeting DNA‐PK and CDK1, with the CDK1‐targeting KIP demonstrating a significant anti‐tumour effect on U87‐MG and GS.607 GB cells. Building on these results, we next created adenosomes loaded with DNA encoding GFP‐KIPs. The adenosomes were characterized using q‐PCR for DNA payload quantification and nanoparticle tracking analysis for particle concentration. Mass spectrometry proteomics confirmed an overall protein profile consistent with exosome‐like characteristics. Cell viability assessments (Cell‐Titer Glo assay) and microscopic morphological analyses demonstrated growth‐inhibitory effects of adenosome‐KIPs on primary GB cultures, with a reduction in cell count compared to controls. Importantly, astrocytes (non‐cancerous cells) remained relatively unaffected. Summary/Conclusion : We have developed adenosomes, we have employed a novel technology (adenosomes) for encapsulating therapeutic DNA within EVs. In our GB model, adenosomes expressing KIPs exhibited selective cytotoxicity toward GB cells, underscoring their potential as a targeted gene therapy tool for cancer treatment applications.

Diagnostic

Hanine E. L. Itawi 1 , Morgan Greig 1 , Indika Mallawaarachchi 1 , Jennie Ma 1 , Clarissa Diamantidis 2 , Hayrettin Yavuz 1 , Luca Musante 3 , Hayden Hutson‐Wiley 1 , Mike Solga 1 , Samantha Upson 1 , Michael A. Harding 1 , Julia Scialla 1 , Agnieszka Swiatecka‐Urban 1 , Uta Erdbruegger 1 1 University of Virginia, Charlottesville, Virginia, USA; 2 Duke University, Durham, North Carolina, USA; 3 University of Pennsylvania, Philadelphia, Pennsylvania, USA Introduction : Urinary extracellular vesicles (uEVs) have been identified as early and sensitive biomarkers in kidney disease; however, their association with diabetic kidney disease (DKD) progression and DKD clinical traits is not yet clear. Characterization of uEVs may confirm the relationship between their glomerular and tubular origin as well as clinical DKD pathophysiological phenotypes. Methods : In this study, we collected uEVs from 24‐h urine collections in 116 adults with diabetes mellitus enrolled in the Simultaneous Risk Factor Control Using Telehealth (STOP‐DKD). To determine uEVs count, size and cargo, we characterized them by spectral flow cytometry (SFC), SP‐IRIS (Exoview R100) for small sample volumes and nanoparticles tracking analysis. We used markers for CD26/DPP4 (PT S1) + , CD35/CR1 (podocyte) + , CD10/MME/NEP (PT S1, S2, S3) + , and tetraspanin, CD9 (EV marker). We used linear regression to assess the cross‐sectional association of SFC data, kidney function and degree of microalbuminuria (UACR). SP‐IRIS analysis was performed on a cohort of 10 healthy and 10 early DKD patients by testing CD63/CD9 and DPP4. Results : Participants were 63.65 ± 9.20 years of age; 50.4% were female. Baseline eGFR was 81 ± 22 mL/min/1.73m 2 and median UACR was 20.26 (IQR 8.38 to 92.97 mg/g). Higher counts of uEVs detected by SFC carrying DPP4 and MME were significantly associated with higher eGFR after adjustment for sex, race, age, log UACR and total urine creatinine. No markers demonstrated significant association with log UACR, and size was not considered further. SP‐IRIS analysed DPP4 and MME subpopulations captured by the CD63 and CD9 probes for exploration of small sample sizes. DPP4/Total Ratio was significantly higher in healthy patients when captured by CD63 ( p = 0.0176), and results of SP‐IRIS demonstrate that sample size can be microscaled. Summary/Conclusion : These results suggest that subgroups of tubular derived uEVs detected by SFC are associated with a significant change in eGFR in a cohort of early DKD. Tubular markers detected by SP‐IRIS are decreased in DKD when compared to HC. Our data shows a significant promise for the uEVs signature detected by single EV analysis. Further investigation is necessary to fully understand if these tubular derived uEVs derive from healthy tubular cells or are expressed more during tubular stress in early DKD. Funding : This study was supported by the American Diabetes Association funding: 7‐22‐ICTSPM‐19.

Dissecting

Presenter: Metoboroghene O. Mowoe Karolinska Institutet, Sweden Introduction : Extracellular vesicles (EVs) are increasingly recognized for their therapeutic potential, yet the role of the EV‐associated protein corona—functional and presumed variable—remains poorly understood. In this study, we aim to delineate the distinct roles of EVs versus their associated corona in cellular interactions and therapeutic applications. Methods : To dissect the distinct contributions of EVs and their corona in cellular interactions, we treated fibroblasts with EVs stripped of their soft corona or corona proteins derived from 12 different cell sources. Doses ranged from 20 to 200,000 EVs per cell or 0.001 and 10 ng of protein per cell. Transcriptional effects were analysed using RNA sequencing. Results : Unbiased global analyses revealed that while corona dose significantly influenced cell responses, cell source had minimal impact. Notably, EVs without their soft corona failed to elicit dose‐ or source‐specific effects, though high doses downregulated exocytosis. corona protein source‐specific dose‐dependence was more pronounced at low doses, likely reflecting increased cellular sensitivity to variations in corona composition when EV concentration is limited. At higher doses, this sensitivity diminished possibly due to the saturation of cellular pathways or receptor systems leading to a convergence in cellular responses regardless of cell source. Summary/Conclusion : These findings underscore the critical importance of optimizing both EV source selection and dose determination for therapeutic applications, emphasizing the functional role of the EV corona in modulating cellular behaviour.

Endothelin

Presenter: Vanessa Biemmi Università della Svizzera Italiana Lugano, Switzerland Introduction : Extracellular vesicles play a pivotal role in intercellular communication, governing diverse physiological and pathological processes. Owing to their stability in various body fluids, these small vesicles establish connections between distant cells, mediating the transfer of small molecules and modulating inflammatory responses. Inflammatory EVs derived from macrophages (M1 EVs) notably exacerbate post‐myocardial infarction (MI) inflammation and increase cardiomyocyte cytotoxicity. The study explores mechanisms behind small EVs release by M1 inflammatory macrophages (M1 sEVs) and evaluates the potential of endothelin receptor A antagonists (ETRA‐a) to impairs macrophage EV sorting. Methods : Buffy‐coat‐derived M0 macrophages were differentiated into the M1 inflammatory phenotype with cytokine stimulation. Through serial centrifugation, large vesicles (lEV) were isolated from the 20K pellet, while small vesicles (sEV) were collected from the 100K ultracentrifugation pellet and characterized per MISEV guidelines. Results : Following polarization, M1 macrophages showed increased sEV secretion, with consistent morphology and size verified by Particle Metrix, TEM, and FC analyses of tetraspanin markers. Polarization did not impact lEV concentration or profile. In vitro, ETRA‐a significantly decreased sEV release in M1 macrophages without affecting lEV release, as shown by Particle‐Metrix and FC. TEM confirmed that EV morphology and size remained unchanged after treatment. ETRA‐a disrupts SRC kinase activation, impairing Syndecan‐Syntenin‐Alix alternative sorting complex formation, as shown by proximity ligation assay. The inability of endosomal vesicles to be committed to the sorting route drives both early endosomes Rab5+ and RAb7+ late endosomes to fuse with lysosomes driving vesicular cargoes to the degradative pathway. This mechanism is not Cacl2 dependent. Furthermore, ETRA‐a ability to impair M1 sEVs offers cardioprotective effects by minimising cytotoxicity on IPS‐derived cardiomyocytes. Summary/Conclusion : The study highlights the role of ETRA in regulating M1 EVs release through Syndecan‐Syntenin‐Alix sorting complex. ETRA‐a impairs complex formation, disrupting the release of pro‐inflammatory EVs and altering endosomal maturation. Our findings connect immune cell‐derived EVs directly to cardiomyocyte health. By clarifying the role of M1 sEVs in myocardial health, this study supports ETRA‐a as a potential therapeutic strategy to protect heart function after MI and mitigate inflammation in various pathological conditions.

Engineered

Julia Rädler 1 , Giulia Corso 2 , Omnia M. Elsharkasy 1 , Antje Zickler 1 , Noriyasu Kamei 3 , Wenyi Zheng 1 , Dhanu Gupta 4 , Samir E. L. Andaloussi 1 1 Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden; 2 Evercyte GmbH, Vienna, Austria; 3 Faculty of Pharmaceutical Sciences, Laboratory of Drug Delivery Systems, Kobe Gakuin University, Kobe, Japan; 4 Institute of Developmental and Regenerative Medicine, Department of Paediatrics, University of Oxford, Oxford, UK Introduction : RNA interference (RNAi)‐based drugs hold significant therapeutic potential due to their high specificity for gene silencing. However, the delivery of naked RNAi agents is limited by their hydrophilicity and low bioavailability. Here, we use extracellular vesicles (EVs) as a delivery vehicle for short‐hairpin RNA (shRNA), by employing a novel approach using Argonaute‐2 protein to actively load the cargo into EVs for efficient delivery to recipient cells. Methods : In order to load shRNA into EVs, EV‐producing cells were engineered to express AGO2 as an RNA binding protein fused to an EV sorting domain, a fusogenic viral glycoprotein, in addition to the shRNA of interest. EVs were isolated from the conditioned media of producer cells and concentrated by spin filtration before further purification by size exclusion chromatography. The absolute copy number of shRNA in EVs was quantified by RT‐qPCR. Additionally, isolated EVs were added to various recipient cells, and knockdown efficiency was assessed based on mRNA levels using RT‐qPCR. Results : EVs were engineered to load shRNA targeting Gapdh (shGapdh). RT‐qPCR analysis revealed a more than 290‐fold enrichment of shGapdh molecules in EVs compared to the controls. Furthermore, quantification of the absolute copy number of shGapdh showed an average of one copy of shGapdh per two EVs. Isolated EVs exhibited an average IC50 of 1.79 pM and a 95% reduction in Gapdh mRNA expression. Additionally, we observed significant knockdown of the target gene in eight different cell lines, highlighting the efficiency and broad applicability of this loading strategy. Summary/Conclusion : Here, we describe a novel platform for loading shRNA into EVs. Using this approach, shRNA was successfully delivered, resulting in efficient gene knockdown. Additionally, this strategy can be adapted for the functional delivery of other RNA therapeutics, such as siRNA. Moreover, these data indicate that engineered EVs offer a promising platform for RNAi agent delivery.

Enrichment

Johanna Puutio 1 , Mari Palviainen 1 , Saara Laitinen 2 , Pia R.‐M. Siljander 1,2 1 Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland; 2 Finnish Red Cross Blood Service, Helsinki, Finland Introduction : Platelets release heterogenous platelet‐derived extracellular vesicle (PEV) subpopulations in response to pathophysiological stimuli. These subpopulations can be classified based on size and structural properties as large EVs (lEVs, > 200 nm), small EVs (sEVs, < 200 nm) and other extracellular particles, such as exomeres (< 50 nm). Quantitation of the subpopulation composition (number of populations, measurement of size and particle number) < 100 nm remains challenging due to technological limitations. Additionally, storage and isolation conditions affect the subpopulation composition of EV samples. We fractionated and analysed differently induced and isolated PEVs with asymmetrical flow field‐flow fractionation (AF4) to evaluate the formation and retention of sPEV populations. Methods : PEVs were generated by stimulating human platelets from platelet concentrates with different agonists and isolated with either iodixanol cushion ultracentrifugation (C‐UC), size exclusion chromatography (SEC) or concentrated with ultrafiltration (UF) without additional purification. PEVs released by unstimulated platelets were used as controls. Subpopulation composition was analysed with AF4 after short ( 12 months) storage at −80°C. Four sPEV subpopulation peaks of interest were identified and fractionated with AF4. Expression of PEV markers and absence of co‐isolated lipoproteins was investigated with ELISA, western blotting and SP‐IRIS from total populations and pooled subpopulation fractions to evaluate sPEV enrichment. Results : AF4 analysis of PEVs isolated with UC, SEC and UF showed that 1) sPEVs were the most abundant subpopulation, and 2) the isolation method significantly impacted their enrichment. SEC isolation removed up to two CD41+, CD63 and CD9+ sPEV populations < 100 nm and masked variations between differently induced PEVs, whereas C‐UC retained the original subpopulation composition of UF samples. A similar loss of sPEVs was observed during long‐term storage at −80°C. Summary/Conclusion : A comprehensive analysis of all EV subpopulations is often overlooked due to methodological challenges. We propose AF4 as a method for monitoring sEV loss and characterising them from complex media. AF4 analysis of PEVs isolated using C‐UC or UF enables quality control and the identification of subtle differences in sPEV subpopulation composition that may have biological significance. Funding : This project is a part of the extracellular vesicle ecosystem (EVE) consortium funded by Business Finland.

Evaluation

Hanna Reßin 1 , Shreyans Chatterjee 1 , Deborah Erhart 2 , Hayrettin Tumani 2,3 , Jan Münch 1 , Rüdiger Groß 1 1 Ulm University Medical Centre, Ulm, Germany; 2 University and Rehabilitation Clinics Ulm, Ulm, Germany; 3 German Centre for Neurodegenerative Diseases (DZNE) Site Ulm, Ulm, Germany Introduction : Extracellular vesicles (EVs) are produced by all cells and contain biomolecules that give clues on the state of the producer cell. Acute infections with SARS‐CoV‐2 are associated with increased EV release in plasma and increased pro‐coagulative properties of these EVs. We here investigated whether patients with persistent fatigue (range 1.0–102.0 months) after infection (post‐COVID, PC) also show a characteristic EV profile in serum or CSF, which could be used for diagnostics and give clues on disease aetiology. Methods : Plasma and cerebrospinal fluid (CSF) samples were collected from patients presenting to the Ulm University Hospital, Dept. of Neurology, Post‐COVID out‐patient clinic, pre‐cleared and stored at −80°C. Reconvalescent (RC) patients with a history of COVID‐19 but no persistent symptoms were used as a control cohort. For all samples, nanoparticle tracking analysis (NTA) was used to determine particle concentration and particle diameter. Bead‐assisted flow cytometry was used to analyse EV surface epitopes using detection of tetraspanin (TSPN) (αCD9, αCD63, αCD81) and phosphatidylserine (PS) (lactadherin). Results : Biophysical parameters (particle concentration and size) were not significantly altered between the PC and RC cohorts for plasma or CSF. Bead‐based flow cytometry showed significant elevation of CD142+ and CD105+ vesicles in CSF of PC patients, hinting at endothelial damage. In plasma, PS‐based detection of vesicles revealed upregulation of several proteins involved in immune activation, including CD25, CD29, CD56 and HLA‐DRDPDQ. Upregulated HLA‐DRDPDQ was also detectable on EVs in the CSF of the PC cohort, suggesting an overall immune‐activated state. Summary/Conclusion : Several EV populations appeared altered in plasma and CSF of PC patients, overall suggesting endothelial damage and/or persistent immune activation. The underlying mechanisms driving these changes, as well as their potential utility as biomarkers for PC is subject to further investigation. Funding : This study was supported by the Ministerium für Wissenschaft, Forschung und Kunst Baden‐Württemberg, COVID Sonderforschung

Ev‐Based

Antri Stefan 1,2* , MartinWolf 1,2 , Rodolphe W. Poupardin 1 , Ulrike Resch 3 , Sarah Hochman 1 , Anna Raninger 1 , Lisa Jakob 1 , Sabine Schmidhuber 4 , Nicole Drexler 5 , Thomas Heuser 5 , Markus Mandler 4 , Achim Schneeberger 4 , Dieter Volc 6 , Dirk Strunk 1,2 1 Paracelcus Medical University, Cell Therapy Institute, Salzburg, Austria; 2 Austrian Red Cross Research (ÖRK), Vienna, Austria; 3 Medical University of Vienna, Vienna, Austria; 4 Tridem Bioscience GmbH & CoKG, Vienna, Austria; 5 Vienna Biocenter Core Facilities, Vienna, Austria; 6 Confraternitaet‐Privatklinik Josefstadt, Vienna, Austria Introduction : Extracellular vesicles (EVs) are key players in cell‐to‐cell communication in health and disease. Their presence in most tissues and bodily fluids makes them excellent targets for biomarker discovery. We aim to develop an EV‐based biomarker assay that will serve as a tool for vaccine response monitoring in Parkinson's disease (PD) patients. We selected PD‐driving alpha‐synuclein (aSyn) as the primary diagnostic target to be challenged by unsupervised omics for additional biomarker discovery. Methods : Tangential flow filtration (TFF), size exclusion chromatography (SEC) and ultracentrifugation were applied to enrich EVs from PD patient's urine and blood. We determined EV quantity by tunable resistive pulse sensing and nanoparticle tracking analysis and EV quality by tetraspanin + aSyn expression in enhanced chemiluminescence dot blots, super‐resolution microscopy, cryo‐electron microscopy and enzyme‐linked immunosorbent assay (ELISA). Subtractive biomarker discovery by multi‐omics has been performed in addition. The level of EV‐associated aSyn in patient samples was compared with respective healthy controls ( n = 20 each). Following MISEV 2023 recommendations, the analysis pipeline was applied in bulk material, EV+ and EV‐ fractions. Results : Various isolation methods resulted in different EV enrichment levels and EV separation from other contaminants. We confirmed EV identity and enrichment by CD9/63/81 dot blots and super‐resolution microscopy. The highest aSyn signals were acquired in patient EV samples isolated by ultracentrifugation compared to SEC for blood samples and TFF for creatinine‐normalized urine samples. Preliminary experiments confirmed the presence of aggregated aSyn in patient EVs by ELISA. Variable aSyn signals were acquired in bulk liquid biopsy material, different SEC fractions, and EV+ and EV‐ preparations. Sensitivity evaluation of our dot blot assay setup using recombinant aSyn revealed a detection limit of ∼10 ng for total and aggregated protein. Bioinformatics analysis of preliminary proteomics data showed differential protein expression within the patient and control groups. Summary/Conclusion : New theranostic biomarkers from easily accessible non‐invasive sources are important for patient acceptance and study performance. Further proteomics analysis is ongoing for assay validation. Based on low sensitivity in dot blots, RT‐QuIC is planned to investigate the association of EVs and aSyn in PD samples. Once established, the biomarker will be validated as a diagnostic tool for monitoring vaccine response.

Functional

Pengfei Wang, Jing Ye, Fei Zhou, Li Pan, Donglei Yang, Weihong Tan Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China Introduction : Tumour cell‐derived small extracellular vesicles (sEVs) contain various molecular ingredients such as membrane proteins and microRNAs (miRNAs) that can serve as critical biomarkers for cancer diagnostics. Nevertheless, conventional detection methods involve extracting proteins and miRNAs out of sEVs, which not only may cause damage/loss of biomarkers, but also demand large amount of effort. There is an increasing need to develop novel methods to enable in situ detection of membrane proteins and miRNAs with sEVs remaining intact. Methods : We developed a sEV membrane protein detection method (PRECISE) by encoding individual protein‐aptamer molecular recognition events into DNA strands of unique sequences via proximity‐induced DNA extension. DNA strands carrying encoded information are subject to amplification, sequencing, and sequence decoding for revealing protein identities and counts. For miRNA detection, we conjugated hydrophobic tethers onto molecular beacons (MB) to promote its transportation efficiency into sEVs to enable in situ miRNA detection. Results : Membrane protein profiles of serum‐derived sEVs from a total of 174 individuals with cancers have been acquired by using PRECISE. Employing machine learning algorithms, we were able to build diagnostic models for distinguishing prostate cancer, gastric cancer, and breast cancer patients from noncancer controls, with a diagnostic accuracy of 100%. Furthermore, we built a multiclass cancer classifier that exhibited potent performance in the classification of three cancer types with a classification accuracy of 82.4%. Cholesterol‐tethered MBs can enable simultaneous detection of multiple miRNA species within serum‐derived sEVs for the diagnosis of prostate cancer, breast cancer, and gastric cancer with an accuracy of 83.3%, 81.8%, and 100%, respectively, in a cohort of 66 individuals. Summary/Conclusion : Our works provide a feasible, robust, and precise technical platform for sEV surface protein and miRNAs fingerprinting that shall hold great application potential in cancer diagnostics and beyond.

Generation

Xiaoling Wu, Susanne Rößner, Katja Blume, Jan Van Deun, Miriam Zentgraf, Andreas S. Baur Department of Dermatology, Unikilinikum Erlangen, Friedrich‐Alexander‐Universität (FAU) Erlangen‐Nürnberg, Deutsches Zentrum Immuntherapie (DZI); Erlangen Germany Introduction : Ex‐vivo amplified autologous Natural killer (NK) cells, similar to CAR T cells, have attracted great interest as an anti‐tumour therapy. The generation of these cells, however, is complex, time consuming and expensive. Injection of designer extracellular vesicles (DesEV) that activate NK cells in vivo may overcome a number of these issues including the most relevant HLA‐mismatch problem. However, still many technical and biological issues need to be addressed before GMP‐grade Designer Vesicles can be generated for in vivo application. This includes the EV‐producer cell, its optimal genetic manipulation, the identification of the subcellular targeting structure and most notably, the potency of DesEV to activate NK cells. Methods : We used different primary cells and cell lines that were genetically transduced with different methods to introduce different GFP‐labelled cytokines, reported to activate primary NK cells. EV were purified from culture supernatants using Ultracentrifugation and incubated with primary NK cells. Confocal microscopy, FACS, proteomics and real‐time PCR analysis were used to analyse and measure the effects on NK cells. Results : While differently generated EV were readily taken up by NK cells, only certain primary cells, stimulated by a defined procedure, resulted in EV able to correctly stimulate NK cells. The cells proliferated and expressed high levels of perforin and granzyme B. One critical factor was the targeting of the internalized vesicle to the perinuclear region. Summary/Conclusion : A number of factors need to be considered when generating DesEV to stimulate immune cells. Such vesicles are able to efficiently generate functional NK cells.

Harnessing

Eylul Gulsen Yilmaz 1,2 , Esma Derin 1,2 , Fatih Inci 1,2 1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey Introduction : Acute kidney injury (AKI) affects approximately 13 million individuals worldwide and is responsible for 1.7 million deaths annually. AKI poses a significant healthcare challenge due to limited biomarkers. Extracellular vesicles (EVs) offer promising potential for diagnostics and therapy, though isolating and characterizing EVs from complex fluids remains challenging in clinical settings. This study aims to isolate and characterize surface markers of EVs released in the urine of AKI patients using a streamlined (5‐step protocol), rapid(1.5‐h), and cost‐effective ($5) method. Herein, we developed an ultrafiltration‐based chip to isolate EVs from both cultured samples and urine samples from healthy individuals and AKI patients. Additionally, we repurposed nanograting plastic surfaces as sensitive metamaterial sensors, further functionalized with EV‐specific antibodies to enable EV detection. Methods : In all experiments, MISEV criteria is closely followed. Through an ultrafiltration chip and a metamaterial sensor, we isolated and detected EVs from both cultured samples and urine samples from healthy controls and AKI patients. EVs were further validated using nanoparticle tracking analysis (NTA), fluorescence NTA, Western blotting, scanning electron microscopy, and x‐ray photoelectron spectroscopy. To evaluate the effectiveness of our platforms, we compared them with standard ultracentrifugation and commercial ELISA methods, followed by statistical analysis to assess performance differences. Results : The ultrafiltration chip yielded results comparable to ultracentrifugation, isolating up to 10 9 EVs/µL. Compared to ELISA, our platform achieved approximately 100‐fold greater sensitivity and a wider dynamic range (spanning four orders of magnitude). Using the metamaterial sensor, we further validated samples from patients and controls, analyzing a broad AKI biomarker panel—including ATF‐3, NGAL, THP, AQP‐1, CD133, and Fetuin A—in addition to tetraspanins. Summary/Conclusion : Our metamaterial sensor exhibited exceptional sensitivity down to 10 3 EVs/µL, complemented by our user‐friendly, cost‐effective, and rapid ultrafiltration chip. In the near future, both platforms have the potential to be seamLessly implemented in clinical settings, significantly advancing the application of EV markers in point‐of‐care diagnostics. Funding : The authors gratefully acknowledge support from the Scientific and Technological Research Council of Turkey 2232 International Fellowship for Outstanding Researchers (118C254), Outstanding Young Scientists Award(GEBİP) from Turkish Academy of Sciences, Young Scientist Awards Program(BAGEP) form Science Academy, and the exRNA‐PATH COST Action CA20110.

High‐Fat

Adam M. Allouch 1,2 , Sarah E. Elzinga 1,2 , Bhumsoo Kim 1,2 , Rosemary E. Henn 1,2 , Mohamed H. Noureldein 1,2 , Faye E. Mendelson 1,2 , John M. Hayes 1,2 , Diana M. Rigan 1,2 , Masha G. Savelieff 1,4 , Junguk Hur 1,4, Kai Guo 1,4 , Yin Zou 3 , Sunitha Nagrath 3 , Eva L. Feldman 1,2 1 University of Michigan, Department of Neurology, Ann Arbor, Michigan, USA; 2 Neuroneetwork for Emerging Therapies, USA; 3 University of Michigan Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA; 4 University of North Dakota Department of Biomedical Sciences, Grand Forks, North Dakota, USA Introduction : Obesity, prediabetes, and diabetes induce adipose tissue inflammation, which contributes to many diabetic complications, including nervous system complications. Adipose tissue inflammation is associated with increased production and release of adipose tissue‐derived extracellular vesicles (ATEVs), which can cross the blood‐brain barrier and promote central nervous system (CNS) inflammation, possibly by activating microglia. As miRNAs are suggested contributors to peripheral and CNS inflammation, we aimed to assess the effect of obesity on ATEV miRNA content and inflammatory gene expression profiles of microglia, the primary immune cells in the CNS. Methods : We induced obesity/prediabetes by feeding 5‐week‐old male C57BL/6 mice standard diet (SD) or high‐fat diet (HFD) for 1 or 3 months, then isolated ATEVs from white epididymal adipose tissue. ATEV size and concentration was confirmed via nanoparticle tracking analyses and miRNA content analysed via NanoString nCounter. Single‐cell RNA sequencing data from hippocampal microglia was used to understand the potential effects of ATEV miRNAs on microglial inflammatory gene expression in vivo. ATEVs were also used to treat a human microglial cell line to assess microglia uptake of ATEVs and inflammatory gene expression after treatment. miRNA mimics and inhibitors of differentially expressed ATEV miRNAs were used to recapitulate ATEV‐mediated neuroinflammation in vitro. Results : Obesity reduced expression of ATEV miRNAs let‐7f, miR‐141, and miR‐504. Enrichment analysis showed that let‐7f and miR‐141 regulate genes in the NF‐κB pathway, and their reduction is associated with NF‐κB activation. In HFD mice, ATEV production increased in white adipose tissue, and microglia uptake of these ATEVs led to elevated pro‐inflammatory gene expression, indicating NF‐κB activation. Summary/Conclusion : These data suggest that HFD alters ATEV miRNA content, disrupting microglial inflammatory responses over time, implicating adipose‐microglia crosstalk as a mechanism for peripheral inflammation spreading to the CNS. Funding : NIH Grant U01AG057562. NIH Grant U24DK115255. Michigan Alzheimer's Disease Research Center P30AG072931. NIDDK Grant T32DK007245. JDRF Grant 5COE‐2019‐861‐S‐B. Edith S. Briskin/SKS Foundation Neuroneetwork Emerging Scholar Fund. Robert E. Nederlander Sr. Program for Alzheimer's Research. Andrea and Lawrence A. Wolfe Brain Health Initiative Fund. A. Alfred Taubman Medical Research Institut. Neuroneetwork for Emerging Therapies. Neuroneetwork for Emerging Therapies Tauber Family Student Internship

Hyaluronan

Presenter: Debashish Paul Shiv Nadar Institution of Eminence, Dadri, Uttar Pradesh, India Introduction : Extracellular vesicles (EVs) are lipid bilayer‐enclosed nanopouches, produced by all cells and abundant in various body fluids. These vesicles mediate the exchange of diverse biomolecules, including nucleic acids, proteins, carbohydrates, and metabolites, between parent and recipient cells. Methods : This study employed high‐resolution atomic force microscopy (AFM) and spectroscopy (AFS) to explore differences between EVs from colon cancer cells and normal colon epithelial cells. Results : Cancer‐derived EVs were more abundant in cell media. They exhibited a higher surface density of hyaluronan (HA), a carbohydrate polymer linked to tumour malignancy, highlighting their potential as colon cancer biomarkers. Contour length measurements using single‐molecule force spectroscopy (SMFS) revealed that these cancer EVs are decorated with low molecular weight HA (LMW‐HA < 200 kDa) and are intrinsically elastic, likely due to the HA and its associated water network on the EV surface. Summary/Conclusion : In addition, AFM‐based techniques such as electrostatic force microscopy (EFM), scanning kelvin probe microscopy (SKPM), and conductive probe force microscopy (CPFM) were used to map the surface electrical properties of HA‐coated cancer EVs, showing promise for non‐invasive liquid biopsy screening.

Inhibition

Presenter: Juan‐Carlos Padilla Institut de Recherches Cliniques de Montreal, Canada Introduction : Extracellular vesicles (EVs) form through regulated biogenesis processes involving sphingomyelinases (SMases), enzymes that metabolize sphingomyelin to produce ceramide—a lipid influencing membrane rigidity and essential for EV generation. Methods : This study explores the impact of inhibiting neutral SMase (NSM) and acid SMase (ASM) on the sorting of EV protein and RNA cargoes in human MCF7 cells. Results : Our results revealed that NSM inhibition reduces EV nanoparticles and diminishes RNA and protein cargoes, including endosomal, spliceosomal, and translation‐related proteins. Conversely, ASM inhibition increased RNA‐binding proteins within and enhanced the expression of ribonucleoprotein complex‐associated RNA in released EVs, including several snRNAs and 7SL RNA. Intriguingly, ASM‐inhibited EVs enhanced the migration and translational activity of recipient MCF10A cells. Summary/Conclusion : These findings suggest an important role for SMase‐dependent vesiculation in governing RNA and protein trafficking to the extracellular space, unveiling potential implications for cellular communication and function. Funding : This research was made possible by grants awarded to E.L. from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Fonds de Recherche du Québec—Santé (FRQS). E.L. is a distinguished research scholar of the FRQS. J.C.A.P. was a recipient of doctoral scholarships provided by the FRQS, McGill University, and the IRCM.

Innovative

Presenter: Hang Zou Southern Medical University, China (People's Republic) Introduction : Wounds with drug‐resistant bacterial infections and impaired healing pose significant threats to public health due to limited treatment options and severe clinical outcomes, including amputation and death. Extracellular vesicles (EVs) from tissues have emerged as novel cell‐free therapeutic strategies to address the dearth of interventions for wound healing and skin regeneration. Yet, they may not fully resolve issues stemming from drug‐resistant bacterial infections. Probes with aggregation‐induced emission (AIE), characterized by excellent optical stability and a range of properties, play a significant role in biomedical applications. Herein, AIE‐active photosensitizers, engineered for diverse ROS‐generating capabilities, have been synthesized. Furthermore, tissue‐derived EV/AIE hybrid nano‐vesicles (AEV) have been successfully created for the treatment of drug‐resistant bacterial infections in wound healing. Methods : EVs were isolated from healthy mice tissue and analysed using proteomics and metabolomics. The AEV was characterized by NTA and TEM. The biocompatibility of the AEV was investigated by blood biochemical safety evaluation experiments and cell experiments. Fluorescence confocal microscopy was used to investigate the ability of the AEV to visualize bacteria. The antibacterial activity of the AEV was evaluated by its inhibition of bacterial growth both in vitro and in vivo. It was applied to a mouse model with drug‐resistant bacteria‐infected full‐thickness skin excisional wounds. Haematoxylin‐eosin, Masson's trichrome, immunofluorescence stainings were performed to explore the effect on skin repair. Results : Transmission electron microscopy showed that AEV were round particles with a double‐layer membrane structure, and NTA showed that the size of AEV was about 120 nm. The AEV had a high binding affinity with the bacteria, and bright red fluorescence was observed on the surface of the bacteria and displayed distinctive photodynamic antibacterial effects under white light irradiation. In vitro and in vivo antibacterial experiments showed that it could effectively inhibit the growth of Gram‐positive bacteria. Histological results showed increased neovascularization, reduced wound scar, and more sebaceous gland and hair follicle formation, suggesting that the AEV could promote wound closure. Summary/Conclusion : In summary, we successfully fabricated novel AIE‐engineered EV hybrid nano‐vesicles (AEV) to realize efficient antibacterial application and microenvironment remodelling in drug‐resistant bacterial infected wound healing. Funding : National Natural Science Foundation of China, 82302647.

Intestinal

Presenter: Chih Yao Hou National Kaohsiung University of Science and Technology, Taiwan, People's Republic of China Introduction : Gut microbiota dysbiosis and altered bile acid metabolism are linked to neurodegenerative diseases like Alzheimer's disease (AD). While most studies focus on colonic bacteria, we propose that the small intestine microbiota, key in bile acid recycling, plays a more significant role in AD pathology. This study examines the impact of small intestine microbiota changes on bile acid metabolism in AD, using amyloid precursor protein knock‐in (APPNL‐G‐F/NL‐G‐F) transgenic mice. Methods : APPNL‐G‐F/NL‐G‐F transgenic mice were used to explore the relationship between small intestine microbiota changes and bile acid metabolism under beta‐amyloid (Aβ) accumulation. The effects of Lactobacillus johnsonii ‐derived extracellular vesicles (EVs) on inhibiting Clostridium scindens and toxic bile acid production were tested at concentrations of 10 1 ⁰ and 10 1 2 /mL. Small RNA removal from EVs was conducted to investigate the mechanism of their inhibitory action. Results : Beta‐amyloid accumulation correlated with a decrease in Lactobacillus johnsonii and an increase in Clostridium species, which produce toxic bile acids. L. johnsonii ‐derived EVs effectively inhibited C. scindens growth and lithocholic acid (TLA) production at non‐cytotoxic concentrations. The removal of small RNA from EVs eliminated their inhibitory effect, suggesting a mechanistic role of small RNA. Summary/Conclusion : The small intestine microbiota appears more impactful than colonic microbiota in AD pathology, where disruptions can alter bile acid metabolism, exacerbating AD. L. johnsonii ‐derived EVs show promise as probiotics for inhibiting toxic bile acid production, presenting a potential therapeutic pathway for AD. Funding : This research was primarily funded by E‐DA Hospital (Grant No. NCKUEDA11008) and the National Science and Technology Council (NSTC), Taiwan (Grant Nos. NSTC 111‐2636‐B‐006‐014, 112‐2221‐E‐992‐002‐MY3, 110‐2320‐B‐992‐001‐MY3, and 111‐2221‐E‐328‐001‐MY3). Additional support was provided by the Higher Education Sprout Project, Ministry of Education, to National Cheng Kung University (NCKU). The authors also acknowledge technical support from the Bioimaging Core Facility of the National Core Facility for Biopharmaceuticals, NSTC, Taiwan.

Leishmania

Presenter: Deborah Brandt‐ Almeida University of São Paulo, Brazil Introduction : The neglected tropical disease leishmaniasis affects different mammals, including humans, and is caused by the intracellular protozoan Leishmania which presents two infective forms, the promastigote and the amastigote forms, presenting different strategies to evade the immune response. Amastigotes release extracellular vesicles (EVs), which induce CD200 from infected macrophages, a ligand that inhibits cellular activation, and the inducible nitric oxide synthase/nitric oxide (iNOS/NO) mechanism. Until now, no information is available on the capacity of promastigotes to induce CD200. Methods : EVs were obtained by incubating the different L. amazonensis forms in their specific medium/temperature for 1 h. Supernatants were filtered through a 0.45‐mm filter, concentrated, ultracentrifugated, and used for the different assays. Nanoparticle tracking analysis (NTA) was included to verify the purity and quantity of EVs. Macrophage stimulation assays for CD200 induction with EVs were analysed by immuno‐precipitation/ western blot (IP/WB) or then analysed by immunofluorescence by using a polyclonal antibody against Leishmania‐EVs and anti‐CD200 antibody. Results : The release of EVs from both promastigotes and amastigotes is similar in quantity and size, reaching a concentrated amount of EVs between 100 and 200 nm. More important, only EVs released by amastigotes induced CD200 in macrophages, analysed by IP/WB and IF. Since both forms are incubated at different pH (5.2 and 7.0), we test the effect of amastigote EVs obtained from different pH. When amastigotes are incubated in another pH different than 5.2, such as the pH of promastigotes culture (7.0), the same level of EVs quantity is obtained, but the capacity to induce CD200 is impaired and associated with a modification of the proteins/genetic content. Summary/Conclusion : These results indicate that amastigotes of L. amazonensis are specialized forms to induce CD200 in infected cells, and the pH during infection could be critical for the release of effector content into EVs that modulate macrophage response, highlighting the unique factors that must be considered in studying parasite EVs. Funding : FAPESP, CNPq, CAPES

Mechanical

Gaia Bianchi 1,2 , Elisa De Lorenzis 3 , Deborah Mattinzoli 4 , Giuseppe Castellano 1,4 , Giovanni Montini 1,2 , Emanuele Montanari 1,3 , Giancarlo Albo 1,3 , Benedetta Bussolati 5 , Federica Collino 1,2 1 Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy; 2 Laboratory of Translational Research in Paediatric Nephro‐Urology, Paediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; 3 Department of Urology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 4 Renal Research Laboratory, Department of Nephrology, Dialysis and Kidney Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 5 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Introduction : Renal cell carcinoma (RCC) accounts for 90% of all kidney malignancies, and nephrectomy represents the mainstay treatment. However, 20%–30% of RCC patients experience recurrence after surgery, with up to 10% reporting a higher risk for distant metastases. Tumour mechanical stresses can induce immune evasion and metastasis formation. Extracellular vesicles (EVs) are relevant players in the tumour microenvironment, with roles in angiogenesis, immune modulation, metastases, and drug resistance. Their composition changes upon external stimuli and stresses, including those occurring during surgical intervention for tumour exportation. Our study aims at defining the phenotypic changes occurring in EVs released by the tumour following a mechanical stress and their potential effects on the surrounding microenvironment during tumour progression. Methods : Twenty‐five RCC patients with tumour grades 2, 3 and 4 (pT1a‐pT3a stage) were recruited after signing an informed consent. The cellular composition of tumour tissues was analysed through spatial proteomics by MICS (MACSima Imaging Cycling Staining), and the presence of EV‐like particles within the tissue was assessed by transmission electron microscopy (TEM). EVs were extracted from the tumour using an optimized enzymatic protocol followed by either simple dropping (matrix‐EVs) or application of a mechanical stress (compression and shears) to the tissue (stress matrix‐EVs). Particle number was determined by nanoparticle tracking analysis, and the expression of 37 surface proteins was analysed by flow cytometry using the MACSPlex kit. Sequencing of matrix‐ and stress matrix‐EVs extracted RNAs was performed. Results : Application of a mechanical stress during the extraction procedure yielded an increased number of released particles. The three tetraspanins were equally expressed on the surface of EVs obtained through either isolation method. Stress matrix‐EVs presented an augmented expression of cellular markers characteristic of the tissue‐resident populations detected through MICS (i.e., immune‐related, inflammatory, and cell adhesion molecules). The epithelial CD326 was the only significantly decreased marker in the comparison with matrix‐EVs. Summary/Conclusion : The application of mechanical stress affected the number of vesicles released from the tissues and the expression of immune‐ and inflammatory‐related markers on their surface. Stress conditions by changing the EVs phenotype can account for their possible role in tumour progression and immune escape.

Mechanisms

Shima Ghoroghi 1 , Louise Merle 1 , Luisa Loconte 1 , Nathalie Nevo 1 , Lea Guyonnet 2 , Jeremy Mesple 1 , Coralie Guérin 2,3 , Lorena Martin‐Jaular 1,3 , Alain Joliot 1 , Clotilde Théry 1,3 A. Joliot and C. Théry are co‐senior authors. 1 Institut Curie Research center, INSERM U932, PSL Research University, Paris, France; 2 Institut Curie Research Center, CurieCoreTech Cytometry, Paris, France; 3 Institut Curie Research center, CurieCoreTech Extracellular Vesicles, Paris, France Introduction : Extracellular vesicles (EVs) are secreted lipid bilayer‐enclosed particles, which can transfer bioactive molecules to target cells and thus modulate their physiology. Tumour EVs can either activate or inhibit anti‐tumour immune responses. Recently, we showed that triple negative breast cancer (TNBC) cells release EVs bearing the macrophage‐colony‐stimulating factor CSF1, which promotes monocyte differentiation towards a distinct macrophage type with pro‐inflammatory features, correlating with better survival and a potentially anti‐tumoural immune infiltrate in TNBC patients (Tkach et al. 2022, PNAS 119: e2107394119). Here, we further studied the molecular bases of CSF1 association to EVs to explore the anti‐tumour therapeutic potential of CSF1‐EVs. Methods : EVs were isolated by size exclusion chromatography from the conditioned medium of MDA‐MB‐231 TNBC cells or HEK293 cells engineered to express different forms of CSF1. EVs were quantified by nanoparticle tracking analysis, tagged CSF1 was analysed by western blotting and non‐tagged CSF1 by commercial LegendPlex. The E0771 C57Bl6 mouse mammary carcinoma was injected orthotopically in the mammary fat pad. Tumour size was measured by calliper, and immune infiltrate by spectral flow cytometry or immunohistochemistry. Results : Injection of CSF1‐EVs from MDA‐MB‐231 reduced progression of E0771 in immunocompetent mice. The CSF1 gene is transcribed as two major isoforms, short S‐CSF1 and long L‐CSF1, both containing a transmembrane domain, but different extracellular glycosylation and cleavage sites. By expressing them separately in HEK293 cells, we demonstrated that S‐ and L‐CSF1 are associated with EVs through different mechanisms: S‐CSF1 is mainly covalently associated as a full‐length transmembrane protein, while L‐CSF1 is associated as a cleaved form through reversible binding to the EV surface without involvement of the transmembrane domain. Functionally, HEK293‐EVs bearing either L‐ or S‐CSF1 induced different differentiation patterns of human monocytes into macrophages in vitro. When injected in vivo, they induced different modifications in the immune cell infiltrates of the tumours, resulting in different effects on tumour growth in immunocompetent hosts. Summary/Conclusion : Our results could lead to future therapeutic uses of CSF1‐bearing EVs as novel strategies of anti‐tumour immunotherapies. Funding : INSERM, CNRS, Institut Curie, Fondation de France, PSL Research University, ANR, INCa, ARC.

Metabolite

Samuel Wachamo, Alisha Thakur, Mallarie Broadway, Brett Moreau, Alban Gaultier University of Virginia, USA Introduction : Alzheimer's disease (AD) is the most common form of dementia affecting 50 million people worldwide, with estimated global costs of $1 trillion annually. AD slowly destroys memory and thinking skills, and subsequently, the ability to perform activities of daily living. The pathological hallmarks of AD include deposition of amyloid beta, formation of neurofibrillary tangles composed of hyperphosphorylated Tau, and neuroinflammation, which result in loss of synapses, neuronal dysfunction, and eventually, neuronal death. However, what triggers the pathological hallmarks of AD remains elusive. Emerging evidence implicates that the alteration of the microbiota and its associated microbial metabolites precedes overt signs and symptoms of AD, but the precise mechanisms by which this contributes to AD pathology are poorly understood. We hypothesized that altered microbiota in AD contributes to the etiology and progression of the disease by releasing bacterial extracellular vesicles (BEVs) that contain bioactive microbial metabolites. Methods : BEVs from the whole intestinal contents of WT (C57BL/6J) and 5XFAD mice (an animal model of amyloid beta plaque deposition) were isolated by ultracentrifugation followed by size exclusion chromatography. BEVs were characterized by BCA assay (total protein), ZetaView nanoparticle tracking analysis (size and concentration), transmission electron microscopy (size and morphology), western blot (BEV specific markers), and metabolomics (cargo). Results : Our preliminary results revealed that the metabolic profiles cluster separately between BEVs from WT and 5XFAD. BEVs also contain metabolites that have been associated with AD. These results will be further validated using plasma samples from mice and AD patients and healthy controls. Furthermore, our studies demonstrated that BEVs can traffic to the brain. Summary/Conclusion : We discovered that BEVs contain bioactive metabolites, which can potentially regulate local and systemic immune response in AD. Ongoing and future studies will leverage Cre‐LoxP system and determine the functional roles of BEVs in the context of AD. These studies will provide valuable insights into the role BEVs in AD pathogenesis and can potentially identify biomarkers and therapeutic targets for AD. Funding : National Institutes of Health (T32 GM007267 and 5T32AI007496‐29), Owens Family Foundation, and the Miller Family.

Microglial

Martina Gabrielli 1,2 , Giulia D'Arrigo 1 , Giulia Cutugno 1 , Maria Teresa Golia 1 , Francesca Sironi 3 , Marta Lombardi 1 , Camilla Halimi 1 , Roberto Frigerio 4 , Marina Cretich 4 , Elisabetta Battocchio 1 , Sara Francesca Colombo 1,2 , Cristiana Barone 5 , Emanuele Azzoni 5 , Roberta Ghidoni 6 , Caterina Bendotti 3 , Rosa Chiara Paolicelli 7 , Claudia Verderio 1,2 Martina Gabrielli and Claudia Verderio are co‐senior authors. 1 Institute of Neuroscience, National Research Council of Italy, Via Raoul Follereau 3, 20854, Vedano al Lambro, Italy. 2 NeuroMI Milan Center for Neuroscience, University of Milano‐Bicocca, Milan, Italy. 3 Research Center for ALS, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Mario Negri, 2, 20156 Milano, Italy. 4 National Research Council of Italy, Institute of Chemical Science and Technologies (SCITEC‐CNR), Via Mario Bianco 9, 20131 Milan, Italy. 5 School of Medicine and Surgery, University of Milano‐Bicocca, Monza, Italy 6 Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, 25125 Brescia, Italy. 7 Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland Introduction : The complement factor C1q is released by microglia, localizes on weak synapses and acts as a tag for microglia‐mediated synaptic pruning, a fundamental process for proper circuit refinement across early postnatal life, and is reactivated in neurodegeneration. However, how C1q tags synapses at specific times remains elusive. We explored the possible involvement of extracellular vesicles (EVs) released by microglia in C1q delivery to synapses designated for removal. Methods : We optimized a protocol to extract large and small EVs from the interstitial fluid of mouse brain tissue. By western blot, TRPS, single molecule arrays (SimoA) and confocal imaging, we measured the amount, cell source and complement cargo of EVs extracted from WT postnatal pups and adult mice brains, and from C9orf72KO adult brains displaying enhanced microglia‐mediated pruning. Then, we performed in vitro and ex vivo studies to enquire about microglial EVs involvement in C1q deposition to the synapse and synaptic engulfment, using approaches to increase or reduce microglial EVs production by EVs supplementation, C9orf72 silencing or pharmacological inhibition (GW4869). Results : We show that microglia release more EVs carrying C1q in C9orf72KO adult mice, providing a link between microglial EVs production and synaptic removal. Moreover, we report that production of C1q carrying microglial EVs peaks during the pruning period in the early postnatal hippocampus (postnatal day P17), confirming a positive correlation between EVs production and synaptic removal in a physiological setting. In neuronee‐microglia co‐cultures, microglial EVs, labelled by the fluorescent dye mCLING, make preferential contacts with synapses, deliver C1q to pre‐synapses that externalize phosphatidylserine and promote synaptic removal. Interestingly, C9orf72KO microglia engulf more synaptic terminals and decrease synaptic density to a greater extent compared to WT microglia, whereas inhibition of EVs release by GW4869 restores normal pre‐synaptic density, providing mechanistic evidence linking EVs release to synaptic remodelling. Summary/Conclusion : This study identifies microglial EVs as delivery vehicles for C1q to synapses targeted for removal and implicates abnormal EVs production from microglia in neurodevelopmental and age‐related disorders characterized by dysregulated synaptic pruning. Analysis of specific subpopulations of brain EVs opens new perspectives to the understanding of EVs roles in physiology and pathology. Funding : RF‐2016‐02361492 Italian Ministry of Health.

Modulating

Charlotte V. Hegeman 1 , Omnia M. Elsharkasy 2 , Tom A.P. Driedonks 2 , Kate R.J. Friesen 3 , Pieter Vader 2 , Olivier G. de Jong 1, 2 . 1 Utrecht University, The Netherlands.  2 University Medical Center Utrecht, The Netherlands.  3 University of Oxford, United Kingdom Introduction : The CRISPR/Cas9 toolbox consists of modular nucleases that can be employed to efficiently modify genomic sequences with high specificity. However, targeted delivery of the large Cas9‐sgRNA ribonucleoprotein (RNP) complexes remains challenging due to immunogenicity, negative charge, and rapid degradation. An approach to overcome these limitations is the use of extracellular vesicles (EVs) as intercellular delivery vehicles. EVs exhibit the natural ability to carry RNA and proteins across biological barriers and can be engineered to load and deliver biotherapeutic molecules and target specific tissues. Methods : To load Cas9‐sgRNA RNPs into EVs, sgRNAs containing MS2 aptamers and a fusion protein of CD63 and tandem MS2 coat proteins (MCPs) were expressed alongside Cas9 and VSV‐G in HEK293T cells. To study the effect of binding affinity on Cas9‐sgRNA delivery, various mutations affecting binding affinity were made in both the interacting sgRNA MS2‐hairpin and the RNA‐binding domain of the MCPs. To separately study the effects of affinity on cargo loading and release, a UV‐sensitive photocleavable protein (PhoCl) was included in the MS2‐CD63 construct to maximize cargo release. We used a previously published fluorescent Cas9 stoplight reporter system to measure Cas9 RNP delivery (De Jong et al. Nature Communications 2020). Results : We confirmed that adaptation of the sgRNAs did not adversely affect their functionality by direct transfection. Comparing Cas9‐sgRNA delivery of the modulated sgRNAs revealed that adapting binding affinity highly affects functional delivery (0.5% to 22.2%). A similar effect on functional delivery was seen after modifying the affinity of the RNA‐binding domain of the MCP. After UV‐treatment, photocleavable MS2‐PhoCl‐CD63 fusion proteins revealed similar Cas9 delivery for most affinities, with the exception of the lowest affinity. This indicates that affinity can be substantially lowered before negatively affecting cargo loading and that cargo release was a limiting factor in aptamer‐mediated Cas9 delivery for most of these sgRNAs. Summary/Conclusion : Here, we describe a novel way to optimize EV‐mediated loading and delivery of Cas9‐sgRNA RNPs. Our results demonstrate that EVs are capable of functional Cas9‐sgRNA complex delivery and that modulation of binding affinity can be used to increase delivery efficiency.

Modulation

Presenter: Yeyu Shen University of Galway, Ireland Introduction : Human mesenchymal stromal cells (MSCs) die by apoptosis when transplanted with biomaterials in preclinical and clinical studies and initiate a bone regeneration cascade. As a pivotal role in fracture healing, osteoclasts initiate bone healing by clearing necrotic tissue, promoting new bone formation, and facilitating bone remodelling, while excessively active osteoclasts can also impact the bone quality following fracture healing. Osteoclast presence has been shown to be a prerequisite for bone healing facilitated by transplanted MSCs. The aim of this study was to investigate if apoptotic bodies derived from MSCs (MSC‐ABs) impact bone regeneration by regulating monocyte‐to‐osteoclast differentiation. Methods : Apoptosis was induced in human bone marrow‐derived MSCs by ultraviolet (UV) irradiation in EV‐free media and verified by flow cytometry of apoptotic MSCs, scanning electron microscopy, and caspase 3 quantification. MSCs‐ABs were isolated from conditioned media via differentiatial centrifugation and characterized by total protein and RNA content, morphology, and surface marker expression by flow cytometry. MSC‐ABs were treated onto human peripheral blood‐derived CD14+ monocytes in an in vitro osteoclast differentiation protocol. Results : Results suggested that the MSCs‐ABs have a pro‐osteoclastic effect, assessed by tartrate‐resistant acid phosphatase (TRAP, predominantly found in osteoclasts) staining and also by measuring the phosphatase concentration assay in the culture supernatant. Conversely, MSCs‐ABs had an inhibitory effect on osteoclast formation in the presence of the receptor activator of nuclear factor kappa‐B ligand (RANK‐L). Additionally, the enzyme‐linked immunosorbent assay revealed that AB‐treated cells secreted significantly higher levels of IL‐6 and IL‐10, which are known to have a dual effect on osteoclastogenesis. Summary/Conclusion : To understand these findings, we are currently sequencing the RNA of the MSC‐ABs, then polymerase chain reaction and western blotting could be used for verifying underlying mechanisms. This study may contribute to clarifying the currently ambiguous mechanisms by which MSC‐ABs regulate osteoclast differentiation and promote bone regeneration, which may provide a promising therapeutic way for clinical bone regeneration. Funding : This research is founded by the European Council Starting Grant, project number 852152. This research is also funded by the Chinese Scholarship Council (CSC).

Multimodal

Presenter: Andrew A. Lin University of Pennsylvania, Philadelphia, Pennsylvania, USA Introduction : Alzheimer's disease (AD) and Lewy body disease (LBD) are the two most common causes of neurodegenerative dementia worldwide. Recent studies have made significant advances in developing targeted therapeutics for AD, but patients and healthcare providers continue to face a lack of robust biomarkers for differentiating LBD versus AD. Extracellular vesicles (EVs) have shown considerable promise as sources of biomarkers in neurodegenerative dementia. However, their clinical application has been limited by technical challenges in isolating brain‐derived EVs and a lack of samples with corresponding post‐mortem pathology for confirming patient diagnoses. Methods : We profiled the miRNA cargoes of neuronal and astrocyte EVs from patient plasma by using a nanofluidic EV isolation platform developed in our group. Specifically, we sequenced the miRNA cargoes of GluR2‐positive (neuronal) and GLAST‐positive (astrocyte) EVs from the plasma of n = 137 dementia patients and clinically‐normal controls with post‐mortem histopathologic confirmation of their diagnoses. We also profiled five plasma protein biomarkers (AB40, AB42, AB42/40, p‐Tau181, p‐Tau231) via commercially‐available digital ELISA as a benchmark comparison. Informed consent was obtained for the collection of all patient plasma samples in this study. Results : We identified multiple differentially‐expressed EV nucleic acid biomarkers alongside plasma proteins which were statistically robust for comparing LBD versus AD. We show that both GluR2‐positive and GLAST‐positive EVs yield unique miRNAs which provide distinct reservoirs of biological information that build on previously‐reported literature. We also demonstrate concordance of our protein biomarker data with previous literature, and perform computational feature selection to compose proof‐of‐concept biomarker panels for differentiating LBD versus AD. This work offers a set of candidate biomarkers for future studies in neurodegenerative dementia. Summary/Conclusion : In this work, we validate the hypothesis of whether a multimodal biomarker panel can meaningfully assist in the differential diagnosis of LBD versus AD from patient plasma alongside clinically‐normal controls. This work demonstrates a multimodal and biomarker‐driven proof‐of‐concept approach towards the precise differentiation of neurodegenerative dementia. Funding : This study was supported by the American Brain Foundation; National Institutes of Health; Robert H. and Clarice Smith and Abigail van Buren Alzheimer's Disease Research Program; Little Family Foundation; Mangurian Foundation; American Academy of Neurology, American Brain Foundation, LBD Center without Walls; Ted Turner and Family Foundation.

Nanospacer

Presenter: Oliver Vanderpoorten UiT The Arctic University of Norway, Tromsø, Norway Introduction : Nanofluidic devices provide great application potential ranging from human health to single molecule metrology. However, their broad application remains largely untouched as microfluidic equipment with pumps, setups and complex chip handling protocols limit their usability—while the research community remains in urgent need of a standardized way of sizing biomacromolecules and vesicles in an easy manner. A method for the detection of nanoscopic specimens and rapid size‐exclusion of larger particles such as blood cells/platelets/cells without time‐consuming steps (e.g., ultracentrifugation) would be of great benefit to the field. Methods : Here we demonstrate the usage of disposable nanofluidic capillaries (NANOSPACERS) for rapid nanoparticle tracking analysis (NTA) of body fluid‐derived EVs and other biomolecules in solution using fluorescence and label‐free microscopy methods (Raman, phase‐contrast, and dark‐field). Results : Within seconds we measure the size of up to thousands of exosomes in parallel using conventional microscopy equipment within a single pipetting step. EVs can be classified into individual subpopulations upon hydrodynamic size and scattering intensity using open‐source software (FIJI, ImageJ). The method is demonstrated on various biomolecules in the micro‐ to nano‐regime: from highly motile bacteria (∼1 µm) to nanocolloids (44–500 nm), extracellular vesicles (40–120 nm), viruses (40–45 nm), DNA (100 bp–1.5 kbp), and down to proteins (1–3 nm) at the single‐molecule level. Summary/Conclusion : We showcase with this approach a versatile method for EV researchers to use their existing lab equipment for rapid EV size analysis, spectroscopy, and classification. Nanospacers allow biological labouratories with limited funds to implement cutting‐edge single‐molecule research in their facilities—enhancing various sectors of life science and biotechnology. Funding : HORIZON‐MSCA‐2021‐PF‐01‐01, 101064246; UiT Talent Innovation Grant 2024‐2026.

Optimising

Elizabeth R. Dellar 1 , Iolanda Vendrell 2 , Roman Fischer 2,3 , Alexander G. Thompson 1 . 1 Nuffield Department of Clinical Neurosciences, University of Oxford, London, UK; 2 Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, London, UK; 3 Nuffield Department of Medicine, Chinese Academy of Medical Science Oxford Institute, University of Oxford, London, UK Introduction : Cells of the central nervous system (CNS) secrete EVs into cerebrospinal fluid (CSF) and thus hold potential as a source of new biomarkers for neurodegenerative diseases. Current methods for purification, such as ultracentrifugation and size exclusion chromatography (SEC), require large starting volumes, but immunocapture methods may help to reduce volume and separate EVs by cellular origin. Our aim is to optimise methods for capture and proteomic profiling of CNS‐EVs from CSF for biomarker discovery in neurodegenerative diseases. Methods : EVs were purified from 200 to 1000 µL CSF by immunocapture with M‐270 Epoxy Dynabeads conjugated to a cocktail of tetraspanin antibodies (CD81, CD63, and CD9), or by SEC. Bound vesicles were lysed with 5% SDS, for subsequent Lys‐C/trypsin digestion with the suspension trapping method (Protifi). Liquid chromatography tandem mass spectrometry with library‐free data‐independent acquisition was used to compare the proteome (1 h gradient on Orbitrap Ascend). Results : Here we demonstrate that the immunocapture methodology is robust at CSF volumes down to 200 µL, with detection of core EV marker proteins CD9, CD81, TSG101, Syntenin‐1, ALIX, and FLOT1 in all samples, and depletion of non‐vesicular proteins such as Apolipoprotein B and Lectin galactoside‐binding soluble 3 binding protein (LGALS3BP) relative to SEC. We show high proteomic depth with 811 ± 14 proteins using immunocapture from 200 µL CSF, increasing to 1285 ± 224 or 1266 ± 18 in 500 µL or 1000 µL, respectively, compared to 812 ± 66 in SEC‐isolated samples from 500 µL. In the total dataset, detected proteins were significantly enriched for choroid plexus and astrocytic markers, but with no significant enrichment for neuronee markers. We identified proposed neuronal‐specific EV capture target proteins L1CAM, NCAM1, ATP1A3 and GAP‐43 in the data, alongside some additional transmembrane neuronal‐enriched proteins. Precursor peptide mapping showed the presence of cytoplasmic regions for NCAM1, ATP1A3 and some novel proteins, providing some evidence for membrane‐bound over soluble protein forms. No known cytoplasmic peptides were detected for L1CAM or GAP‐43. Summary/Conclusion : This work demonstrates that the immunocapture methodology allows for robust EV purification from small volumes of CSF using generic EV markers. This approach can be used to test alternative neuronal, microglial and astroglial EV capture targets, which we have identified in the dataset.

Optimizing

Presenter: Paula Horta Hugues King's College London, London, UK Introduction : The skin and oral cavity both undergo the same wound healing process, yet the oral mucosa heals without scarring, with reduced inflammation and faster re‐epithelialization. These differences may arise from cell‐intrinsic fibroblast properties. Extracellular vesicles (EVs), which transport biomolecules like DNA, RNA, lipids, and proteins, have been increasingly studied for their regenerative potential. While mesenchymal stem cell‐derived EVs have been shown to promote wound healing, invasive isolation methods and the wound‐healing relevance of fibroblasts suggest the need to investigate fibroblast‐derived EVs. We aim to explore the role of gingival fibroblast‐derived EVs in wound healing and compare transcriptomic and proteomic profiles with dermal fibroblast‐derived EVs. Here, we optimize isolation conditions and techniques to develop protocols for both cell types. Methods : Primary gingival fibroblasts from patients were cultured under three conditions (serum‐free, 10% ultralow endotoxin FBS, and 2% Ultroser G) for 24 h following a 10% FBS culture period. Proliferation and morphology were monitored for 72 h using CellCytex. EVs were isolated by ultracentrifugation and ExoQuick‐TC, following the manufacturer's protocol. EVs were characterized by Exoview R200 and NanoSight LM10. Results : Serum‐free conditions showed no growth after 72 hs, while cells in 10% FBS, 2% Ultroser G, and ultralow endotoxin FBS reached 35%, 28%, and 30% confluency, respectively, with no morphological changes. ExoQuick‐TC yielded 4.4 times higher protein concentration than ultracentrifugation. Exoview R200 results indicated a low particle range, suggesting subpopulations may be lost with these purification methods. In unpurified serum‐starved samples, CD81 was the most abundant exosome marker (44%), followed by CD63 (33.7%) and CD9 (22.3%). Using NanoSight, the mean particle size from ExoQuick‐TC was 202.3 nm with a concentration of 4.942 × 10 9 particles/mL, whereas ultracentrifugation yielded 361 nm particles at 3.4 × 10 8 particles/mL. Summary/Conclusion : Low endotoxin FBS and Ultroser G conditions promoted greater cell growth for EV isolation. ExoQuick‐TC provided faster, higher‐concentration particle yields compared to ultracentrifugation, with more precise purification. These optimized conditions will be applied to purify EVs from dermal fibroblasts in future stages.

Peripheral

Presenter: Leandra K. Figueroa‐Hall Laureate Institute for Brain Research, USA Abstract unavailable

Phenotypic

Presenter: Maria Teresa Golia CNR Institute of Neuroscience, Italy Introduction : Neurones are highly dependent on microglia. In several brain diseases microglia display a disease‐associated microglia (DAM) transcriptional state that initially protects neuronees but then becomes detrimental due to downregulation of homeostatic genes. Microglia normally lose their protective function during senescence. Senescent microglia exhibit cell cycle arrest, dystrophic morphology, deficits in phagocytic and lysosomal activities, impaired metabolism, and senescence‐associated secretory phenotype (SASP) with enhanced EV secretion. In various tissues, accumulation of intracellular iron and ferritin are key features of cellular senescence. Accordingly, immunopathological studies have identified dystrophic, iron/ferritin‐positive microglia in the aged and diseased human brain. However, RNAseq of iron‐laden microglia suggests enhanced activation, proliferation and metabolic activity. Therefore, how microglia respond to iron accumulation is still unclear. This study aimed to study the temporal response to iron of murine microglia in vitro and to explore the impact of their secretome/extracellular vesicles (EVs) on neighbouring cells. Methods : The impact of iron (500 µM) on microglia functions and senescence was assessed by immunofluorescence assays. RNA‐Seq was used to define the most deregulated transcriptional pathways. Finally, metabolic alterations were evaluated using biochemical assays, western blot, RT‐PCR techniques, and metabolomic analyses. Results : We found that short exposure (3 days) to iron activates a fast proliferative and phagocytic response in microglia cultured with astrocytes. Conversely, microglia chronically exposed to iron (30 days) show a senescent‐like phenotype characterized by flattened morphology, proliferation arrest, decreased phagocytosis, increased ß‐galactosidase activity, cell death resistance and enhanced EV production. Furthermore, the cells show increased activity of the NAD‐consuming enzyme CD38, decreased NAD content and downregulation of NAD‐dependent sirtuins. Interestingly, iron‐fed senescent microglia decrease NAD content and induce senescent traits in healthy microglia in non‐contact coculture but not in microglia in which NAD levels were boosted by pharmacological treatments. The impact of iron‐fed microglia on healthy microglia in vivo is under investigation. Summary/Conclusion : Collectively these data advance our understanding of the microglial response to iron and identify iron‐loaded microglia as a powerful in vitro tool to study mechanisms governing microglial transition from the proliferative to the hypofunctional/senescent state typical of late‐stage DAM and to screen for molecules able to counteract microglia‐mediated paracrine senescence.

Pioneering

Yee‐Hsien Lin 1 , Han‐Tse Lin 1 , Wei‐Tung Wang 1 , Shih‐Hwei Liu 1 AventaCell BioMedical Corp., Kent, WA 98031, USA Introduction : The therapeutic potential of extracellular vesicles (EVs) has emerged as a key focus in the biomedical field, owing to their crucial role in intercellular communication. EVs contribute to various disease processes, cancer progression, and tissue regeneration. Among them, exosomes, a specific subclass of EVs, are distinguished by their size and rich content of bioactive molecules and genetic material. These unique biological features have generated significant interest in using exosomes as advanced drug delivery systems for tissue repair and cancer therapies. Therefore, precise methodologies are vital to ensure the production of high‐quality exosomes, particularly for cancer treatment and regenerative medicine. Platelet‐derived exosomes, enriched from human platelets, demonstrate a remarkable capacity to promote cell proliferation and inhibit apoptosis. With their high biocompatibility, low cytotoxicity, minimal tumourigenicity, and reduced immunogenicity, these exosomes show significant therapeutic potential, especially for homologous administration in clinical settings. The main objective of this study is to provide a comprehensive molecular and morphological characterization of exosomes derived from human platelets. By isolating and purifying exosomes from human platelet lysate, we introduce them as a promising candidate for future therapeutic applications in exosome research and clinical use. Methods : Human platelet‐derived exosomes were successfully isolated from human platelet lysate using a tangential‐flow filtration system equipped with a hollow fibre for efficient depletion. Following isolation and purification, key CD markers, including CD9, CD63, and CD81, were effectively characterized. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) confirmed the average particle size. Results : Additionally, when these exosomes were introduced into a 72‐h cell culture with mesenchymal stem cells, no cytotoxicity was observed throughout the culture period. Consequently, human platelet‐derived exosomes present themselves as a promising therapeutic candidate, offering significant potential for both exosome research and various clinical applications. Summary/Conclusion : Human platelet‐derived exosomes show great potential as therapeutic agents due to their biocompatibility, low immunogenicity, and ability to promote cell growth. This study provides a foundation for further research into their clinical applications in tissue regeneration and cancer treatment.

Preserving

Invited Speaker: Stefan Momma Goethe University Frankfurt, Frankfurt, Germany

Proteomics

Shanaya Haque 1 , Morgan Rouse 1 , Francis Li 1 , Purva Nayam 1 , Kaelan Samoranos 1 , Elexa Rallos 1 , William Glembocki 1 , Vanessa Correll 1 , Shimin Chen 1 , Julius Nyalwidhe 1 , Eric Feliberti 1,2 , Lifang Yang 1 1 Macon & Joan Brock Virginia Health Sciences at Old Dominion University, Norfolk, Virginia, USA; 2 Sentara Healthcare, Virginia Beach, Virginia, USA Introduction : Oestrogen receptor‐positive (ER+) breast cancer represents the most common breast cancer subtype. Tamoxifen and fulvestrant, both directly targeting the ER, have considerably reduced recurrence and mortality from ER+ breast cancer. Unfortunately, acquired resistance to these and other antiestrogens eventually becomes a major challenge, especially in the metastatic setting. Accumulating evidence suggests small extracellular vesicles (sEVs) that are employed by cancer cells for intercellular communication have been involved in mediating endocrine resistance in breast cancer. But the specific sEV molecular cargo and sEV‐mediating intercellular signalling to drive this phenotype remain largely unknown. The aim of this study was to address these issues by conducting an in‐depth characterization of the proteomes of sEVs derived from a breast cancer endocrine resistance model. Methods : Normal breast epithelium MCF10A, endocrine therapy‐sensitive MCF7 breast cancer cells, and dual tamoxifen‐ and fulvestrant‐resistant MCF7 variant LCC9 cells were cultured. sEVs were isolated from cell supernatants via a differential ultracentrifugation approach. The morphology, size, and purity of isolated sEVs were confirmed by transmission electron microscopy, NanoSight, and western blot. sEV proteins were extracted by the RIPA buffer and quantified by the micro‐BCA protein assay. Label‐free mass spectrometry and bioinformatic analysis were conducted to compare cellular and sEV proteomes across three cell lines. Results : Compared to MCF10A and MCF7 cells, LCC9 cells have demonstrated a significant reduction in sEV secretion and sEV protein content. These changes are also revealed by unbiased proteomics analysis in which the LCC9 cellular proteome has shown alterations in protein sorting signalling and EV biogenesis pathways. Compared to normal counterparts, sEVs derived from both cancer cells are enriched in proteins that play important roles in cancer growth and metastasis. Further analysis indicated that proteins regulating interferon and PTEN signalling are more upregulated in sEVs derived from drug‐resistant cells than drug‐sensitive versions, suggesting their roles in fostering an endocrine resistance phenotype. Summary/Conclusion : Our study highlights the mechanistic insights of sEVs in the endocrine resistance in breast cancer. Further investigations are warranted, paving the way to identify novel endocrine resistance biomarkers and druggable therapeutic targets to monitor and reverse this deadly disease. Funding : This work was supported by Eastern Virginia Medical School Breast Cancer Research funds.

Reciprocal

Presenter: Janusz Rak McGill University, Canada Introduction : Glioblastoma (GBM) remains incurable in spite of decades of intensive research. Florid neovascularization, a morphological hallmark of GBM, is still poorly understood in terms of mechanisms and function. While oncogenic pathways and tumour microenvironments are recognized as forces driving vascular responses in GBM, the identity of mediators that control endothelial growth, regulatory effects, barrier function and immunomodulation are still to be fully elucidated. Here we present evidence suggesting that extracellular vesicle (EV) pathways play a major role in vascular growth, patterning, paracrine (angiocrine) effects of endothelial cells and immunoregulation. Methods : Small EVs from conditioned media of human glioma stem cells (GSC) were isolated by ultracentrifugation and size exclusion chromatography and tested for endothelial cell responses in vitro (migration) and in vivo (xenografts). Similarly, EVs from endothelial cells were assayed for impact on GSCs. We also examined vascular properties of gliomas in the context of genetically disrupted vesiculation pathways. Results : We observed that while proneural GSCs produced mainly soluble angiogenic factors (VEGF), their mesenchymal counterparts shed EVs enriched for oncogenic epidermal growth factor receptor (EGFRvIII) while engaging several different pathways of vesiculation. These EGFRvIII‐EVs triggered a non‐angiogenic vascular growth process (vasectasia) with distinct molecular features. Endothelial EVs promoted a switch from a proneural to a mesenchymal phenotype in GSCs. Genetic disruption of vesiculation pathways altered the interactions between intracranial tumours, vasculature and immune effector cells. Targeting EV‐mediated vascular effects resulted in prolonged survival of mice with GBM. Summary/Conclusion : EVs contribute to interactive networks that couple cancer progression and vascular and immune responses in GBM. Funding : Fondation Charles Bruneau and CIBC, Canadian Institutes of Health Research.

Regulation

Presenter: Dokyung Jung Kyungpook National University, Republic of Korea Introduction : Cancer Seed and soil hypothesis EV Cytokines Methods : Cell lines and cell culture Isolation of EVs Nanoparticle tracking analysis (NTA) Cell viability assay Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) Western blot assay Immunofluorescence staining Protein profile analysis Animal study H&E staining EV miRNA data analysis Kaplan‐Meier survival analysis Statistical analysis. Results : 1. Discovery of a cytokine for inhibition of EV secretion 2. Cytokine receptor‐dependent regulation of EV secretion in breast cancer 3. An immune‐activating cytokine reduces lung metastasis mediated by cancer‐derived EVs 4. An Immune‐activating cytokine modulates cancer cell‐derived EV miRNA profiles 5. Correlation between immune‐activating cytokine expression in human patient survival. Summary/Conclusion : This study highlights this immune‐activating cytokine as a promising therapeutic agent with anti‐metastatic effects, capable of regulating EV secretion and modulating EV characteristics. These findings underscore its potential utility as both a prognostic biomarker and a novel therapeutic strategy for metastatic cancer, paving the way for its broader application in clinical settings. Funding : This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT).

Regulatory

Presenter: Jun Shirai Tokyo Metropolitan University, Japan Introduction : Valosin‐containing protein (VCP)/p97/CDC48 is an AAA+‐type ATPase that is crucial for endosomal sorting. Despite its importance in multivesicular body (MVB) biogenesis, the regulatory mechanisms of small extracellular vesicle (sEV) secretion mediated by VCP remain poorly understood. VCP demonstrates functional and spatial specificity through interactions with various adapters, including UBX domain‐containing proteins (UBXNs). Therefore, we investigated the potential roles of VCP and the UBXN family proteins in regulating sEV secretion in cultured cells. Methods : sEVs were purified from HeLa cells using ultracentrifugation or the phosphatidylserine affinity method. Purified sEVs were validated by detecting multiple markers by Western blotting. Characterization of sEVs was conducted using nanoparticle tracking analysis and transmission electron microscopy. MVB imaging focused on the marker CD63, employing confocal microscopy. Results : To determine whether VCP regulates sEV secretion, we analysed sEVs from HeLa cells treated with the VCP inhibitor NMS‐873 and found that VCP inhibition significantly enhances sEV secretion. To identify VCP adapters involved in sEV secretion, we performed a knockdown screening of UBXN family proteins. As a result, the depletion of UBXN3B increased sEV marker secretion. Importantly, there were no significant changes in particle size following UBXN3B knockdown, suggesting that UBXN3B regulates sEV secretion. Additionally, the knockdown of Rab27a decreased sEV secretion in UBXN3B‐depleted cells, indicating that UBXN3B regulates sEV secretion in a Rab27a‐dependent manner, which involves MVB trafficking. UBXN3B is known to function in endoplasmic reticulum‐associated degradation (ERAD). However, since the suppression of UBXN3B led to an increase in sEV secretion even under conditions of ER stress induced by tunicamycin, it is possible that UBXN3B regulates sEV secretion in an ERAD‐independent manner. Localization analysis revealed that endogenous UBXN3B is expressed at higher levels in close proximity to the MVB marker CD63, and its roles in MVB positioning and maturation are under investigation. Summary/Conclusion : We demonstrate that VCP negatively regulates sEV secretion and identify UBXN3B, a VCP adapter, as a novel regulator of sEV secretion. In this presentation, we will report the latest findings regarding the regulatory mechanisms of MVB via UBXN3B and the role of other UBXN adapters in sEV secretion. Funding : This work was supported by JSPS KAKENHI Grant Number JP24KJ1860.

Separation

Gonzalo Bustos‐Quevedo 1,2 , Jerome Nouvel 1,2,3 , Tony Prinz 1,2 , Ramsha Masood 1,2 , George Daaboul 4 , Tanja Gainey‐Schleicher 1,2 , Uwe Wittel 2,5 , Sophia Chikhladze 2,5 , Bence Melykuti 1,2 , Martin Helmstaedter 2,6 , Gerhard Pütz 2,3 , Irina Nazarenko 1,2,7,8 1 Institute for Infection Prevention and Hospital Epidemiology, Freiburg, Germany; 2 Faculty of Medicine, Medical Center, University of Freiburg, Freiburg, Germany; 3 Institute of Clinical Chemistry and Laboratory Medicine, Freiburg, Germany; 4 NanoView Biosciences, Boston, Massachusetts, USA; 5 Department of General and Visceral Surgery, Freiburg, Germany; 6 IMITATE EM Core Facility, Freiburg, Germany; 7 Hahn‐Schikard, Freiburg, Germany. 8 German Cancer Consortium (DKTK), Partner Site Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany Introduction : EVs are particles released from all types of cells sharing similarities with the cells from where they were released. Many isolation techniques are being developed for EV isolation because of their applicability as liquid biopsies, and specifically as a way of understanding their role in the progression of different diseases. Blood is one of the biofluid more used, where other EV‐like particles are found, such as lipoproteins. To obtain EV samples without lipoproteins is quite used in the research community consecutively EV isolation based on different approaches, having a first step of size exclusion isolation followed by a step of ultracentrifugation. Resulting in a high purity of EVs but a low EV recovery. We addressed this problem using fast protein liquid chromatography (FPLC) as an EV isolation method. Methods : NTA, TEM, MACSPLEX, TECAN, cell culture, lipoproteins isolation, spiking blood samples with EVs derived cell culture, western blot. Results : FPLC was validated using pre‐isolated EVs from cell culture and lipoproteins from blood. EVs were visualized in fraction 16, and lipoproteins in fraction 25. TEM and western blot were used to corroborate integrity from fraction 16 and the presence of CD63 as a surface marker and ALIX, TSG101, and HSP70 as associated EV markers. Those markers were missing in the rest of the fractions. Then, plasma samples from healthy donors were run using antibodies using CD9 and CD63 for EVs, and ApoB100 and ApoA1 for lipoproteins. Fraction 16 being the highest in EV particles and fraction 25 for lipoproteins. Then, EVs from four blood samples with Pancreas adenocarcinoma (PDAC) were isolated by FPLC to validate that the EV‐rich fractions are useful for detecting EV‐derived cancer cells. Isolated particles were measured by NTA and MACSPLEX. Fraction 16 did not contain a high particle number, but EV biomarkers such as CD63 and EV cancer biomarkers such as TSPAN8, and EpCAM were detected with the maximum signal in fractions 16 and 17. Summary/Conclusion : The applicability of the FPLC for EV isolation technique was corroborated having a higher resolution of EV rich fractions with a lower number of lipoproteins co‐isolated, and also isolating EVs carrying EV cancer biomarkers.

Spectrally

Imran Mahmud*, Md. Khirul Islam, Iida Martiskainen, and Janne Leivo Department of Life Technologies, University of Turku, Turku, Finland Introduction : Extracellular vesicles (EVs) are highly enriched with tetraspanins like CD9 and CD63, which are widely used for EV analysis and characterization and serve as key markers for the development of EV‐based detection technologies. This study aimed to develop a highly sensitive dual‐label lateral flow immunoassay (dual‐LFIA) for multiplex detection of CD9+ and CD63+ EVs using upconverting nanoparticles (UCNPs) as reporters. This dual‐LFIA employs two different UCNPs as luminescent reporters, enabling spectrally distinct and simultaneous detection of CD9 and CD63 targets within a single assay. Methods : EVs were isolated from prostate cancer Du145 cell culture medium (CCM) by using ultracentrifugation (UC) and size exclusion chromatography (SEC). To capture EVs, anti‐CD9 and anti‐CD63 antibodies were printed onto the nitrocellulose membrane of lateral flow cards as test lines. The cards were assembled with sample and absorbent pads and cut into 4.8 mm strips. Next, the isolated EVs were added on strips for their simultaneous detection using the same CD9 and CD63 antibodies, which were conjugated to erbium‐doped (NaYF4:Yb3+, Er3+) and thulium‐doped (NaYF4: Yb3+, Tm3+) UCNPs, respectively. These UCNPs emit spectrally distinct, narrow emission peaks, enabling optical separation. Specifically, thulium‐doped UCNPs produce a blue emission at 450 nm, while erbium‐doped UCNPs emit green light at 540 nm, facilitating multiplex EV detection and differentiation. Results : This developed proof‐of‐concept dual‐LFIA was able to identify both CD9‐ and CD63‐positive EVs in Du145‐CCM, ucEVs, and secEVs. It demonstrated high sensitivity and linearity for EV detection. Summary/Conclusion : Our preliminary studies demonstrate that this novel UCNP‐based dual‐LFIA approach offers superior sensitivity due to the simultaneous detection of CD9+ and CD63+ EVs. Future studies will focus on comparing the sensitivity of this dual‐LFIA with our previously developed single‐label LFIA and investigating disease‐specific markers for clinical applications.

Squeezable

Presenter: Jina Ko University of Pennsylvania, Philadelphia, Pennsylvania, USA Introduction : Extracellular vesicles (EVs) are promising for molecular diagnostics, but current analyses are limited by the rarity and compositional heterogeneity of EV protein expression. Therefore, single EV profiling methods require high sensitivity, multiplexing, and throughput to address these issues. Here a single EV analysis technique that utilizes squeezable methacrylated hyaluronic acid hydrogel microparticles (MHPs) is described as a scaffold to immobilize EVs and perform an integrated rolling circle amplification (RCA) assay for an ultra‐sensitive and multiplex analysis of single EV proteins. Methods : To perform ultra‐sensitive and multiplex protein analysis of single EVs, uniform droplets consisting of methacrylated hyaluronic acid (MeHA) polymer precursor and bulk EVs were generated with a flow‐focusing microfluidic droplet generator. EVs are physically arrested within hydrogel microparticles by photopolymerization. Consequently, captured EVs are labelled with DNA barcoded antibodies (Ab‐DNA), and multiplex protein signals are amplified by incorporating RCA into the hydrogel microparticle assay without Poisson and steric limitations. Results : We validated that MeHA hydrogel microparticles (MHPs) exhibit high compressibility upon physical squeezing, which can align RCA products in a single plane to uniquely enable imaging without the need for high‐magnification z‐stack imaging. As a result, image acquisition is cost‐effective, simple, and fast, and EV analysis is high throughput, as a single image contains > 1000 EVs. An automated pipeline was developed to describe the presence/absence and co‐expression of protein markers for single EVs from different cancer cell lines. For validation, we profiled the protein expression patterns of highly expressed cancer cell markers among single EVs from different cancer lines and elucidated their heterogeneity. As a result, this method bypasses the Poisson distribution and multiplexing limits of digital ELISA platforms, enhances sensitivity by enabling a larger surface area of interactions with epitopes, and provides a scaffolding for conducting RCA steps to enhance visualization of rare protein signals. Summary/Conclusion : We present a simple, robust, and scalable platform that can enable the identification of rare EV subpopulations with diagnostic potential and advance the development of molecular assay technologies.

Systematic

Presenter: Hajar Yaakoub Nantes Université, France Introduction : Early‐life gut microbiota is a key determinant of lifelong health, influencing intestinal function and mediating interactions with distant organs, a process increasingly linked to bacterial extracellular vesicles (BEVs). However, fecal extracellular vesicles (fEVs) remain understudied, with no standardized extraction protocol, limiting insights into microbiota‐host interactions. Using a simplified gut microbiota murine model, this study aims to characterize fEVs by examining their origin, contributions of G‐ and G+ bacteria, and metabolite profile, while assessing the impact of gut colonization type, sample type, sex, and age. Methods : Two controlled colonization models of 7‐week‐old mice were used: one group monocolonized with a G‐ bacterium (Escherichia coli) and another bicolonized (dixenic) with both G‐ (E. coli) and G+ (Bifidobacterium longum) species. Control groups included germ‐free (axenic) and naturally colonized mice. A 15‐week‐old dixenic group was included to assess age effects. EVs were extracted from cecum, colon, feces, and plasma using an optimized protocol. In addition, culture‐derived BEVs were used for comparison. Characterization involved protein quantification, nanoparticle tracking analysis, dot blotting, mass spectrometry, and transmission electron microscopy (TEM). Results : Cecum samples contained half a log more fEVs than colon or feces, with higher protein enrichment and larger size, making them the optimal sample for fEV studies. Colonization model had a slight effect on fEV abundance, with the dixenic group showing the lowest values. However, fEVs were significantly smaller in axenic mice and highly protein‐enriched in the normal group, highlighting colonization's impact on fEV size and production. The fEV properties were similar, irrespective of age and sex. Dot blotting of fEVs confirmed the presence of BEVs and host‐derived EV components. Data suggest bioactive molecule enrichment in culture‐derived BEVs, with ongoing analyses determining whether this extends to fEVs. TEM observations confirmed the integrity of fEVs. Summary/Conclusion : This study establishes a framework to explore fEV production, origin, composition, and circulation. The findings presented here spur further investigational efforts and pave the way for more comprehensive research, potentially advancing research on their impact on health and disease.

Technology

Eylul Gulsen Yilmaz 1,2 , Gamze Kara‐Magden 3 , Yeseren Saylan 4 , Fatih Inci 1,2 1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey; 3 Department of Chemistry, BUGAMED Biotechnology Industry and Trade Inc., Eskişehir, Turkey; 4 Hacettepe University, Ankara, Turkey Introduction : Cancer cells encounter diverse fluid shear stresses (FSS) within the tumour microenvironment, but existing in vitro 3D models are not adequate for examining how these forces influence cancer progression and resistance to chemotherapy. To address this gap, new approaches have emerged, including the use of microfluidic systems. These systems are gaining popularity due to their affordability, high‐throughput capabilities, efficient use of resources, and ease of manipulation. Furthermore, they can simulate ECM‐like conditions with biomaterials such as silk fibroin (SF), which is ideal for 3D cell cultures because of its strength, biocompatibility, and biodegradability. Additionally, research has revealed that tumour‐derived extracellular vesicles (EVs) play a significant role in promoting cancer growth, drug resistance, and metastasis. Methods : In this research, MCF‐7 cells were cultured under static 2D, static 3D, and dynamic environments. EVs were collected from these cultures and isolated using a microfluidic device based on ultrafiltration, featuring 200 and 50 nm filters. The EVs were subsequently characterized using western blot, NTA, and SEM. Then, MCF‐7 cells were treated with the anticancer drugs doxorubicin and docetaxel to examine whether these treatments induce changes in EVs under FSS. Lastly, RT‐qPCR was utilized to analyse variations in miRNA levels. Results : EVs from the control groups exhibited average sizes of 177 nm (static 2D), 91 nm (static 3D), and 128 nm (dynamic conditions). EVs generated under static environments appeared more aggregated compared to those from dynamic cultures. Increasing doses of Doxorubicin led to a reduction in EV size. SEM images supported these findings, showing consistent effects with both Doxorubicin and Docetaxel treatments. Western blot analysis confirmed successful EV isolation, with a strong CD63 band detected between 30 and 80 kDa, notably around 70 kDa, and a CD81 band observed near 60 kDa. Additionally, miRNA expression was assessed for EVs derived from MCF‐7 cells. Summary/Conclusion : In summary, we effectively isolated and characterized EVs under various conditions. These EVs hold significant potential as biomarkers for cancer and may serve as a promising tool for early cancer detection. Funding : The authors gratefully acknowledge support from the Scientific and Technological Research Council of Turkey (TÜBİTAK) 2232 International Fellowship for Outstanding Researchers (Project No: 118C254).

Unraveling

Astrid Laimer‐Digruber, Tanja V. Edelbacher, Caroline Lassnig, Silvio Kau‐Strebinger, Belinda Maw, Karoline Kollmann, Birgit Strobl, Monika Ehling‐Schulz University of Veterinary Medicine Vienna, Austria Introduction : Bacillus cereus is a Gram‐positive spore‐forming bacterial pathogen that is well‐known for its ability to cause food poisoning [1]. Notably, this opportunistic pathogen is gaining increasing prominence as causing agent of non‐GI related illnesses, including systemic and local infections [2]. Recently, we showed that B. cereus secretes biologically active extracellular vesicles (EVs). In contrast to EVs of Gram‐negative bacteria, EVs in Gram‐positive bacteria are far less studied and their role in bacterial (patho)biology is largely unknown. By purifying and characterizing B. cereus EVs we could show that EVs allow to deliver its virulence factors in a protected manner to host cells [3]. Methods : Bacterial EVs were purified by employing differential centrifugation. In addition, EV‐preparations were characterized by transmission electron microscopy, nanoparticle tracking analysis and flow‐cytometry. We further analysed EV‐mediated pathogenicity using in vitro and in vivo approaches. Application of EVs on bone‐marrow derived macrophages (BMDMs) resulted in dose‐dependent release of proinflammatory cytokines and cell‐damage. By employing confocal microscopy, we were also able to closely follow the uptake of EVs with BMDMs. Results : Systemic application of B. cereus EVs in a murine mouse model—mimicking bacterial EV release during systemic infections—led in a time and content dependent manner to a massive release of proinflammatory cytokines and inner organ damage. Summary/Conclusion : In this study, we show for the first time that the presence of B. cereus EVs without its bacterial cells is sufficient to cause systemic inflammatory response symptoms. Therefore, it is anticipated that this research will provide new ways for understanding EV‐contribution of pathogenic Gram‐positive bacteria to systemic bacterial infections, as well as aid in the development of innovative defense mechanisms and novel therapeutic approaches.

Validation

Biancamaria Pierri 1 , Gabriela L. Jackson 1 , Fang Wang 1 , Erez Eitan 2 , Olga Volpert 2 , Vrinda Kalia 1 , Andrea Baccarelli 3 , Haotian Wu 1 1 Columbia University, New York, New York, USA; 2 NeuroDex Inc., Cambridge, Massachusetts, USA; 3 Harvard University, Cambridge, Massachusetts, USA Introduction : Extracellular vesicles (EVs) in circulation are informative tissue‐specific biomarkers that potentially reflect the phenotypic state of the parental cells, but their high heterogeneity poses a challenge for the enrichment of tissue‐specific EVs, limiting their utility. The absence of standardized, validated methods undermines the reliability of isolated materials and data reproducibility, causing significant obstacles to the translation of EVs as biomarkers. We propose a general workflow to enrich tissue‐specific EVs from human plasma, targeting tissue/cell‐specific surface proteins and validating EV subpopulations through multiple cytosolic targets. Methods : Total EVs were purified by ultracentrifugation, size exclusion chromatography, or polyethylene glycol isolation reagent. An immunoprecipitation (IP) protocol followed by immunoassay validations was performed to enrich tissue‐specific EV subpopulations derived from brain, lung, liver, and placenta cells. In accordance with MISEV guidelines, we visualized the resulting EVs, measured particle concentrations and sizes, and verified the presence of canonical EV tetraspanins markers. The enrichment of target fractions was validated using ELISA assays for two tissue‐specific cytosolic proteins per tissue. Total EVs, IP unbound fractions, and IgG were considered as controls to compare the relative enrichment obtained for each EV subpopulation. Results : ELISA assays showed >10× enrichment for all tissue‐specific cytosolic protein markers in IP‐purified subpopulations compared to total EVs (all p values < 0.05). General EV characterization confirmed the successful enrichment of the targeted EVs, demonstrating the colocalization of tetraspanins alongside tissue‐specific markers. Summary/Conclusion : Our results show the effectiveness of an immunoprecipitation‐based protocol targeting tissue/cell‐specific surface antigens to enrich subpopulations of EVs from human plasma. The use of multiple cytosolic cell‐specific markers to validate the enrichment of the targeted EVs strengthens the reliability of our findings. This approach can be replicated to enrich other EV subpopulations, providing further insights into the physiology or pathology of the source tissue, and enhancing the application of EVs through a liquid biopsy perspective. Funding : This research is supported by the National Institute of Environmental Health Sciences grant 1R35ES031688‐01A1.

Agri‐Food

R. Latella 1 , L. Urbanelli 1,5 , E. Chiaradia 2 , L. Mezzasoma 3,5 , R. Romani 3,5 , M. Gargaro 4 , R. Pellegrino 1 , H. B. R. Alabed 1 , C. Emiliani 1,5 , S. Buratta 1 1 Department Chemistry, Biology and Biotechnology, University of Perugia (Italy); 2 Department of Veterinary Medicine, University of Perugia (Italy); 3 Department of Medicine and Surgery, University of Perugia (Italy); 4 Department of Pharmaceutical Sciences, University of Perugia (Italy); 5 Extracellular Vesicles network (EV‐net) of the University of Perugia (Italy) Introduction : The growing demand for food production due to the rapid increase in the world population generates a huge amount of agricultural by‐products that might be valorized through the extraction of biomolecules and preparation of value‐added products useful for various biomedical applications. Here, we evaluated the possibility that agricultural by‐products could represent a valuable source of bioactive nanovesicles (NVs), reinforcing the concept of waste valorization within the context of the circular economy. In particular, we successfully isolated NVs from olive vegetation water (OVW), a by‐product of the olive oil industry, and serum milk (SM) generated during the cheese‐making process. NVs have been biophysically and biochemically characterized before the evaluation of their biological effects on mammalian cell lines. Methods : OVW‐NVs were isolated through differential centrifugation followed by density‐gradient centrifugation [1], whereas SM‐NVs were isolated by a protocol currently used to isolate vesicles from milk [2]. NVs were biophysically characterized through scanning electron microscopy, nanoparticle tracking analysis, cryo‐transmission electron microscopy and dynamic light scattering. Since information on biochemical composition is essential to rationalize the biological effects of NVs on mammalian cells, a complete characterization of NVs was obtained by integrated lipidomic, metabolomic, and proteomic procedures. Finally, biological effects (cytotoxicity, internalization ability, antioxidant and anti‐inflammatory effects) of these vesicles on mammalian cell lines (PC3 and THP‐1) have been explored. Results : Biophysical data and information on lipid, metabolite and protein composition demonstrated that the features of OVW‐NVs and SM‐NVs overlap with that of bioactive NVs isolated from edible plants/fruits and from milk, respectively. Both NVs are internalized by mammalian cells and protected them from cytotoxicity and oxidative stress induced by H 2 O 2 exposure. Further, OVW‐NVs and SM‐NVs suppressed NLRP3 inflammasome activation, warranting further investigation as potential anti‐inflammatory tools. Summary/Conclusion : Overall these results demonstrate, for the first time, that agricultural residues might represent a source of bioactive NVs that can be internalized by mammalian cells, protecting them by oxidative stress and inflammatory stimuli. This evidence strengthened the idea that agricultural by‐product‐derived NVs might become a promising nutraceutical tool. Funding : European Union—NextGenerationEU under the Italian Ministry of University and Research (MUR) National Innovation Ecosystem grant ECS00000041—VITALITY.

Amyotrophic

Presenter: Suzy Varderidou‐Minasian UMC Utrecht, The Netherlands Introduction : Amyotrophic lateral sclerosis (ALS) is an adult‐onset neurodegenerative disorder characterized by the degeneration of both upper and lower motor neuronees. Despite the promise of new treatments, clinical trials face high failure rates. Mesenchymal stem cells (MSCs) and the extracellular vesicles (EVs) these cells release have gained significant interest due to their accessibility, capacity for migration to damaged tissues, and involvement in reparative processes. We utilize induced pluripotent stem cell (iPSC)‐derived motor neuronees, which provide a valuable method to study disease signatures derived from patient‐specific cells to get insight into the mechanisms underlying ALS and enable the testing of novel therapeutic agents. Several ALS iPSC‐derived motor neuronees were treated with MSC‐EVs and analysed with high‐throughput mass spectrometry (MS) approaches for the in‐depth characterization of relevant ALS disease pathways. Methods : Thirty‐six motor neurone samples (9 ALS patients (carrying FUS, TDP‐43 or C9ORF72 mutations), 6 isogenic lines and 3 healthy controls) were analysed by MS for proteomic alterations. EVs were isolated from human bone marrow MSCs using ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot for EV markers. Motor neuronees were treated with EVs derived from 4x106 MSCs per well (6 wp) for 2 days. The isolated EVs and treated samples were analysed by MS and neurite length was measured using bright‐field microscopy. Results : Principal component analysis across all lines and differentiation batches showed that individual variation is higher than technical variation. Global proteomic analysis revealed dysregulated ‘oxidative phosphorylation’ for C9ORF72‐ALS, ‘cytoplasmic translation’ for FUS‐ALS and ‘coding region instability determinant‐mediated mRNA stabilization’ for TDP‐43‐ALS mutation. Additionally, we identified universal proteomic signatures characteristic of ALS in general, including alterations in ‘ATP biosynthetic processes’ and ‘DNA replication.’ MSC‐EV treatment improved neurite outgrowth, with the observed proteomic signatures corresponding to recovery in both mutation‐specific and general ALS conditions. Summary/Conclusion : By generating patient‐specific motor neuronee cultures, we highlight the proteomic variances observed during the differentiation and between distinct iPSC lines. In addition, we found both mutation‐specific and common ALS‐related alterations, such as dysregulation of oxidative phosphorylation and mRNA stabilization. MSC‐EV treatment restored protein expression, offering hope for future ALS therapies.

Application

Presenter: Jing Zhang Huazhong University of Science and Technology, Wuhan, China Introduction : The challenges of traditional bone grafts and cell therapies in treating critical bone defects have led to increased interest in extracellular vesicles (EVs)‐based therapy. This thesis aims to systematically review the current progress in engineered EVs for bone defect regeneration, focusing on isolation methods, characterization techniques, therapeutic mechanisms, engineering strategies, and their applications in bone regeneration. Methods : A systematic review of published literature on engineered EVs in bone defect repair was conducted. The review comprehensively analyses various isolation and characterization methods of EVs, therapeutic mechanisms, engineering modification strategies, and their therapeutic applications in bone regeneration. Results : Current isolation methods mainly include ultracentrifugation, ultrafiltration, size exclusion chromatography, and immunoaffinity capture. Characterization techniques such as electron microscopy, nanoparticle tracking analysis, and dynamic light scattering have been developed to analyse EV properties. EVs exhibit therapeutic potential through immunomodulation, angiogenesis promotion, and enhancement of target cell functions. Surface modification strategies comprise genetic engineering, metabolic engineering, and membrane engineering for pre‐isolation approaches, while post‐isolation methods include membrane fusion, hydrophobic insertion, and chemical conjugation. Cargo loading encompasses endogenous methods through cell transfection and co‐culture, as well as exogenous approaches via electroporation, sonication, and surfactant‐assisted permeation. In applications, bone morphogenetic protein‐2 over‐expressing EVs enhanced osteogenic differentiation through signalling cascade microRNAs, showing superior bone regeneration when combined with various scaffolds. MicroRNA‐engineered EVs, especially those over‐expressing miR‐424, enhanced osteogenic differentiation by activating SMAD1/5/8 phosphorylation. Other bioactive factor‐modified EVs, such as mutant hypoxia‐inducible factor‐1α modified EVs, promoted osteogenesis and angiogenesis in steroid‐induced osteonecrosis, while vascular endothelial growth factor plasmid‐loaded EVs enhanced osteoinduction and vascular remodelling. Summary/Conclusion : Engineered EVs represent a promising cell‐free therapeutic approach for bone defect regeneration, offering advantages in biocompatibility, tissue targeting, and therapeutic efficacy. Future research should focus on standardizing isolation techniques and elucidating therapeutic mechanisms to facilitate clinical translation. The integration of various engineering strategies with scaffold materials shows particular promise for treating critical‐sized bone defects.

Arabidopsis

Presenter: Benjamin Koch Indiana University, USA Introduction : Extracellular vesicles (EVs) secreted by mammalian cells are heterogenous in content, biogenesis, and function. Whether this is also true for EVs secreted by plant cells is not yet known. Though plant EVs have been observed and associated with immune response for 50 years, the precise role of plant EVs in immunity is unknown. Methods : We used high‐resolution density gradient ultracentrifugation, protection assays, and total internal fluorescence microscopy (TIRF‐M) to separate, purify, and distinguish distinct subpopulations of Arabidopsis EVs. We characterized the response of plant EV subpopulations to biotic and abiotic stresses, including fungal infection, phytohormone treatment, and changes in temperature. We also analysed EV subpopulation‐specific defects of Arabidopsis mutants, including ESCRT, RAB, EXOCYST, and VAMP‐ASSOCIATED PROTEIN (VAP) family proteins. We studied the effect of plant EVs on the growth, development, and virulence of the fungal pathogen Colletotrichum higginsianum . Results : The EV marker protein TETRASPANIN 8 (TET8) was detected specifically in medium‐density EVs and was uniquely not associated with cell wall polysaccharide nanofilaments that co‐pelleted with high‐density EVs. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted in separate EV populations using TIRF‐M, while PEN1 was co‐secreted with PENETRATION 3 (PEN3) much more often. RNA and ARGONAUTE proteins were not associated with EVs of healthy plants. Mutant analysis indicated a general role in EV secretion for EXO70 proteins, as well as a specific role of VAPs and RabA2a in the secretion of TET8+ and PEN1+ EV‐subpopulations respectively. In addition, exo70 family mutants were more susceptible to Colletotrichum infection. We found that EV subpopulations marked by TET8, PEN1, and RPM1‐INTERACTING PROTEIN 4 (RIN4) are secreted to the leaf extracellular space and the leaf surface in response to biotic and abiotic stress, while other EV subpopulations were less responsive. Treatment of Arabidopsis seedlings with plant EVs delayed the progression of Colletotrichum infection by altering fungal germ tube development and morphology. Summary/Conclusion : Our data indicate that Arabidopsis EVs are highly heterogenous, that EV subpopulations have differing mechanisms of biogenesis, and that specific EV subpopulations contribute to plant immunity by interacting with fungi during early stages of infection on the leaf surface.

Association

Presenter: Anca D. Dobrian, Eastern Virginia Medical School, USA Introduction : African American (AA) women have disproportionate cardiovascular disease (CVD) risk due to increased exposure to adverse social determinants of health (SDoH), including lower individual‐ and neighborhood‐level socioeconomic status (SES). Physical activity (PA) and plasma extracellular vesicles (EVs) are known to modulate CVD risk, but their relationship with SDoH remains unclear. This study investigates associations between SDoH, PA, and plasma EVs in a pilot cohort of at‐risk AA women. Methods : Participants joined the Step It Up pilot study, utilizing a place‐tailored digital app to increase PA. Objective PA was assessed through daily step counts via Fitbit. Neighborhood socioeconomic deprivation (NSD) was determined from U.S. Census data. Individual household SES was self‐reported. EVs were isolated using size‐exclusion chromatography from fasting EDTA plasma samples and characterized using nanoparticle‐tracking analysis. EV miRNA cargo was determined using the NanoString platform. Functional effects of EVs on endothelial barrier function and cell migration were measured using electric cell‐substrate impedance sensing methodology. Multivariable regression analysis was conducted, adjusted for BMI and ASCVD 10‐year risk score. Results : In AA women ( N = 24, age: 57 ± 12, BMI: 35 ± 6, ASCVD: 9 ± 5), NSD associated with decreased EV size ( β = −0.49, p = 0.01). Higher daily step counts associated with increasing EV size ( β = 0.48, p = 0.02). EV miR‐1246, miR‐28‐5p, and miR‐765 exhibited distinct expression patterns associated with EV size, NSD, or daily step count. In vitro, endothelial cell (EC) barrier integrity associated with EV miR‐28‐5‐p ( r = 0.73, p = 0.007) and miR‐765 contents ( r = −0.58, p = 0.049) while EC migration potential after wounding were associated with miR‐28‐5‐p content ( r = −0.67, p = 0.02). Summary/Conclusion : Our findings show that smaller EV size associates with high NSD and low PA. Increased miR‐28‐5p cargo in EVs may support EC function, influencing CVD pathogenesis. By examining the relationships between NSD, PA, and EV characteristics in this high‐risk population, we aimed to uncover novel biological mechanisms underlying health disparities. Our comprehensive approach, combining community‐based sampling with advanced EV and miRNA profiling, provides unique insights into how SDoH may influence CV risk at the molecular level. Furthermore, through in vitro experiments, we determined the impact of the isolated EVs and their cargo on endothelial function, a hallmark of CVD development and progression.

Biophysical

Presenter: Rui Hao Shenzhen Institute of Advanced Technology, China (People's Republic) Introduction : Small extracellular vesicles (sEVs) are cell‐derived lipid‐bound nanoparticles typically smaller than 200 nm, serving as vital intercellular mediators in various (patho)physiological processes. Their potential in drug delivery is immense, yet efficient cargo loading remains a significant challenge. Conventional engineering loading methods, such as cuvette electroporation, suffer from limitations including mismatched feature dimensions, inconsistent stimulation, and sEV damage. Nanofluidic mechanoporation, which involves generation of transient nanopores in sEV membranes to facilitate exogenous substance uptake while minimizing disruption, offers a promising alternative. However, the underlying mechanisms governing nanopore dynamics during this process remain inadequately understood. Methods : This study proposes a nanofluidic device tailored for the biophysical study of sEV mechanoporation via hydrodynamic stretching. Initially, a computational fluid dynamics model was developed to simulate sEV deformation and force distribution as they navigate through a contraction‐expansion nanochannel, deepening our understanding of fluid‐sEV interactions. Delving into the biophysical properties of sEVs during this process lays a crucial theoretical foundation for optimizing nanofluidic designs to provide new engineering methods. Subsequently, the simulation model was experimentally validated by observing membrane mechanoporation and quantifying cargo loading efficiency. Results : Cryogenic transmission electron microscopy confirmed that fluid‐dynamic stresses within the nanochannels induce sEV deformation, leading to membrane permeabilization and the generation of transient nanopores. Additionally, nano‐flow cytometry analysis, using Alexa Fluor 488‐labelled dextran (10 kDa, ∼5 nm) as a model cargo, revealed that the proportion of dextran‐loaded sEVs increased with higher sample flow rates. This finding aligns with theoretical hydrodynamic studies, suggesting that elevated flow rates enhance fluid‐induced stresses, thereby promoting membrane deformation and permeability. Detailed experimental data and mechanistic insights will be elabourated upon at the conference. Summary/Conclusion : In summary, the nanofluidic mechanoporation platform effectively permeabilizes sEV membranes by precisely modulating membrane deformation and transient nanopore formation through hydrodynamics, facilitating controlled cargo loading. This technology not only serves as a robust tool for EV engineering but also holds promise as a versatile platform for the exploring biophysical properties of EVs. By integrating theoretical modelling with experimental validation using the nanofluidic platform, we could pave the way for advancing the understanding and application of sEVs in nanomedicine and beyond.

Ceftriaxone

Kulzhan Berikkhanova, Alexandr Gulyayev, Ernur Zakirov, Shynggys Sergazy, Zarina Shulgau Nazarbayev University, Kazakhstan Introduction : Encapsulating therapeutic agents within erythrocyte‐derived pharmacocytes provides an innovative approach to achieve sustained drug release while potentially minimizing systemic toxicity. This study explores the encapsulation and controlled release profile of ceftriaxone loaded into erythrocyte‐derived pharmacocytes through a hypoosmotic hemolysis technique, offering a new avenue for antibiotic delivery. Methods : Blood samples were collected and erythrocytes isolated under sterile conditions. Using a hypoosmotic solution, erythrocytes underwent hemolysis, allowing for ceftriaxone loading (1.0 g per 7.8 mL pharmacocyte suspension). Post‐loading, the pharmacocytes were resealed and subjected to a temperature‐stabilized environment. Controlled release of ceftriaxone from pharmacocytes was assessed over 24 h using equilibrium dialysis in NaCl (0.9%) solution at intervals (15 min to 24 h). Spectrophotometric analysis determined ceftriaxone concentration at each interval. Results : The pharmacocytes demonstrated consistent ceftriaxone release over the 24‐h period, with concentrations ranging from 1.8534 ± 0.033 mg/mL at 15 min to 0.0130 ± 0.007 mg/mL at 24 h. Peak release was observed within the first 3 h, with a progressive decline, indicating a stable release profile. Average ceftriaxone release across five pharmacocyte samples totaled 5.75–6.41 mg/mL over 24 h, confirming efficient loading and sustained release potential. Summary/Conclusion : This study establishes erythrocyte‐derived pharmacocytes as effective carriers for ceftriaxone, providing a controlled release profile that could enhance therapeutic outcomes for antibiotic therapy. Future investigations will focus on pharmacokinetic modelling and in vivo evaluation of ceftriaxone pharmacocyte efficacy and biocompatibility. Funding : This research is funded by Nazarbayev University under Collabourative Research Program Grant № 211123CRP1614, A.G.

Cell‐Free

Christian Grätz 1 , Prashant Changoer 2 , Dapi Menglin Chiang 1 , Johannes Kersting 1 , Martin Jaeger 2 , Romana Netea‐Maier 2 , Markus List 1 , Benedikt Kirchner 1,3 , Marlene Reithmair 4 , Michael W. Pfaffl 1 1 Technical University of Munich, Munich, Germany; 2 Radboud University Medical Center, Nijmegen, The Netherlands; 3 German Cancer Research Center, Heidelberg, Germany; 4 Ludwig‐Maximilians‐University Munich, Munich, Germany Introduction : Anaplastic thyroid cancer (ATC) is a rare but very aggressive and undifferentiated form of thyroid cancer with a high metastatic tendency and poor prognosis. It lacks targeted therapy options, but studies have shown therapeutic potential in off‐label use of drugs like vandetanib. Since off‐label cancer therapy monitoring is not yet established in clinical practice, we assessed whether EVs are suitable sources of protein and transcriptional biomarkers for this purpose in an in vitro approach, evaluating vandetanib treatment response of the ATC cell line Cal62. Methods : Cal62 cells were grown in EV‐depleted medium and treated with three doses of vandetanib (EC10, EC50 and EC80, as previously determined) besides the DMSO control. EVs were enriched from the supernatant using PEG precipitation to include as much cell‐free transcriptional and translational information as possible. The EV preparation was characterized by nanoparticle tracking analysis (NTA) for size and particle concentration and flow cytometry for EV marker expression. RNA and proteins from cells and the EV preparation were isolated and analysed by total RNA sequencing and LC‐MS/MS proteomics to identify biomarker signature candidates. Results : Treatment with the EC80 dose of vandetanib led to significantly increased particle concentrations in the culture supernatant, while the treatment did not influence the particle size. The percentages of membranous EV‐marker positive particles (CFSE+, CD9/CD63/CD81+) were significantly increased through vandetanib treatment (EC50 and EC80 doses). Through differential gene expression analysis, dimensional reduction (sPLS‐DA) and pathway analysis, we identified sets of 4–8 protein and RNA biomarker candidates each from the proteomics and RNA‐Seq datasets that could be detected both intracellular and in the cell culture supernatant. The transcriptional biomarker signature was further validated by RT‐qPCR. Summary/Conclusion : The identified cell‐free biomarker signatures enable the assessment of Cal62 cell response to vandetanib treatment in vitro. They may serve as a basis for follow‐up clinical studies with blood samples from ATC patients receiving vandetanib therapy. Given the limited number of such patients, our pre‐clinical findings address a significant knowledge gap in the use of vandetanib for ATC treatment. Further, these results underscore the potential of cell‐free EV‐associated RNA and proteins as promising targets for non‐invasive monitoring of cancer therapy.

Circulating

Presenter: Nagesha Guthalu Kondegowda City of Hope, Duarte, California, USA Introduction : Type 1 diabetes (T1D) results from diminished functional β‐cell mass due to autoimmunity. Plasma‐derived circulating extracellular vesicles (cEVs) contribute to multiple diseases. However, their impact on T1D remains unknown. Our finding that serum from T1D subjects is cytotoxic to human β‐cells led to our hypothesis that cEVs mediate β‐cell cytotoxicity through their distinct molecular cargo and thus contribute to T1D pathogenesis. Methods : cEVs isolated from T1D (< 5 years since diagnosis), multiple autoantibody‐positive (Aab+), age, sex, and ethnicity‐matched non‐diabetic healthy donor (HD), and T1D subjects pre‐ and post‐islet transplantation, were characterized physically, and protein cargo identified. Human islet cells cultured with T1D or HD plasma (10% vol/vol) or cEVs (≥ 50 µg/mL) were examined for cell death by TUNEL staining. Glucose and β‐cell homeostasis was assessed in NOD/ShiLtJj female mice, an autoimmune T1D mouse model, treated with GW4869, an EV secretion inhibitor. PBMC‐derived EVs from T1D and HD subjects were evaluated for β‐cell cytotoxicity. Results : Plasma and cEVs from T1D but not HD subjects ( n = 10/group) significantly increased human β‐cell but not α‐cell death, mimicking disease pathology. Proteomic analysis ( n = 5/group) identified differential cargo in T1D vs HD cEVs. Proinflammatory cytokine, IFN‐gamma, was increased in T1D cEVs, mediating its β‐cell cytotoxic effect ( n = 5/group). GW4869‐treatment in NOD mice ( n = 8‐9/group) reduced cEVs, improved glucose and β‐cell homeostasis in vivo, and diminished β‐cell cytotoxicity of serum ex vivo, supporting cEV contribution to T1D pathology. Of clinical relevance, serum and cEVs ( n = 7–8/group) from Aab+ subjects also induced human β‐cell cytotoxicity, implying that humoral β‐cell cytotoxicity precedes clinical disease‐onset. cEVs from post‐islet transplant T1D patients under immunosuppressive‐regimen showed reduced β‐cell cytotoxicity, suggesting that immune cells may be one source of cytotoxic cEVs in T1D. Supporting this, PBMC‐derived EVs from T1D but not HD subjects ( n = 4/group), like cEVs, induced human β‐cell cytotoxicity, which was reduced with in vitro immunosuppression. Summary/Conclusion : Our findings demonstrate a critical role of T1D‐cEVs in disease pathology through their, selective cytotoxicity to human β‐cells; distinctive proinflammatory protein cargo mediating the cytotoxicity; in vivo role in T1D pathogenesis; cytotoxic phenotype acquired prior to disease‐onset; cytotoxicity modulated by immunosuppression; and potential source being PBMC. Funding : This study was supported by NIH/NIDDK, Grant Number: R01DK125856.

Comparative

Hannah Aris 1 , Pamina Contreras Kallens 1 , Yeyu Shen 1 , Andrew Daly 1 , Xandra O. Breakefield 2 , Garry D. Duffy 1 , Meadhbh Á. Brennan 1 1 University of Galway, Galway, Ireland; 2 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) offer tremendous potential in tissue repair as acellular alternatives to MSC therapies due to their regenerative properties. Numerous studies show EVs administered to injury sites via biomaterials are retained longer than those delivered via saline solution, facilitating greater therapeutic impact. As such, researchers are implementing intrinsic and modified hydrogel properties to generate scaffolds with sustained EV release. Various methods are employed during hydrogel development to quantify EVs as they are released from scaffolds to understand the EV‐retention capacity of the gels. However, no work has been conducted to assess if hydrogels interfere with EV quantification, potentially limiting the accuracy of hydrogel characterization. Here, we have assessed the interference of three hydrogels commonly used for EV delivery on multiple EV quantification techniques. Methods : Hydrogels were cross‐linked to the bottom of well‐plates and bathed in double‐filtered PBS. At various time points, the hydrogel‐conditioned PBS (HG‐CPBS) was collected. Naïve, fluorescent, or bioluminescent/fluorescent EVs were obtained from murine bone marrow‐derived MSCs, the latter two EV types from cells transduced with genes encoding for palmitoylated tandem dimer Tomato (PalmtdTomato) or palmitoylated GFP‐nanoluciferase bioluminescence resonance energy transfer reporter (PalmGRET) for EV membrane labelling. EVs were isolated via differential centrifugation followed by size exclusion chromatography. EVs were serially diluted in HG‐CPBS and PBS for quantitative assessment by Nanoparticle Tracking Analysis (NTA), Bicinchoninic Acid (protein) assay (BCA), phospholipid assay, RNA quantification, CD9 ExoELISA, and fluorescence/bioluminescence measurements of PalmtdTomato or PalmGRET EVs. Results : Results suggest hydrogels interfere considerably with NTA, BCA, ExoELISA, and RNA measurements, and to a lesser but noteworthy extent with phospholipid quantification, making these methods unreliable in this context. Fluorescence measurements of PalmtdTomato EVs offer accurate quantification. Bioluminescent and fluorescent measurements of PalmGRET EVs are being investigated. Summary/Conclusion : Based on these results, it is recommended that fluorescent EVs are used for accurate EV quantification in the presence of hydrogels. It is essential to develop an accurate depiction of EV release in the development of EV carriers with optimized release profiles for tissue regeneration, and precise EV quantification is central to achieving this goal. Funding : This study was supported by the Science Foundation Ireland Starting Investigator Research Grant.

Correlation

Giuseppe Cammarata 1 , Giulia di Fazio 2 , Alessia Lo Curto 1 , Giuseppina Poppa 2 , Sandra D'Ascenzo 2 , Francesca Megiorni 3 , Simona Ceccarelli 3 , Vincenza Dolo 2 , Ilaria Giusti 2 , Simona Taverna 1 1 National Research Council, Rome, Italy; 2 University of L'Aquila, L'Aquila, Italy; 3 Sapienza University, Rome, Italy Ilaria Giusti and Simona Taverna are co‐senior authors . Introduction : Ovarian cancer (OC) is a common malignancy with high mortality. Highlighting the mechanisms of OC progression and discovery of new biomarkers may improve OC diagnosis and therapy. OC is often caused by germLine mutations in BRCA1 genes. Circular RNAs (circRNAs) are a class of novel non‐coding RNAs that undergoe back‐splicing and form a covalently closed loop. circRNAs have regulative roles in cancer. Extracellular vesicles (EVs) are natural intercellular shuttles for circRNAs. Our study aims to correlate circRNAs shuttled by OC‐EVs with BRCA mutational status. Methods : Human OC cell lines UWB1.289 (BRCA1‐null), UWB1.289 + BRCA1 (BRCA1‐restored) were cultured in RPMI/HUMEC medium (3% FBS); ES‐2 (BRCA wild type) was cultured in McCoy's 5A medium (10% FBS). EVs were isolated by ultracentrifugation from both UWB cell lines and characterized according to MISEV 2023. To analyse circRNAs in UWB cells and EVs, RNA was extracted with a commercial kit, and complementary DNA was synthesized by reverse transcription. circRNA was evaluated using divergent primers by RT‐qPCR with SYBR Green. GAPDH was used as a reference. Relative changes in circRNA expression were determined by the ΔΔCt method. The effects of UWB‐EVs on ES‐2 cell proliferation and migration were evaluated by MTS/CCK8 and wound healing assay, respectively. Results : EVs were isolated from both UWB cell lines and characterized: they appear intact and rounded (TEM), with a mean size ranging around 160–190 nm (NTA), and are positive for CD63, CD9, TSG‐101 and negative for CNX (WB). A selection of circRNAs (CDR1AS, HIPK2, HIPK3, FAM120a, ITCH, AFF1, MUC16) was tested on cells and EVs in both UWB cell lines. Among these circRNAs, MUC16 and FAM120a were represented differently in UWB cells and EVs. Notably, UWB‐EVs were able to modulate ES‐2 proliferation and migration. Summary/Conclusion : Multiple genes with germLine mutations have been implicated in hereditary OC. Our results indicate that, in OC cell lines, circRNAs contained in EVs could be linked to BRCA mutational status and OC progression. This study confirms the potential of circRNAs shuttled by EVs as biomarkers for OC diagnosis and therapeutic targets. Funding : Project funded from Program PRIN 2022 under grant agreement N°20224XB79P by the European Union, Next‐generation EU, PNRR, M.4‐C2‐1.1.

Deciphering

Monica De Palma 1 , Michele Bifolco 2 , Olga Cannavacciuolo 1 , Emanuele Rosa 2 , Marisa Conte 2 , Elisa Cappetta 2 , Francesca Mensitieri 3 , Carmine Del Regno 2 , Adriana Sacco 4 , Liberata Gualtieri 4 , Maria Moros 5 , Nunziatina De Tommasi 2 , Fabrizio Dal Piaz 3 , Alfredo Ambrosone 2 1 National Research Council (CNR), Institute of Biosciences and BioResources (IBBR), Italy, 2 Department of Pharmacy, University of Salerno, Italy, 3 Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Italy, 4 National Research Council (CNR), Institute for Sustainable Plant Protection (IPSP), Italy, 5  Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC‐Universidad de Zaragoza, Spain Introduction : Plant extracellular vesicles (EVs) are key components in plant‐microbe interactions, facilitating complex intercellular communication and defense mechanisms. However, the molecular composition and functional roles of EVs in these processes remain largely unexplored. This study explores the plant “secrEVome”, the array of molecules carried by EVs that transmit molecular messages to both beneficial and pathogenic fungi. Methods : We investigated a model of plant‐fungi interactions, employing tomato (Solanum lycopersicum) as a host system to elucidate the role of EVs in the beneficial associations with Trichoderma spp and in the interaction with the phytopathogen Botrytis cinerea. More in detail, Trichoderma spores were inoculated at the roots, while B. cinerea was applied to the leaves. EVs were collected from different sources: leaf apoplast EVs (apoEVs) were purified using vacuum infiltration, and root exudate EVs (reEVs) were obtained from a split‐root hydroponic floating system designed to prevent co‐purification of fungal EVs. Additionally, hairy roots (HRs) were utilized as a biotechnological platform to produce EVs (hrEVs) under control and elicited conditions. All EV types were isolated using differential ultracentrifugation. EV size and concentration were assessed by nanoparticle tracking analysis. Ultrastructural imaging was conducted using a transmission electron microscope (TEM). Metabolomic profiling was performed by mass spectrometry (MS), while proteomic and miRNome analyses are ongoing by MS and next‐generation sequencing, respectively. Results : apoEVs, reEVs and hrEVs exhibit similar size (150‐200 nm, mean). HR cultures yielded the highest of EVs, likely due to their high metabolic activity, while apoEVs showed the lowest concentration, limited by apoplastic space. TEM confirmed the presence of intact round‐shaped EVs, indicative of consistent isolation quality. Metabolomic analysis identified tomatine and tomatidineol as primary bioactive compounds, suggesting EVs' potential in plant defense, especially against fungal pathogens, by enabling targeted distribution of antimicrobial molecules. Summary/Conclusion : By characterizing the molecular composition of plant EVs and integrating multi‐omics data, this study aims to elucidate how EVs regulate defense responses, modulate host‐pathogen interactions, and impact the surrounding microbiome, unveiling fascinating evolutionary dynamics. These findings pave the way for novel agronomic strategies that exploit EV‐mediated pathways to enhance plant resilience and cope with phytopathogenic challenges. Funding : SecrEVome project (Grant number: 202224M943)—European Union‐ NextGeneration EU within the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.1 ‐ National Research Programme Fund (NRP) and Research Projects of National Relevant Interest (PRIN), CUP: D53D23011500006, B53D23017030006.

Development

Yung‐Hsin Cheng, Hao‐Zhong Lu, Tzu‐Hsiang Chen, Anh Nhat Thuy Ta National Taiwan University of Science and Technology, Taipei, Taiwan Introduction : In chronic wounds, excessive levels of inflammatory cytokines and reactive oxygen species affect the wound healing process. Recent studies showed the positive therapeutic effects of using extracellular vesicles (EVs) in the wound healing. Loading EVs into carriers can improve the bioavailability and prolong the half‐life, maintaining the effective therapeutic concentration at the target site. Mesenchymal stromal cells derived from umbilical cord Wharton's jelly (WJ‐MSCs) are easily accessible, with no invasive isolation procedures or ethical concerns. In this study, the conductive tetra‐aniline (TA)‐grafted chitosan (TAC) hydrogels containing EVs were developed and utilized as a wound dressing. The conductive TAC hydrogels may promote the efficiency of electrotherapy and sustain the release of EVs to accelerate wound healing. Methods : In the study, EVs were derived from WJ‐MSCs (WJ‐MSC‐EVs) and isolated by size exclusion chromatography column. The isolated EVs were characterized with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot. Conductive TAC hydrogels were evaluated by 1H‐nuclear magnetic resonance spectroscopy (NMR), Fourier‐transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), conductivity, swelling and in vitro biocompatibility test. The possible therapeutic effects of the optimized TAC hydrogels containing WJ‐MSC‐EVs in L929 fibroblasts under H2O2‐induced oxidative stress were investigated. Results : The isolated WJ‐MSC‐EVs, with the mean diameter ∼131 nm, exhibited bilayer membrane structure and were positively expressed for CD81 and Flotillin‐1. The characteristic peaks of synthesized materials were confirmed by 1H‐NMR and FTIR. The TAC hydrogels showed interconnected porous structure, allowing the encapsulated EVs to diffuse freely through the hydrogels matrix. The conductivity of developed hydrogels was increased with the rising TA content. The swelling capability of TAC hydrogels containing 15% and 20% of TA was dramatically reduced, which may affect the diffusion of EVs. The results of cell viability showed no cytotoxicity of TAC hydrogels containing 5% and 10% of TA. In the oxidative damage model of fibroblasts, WJ‐MSC‐EVs‐loaded TAC hydrogels were able to rescue the damaged cells. Summary/Conclusion : The results suggested that WJ‐MSC‐EVs‐loaded TAC hydrogels with conductivity and sustain release properties may have potential applications in the future for treatment of chronic wounds. Funding : This study was supported by the National Science and Technology Council (NSTC 113‐2221‐E‐011‐024), Taiwan.

Differences

Monika Sypecka 1,2 , Sylwia Katarzyna Król 3 , Anna Sarnowska 1 1 Translational Platform for Regenerative Medicine, Mossakowski Medical Research Institute, PAS, Poland. 2 Doctoral School of Translational Medicine, Centre of Postgraduate Medical Education, Poland. 3 Department of Neurooncology, Mossakowski Medical Research Institute, PAS, Poland. Introduction : Mesenchymal stem/stromal cells derived from Wharton's jelly of human umbilical cord (WJ‐MSCs) exhibit unique immunomodulative, regenerative and neuroprotective properties and are therefore considered as a potential tool in the treatment of various disorders. Their therapeutic potential mostly relies on their secretome—WJ‐MSCs secrete different factors that are either secreted into the environment as a soluble secretome or are encapsulated within extracellular vesicles (EVs). As the ability to secrete immunomodulative and neuroprotective factors usually varies significantly between donors, we hypothesize that this correlates with different numbers of EVs released by these cells. To confirm this thesis, we therefore performed a comparative study of the ability to secrete EVs by WJ‐MSCs obtained from different donors. Methods : Umbilical cords were obtained from full‐term deliveries [according to the Ethics Committee of Warsaw Medical University guideline (KB/213/2016) and with mothers’ written consents] and MSCs were then mechanically isolated from Wharton's jelly. The cells were then cultured until 3rd passage in standard culture medium (DMEM + 5% human platelet lysate + 1% antibiotic‐antimycotic) in 5% O2, 5% CO2, 37⁰C. Next, the standard medium was removed, WJ‐MSCs were rinsed with double‐filtered PBS (0,1 µm) and a medium without platelet lysate was added for 24 h. After 24 h, cell culture medium containing EVs secreted by WJ‐MSCs was collected and EVs were isolated by ultracentrifugation (300 × g for 10 min, 4⁰C; 2000 × g for 30 min, 4⁰C; 100,000 × g for 90 min, 4⁰C). The isolated EVs were suspended in double‐filtered PBS and NTA analysis was performed. Results : The analysis revealed that WJ‐MSCs obtained from different donors secrete significantly different numbers of EVs. Summary/Conclusion : As significant differences in the numbers of EVs released by WJ‐MSCs obtained from different patients have been discovered, it may potentially have an impact on the efficiency of cellular‐free therapies dedicated for individual patients—in the future it may become a potential drawback considering personalized cellular‐free therapies. Funding : This research was funded by National Science Centre grant number 2022/47/O/NZ3/01739.

Dual‐Mode

Presenter: Seonghyun Kim SHIFTBIO Inc., Seoul, Republic of Korea Introduction : Acute liver failure (ALF) is a critical inflammatory condition characterized by rapid hepatocyte death, impaired liver regeneration, and high mortality rates. This study introduces a novel dual‐mode action therapeutic approach using extracellular vesicles expressing Signal Regulatory Protein Alpha (SIRP‐EVs) derived from genetically engineered mesenchymal stem cells (MSCs). These SIRP‐EVs are designed to concurrently resolve necroptosis and promote liver regeneration. Methods : We evaluated CD47 expression across diverse ALF models using histological analysis, western blot, confocal microscopy, and spatial transcriptomics. EVs were harvested through a 3D bioreactor‐based process and subsequently purified. These purified EVs underwent established characterization assays. After a single systemic injection, CD47‐dependent accumulation of SIRP‐EVs in liver tissues was evaluated using the in vivo imaging system. The therapeutic efficacy of SIRP‐EVs was determined by measuring liver enzyme levels, examining histological changes, tracking survival rates, assessing principal component analysis of RNA sequencing data, and analysing inflammatory cytokine profiles. Results : Our studies identified CD47 and SIRPα as promising therapeutic targets for ALF. We developed a scalable 3D bioreactor process for producing high‐purity SIRP‐EVs with significant yields of SIRP‐EVs, while preserving MSC properties. SIRP‐EVs effectively eliminate necroptotic hepatocytes by targeting CD47 and simultaneously enhance the regenerative capabilities of myeloid cells through delivery of MSC‐derived cargo. Comprehensive in vivo studies demonstrate that SIRP‐EVs exert significant therapeutic effects in ALF. Summary/Conclusion : These findings highlight SIRP‐EVs' potential as a dual‐mode action therapeutic for ALF, offering prospects for application in other inflammatory diseases. Moreover, these results pave the way for advancing EV‐based therapies toward clinical implementation. Funding : This research was supported by SHIFTBIO INC. and funded by Grant Numbers: 23C0111L1, H123C0883, HI23C154100, and S3252275.

Elucidating

Eylem Baysal 1 , Niyaz Al‐Sharabi 1 , Neha Rana 1 , Daniela E. Costea 1,2 , Meadhbh Brennan 3 and Salwa Suliman 1 1 University of Bergen, Norway. 2 Haukeland University Hospital, Norway. 3 University of Galway, Ireland. Introduction : Regulatory T cells (Tregs), a specialized subset of CD4+ T cells, play a crucial role in immune regulation and have also been implicated in promoting bone repair. Emerging evidence highlights the immunomodulatory potential of extracellular vesicles derived from Tregs (Treg‐EVs), however, their precise mechanism of impact on osteogenesis remains understudied. Therefore, our study aimed to assess the role of Treg‐EVs and the phosphatidylinositol 3‑kinase (PI3K)/AKT, a crucial pathway in osteogenesis, in modulating the osteogenic potential of bone marrow‐derived mesenchymal stromal/stem cells (BMSCs). Methods : Tregs were isolated from the peripheral blood of healthy donors ( n = 6) following informed consent. Tregs were expanded for 13 days, followed by 24 h of serum starvation. Thereafter, conditioned media were collected, pooled and concentrated. Treg‐EVs were isolated using size‐exclusion chromatography and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. In the presence or absence of PI3K/AKT pathway inhibitor, BMSCs were treated with Treg‐EVs (80 µg/mL), and morphology, metabolic activity, migration, and osteogenic potential of BMSCs were evaluated at mRNA and protein levels. Results : Treg‐EVs were shown to express CD25, CD73, HLA‐DR, CD63, and CD81 and uptake of Treg‐EV by BMSCs was confirmed by confocal imaging after 24 h. Treg‐EVs significantly enhanced BMSCs metabolic activity after 4 and 7 days, albeit insignificant when PI3K/AKT pathway was inhibited. Treg‐EVs reduced BMSCs migration after 48 and 72 h, also during PI3K/AKT pathway inhibition. During osteogenic differentiation, PI3K/AKT pathway was highly activated in BMSCs from 4 days when treated with Treg‐EVs compared to BMSCs not treated with Treg‐EVs. Moreover, Treg‐EVs significantly enhanced alkaline phosphatase enzyme activity and osteogenic markers at mRNA and protein level in BMSCs after 7 days with and without PI3K/AKT pathway inhibition. Mineralization after 10 days was enhanced, even with pathway inhibition. Summary/Conclusion : Treg‐EVs promote BMSCs viability and osteogenic differentiation while reducing migration. This positive impact of Treg‐EVs on osteogenesis was sustained despite PI3K/AKT pathway inhibition, suggesting a potential PI3K/AKT independent pathway for enhancing osteogenesis. These findings provide insights into Treg‐EVs mechanisms for enhancing osteogenesis and highlight their therapeutic potential in bone regeneration. Funding : Funded by Trond Mohn Foundation (TMS2021STG03) and Norwegian Research Council (314473).

Endothelial

Kamil Wawrowicz 1,2 , Martyna Durak‐Kozica 1,2 , Marcin Marzejonc 3 , Maciej Trusiak 3 , Ewa Ł. Stępień 1,2 1 Faculty of Physics, Department of Medical Physics, M. Smoluchowski Institute of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland; 2 Center for Theranostics, Jagiellonian University, Kraków, Poland  3 Institute of Micromechanics and Photonics, Warsaw University of Technology, Warsaw, Poland Introduction : Produced by different types of cells, extracellular vesicles (EVs) have many important functions. The natural characteristics of these protein‐lipid structures make them extremely interesting potential drug delivery systems, combining precise interaction with the target cell and a carrier function for substances with therapeutic effects. Designing vesicle‐based carriers, however, involves addressing two major requirements: large‐scale production of EVs (1) and understanding the natural behaviour of these structures (2) under different clinical conditions. Current methods for producing EVs are well understood, but due to a number of limitations, they require further improvements. The most desirable direction for the development of these technologies should address more efficient production resulting in a highly concentrated product. Thus, the purpose of our study was to verify whether EVs produced by endothelial cells in a bioreactor exhibit biological activity and native internalization capacity in target cells. Methods : We implemented and evaluated the applicability of hollow fibre membranes (HFMs) bioreactors as a potentially beneficial approach for large‐scale EVs production. The obtained EVs were comprehensively characterized with transmission electron microscopy (TEM), spectral flow cytometry and tunable resistive pulse sensing. Subsequently, we examined the obtained small and large EVs uptake by target cells. For this purpose, state‐of‐the‐art label‐free imaging techniques were implemented in order to assess intercellular uptake (optical diffraction tomography) and morphological changes in cells after EVs internalization (Fourier ptychography microscopy). Results : Morphological and physiological evaluation of the resulting vesicles confirmed the expression of their characteristic proteins, such as CD9, CD63 or CD81, and typical structure. Using modern label‐free imaging techniques, an increased accumulation of small ( 200 nm) vesicles was observed in target cells. Summary/Conclusion : The results provide a promising introduction to further research into the development of methods for producing extracellular vesicles and their application as drug carriers. A new EV visualization technique based on optical diffraction tomography allowed for obtaining new types of microscopic images. Funding : This work was supported by the National Science Centre of Poland through grant no. NCN OPUS 2022/45/B/NZ7/01430 to E.Ł.S. and the Jagiellonian University via projects CRP/0641.221.2020, SciMat and qLife Priority Research Areas budget under the programme Excellence Initiative—Research University.

Engineering

Peivand Sadat Mousavi, Lucie Thomas, John C. Bell, Carolina S. Ilkow University of Ottawa, Ottawa Hospital Research Institute, Canada ABSTRACT UNAVAILABLE

Enhancement

Presenter: Chadaporn Attakitbancha Chulalongkorn University, Thailand Introduction : Mesenchymal stem cells (MSCs) are known for their paracrine effects, particularly in immunomodulation, where extracellular vesicles (EVs) play an important role in signal transmission across cells. Recent evidence suggests that the preconditioning of MSCs with various supplements can alter EV profiles, enhancing their functional activities in response to specific environments. In this study, MSCs were preconditioned with individual and combined inflammatory cytokines, including Interleukin‐1β (IL‐1β), Interleukin‐6 (IL‐6), Tumour necrosis factor‐alpha (TNF‐α), and Interferon‐gamma (IFN‐γ) to understand MSCs responses to inflammatory conditions from an EVs perspective. Methods : Human Umbilical cord‐derived MSCs (UC‐MSCs) were cultured in xeno‐free conditions until they reached 80% confluence. Then, they were preconditioned with specific inflammatory cytokines with specific concentrations individually and in combination. Following preconditioning, EVs were concentrated by centrifugal ultrafiltration and isolated using size‐exclusion chromatography. The total protein concentrations of EV samples were quantified using the micro‐BCA assay, and EV densities were determined by nanoparticle tracking analysis (NTA). The secretion profile of critical cytokines and growth factors was analysed via the human‐customized multiplex assay. In addition, anti‐inflammatory effects were assessed by measuring nitric oxide (NO) levels in IL‐1β induced‐fibroblasts treated with MSCs‐derived EVs. Results : MSCs preconditioned with various inflammatory cytokines demonstrated distinct protein concentrations, EV counts, secretion profiles, and functional activities. EVs from cytokine‐preconditioned MSCs exhibited higher anti‐inflammatory cytokine levels than the negative control. In cytokine‐primed conditions, EV‐treated fibroblasts showed significantly reduced nitric oxide (NO) levels, indicating enhanced anti‐inflammatory activity. Summary/Conclusion : Preconditioning MSCs with specific inflammatory cytokines is a promising strategy for generating EVs with tailored secretion and functional profiles. Funding : This research was supported by the National Research Council of Thailand (NRCT)

Evanalyzer2

Melanie Schürz 1,2 , Joachim Danmayr 1 , Maria Jaritsch 1 , Tanja Plank 1,2 , Patricia Hrasnova 1,3 , Vesna Stanoljovic 1,2 , Heloisa Melo‐Benirschke 1 , Cristian Matea Tudor 1 , Nicole Meisner‐Kober 1,2 1 Paris Lodron University of Salzburg, Austria; 2 Ludwig Boltzmann Institute for Nanovesicular Precision Medicine at the PLUS, Salzburg, Austria; 3 Paracelsus Medical University Salzburg, Austria Introduction : With the rapid growth of the EV field, rigorous characterization at the single vesicle level has become increasingly important to assure comparability and reliability of published data. To facilitate standardization, we recently developed ‘EVAnalyzer,’ an ImageJ plug‐in optimized for automated, quantitative image analysis from single vesicle imaging and cellular EV uptake data, which we published together with a robust protocol for routine EV applications. With over 3400 downloads, the program has become a tool of widespread use within the EV community. Methods : Given this evident need in the field, we further progressed EVAnalyzer into EVAnalyzer2, which additionally enables tracking of EVs in huge, complex in vitro and in vivo models. Usage of near infrared dyes together with flexible image processing pipelines and rigorous object quantification enables the detection of EV signals over diverse autofluorescent structures and thereby enables sensitive tissue and serum pharmacokinetics. In addition, advanced object recognition and segmentation are enabled by the support of AI models that are entirely based on open‐source technology and can be easily trained and used without any deep AI knowledge. Moving into in vivo high‐content vesicle imaging, the increase in image sizes and the inherent growth in data volume make automated image and fast data processing inevitable. As these requirements hit the limits of the Java‐based image analysis software ImageJ, we changed to the more efficient programming language C++. Results : New developments for EVAnalyzer2 will be presented together with application examples for quantitative serum and tissue pharmacokinetics. Additionally, inspired by the rigour and standardization work done by ISEV, especially in the field of flow cytometry, we also evaluate calibration beads in single vesicle imaging for the comparisons of EV numbers and brightnesses between different instruments and imaging platforms. Summary/Conclusion : EVAnalyzer2, an open‐source high‐throughput image processing tool with a comprehensible user interface, allows the creation of highly flexible pipelines for standard image processing and thresholding and the inclusion of AI‐driven object detection and automated data processing options. This makes the analysis of EVs in complex in vitro and in vivo images accessible to everyone. Funding : EV‐Quant (Salzburg, WISS2025, 20102‐F2100572‐FPR); EVTT (EU, EFRE/IWB 20102‐F1900731‐KZP); CONSONANT (Salzburg, WISS2025; F2200397‐KZP).

Fluorescent

Lara Pierantoni, Teresa Lage, Lorena Diéguez, Carlos Honrado, Sara Abalde‐Cela INL‐International Iberian Nanotechnology Laboratory, Braga, Portugal Introduction : Liquid biopsies are emerging as non‐invasive methods for novel biomarkers discovery in cancer research. Extracellular vesicles (EVs) have gained significant interest due to their capacity to carry valuable cargo derived from active tumours. This can provide critical information about tumour phenotype and metastasis initiation. For this reason, it is important to find reliable methods to label and image EVs, not only for tracking during isolation protocols, but also to better understand cellular communication processes. Typical EVs dyes, like PKH67, self‐form dye micelles, thus interfering with EVs staining and making it imperative to find alternative methods. Methods : Here we propose two different strategies for EVs fluorescent labelling. The first uses internalized cell‐permeant fluorescent dyes (e.g., calcein) added during cell culturing. The second uses cell transfection with plasmid DNA encoding a fluorescently tagged EV membrane marker (e.g., CD63). For both cases, cancer cell lines (Hec1A, SW480) are seeded, cultured with EVs‐depleted media for 72 h, and the conditioned media is collected. EVs are isolated by centrifugation (10 min 500 ×  g ; 10 min 2000 ×  g ; 20 min 10,000 ×  g at 4°C) and ultracentrifugation (90 min 100,000 ×  g at 4°C, wash with PBS, and repeat). The generated EVs are characterized by nanoparticle tracking analysis (NTA), confocal and total internal reflection fluorescence (TIRF) microscopy imaging. Results : Preliminary results showed the successful isolation by ultracentrifugation of EVs from both labelling strategies. Confocal and TIRF imaging confirmed that EVs maintained the acquired fluorescence. To assess the fluorescence efficiency, and whether the two methods influence EVs production, further analyses are being performed, namely checking size and yield by NTA, protein presence by BCA and Western blot, and DNA quantification and characterization by Qubit assays and BioAnalyzer DNA integrity assessment. Summary/Conclusion : With fluorescent EVs we will better visualize processes such as microfluidic‐based isolation, permitting immediate assessment of capture efficiency; and intercellular communication within complex organ‐on‐chip models, such as the interplay between cancer and stroma cells (e.g., cancer‐associated fibroblasts). Funding : This study was supported by the Health from Portugal (C630926586‐00465198), through the NextGenerationEU Fund.

Genetically

Colin Esmonde 1 , Li Sun 1,2 , Yun Chang 3 , Changchun Zeng 4 , Alfredo Quiñones‐Hinojosa 5 , Xiaoping Bao 3 , Yan Li 1 1 Department of Chemical and Biomedical Engineering, FAMU‐FSU College of Engineering, Tallahassee, Florida, USA; 2 Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA; 3 Purdue University, West Lafayette, Indiana; 4 Department of Chemical and Biomedical Engineering, FAMU‐FSU College of Engineering, Tallahassee, Florida, USA; 5 Mayo Clinic, Jacksonville, Florida, USA Introduction : Glioblastoma multiforme (GBM) is the most commonly occurring tumour in the central nervous system, accounting for 80% of brain tumours. Work in gene editing and immunology has led to the development of chimeric antigen receptors (CARs). CARs employing a chlorotoxin receptor domain have recently been incorporated into neutrophils for GBM treatment. However, such cells are liable for inducing body wide off‐ target effects that can be deadly. Extracellular vesicles (EVs) secreted by the cells have been shown to share properties with their parent cells including the expression of CARs and tumour‐lytic effects. However, this has never been investigated in CAR neutrophils. Methods : The DNA for the CAR‐CLTX was inserted into the AAV1 safe locus of induced pluripotent stem cells, which were differentiated into neutrophils. Conditioned media were collected along the differentiation and went through the extraPEG EV isolation. EVs were quantified by nanoparticle tracking analysis, electron microscopy, and western blot. Proteomics analysis of protein cargo and microRNA (miRNA) sequencing of the CAR EVs was performed. The cytotoxicity of the EVs was performed with U87MG and LN229 glioblastoma cells in 2D culture and 3D organoids, as well as in vivo. Results : EVs are secreted in high quantities in the size range of 100–200 nm. The EVs expressed exosomal markers and displayed exosomal morphology. Proteomics analysis indicate that neutrophil markers and proteins related to regulation of neutrophil migration, chemotaxis, and degranulation were identified in EVs. miRNA profiling reveals high levels of miRNAs (e.g., miR‐182) active in apoptotic and tumour suppressive pathways, including T cell receptor signalling pathway, cytokine‐cytokine receptor interactions, Jak‐STAT, Hedgehog signalling pathways, and natural killer cell‐mediated cytotoxicity. EV uptake in an in vitro glioblastoma model was demonstrated. CAR Neutrophil EVs are cytotoxic to multiple glioblastoma cell lines and organoids, and in vivo. Summary/Conclusion : EVs of CAR Neutrophils contain anti‐tumour cargo. The CAR EVs have shown uptake in glioma cells and induced cytotoxicity of the glioblastoma cells. These results demonstrate the feasibility to establish correlation between protein and miRNA cargo of CAR Neutrophil EVs and the function of EVs in GBM treatment. Funding : This project is supported by NSF 1917618 and NIH of USA.

Gmp‐Grade

Alice Zaramella 1,2 , Paola Bisaccia 1,2 , Agner Henrique Dorigo Hochuli 1,2 , Raquel Moll‐Diaz 1,2 ,Miriam Duci 1,2 , Rudra Kashyap 3 , Marcin Jurga 3 , Maurizio Muraca 2 , Andrea Porzionato 4 , Michela Pozzobon 1,2 1 Department of Women's and Children's Health, University of Padova, Italy; 2 Institute of Pediatric Research Città della Speranza, Padova; 3 EXO Biologics, Liège, Belgium; 4 Department of Neurosciences, University of Padova, Padua, Italy Introduction : Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that has a multifactorial nature, including the environment, microbiome, genetics and immune suppression/stimulation balance. The inflammation of colon tissue resulting from impaired immune system regulation affects the integrity of the intestinal barrier. The loss of intestinal barrier integrity leads to increased intestinal permeability and, in turn, mucosal barrier dysfunction. Since a definitive cure for IBD is still not available, the need for new therapeutic targets remains fundamental. Our group already demonstrated the therapeutic effect of murine mesenchymal stromal cell‐derived Extracellular Vesicles (EVs) in IBD. Here, good manufacturing practice‐grade (GMP‐grade) produced EVs from human mesenchymal stromal cells were used to evaluate their anti‐inflammatory and pro‐regenerative effects in an in vivo model of IBD. Methods : For six days (from day one to day six), 3% dextran sulphate sodium (DSS) was administered in drinking water to induce colitis, and on day ten, the animals were sacrificed. Human GMP‐grade EVs were administered by five intraperitoneal (ip) injections (daily from Day 3 until Day 7) in mice with acute colitis induced by DSS. The experiments included a control group (CTRL, no DSS and EVs administration, n = 15 mice), a DSS+placebo (PBS) group ( n = 25), and a DSS + EVs group ( n = 30). EVs were characterized by Nanosight, CryoTEM, and cytofluorimetric analysis of antigen surface. The clinical efficacy, the histological score and protein expression by immunofluorescence were evaluated. Results : Mice's weight in experimental groups was reduced compared to the CTRL without significative differences. The percentage of damaged mucosa was significant decreased in the EV‐treated group compared to the DSS group. The colon length of the EV‐treated mice significantly improved. The secretion of MUC2, MUC5, iNOS and CD163 was evaluated by immunofluorescence. The EV administration increased the secretion of regenerative proteins such as mucins. Moreover, a reduction of iNOS and a parallel increase of CD163 were also observed. Summary/Conclusion : We proved for the first time that human GMP‐grade EVs have significant pro‐regenerative and anti‐inflammatory activity in a murine model of IBD.

Identifying

Yue Su 1,2 , Kekoolani S. Visan 1,2 , Andreas Möller 1,2,3 1 JC STEM Lab for Personalised Cancer Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China; 2 Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China; 3 QIMR Berghofer Medical Research Institute, Brisbane, Australia Yue Su and Kekoolani S. Visan are co‐senior authors . Introduction : Head‐and‐neck cancer (HNC) is one of the most common cancers worldwide. The majority of HNC cases (90%) are Head and Neck Squamous Cell Carcinoma (HNSCC), with particularly high prevalence in Asia. Diagnosis at advanced stages (stage III or IV), as well as high rates of metastasis and recurrence, largely contributes to the poor survival rate of HNSCC patients. Therefore, there is an urgent need to develop methods for early diagnosis of HNSCC. Small extracellular vesicles (sEVs) are membrane‐enclosed lipid bilayer vesicles of endocytic origin and of less than 200 nm in diameter. They are released by cells and contain subsets of lipids, nucleic acids and proteins specific to their parental cells. Plasma sEVs are mostly derived from blood and vessel cells but are also contain sEV derived from various tissues, including cancer masses. Therefore, plasma sEVs may be an ideal source of specific diagnostic biomarkers of HNSCC, with the added benefits of enabling real‐time monitoring of disease, and their non‐invasive and therefore repeatable nature making them accessible for convenient and efficient collection. Methods : In this study, sEVs were isolated from plasma samples from cancer‐free healthy controls ( n = 5) and HNSCC patients ( n = 5) using size‐exclusion chromatography. Plasma sEVs were characterised using nanoparticle tracking analysis, western blot assays and transmission electron microscopy. Specific proteins in plasma sEVs of HNSCC patients were analysed and quantified by mass spectrometry. Results : This approach identified a total of 603 proteins, of which 70 were significantly higher and 86 were lower in HNSCC patients’ blood sEV samples. Validation of the sEV biomarker proteins in a large, independent confirmation cohort of HNSCC patients and matched healthy subjects is being conducted, with initial data showing high reproducibility. Summary/Conclusion : Overall, this study provides a series of novel, potential biomarkers in plasma sEV for early HNSCC identification and monitoring of therapeutic efficacy.

Integrating

Presenter: Shuhan Jiang Max Planck Institute for the Science of Light, Erlangen, Germany Introduction : Accurate characterization of biological particles, especially extracellular vesicles (EVs), is essential for uncovering their roles in various biological functions and diagnostic applications. The heterogeneous nature of EVs complicates the precise measurement of their biophysical and biochemical characteristics. Methods : We recently introduced interferometric nanoparticle tracking analysis (iNTA) as a label‐free technique for analysing nanoparticles like EVs. Building on this, we present a method that integrates iNTA with single molecule sensitive fluorescence detection, enabling characterization of EVs alongside their biomolecular content, in solution. This approach enables detection of fluorescence labels on the EV surface, including lipid markers and surface proteins tagged with specific antibodies, as well as internal proteins within EVs labelled by carboxyfluorescein succinimidyl ester (CFSE). To prevent fluorescence bleaching, a flow system is used for sample introduction. Our method facilitates the simultaneous measurement of particle size, refractive index, and fluorescence intensity at the single‐particle level. Results : To demonstrate our method, we used a mixture of 40 nm non‐fluorescent polystyrene beads and 100 nm fluorospheres, effectively distinguishing between nanoparticles with and without fluorescence labels. We further evaluated the fluorescence sensitivity of our setup with liposomes labelled with two lipid dyes, MemBright (488 nm excitation) and R18 (561 nm excitation), and confirmed that fluorescence intensity was proportional to liposome size and dye concentration. Finally, we applied our method to HEK293‐derived EVs, labelling them with anti‐CD9 and anti‐CD81 fluorescent antibodies to quantify the proportion of EVs expressing these biomarkers and labelling internal proteins with CFSE to assess internal molecular content. Summary/Conclusion : This single‐molecule‐sensitive, multimodal approach enhances the ability to differentiate EV populations within complex samples, offering significant potential for advancing biomarker discovery and detailed analysis of heterogeneous particle systems. Funding : Max Planck Society.

Kinesin‐3

Henrike Tietz 1,2 , Leonie Witte 2 , Davina Störmer 1 , Christof Lenz 3 , Mona Honemann‐Capito 2 , Wilhelm Walter 1 and Julia Christina Gross 1,2, 1 Institute of Molecular Medicine, HMU Health and Medical University Potsdam, Potsdam 2 University Medical Center Goettingen, Goettingen 3 Department of Clinical Chemistry, University Medical Center Goettingen, Goettingen Introduction : Extracellular vesicles (EVs) are critical for intercellular communication, reflecting their cell of origin's molecular profile. In the last years, the machenery of small EV (sEV) biogenesis and secretion has been studied in great detail, yet a remaining question is, how secretory multivesicular bodies (sMVBs) are transported towards the plasma membrane for release. Intracellular anterograde transport along microtubules is mediated by the kinesin motor protein family. Specifically the kinesin‐3 family acts in the directed movement of endosomal compartments. Understanding which of these kinesins mediate sMVB transport is a key step towards unterstanding the balance between secretory versus degradative MVB trafficking, the release of EV subpopulations and the underlying regulation of sEV biogenesis. Methods : The involvement of the kinesin‐3 family in endosomal trafficking and sEV release was analysed by siRNA mediated knockdowns (KD) of different kinesin candidates in combination with immunofluorescent staining (IF), western blotting (WB) and nanoparticle tracing analysis (NTA) of purified sEVs. In addition, using motorless kinesin constructs linked to a minus end‐directed dynein motor, we generated systematic kinesin‐endosome interaction profiles of 7 kinesins candidates with 13 different endosomal/sEV‐markers. Indeed, individual kinesin‐3 members were distinctly recruited to specific sEV marker‐positive endosomal compartments and we found other kinesins interacting with early Rab11‐endosomes then CD63‐bearing late endosomes. As both of these compartments participate in the release of sEVs, we consequently probed for the involvement in secretory MVB trafficking and found their knockdown associated with partially reduced sEV secretion levels. Results : The linkage assay revealed cooperative effects between the kinesin members themselves or between multiple kinesins and members of the EV secretion machinery, such as nsmase‐2 and SYTL4. Summary/Conclusion : We will present our findings showing the kinesin‐3 family cooperatively involved in the secretion of sEVs and thus integrating the intracellular transport of sMVB into the biogenesis pathway of sEVs.

Leverageing

Presenter: Seung Ah Choi Seoul National University Hospital, Republic of Korea ABSTRACT UNAVAILABLE

Long‐Term

Presenter: Soyoung Son Sungkyunkwan University, Republic of Korea Introduction : The clinical application of extracellular vesicles (EVs) is constrained by their poor stability during storage. The current preservation methods, including cryopreservation and lyophilization, either fail to maintain stability at low concentrations or cause cytotoxicity at high levels. Furthermore, effective EV delivery remains a significant challenge due to the skin barrier and the necessity for precise intradermal administration. Microneedles (MN) offer a minimally invasive, self‐administered solution for stable, controlled intradermal delivery. They can retain the activity of encapsulated biomolecules over extended storage and under extreme conditions. In this study, we developed hyaluronic acid (HA)‐based MN loaded with EVs (EV@MN) to explore their potential for the long‐term storage and effective delivery of EVs. Methods : After EV@MN was prepared, the biological activity of EVs in EV@MN were evaluated at various stages of the fabrication process and over 6 months of storage by examining their functional impact on fibroblasts. CLSM and in vivo imaging were employed to analyse the precise delivery and sustained release of EVs within the dermis. Moreover, the biological effects of EV@MN on dermal tissue in vivo were investigated. Results : EV@MN demonstrated the ability to maintain EV stability, exhibiting high particle retention and bioactivity over a 6‐month period at 4°C. The mechanical testing confirmed that the EV@MN could penetrate the skin, thereby achieving the targeted release of EVs into the dermis. In vivo, EV@MN demonstrated sustained EV retention at the injection site for seven days, significantly longer than intradermal injections, with extended fluorescence. Functional analysis revealed that EV@MN‐treated skin exhibited increased dermal thickness, enhanced collagen and elastin levels, and promoted fibroblast proliferation. Summary/Conclusion : EVs were effectively isolated and incorporated into EV@MN, with their biological activity preserved for over 6 months under mild storage conditions. Furthermore, EV@MN facilitated precise and straightforward intradermal administration, allowing for sustained EV release within the dermal layer. This formulation augmented the functional effects of EVs on dermal fibroblasts. This study presents a viable approach for EV clinical applications, addressing challenges in storage and delivery while enhancing usability for potential skin care applications.

Lyophilized

Presenter: Yeimi Herrera‐Luna Universidad de los Andes, Chile Introduction : Osteoarthritis (OA) affects the articular cartilage, causing an increase in oxidative stress leading to cartilage damage. It has been shown that mesenchymal stem/stromal cells (MSCs) therapeutic effects depend on their small extracellular vesicles (sEVs). However, for optimal clinical outcomes there is a need to enhance their therapeutic efficacy and to improve their storage for potential clinical application. We propose that sEVs derived from metabolically reprogrammed glycolytic‐MSC (Glyco‐sEVs) have a better therapeutic effect than those under basal conditions (Basal‐sEVs) and that after a lyophilized process they maintain their phenotype and function against OA progression. Methods : Chondrocytes were isolated from the knee of OA patients. The chondroprotective effect of frozen or lyophilized Glyco‐sEVs was measured by an apoptotic assay with menadione through an annexin/PI kit and flow cytometry. To determine the clinical effect of frozen or freeze‐dried Glyco‐sEVs, both were injected intraarticularly in a collagenase murine model of OA (CIOA) and evaluated by microCT and histopathology. Results : Our results showed that lyophilized Glyco‐sEVs do not change their sEV characteristics; therefore, they maintain their chondroprotective effect on chondrocytes when exposed to menadione as a source of oxidative stress. Moreover, CIOA mice treated with either frozen or lyophilized Glyco‐sEVs showed a significant reduction in bone mineral density and the clinical damage score obtained from joint histology. Summary/Conclusion : Lyophilized Glyco‐sEVs maintain their phenotype by expressing CD63, CD81 and CD9 and their size and shape with a yield of 86.5%. Moreover, they do not change their chondroprotective effect in vitro since, as frozen Glyco‐sEVs, they display higher resistance to apoptosis induced by oxidative stress as compared to basal sEVs. Moreover, CIOA mice treated with both Glyco‐sEVs showed a regenerative effect with significance evidenced in the histomorphometry parameters and the clinical joint score by histology. These results show that lyophilized Glyco‐sEVs represent a potential new strategy for treating OA through their chondroprotective and regenerative properties. Funding : ANID FONDECYT Regular N°1211353; ANID FONDECYT Iniciación N°11220549; ANID FONDEF N°21110194; and ANID Fondo Basal 210024 (IMPACT).

Mechanistic

Presenter: Xiang Luo King's College London, UK Introduction : Human milk extracellular vesicles (EVs) are crucial mother‐to‐baby messengers that transfer biological signals. These EVs are reported to survive digestion and transport across the intestine. The mechanisms of interaction between human milk EVs and the intestinal mucosa, including epithelial uptake remain unclear. Here, we studied the interaction of human milk EVs with the gut barrier components, including intestinal biofluids, enzymes, mucus and epithelium. Methods : EVs were isolated from human milk by a pH adjustment and differential ultracentrifugation. The characterization of EVs were determined by BCA, NTA, DLS, and TEM. The stability study in the intestinal environment was conducted in FaSSIF, FeSSIF and enzyme fluids. Cellular uptake of EVs was measured by flow cytometry and confocal microscopy. The transport of EVs across intestinal monlayers was conducted with Caco‐2 transwell model. The pharmacological inhibitors of endocytic pathways was used to determine the relative contribution of specific pathways (chlorpromazine, genistein, nocodazole, 5‐(N‐ethyl‐N‐isopropyi)‐amiloride, methyl‐β‐cyclodextrin and dynasore). The proteomic analysis was done by LC‐MS/MS. The nucleic acids were loaded into EVs with electroporation and the cellular study and transport were done. Results : We show that human milk EVs are largely stable in the biochemical gut barriers and demonstrate high mucus diffusivity. EVs show a high level of epithelial cell uptake (∼70%), and efficient transport across Caco‐2 monolayers. Whilst cell uptake of EVs was mediated by multiple routes, none of the pathway‐specific inhibitors inhibited their epithelial translocation. Proteomic analysis of EVs transported across Caco‐2 monolayers identified 14 enriched EV proteins that may facilitate intestinal transport. These findings significantly expand our understanding of the interactions between human milk EVs and the gut barriers, including their intestinal uptake. The siRNA was loaded into EVs with electroporation and the permeability of EVs‐associated siRNA through the intestinal barrier was significantly higher than free siRNA. Summary/Conclusion: The work strengthens the existing evidence that EVs in human milk may be important signalling mediators based on their ability to carry biological cargo into and across intestinal epithelial cells within the hostile biochemical environment of the gut.

Mesenchymal

Presenter: Enza Torino University of Naples Federico II, Italy Introduction : Chondrosarcomas (CHS) account for approximately 20% of all primary malignant bone tumours, characterized by slow progression and minimal early symptoms. Despite their heterogeneity, high‐grade and dedifferentiated CHS subtypes pose significant therapeutic challenges due to their resistance to conventional chemotherapy and radiotherapy. Consequently, there is an urgent need for innovative treatment strategies, particularly for aggressive CHS forms with metastatic potential. Methods : In this study, we explore the use of mesenchymal stem cell‐derived small extracellular vesicles (MSC‐sEVs) as a nanocarrier system to enhance drug delivery in a 3D in vitro CHS model. Using high‐pressure homogenization (HPH), we achieved exceptional encapsulation efficiency of doxorubicin (DXR) within MSC‐sEVs (DXR‐MSC‐EVs). Results : A comparative analysis of free DXR versus DXR‐MSC‐EVs revealed significantly improved drug penetration and cellular uptake in the 3D tumour model. Notably, DXR‐MSC‐EVs induced a higher rate of necrosis and exhibited greater cytotoxic effects than free DXR, underscoring their potential as an advanced drug delivery platform for treating aggressive CHS. Summary/Conclusion : These findings highlight MSC‐sEVs as a promising strategy for overcoming drug resistance and improving therapeutic outcomes in CHS patients.

Metabolomic

Estelle Maret 1,2 , Sylvain Le Gludic 3 , Caroline Samer 1,2 , Aurélien Thomas 3 , Youssef Daali 1,2 1 Division of Clinical Pharmacology and Toxicology, Geneva University Hospital, Geneva, Switzerland; 2 Department of Anaesthesiology, Pharmacology, Intensive Care and Emergency Medicine, Faculty of Medicine UNIGE, Geneva, Switzerland; 3 University Center of Legal Medicine, Unit of Forensic Toxicology and Chemistry, Lausanne, Switzerland Introduction : In the past few years, plasma‐derived extracellular vesicles (EVs) have emerged as a promising source of biomarkers for various pathological conditions, particularly in cancer research (Witwer et al.). However, limited research has been conducted on EVs from healthy individuals, despite their potential value in clinical studies. This study aims to characterize the metabolome of EVs from healthy donors, addressing the challenge of lower EV secretion rates in non‐pathological states and exploring their potential as a source of biomarkers for future research in drug exposure prediction. Methods : Six mL of whole blood from 42 healthy donors was processed within 2 h via 3 centrifugation steps. Plasmas from 6 donors were pooled together, reaching a final volume of 10 mL. EVs were isolated using size exclusion chromatography (QEV10, Izon), either from 10 mL plasma loading ( n = 3) or from 5 mL plasma loading in two separated columns ( n = 4). Particles were eluted with 5 mL PBS, and fractions were characterized for total protein concentration (Micro BCA), EVs (LAMP2), and lipoproteins (APOC3 and APOB100) using commercially available ELISA kits. Untargeted metabolomics analysis was performed using LC‐HRMS. Results : Particles elution profiles shifted by one fraction when 10 mL plasma was loaded compared to 5 mL. The 5 mL loading showed significantly reduced APOC3 contamination in fraction 8 ( p = 0.002). APOB100 and APOC3 showed an elution ratio at 5%–7% in fractions 8–9 (5 mL) or 9–10 (10 mL), while LAMP2 showed 15%–21% elution. Untargeted metabolomics of these fractions revealed particle‐enriched features when compared to plasma. Fraction clustering was found to be independent of the volume loaded onto the column, suggesting a different metabolome across early and late fractions. Several features showed positive correlation with LAMP2, indicating EV‐associated metabolites. LAMP2‐correlated features will be further identified. Summary/Conclusion : This research highlighted features potentially associated with or encapsulated within EVs, thus providing a methodological framework for exploring the subtle yet significant metabolic signatures carried by EVs which could be used for clinical studies involving healthy cohorts. Funding : This research is funded by the Swiss National Science Foundation.

Micro‐Tff

Presenter: Elena Hoffmann Fraunhofer IMM, Mainz, Germany Introduction : In recent years, extracellular vesicles (EVs) have attracted considerable attention within medical research due to their critical role in intercellular communication and their potential as diagnostic and therapeutic avenues. EVs are released by various cell types, and they are known to carry a wide range of bioactive molecules, including proteins, lipids, and nucleic acids. These components enable EVs to convey information between cells, thereby influencing numerous physiological and pathological processes. Despite their biological significance, the isolation of EVs remains a considerable challenge. The most widely published method for EV isolation is ultracentrifugation, which, while effective, presents several drawbacks that have also been documented in the MISEV guidelines. This technique requires specialized and expensive equipment, making it less accessible to many laboratories. Additionally, ultracentrifugation is a time‐consuming process that involves multiple steps, leading to potential losses of relevant EVs and their associated cargo. Methods : To address these limitations, the present study focuses on the isolation of EVs using a micro‐tangential flow filtration (Micro‐TFF) system, which has been specifically designed by us for this purpose. The Micro‐TFF technique offers a more efficient and versatile alternative to ultracentrifugation. It allows for the customization of the filtration process according to the specific cell type under investigation by utilizing various filters with differing membrane permeabilities. This adaptability facilitates the effec‐tive isolation of EVs of different types and sizes. Though adjusting the filtration rate, it is possible to isolate the EVs under gentle conditions. Additionally, this method saves a lot of time and space. Results : Preliminary results demonstrate that the Micro‐TFF method has successfully isolated EVs from a A549 cell line, highlighting its potential as a reliable and scalable approach for EV isolation in both research and clinical settings. Summary/Conclusion : By advancing the methodologies for EV isolation, this study aims to contribute to a more gentle and faster way to isolate EVs for improved ac‐cessibility. This enables an acceleration of scientific EVs research, particularly in their application in the medical field, as therapeutics or bi‐omarkers.

Multiplexed

Presenter: Moein Talebian Gevari Uppsala University, Uppsala, Sweden Introduction : Here, we demonstrate the fabrication, characterization, and application of a novel silicon microchip‐based biosensor that exploits streaming current (I_s) for the detection and characterization of small extracellular vesicles (sEVs). The method was further optimized and compared with a fluorescence‐based single EV technology for a semi‐quantitative assessment of EV count, membrane‐protein expression, and relative abundance of various EV‐subpopulations. Methods : sEVs were isolated from cell culture media of the non‐small cell lung cancer H1975 cell line cultured in RPMI‐media with FBS depleted from exogenous exosomes. The media was concentrated and sEVs were isolated by SEC. The sEVs were characterized by NTA and earlier western blot confirmed expression of the targeted proteins. The CD81 and CD9 membrane proteins of the sEVs were profiled using a novel microchip technology. The microchips, composed of four interconnected microchannels, were designed with an open‐top geometry where each of the channels could be functionalized with different antibodies for multiplexed measurement. The expression level of target markers was detected by immunocapture of sEVs on the sensor surface and measuring the changes in I_s. Results : We assessed poly‐L‐lysine and silane‐based surface functionalization strategies to capture sEVs on silica. As a proof of concept, CD81 and CD9 tetraspanins from H1975‐derived sEVs were targeted and compared to state‐of‐the‐art methods. The results showed an LoD of 9.5 × 10 3 sEVs/mL and 7.6 × 10 4 sEVs/mL for CD81 and CD9 tetraspanins, respectively. Using membrane‐sensitive peptides, our platform achieved an LoD of 1 × 10 4 sEVs/mL. sEVs were isolated by size exclusion chromatography, and their surface proteins were profiled using single‐EV fluorescence. Nanoparticle tracking analysis (NTA) characterized size and charge distribution prior to biosensing. Summary/Conclusion : This biosensing platform demonstrated sensitive, multiplexed detection of sEVs, outperforming other sensitive platforms. With a quick 30‐min detection time and minimal sample requirement of 30 µL, this device holds promise for clinical and research applications in studying sEVs. Funding : This study was supported by the Swedish Research Council; The Swedish Cancer Foundation

Nanochannel

Viktoria de Carvalho, Elin Persson, Aaron Domenzain Del Castillo Cerecer, Alex Lech, Sriram KK, Daniel Midvedt, Giovanni Volpe, Fredrik Westerlund, Elin Esbjörner Chalmers University of Technology, Gothenburg, Sweden Introduction : Extracellular vesicles (EVs) are membrane‐enclosed biological nanoparticles that play crucial roles in cell‐to‐cell communication in both health and disease. A major problem in understanding EV biology relates to their vast heterogeneity in terms of both physical and biochemical properties. To address this problem, single‐particle methods are needed. This project uses and further develops a unique single particle analysis platform, combining nanofluidics with fluorescence microscopy, to shed light on EV heterogeneity. Methods : The nanofluidic device, with nitrogen inlets allowing for pressure‐driven flow through microchannels and nanochannels, is mounted on an inverted fluorescence microscope. Fluorescent particles diffusing in the nanochannels are recorded with single or multicolour fluorescence detection. Using a machine learning algorithm, particle trajectories are retrieved, from which diffusivity and, based on that, size are determined. The nanochannels, with a cross section of 300 × 300 nm 2 , allow tracking of particles in the focal plane, while avoiding particle trajectories from crossing each other. EVs from engineered HEK293t cells overexpressing CD63 fused to mCherry are extracted from cell culture media and isolated via tangential flow filtration (TFF) and ultrafiltration (UF). We also investigate EVs collected from cell media directly in the nanochannels, avoiding sample preparation biases. Results : Previous results have demonstrated that our method can distinguish between 50 and 100 nm polystyrene bead populations with high specificity in a mixed sample. Here, we show that EVs isolated with TFF and UF can freely diffuse in the nanochannels, their emission intensity can be recorded, and their hydrodynamic size can be determined. We also show that EVs taken directly from cell media can be analysed in the nanochannels. Summary/Conclusion : The nanofluidic platform can be used to determine the hydrodynamic size of EVs in solution using fluorescence microscopy and pressure‐driven flow. Subsequent projects will use this technique to further investigate EV populations from engineered HEK293t cells with CD69‐mCherry undergoing different treatments, such as oxidative stress, which may affect EV population characteristics. Funding : Knut and Alice Wallenberg Foundation (KAW).

Nano‐Flow

Yunyun Hu, Haonan Di, Ye Tian, Xiaomei Yan Xiamen University, China Introduction : Recent advances in isolation and analytical methods have highlighted the complexity and diversity of extracellular particles (EPs). Among these, non‐vesicular extracellular particles (NVEPs) are distinct from extracellular vesicles (EVs) as they lack lipid bilayer membranes, and are more abundant in extracellular spaces and body fluids. NVEPs exhibit higher pro‐metabolic activity and hold significant promise in clinical diagnostics. However, traditional EP separation methods are often labour‐intensive and inefficient, and many characterization techniques lack the specificity to differentiate EVs from smaller NVEPs. Developing a rapid and robust approach to discriminate EVs and NVEPs is therefore critical for identifying extracellular carriers of specific biomolecules and understanding their biological functions. Methods : EPs were isolated from colorectal cancer cell lines using differential ultracentrifugation (UC), followed by high‐resolution iodixanol density gradient centrifugation for separation of EVs and NVEPs. A Laboratory‐built nano‐flow cytometer (nFCM) with two spatially arranged lasers was applied for single‐particle characterization of size, purity, particle concentration, DNA content, lipid membranes, and specific proteins for EVs, NVEPs, and mixed EP samples. Side scattering and fluorescence detection were performed after SYTO 16, di‐8‐ANEPPS, Cy5‐NHS, and/or immunofluorescent staining. Additionally, EPs obtained via UC were processed using UC, ultrafiltration (UF), and size exclusion chromatography (SEC) to evaluate their efficacy in recovering NVEPs. Results : Significant differences in morphology (via TEM), biophysical properties, and molecular compositions were observed between EVs and NVEPs. DNA staining together with enzyme treatment enabled precise single‐particle discrimination of EVs and NVEPs. Through multiparameter analysis of DNA and protein/lipid membrane, NVEPs lacked EV‐specific protein markers and lipid bilayers. However, NVEPs could labelled with Cy5‐NHS and sensitive to proteinase K, suggesting NVEPs are protein‐nucleic acid complexes distinct from EVs. Enhanced centrifugation force and duration in UC, as well as SEC, were effective for improving NVEP recovery. Summary/Conclusion : This study demonstrates that nFCM is a powerful platform for distinguishing EVs from NVEPs through DNA analysis on single EPs. Multiparameter analysis of DNA and proteins provide deeper insights into the intra‐subtype heterogeneity of NVEPs and EVs, paving the way for further fundamental and translational research on EP subtypes. Funding : This research was supported by the National Natural Science Foundation of China (Grants 21934004)

Ngs‐Based

Presenter: Elodie Simphor CNRS/ALCEN, France Introduction : Extracellular vesicles are lipid‐bound nanoparticles that play a crucial role in cell‐to‐cell communication, influencing both physiological and pathological processes. Their presence in peripheral biofluids, including saliva, an easily accessible and non‐invasive sample source, makes them promising candidates for biomarker discovery in diagnostics and prognostics. Additionally, microRNAs (miRNAs) are increasingly recognized as valuable diagnostic tools for various diseases. Although multiple methods exist for isolating and characterizing salivary EVs (sEVs), further optimization and standardization are necessary to facilitate their clinical translation. This study aims to characterize and compare the miRNA content of salivary EVs isolated using two rapid and scalable techniques, co‐precipitation (Q) and immunoaffinity (M), against the gold standard ultracentrifugation (UC) method. Methods : Saliva samples were collected from three healthy volunteers. sEVs were isolated using UC, Q, and M. Following a comprehensive characterization, miRNAs were extracted from both sEVs and whole saliva (WS) and subsequently sequenced using next‐generation sequencing (NGS). Results : Our analysis revealed that while saliva and EVs share common sets of miRNAs, several miRNAs exhibit differential expression between WS and sEVs. Principal component analysis demonstrated a clear separation between WS and the three isolation methods. UC and Q showed a strong similarity, clustering primarily by individual rather than by isolation technique, whereas M exhibited a separation pattern driven more by the isolation method than by individual variation. Despite these differences, all three methods yielded highly consistent miRNA enrichment profiles when compared to WS. Gene Ontology and KEGG pathway analyses indicated significant enrichment of miRNA‐related processes, including small RNA pathways and splicing regulation, particularly in the M condition. Summary/Conclusion : This study highlights the significance of sEVs as a source of miRNA and demonstrates that certain miRNA species are preferentially enriched in WS, while others are predominantly contained within sEVs. The three isolation techniques provided robust and consistent miRNA profiles, with UC and Q exhibiting strong similarities and M showing superior enrichment in specific pathways. These findings underscore the importance of EV isolation before miRNA analysis and confirm that all three methods are suitable for clinical salivary sample processing. Funding : This research was founded by French Centre National de la Recherche Scientifique (CNRS) and ALCEN

Non‐Lytic

Presenter: Thupten Tsering University Health Centre, Montreal, Canada Introduction : Migrating cancer cells emit a variety of extracellular vesicles (EVs), including small EVs (sEVs), large EVs known as migrasomes and cellular fragments. These EVs may carry bioactive molecules that play roles in intercellular communication and tumour progression. We previously developed EV‐ADD, a database on EV‐associated DNA (Tsering et al. 2019, JEV), and we also demonstrated the presence of EV‐associated DNA at the single EV level using transmission electron microscopy (TEM) (Tsering et al. 2024, Current Protocols). However, the molecular mechanisms underlying the packaging and release of DNA within these EVs remain unclear. Methods : In this study, we investigated the molecular composition of cellular fragments released during migration in cancer and non‐cancer cell lines. These non‐lytic cellular fragments were isolated using ultracentrifugation and characterized using NanoSight, atomic force microscopy, TEM and western blots. To determine their contents, we performed whole‐genome sequencing (WGS) and proteomics analysis. WGS findings were validated using droplet digital PCR and Sanger sequencing. To further understand the migration‐dependent release of these fragments, we treated them with migration‐inhibitory drugs (cytochalasin B, CK‐636, dynasore). Results : Scanning electron microscopy and live‐cell time‐lapse imaging revealed distinct cellular fragments released by breast cancer cells, but not by normal epithelial cells. WGS analysis showed that these cellular fragments carry DNA representing the complete genomic sequence of the parent cells and reflecting their mutational status. Proteomics analysis identified several membrane, cytoplasmic, and mitochondrial proteins, including integrin alpha‐2 (ITGA2), integrin alpha‐3 (ITGA3), integrin beta‐1 (ITGB1), basigin (BSG), filamin‐C (FLNC) and malate dehydrogenase 2 (MDH2). Analysis of the top 100 proteins revealed distinct protein profiles in these cellular fragments compared to other EV subtypes (small and large EVs). Notably, migration‐inhibitory drugs significantly reduced the biogenesis of cellular fragments, while the exosome inhibitor GW4869 had no effect. We have termed these cellular fragments ‘Motile Vesicles’ (MoVes). Summary/Conclusion : This study is the first to demonstrate that non‐apoptotic and active emission of cellular fragments carrying DNA mirror the complete genome of the host cancer cells. Our findings provide novel insights into the molecular cargo of cellular fragments released during cell migration and highlight their potential role in cell‐to‐cell communication and metastasis.

Peg‐Based

Leticia A. Fernandes 1 , Pedro E. S. Lima 1 , Milton Y. Nishiyama‐Jr 2 , Umut A. Gurkan 3 , Ana Claudia O. Carreira 1,4 1 University of São Paulo, São Paulo, Brazil; 2 Butantan Institute, São Paulo, Brazil; 3 Case Western Reverse University, Cleveland, Ohio, USA; 4 Federal University of ABC, Santo André, Brazil Milton Y. Nishiyama‐Jr and Umut A. Gurkan are Co‐senior authors . Introduction : Extracellular vesicles (EVs) have revolutionized the biological field, prompting the development of diverse isolation methods based on size, density, immune assays, polymer precipitation, and microfluidics. However, a single method that achieves high yield, quality, cost‐effectiveness, easy hands‐on, and clinical applicability remains elusive. We developed an all‐in‐one PEG‐based methodology for EV isolation from multiple sources to address this gap. Methods : EVs were isolated using the Polyethylene Glycol (PEG) mixture protocol. Sources standardized included (a) human bone marrow mesenchymal stem cells (hBM‐MSC); (b) human umbilical vein endothelial cells (HUVEC); (c) breast cancer cells (MDA.MB.231); and blood components (Ethical committee (05‐14‐07C) as (d) poor‐platelet plasma (PPP), (e) red blood cells (RBC), and (f) white blood cells (WBC). Blood fractions were prepared from 2 mL of fresh whole blood, concentrated using Amicon Ultra‐15 Centrifugal Filter 10 kDa cut‐off, and incubated with our PEG mixture. Conditioned media from cell lines were processed without foetal bovine serum (FBS) or exosome‐depleted serum. EV characterization included Nanoparticle Tracking (NTA), Cryo‐electron Microscopy, and label‐free quantitative liquid chromatography‐mass spectrometry (LC‐MS). The ultracentrifugation (UC) method was the comparative standard protocol. Results : Our PEG method consistently preserved EV integrity, maintaining their intact lipid bilayer, and yielded enhanced enrichment compared to UC. HUVEC and hMB‐MSC EVs yields were nearly five times higher using our method. Interestingly, our methodology yielded 15 times more than the UC protocol for all blood components EV isolation. To challenge the effectiveness of our method, we performed the isolation using only 50 uL of plasma, exceeding a yield of over 2 × 10 11 particles per mL. Cryo‐EM confirmed better structural preservation across all blood samples using PEG. LC‐MS demonstrated, in HUVEC, that PEG method enriched 1763 proteins demonstrating that PEG maintains protein cargo integrity. Summary/Conclusion : Our PEG precipitation method represents a significant advancement with outstanding performance in terms of yield ensuring scalability and reproducibility across diverse biological samples. Its user‐friendly principle and cost‐effectiveness make it a robust candidate for widespread application in EV research and diagnostics. Funding : Coordination for the Improvement of Higher Education Personnel (CAPES)—Grant no. 88887.666887/2022‐00 and 88887.916425/2023‐00 (Fernandes, L.A. fellowship).

Persistence

Presenter: Hongyue Zhang George Washington University Introduction : Antibiotic overuse has made wastewater a major reservoir for antibiotic‐resistant bacteria (ARB) and antibiotic resistance genes (ARGs). The growing demand of wastewater reuse further expands the spread of ARBs and ARGs, posing risks to public health. ARGs can be encapsulated in bacterial membrane vesicles (BMVs) released during ARB growth and apoptosis, enhancing their environmental persistence and potential for bacterial transformation compared to free ARGs. However, the presence, prevalence, and transmission of BMV‐associated ARGs was largely underexplored. Additionally, isolating and purifying BMVs from complex environments like wastewater suffer from a low yield and selectivity, which requires further advancement. Methods : Immunomagnetic selection was adopted for isolating BMVs from wastewater, by using antibodies targeting lipoteichoic acid (LTA) and lipopolysaccharides (LPS) that are biomarkers of Gram‐positive and Gram‐negative bacterial membrane vesicles, respectively. Wastewater samples were collected from various stages of the treatment process at a local wastewater treatment plant (WWTP). RT‐qPCR and metagenomics sequencing were applied to evaluate the presence and abundance of ARGs throughout the treatment process. Results : We cultured Methicillin‐resistant Staphylococcus aureus (MRSA) and ARG‐loaded E. coli, and pelleted down BMVs through ultracentrifugation. BMVs were spiked into wastewater, and the recovery rate was 51.8% and 32.4% respectively for Gram‐positive and Gram‐negative BMVs. Minimal impurities were co‐isolated from the wastewater, including free DNAs (in the form of plasmids) and viruses. Transmission electron microscopy and nanoparticle tracking analysis confirmed that the isolated specimen were BMVs. During WWTP surveillance, BMV‐associated kanamycin resistance gene (KanR) reached up to 5.49 log10 gene copies per litre, contributing to 11.9% of total KanR. The removal efficiency of total KanR was 3.95 log10 through all treatment chains, while BMV‐associated KanR showed a lower removal of only 2.35 log10. The ratio of BMV‐associated KanR to the total KanR increased from < 1% to 11.9% through treatment chains. Summary/Conclusion : Our study introduced a new approach to isolate BMVs from complex environment that can outperform conventional methods. This isolation method was successfully applied for BMV isolation and purification from wastewater. BMVs can facilitate ARG transmission due to increased persistence of ARGs.

Preclinical

Susanne Sasse 1 , Odett Kaiser 1 , Jennifer Harre 1 , Mario Gimona 2,3 , Eva Rohde 2 , Daniela Auer 2 , Stefan Rund 2 , Tanja Schally 2 , Carina Kals 2 , Christina Folie 2 , Elisabeth Bayer 2 , Hinrich Staecker 4 , Athanasia Warnecke 1 1 Hannover Medical School, Hannover, Germany; 2 Paracelsus Medical University Salzburg, Salzburg, Austria; 3 Paris Lodron University, Salzburg, Austria; 4 University of Kansas School of Medicine, Kansas City, Kansas, USA Susanne Sasse and Odett Kaiser are co‐first authors . Introduction : Patients with profound hearing loss undergoing cochlear implantation often lose their residual hearing due to implantation trauma, which impairs hearing performance. So far, there are no approved drugs available for this purpose. Extracellular vesicles (EV) derived from human umbilical cord mesenchymal stromal cells have been tested as novel drug candidates exerting immunomodulatory and/or neuroprotective effects in the inner ear after implantation trauma. To prepare a clinical trial, the safety and biodistribution of EV in different cochlear implantation trauma animal models were investigated. Methods : Following a series of initial in vitro and in vivo experiments supporting the biological concept, preclinical safety was evaluated in NMRI mice after a single intracochlear application of various doses of EV under GLP compliance. Safety and efficacy were investigated in guinea pigs after cochlear implantation trauma, and long‐term safety was tested in guinea pigs after EV application. Biodistribution of DiD‐labelled EV was evaluated initially in guinea pigs and was extended under GLP‐like conditions in NMRI mice. All data were compiled in various documents for clinical trial application. Results : Applied EV was well tolerated, and no abnormalities or drug‐related clinical symptoms were observed at any time in either mice or guinea pigs. Application improved hearing after implantation trauma in the guinea pig cochlear implant model. Six months after the EV application, hearing thresholds remained at a similar level to the control group. Regardless of the species, DiD‐labelled EVs could be tracked depending on their duration of action and detected at the applied cochleae (hair cells, supporting cells and spiral ganglion neuronees) and in the kidney, indicating a physiological degradation. Summary/Conclusion : Preclinical testing of a single EV application in the inner ear for trauma reduction did not raise any specific safety concerns in any of the described animal models. Submission of the study via the Clinical Trials Information System (CTIS) resulted in the approval of a Phase I/II clinical trial (ESCRT) for safety and efficacy in October 2024. Funding : This work was funded by MED‐EL, the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy—EXC 2177/1 (Project ID 390895286) and the Project “ExtraNeu” from the State of Salzburg, Austria.

Pseudomonas

Priyakshi Gogoi, Ambalika Roy, Dipankar Ghosh Special Centre for Molecular Medicine, Jawaharlal University, New Delhi, India Introduction : The cross‐talk between alveolar macrophages and pneumocytes is essential to deploy circulating monocytes to infection sites. This study reveals that PQS trafficked by BMVs significantly impairs monocyte recruitment and activation into M1 macrophages, disrupting immune homeostasis. Methods : BMVs were purified using density gradient from clinical strains of P. aeruginosa biofilms and characterized using NTA and TEM and Q‐TOF LCMS. PQS trafficked within BMVs were quantitated using Triple Quad LC‐MS/MS. AT2 ‐ Macrophage ALI co‐culture model was developed using A549 pneumocytes and THP‐1 derived macrophages. PQS extracted from predetermined BMV numbers were exposed to the apical surface of the ALI co‐cultures with LPS. The cells and culture supernatants were quantitated for chemokines CCL2, CCL4, CXCL8, CXCL10 and CXCL11 using qRT‐PCR and CBA Array respectively. Parallel sets were used to study chemotaxis of THP‐1 monocytes and CD14+ sorted cells from human blood using a Combined Boyden‐Flow Cytometry Assay. Monocytes were exposed to PQS under M1‐polarizing conditions and assessed for differentiation through surface marker expression, cytokine profiling, and functional assays. Results : Biofilm‐associated BMVs from clinical P. aeruginosa strains contained elevated PQS levels than the type strain PA‐O1. In the AT2 ‐ Macrophage co‐culture model under ALI conditions, LPS activation induced significantly higher chemokines production (CCL2, CCL4, CXCL8, CXCL10, CXCL11) compared to a 2‐D model, with CCL2 and CXCL8 being most expressed. Notably, PQS suppressed the expression of the chemokines at both mRNA and protein levels by inhibiting the nuclear translocation of NF‐ĸB p65. Suppression of chemokines led to impaired chemotaxis of THP‐1 and CD14+ monocytes. Furthermore, PQS exposure under antigenic stimulation attenuated monocyte activation into M1 macrophages by inhibiting surface markers expression, cytokines and dysregulating energy metabolism. This significantly reduced the overall antimicrobial potential in these cells. Summary/Conclusion : Pulmonary macrophages and pneumocytes work in tandem with circulating monocytes for effective host defense against P. aeruginosa biofilm infections. P. aeruginosa BMVs present unique pathogenic mechanisms that can remotely compromise this first line of defense and exacerbate infections in the lung. Funding : This research is part of Priyakshi Gogoi's and Ambalika Roy's PhD projects at Jawaharlal Nehru University, funded by the Department of Biotechnology, Government of India.

Quantifying

Timea Boroczky 1,2,3 , Maria Harmati 1 , Matyas Bukva 1,2 , Gabriella Dobra 1,2 , Edina Sebestyen‐Gyukity 1,2 , Edit Kotogany 4 , Gabor Szebeni 4 , Peter Horvath 1 , Krisztina Buzas 1,2 1 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, HUN‐REN Biological Research Centre, Szeged, Hungary; 2 Department of Immunology, University of Szeged, Szeged, Hungary; 3 Doctoral School of Interdisciplinary Medicine, University of Szeged, Szeged, Hungary; 4 Laboratory of Functional Genomics, HUN‐REN Biological Research Centre, Szeged, Hungary Introduction : Tumour progression and metastasis are strongly influenced by the intercellular communication between tumour cells and the surrounding tumour microenvironment (TME), with extracellular vesicles (EVs) as key players. EVs carry bioactive molecules that can alter the recipient cells’ behaviour, supporting tumour development. However, a comprehensive understanding of EV dynamics—including both release and uptake by various cell types—remains challenging due to EV heterogeneity and the complex interactions within the TME. Therefore, our goal was to develop a robust, flow cytometry‐based method to quantitatively measure EV dynamics across diverse cell populations in the TME. Methods : To track vesicle release and uptake with precision, EVs were labelled within the donor cells rather than isolating and staining them, preventing artificially high EV concentrations. Tumour and stromal cells were stained with different DiO and DiD dyes respectively, causing each cell to produce vesicles in its corresponding colour. After one day, media were swapped allowing DiO vesicle‐producing cells to take up DiD vesicles, and vice versa. Using flow cytometry, EV dynamics were analysed measuring the uptake and release of dyed vesicles. Vesicle size, concentration and surface markers were also assessed by nanoparticle tracking analysis and flow cytometry. To quantify the EV uptake‐release balance over time, ‘EV‐rate’ was developed. This reproducible and quantifiable measure is suitable for comparing EV dynamics across different cell types, including fibroblasts, endothelial cells, and cancer cells. Moreover, to mimic chemotherapeutic stress, low‐dose doxorubicin was used, and the samples were compared based on EV‐rate. Results : Our data show that the EV‐rate was similar across cell types, indicating balanced uptake and release, although different cell types maintained different EV concentrations. Furthermore, doxorubicin treatment induced a notable shift in EV‐rate, with treated tumour cells showing increased EV production. These findings suggest that chemotherapy may modulate EV‐mediated communication, potentially impacting therapeutic resistance. Summary/Conclusion : This study presents a novel, quantifiable approach, the EV‐rate, to assess EV uptake and release in the TME through flow cytometry, providing a valuable tool for investigating intercellular communication in cancer. Our approach holds promise for future research on targeted EV‐based cancer therapies. Funding : This study was supported by the TKP‐EGA‐09; OTKA‐K143255, EKÖP‐24‐3; MEC_R_149867.

Rna‐Based

Sara Biagiotti, Elena Perla, Debora Libetti, Barbara Canonico, Mattia Tiboni, Francesca Pierigè, Stefano Papa, Luca Casettari, Marzia Bianchi, Luigia Rossi, Michele Guescini, Mauro Magnani University of Urbino, Urbino, Italy Introduction : Extracellular vesicles (EVs) are promising nanocarriers for the delivery of drugs and biologics. Red blood cells (RBCs) are one of the most interesting sources for EV generation since they are easy to obtain, safe, non‐immunogenic and do not contain DNA. However, producing RBC‐derived extracellular vesicles (RBCEVs) loaded with therapeutics is still challenging for several reasons (e.g., production yield, loading efficiency, and translatability to the clinics). Methods : RNA‐loaded RBCEV mimetics were produced by a newly developed and patented physical method starting from preloaded RBCs. Cargo preloading in RBCs was obtained by a process already established and used in clinics. Vesiculation was carried out by ‘soft extrusion,’ a non‐disruptive process that mimics what naturally happens in the spleen. Results : Thanks to their unique biological and physicochemical features, RBCs were efficiently preloaded with several molecules and further used to generate a very high yield of cargo‐loaded RBCEV mimetics. The obtained RBCEV population was deeply characterized according to the MISEV2023 guidelines, and it showed great homogeneity in terms of size, biochemical features, surface antigens and cargo. Feasibility studies have been accomplished at the lab scale using dextran‐conjugates, while the next experiments were carried out with RNAs. In the first application, RBCEVs were used to deliver a small RNA (i.e., miR‐210‐3p) to treat endothelial dysfunctions in Type 2 Diabetes. In vitro results showed a good uptake in HUVEC, efficient delivery, and biological effect, as proved by PTP1B protein inhibition and reduction of mitochondrial metabolism. A second application for delivering a long RNA to treat a rare metabolic disease is at the preclinical level. A synthetic mRNA coding for the GAMT enzyme was efficiently loaded in RBCs and retained in the produced RBCEVs. Here, we successfully scaled up the procedure using an automated extruder, and GAMT mRNA‐loaded RBCEVs were administered to GAMT‐/‐ mice. Preliminary results showed a half‐life in circulation longer than expected. Biodistribution and efficacy studies are ongoing. Summary/Conclusion : Our RBCEV mimetics can be efficiently used for the loading and delivery of biologics and represent a promising tool for the development of RNA‐based therapies, being easily translatable, devoid of chemicals and fully automated. Funding: PRIN 2022 PNRR under Grant P202284WZH.

Stimulatory

Presenter: Peng Wang Radboud University Medical Center, Nijmegen, The Netherlands Introduction : Bovine milk‐derived EVs (BMEVs) have gained interest in recent years for the treatment of bone loss. However, little is known about BMEVs homogeneity and the functionality of subpopulations. Emerging evidence indicates that EVs inherently interact with the meshwork of the extracellular matrix (ECM) through surface molecules. In this case, we utilized the collagen‐binding property of BMEVs for selection and to explore the effects of this ECM‐binding subpopulation in 2D and 3D models of osteogenesis. Methods : BMEVs were isolated by ultracentrifugation and size exclusion chromatography, and their effects on osteogenesis were evaluated using osteogenic differentiation of human bone marrow stem cells (BMSCs) cultured on 2D plastic plates and 3D human bone organ culture models. Collagen‐binding of EVs was observed using IVIS optical imaging. The cargo protein content of BMEVs and collagen‐unbound BMEVs was determined through LC‐MS/MS proteomics and validated by western blotting. A subpopulation of BMEVs was obtained by immunomagnetic separation, and its effect on human BMSCs osteogenic differentiation on 2D collagen plates and 3D collagen hydrogel constructs was investigated through calcium assay and alizarin red staining. Results : BMEVs increased type I collagen expression, alkaline phosphatase level and calcium mineralization of BMSCs and stimulated new bone formation in the defect site of human bone organ cultures as determined by fluorochromes calcein green and alizarin red. Approximately 5% of BMEVs adhered to the collagen plate, and those strongly promoted human BMSCs mineralization. However, the unbound BMEVs showed no enhancing effect on mineralization by human BMSCs when cultured on a 2D collagen plate or in 3D collagen hydrogel culture. Furthermore, removal of the collagen‐binding BMEVs led to a reduction of CD9 and Annexin V protein levels in BMEVs, and Annexin V‐depleted EVs diminished BMEV‐induced mineralization by human BMSCs. Summary/Conclusion : Utilizing the ECM‐binding property of EVs enables selection of a subpopulation of BMEVs that is positive for annexin V and has robust stimulatory effects on osteogenesis.

Subclinical

Mara D. Saenz‐de‐Juano 1 , Giulia Silvestrelli 1,5 , Vadim Krivitsky 2,4 , Adva Krivitsky 2,4 , Sibilla Sander 3 , Jean‐Christophe Leroux 2 , Jörn Dengjel 3 and Susanne E. Ulbrich 1 1 Institute of Agricultural Sciences, ETH Zurich, Switzerland. 2 Institute of Pharmaceutical Sciences, ETH Zurich, Switzerland. 3 Department of Biology, University of Fribourg, Switzerland 4 Acytronix GmbH, Switzerland. 5 Institute of Science and Technology Austria, Austria. Introduction : Subclinical mastitis is a form of mammary gland inflammation that occurs without visible clinical symptoms, explaining its high prevalence and why it can easily spread to other animals in the herd during routine milking. Despite its hidden nature, it negatively impacts milk yield and quality, reduces reproductive performance, and compromises animal welfare. Many studies have demonstrated that mastitis induces changes in milk extracellular vesicles (EVs) cargo. However, common milk EV isolation techniques do not allow the purification of specific milk EV subpopulations. In this study, we used a novel portable microstructured electrochemical device (PMED) to isolate specific CD81‐positive EVs and to assess the differences in particle concentration and proteomic cargo in healthy quarters and quarters affected by subclinical mastitis. Methods : Milk samples were manually obtained from 3 dairy cows with subclinical mastitis. Based on the somatic cell count (SCC), two‐quarters of each cow were selected and classified as either inflamed (high SCC) or healthy (low SCC). Milk fat, cells, and caseins were removed using differential centrifugation, acid precipitation, and filtration. Then, EVs were extracted from skimmed milk using both ultracentrifugation (UC) and the PMED modified to isolate CD81‐positive EVs. Particle size and concentration were obtained using Nanoparticle Tracking analysis (NTA), and proteomic analysis of the EV cargo was performed using bottom‐up proteomics coupled with Data‐Independent Acquisition (DIA). Results : Subclinical mastitis increased the concentration of CD81‐positive milk EVs. When comparing high and low SCC milk EVs, CD81‐positive EVs showed a distinct cargo associated with inflammatory responses, complement cascade activation, and plasma lipoprotein regulation. Our results confirm the role of milk EVs in mastitis pathology and emphasize the importance of examining specific milk EV subpopulations. Summary/Conclusion : The observed alterations in protein cargo between high and low SCC quarters underscore milk EVs' potential diagnostic and prognostic utility in mastitis management. The use of PMED represents a promising advancement for studying milk EV subpopulations.

Suppression

Presenter: Kotb Abdelmohsen NIH, USA Introduction : Cellular senescence, a state of irreversible cell cycle arrest, plays a pivotal role in ageing and age‐related disorders. Understanding senescence and identifying novel therapeutic strategies are critical for mitigating its pathological effects. In this study, we investigated the secretome and extracellular vesicles (EVs) derived from human trophoblast stem cells (hTSCs) for their potential anti‐senescence properties. We found that the hTSC‐S and EVs significantly suppressed the expression of key SASP‐related mRNAs, including CXCL1, CXCL8, and GDF15. We found that the hTSC‐S and EVPs are enriched in proteins involved in extracellular matrix remodelling, cell adhesion, and tissue repair, underscoring their anti‐inflammatory impacts. The hTSC‐S TEMPTEMP effectively reduced both the expression of SASP‐related mRNAs and the secretion of their encoded proteins. These TEMPTEMP effects may be linked to decreased senescence‐associated β‐galactosidase (SA‐β‐Gal) activity and reduced DNA damage. Furthermore, hTSC‐S attenuated inflammatory signalling by inhibiting NF‐κB phosphorylation, a major SASP regulator. In vivo analysis indicated that hTSC‐S treatment decreased the levels of the pro‐inflammatory marker CXCL1 and the ageing marker GDF15. Our findings establish hTSC‐S and EVs as promising candidates for senescence‐targeted therapies, offering a novel strategy to address ageing and age‐related diseases. Methods : Size exclusion chromatography (SEC), EVP isolation by ultraceltifugation, nanoflow cytometry (NFCM), ExoView tetraspanin analysis, transmission electron microscopy Results : hTSC‐secretome suppresses SASP mRNAs, hTSC‐EVs suppress SASP mRNAs, hTSC‐secretome promotes growth and ECM remodelling in IR‐induced senescence, Potential restoration of key proteins in senescence by hTSC‐derived EVP hTSC‐secretome reduces DNA damage and NF‐κB signalling activity hTSC‐secretome reduces inflammatory SASP markers in aged mice. Summary/Conclusion : hTSC‐Secretome suppresses SASP mRNAs; hTSC‐EVs suppress SASP mRNAs; hTSC‐secretome promotes growth and ECM remodelling in IR‐induced senescence; potential restoration of key proteins in senescence by hTSC‐derived EVPs; hTSC‐secretome reduces DNA damage and NF‐κB NF‐κB signalling activity; hTSC‐Secretome reduces inflammatory SASP markers in aged mice. Funding : This research was supported by the National Institute on Aging Intramural Research Program of the National Institutes of Health. The TSC‐secretome was provided by Accelerated Biosciences.

Symptomatic

Alice Hodge 1 , Emily Clarke 1 , Anders Jensen 1 , Olivia Pigden 1 , Paula Briggs 2 , Dharani Hapangama 2 , Mandy J Peffers 1 University of Liverpool, United Kingdom. 1 Liverpool Women's NHS Foundation Trust, United Kingdom.  2 Introduction : The variability in hormone replacement therapy (HRT) efficacy among menopausal women necessitates early identification of responders to optimise costs and outcomes. Extracellular vesicles (EVs), intercellular messengers released in response to triggers, including hormones, may serve as indicators of physiological response to HRT. This study is the first to explore the proteome of plasma EVs in menopausal women and investigate resistance to HRT, aiming to elucidate reasons for non‐responsiveness and to determine if EV protein cargo can predict HRT outcomes. Methods : A cross‐sectional observational study was conducted ( n = 12) with four groups ( n = 3) stratified based on the MENQOL symptom questionnaire: ‘No HRT,’ ‘Implants,’ ‘Standard HRT responders’ (SHRT.R), and ‘Non‐responders’ (SHRT.NR). EVs were isolated using differential ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy (TEM), and western blotting. Proteomic analysis was performed using liquid chromatography‐tandem mass spectrometry, followed by bioinformatics. Results : Characterisation revealed a heterogeneous EV population with sizes ranging from 146.5 to 223.5 nm and concentrations of 3.63 × 10 8 to 5.95 × 10 9 particles/mL across all samples. TEM confirmed phospholipid bilayer morphology, and the western blot confirmed ALIX and CD9 surface markers in all samples. A total of 333 proteins were identified in plasma EVs; 67 were differentially abundant across groups. SHRT‐NR had a different proteome compared to all other groups. Functional enrichment analysis resulted in significantly different canonical pathways, in SHRT‐NR compared to other groups. Regarding predicted upstream analysis, TFGβ was downregulated in SHRT‐NR compared to No HRT ( p = 0.036, z = 1.114) and Implant ( p = 0.004, z = −2.172), and IL‐4 was downregulated in SHRT‐NR compared to No HRT ( p = 0.016, z = 0.447) and SHRT‐R ( p = 0.033, z = −0.343). Significant biofunctions included ‘Production ( p = 0.032, z = 0.830) and synthesis ( p = 0.048, z = 0.916) of reactive oxygen species (ROS) and necrosis’ ( p = 0.045, z = 1.525) upregulated in SHRT‐NR compared to Implant. KRT1, IGKV1‐27 and ADARB1 are potential biomarkers for SHRT‐NR. Summary/Conclusion : SHRT‐NR plasma EVs had a different proteome from other groups. These patients may face increased harm and forgo the bone and cardiovascular protection typically provided by HRT. Identifying specific protein markers could enhance predictive capabilities regarding HRT responsiveness. Funding : University of Liverpool funded.

Synergistic

Presenter: KIM YongWoo Korea University, Seoul, South Korea Introduction : As the importance of extracellular vesicles (EVs) grows in diagnostics and therapeutics, there is a pressing need for technologies that enable both large‐scale EV extraction and high‐purity isolation. Conventional methods struggle to effectively separate EVs from other particles and soluble proteins, creating challenges in clinical and research applications. The ExoTFF system is introduced as a solution, combining electrokinetic mesh filtration (ExoFilter) with size‐exclusion tangential flow filtration (TFF) to enhance EV isolation efficiency. Methods : ExoTFF leverages two complementary filtration techniques. First, TFF removes particles smaller than 30 nm, effectively filtering out impurities. Second, the ExoFilter applies electrokinetic filtration to capture negatively charged EVs, resulting in an optimized separation process. This hybrid system was tested to evaluate improvements in yield and purity over traditional TFF methods. Results : In proof‐of‐concept experiments, ExoTFF successfully processed 10 mL of plasma within 10 min, achieving near‐total removal of albumin and HDL. The hybrid system demonstrated nearly 100% yield and an 800% increase in purity compared to conventional TFF. Further tests with an automated ExoTFF system confirmed these results, maintaining the same yield and purity levels when processing 500 mL of plasma in 50 min. Summary/Conclusion : small‐scale research and industrial EV production. The platform's ability to produce high‐quality EVs efficiently supports the growing demands in precision medicine and therapeutic applications, positioning it as a valuable tool for advancing EV‐based diagnostics and treatments. Funding : This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government, MISP (2016RIA5A1010148).

Telomerized

Regina Grillari 1,2 , Marieke Roefs 1 , Matthias Wieser 1 , Alessia Brancolini 1 , Johanna Gamauf 1 , Matthias Postl 1 , Marianne Pultar 3 , Giulia Corso 1 , Matthias Hackl 3 , Madhusudhan Reddy Bobbili 2,4,5 , Johannes Grillari 2,4,5 1 Evercyte GmbH, Austria; 2 Austrian Cluster for Tissue Regeneration, Austria; 3 TAmiRNA GmbH, Austria; 4 BOKU University, Austria; 5 Ludwig Boltzmann Institute for Traumatology, Austria Introduction : Human MSC‐EVs are widely recognized for their potential therapeutic applications in age‐related diseases and tissue regeneration due to their immunomodulatory and pro‐regenerative properties. However, their clinical use is constrained by the lack of standardized and scalable EV production. Primary MSCs have a limited replicative lifespan and progressively lose differentiation capacity at early population doubling levels (PDLs), casting doubt about the resulting bioactivity of later PDL‐derived MSC‐EVs. Telomerized cell lines can circumvent this limitation; they offer indefinite proliferation potential while maintaining cell‐type specific characteristics. Here, we established different telomerized MSC lines and investigated their potential for therapeutic EV production. Methods : MSCs were isolated from different tissues, including umbilical cord, bone marrow, adipose tissue, placenta, endometrium and dental pulp under xeno‐free conditions with full documentation. Thereafter, the replicative life span was extended by non‐viral overexpression of the catalytic subunit of human telomerase (hTERT). Cell‐type‐specific characteristics were analysed and mRNA profiles were evaluated by NGS. EVs were produced under xeno‐free 2D or 3D culture conditions and isolated using tangential flow filtration. EVs were characterized for their in vitro anti‐inflammatory and anti‐fibrotic properties. Moreover, EVs were characterized for safety aspects, especially in terms of the presence of hTERT. Results : Telomerized MSCs maintained the typical markers and function of primary MSCs while exhibiting an unlimited replicative lifespan. mRNA profiling of the top 10% of genes revealed tissue‐specific differences in MSCs isolated from various human organs, with MSCs derived from different parts of the placenta clustering together. EVs isolated from all telomerized MSCs showed particle sizes in the typical EV range (100–200 nm) and expression of tetraspanin markers. Moreover, they displayed anti‐inflammatory and anti‐fibrotic activity, showing slight differences depending on their MSC origin. Importantly, EVs from primary and telomerized cells do not carry hTERT DNA, full length hTERT mRNA or hTERT protein. Summary/Conclusion : This study demonstrates that telomerized MSCs retain the essential properties of primary MSCs and produce EVs with biological activity without any risk of transferring hTERT to the recipient cells. These findings suggest that telomerized MSCs are a reliable and safe cell factory for producing clinical‐grade EVs, circumventing the limitations of primary MSCs for EV production. Funding : EU‐Horizon‐2020 (#101072766; #721975). Evercyte GmbH; TAmiRNA GmbH.

Therapeutic

Presenter: Young‐Eun Cho Andong National University, Republic of Korea Introduction : Metabolic dysfunction‐associated fatty liver disease (MASLD) and alcohol‐associated liver disease (ALD) represent chronic liver diseases that can progress to steatohepatitis (inflammation), liver fibrosis/cirrhosis, liver failure, and death. Unfortunately, there are few medicines approved for treating liver fibrosis/cirrhosis. Therefore, in this study, we investigated the protective effects of ginseng‐derived extracellular vesicles (GDEVs) administration on liver fibrosis. Methods : We administered GDEVs to mouse and cell models of liver fibrosis. The effects were assessed through histological analysis and biomarker measurements to evaluate fibrosis mitigation. Results : GDEVs successfully reached the gut or liver in both in vitro and in vivo models without much degradation or cytotoxicity. GDEVs administration decreased serum endotoxin levels and restored intestinal barrier proteins with gut microbiome changes in both MASLD and ALD mouse models. Additionally, GDEVs administration reduced oxidative stress and fibrosis maker proteins to baseline levels in both models of MASLD and ALD mice. GDEVs also ameliorated liver fibrosis through regulating TIMP2/MMPs pathways in LX‐2 human stellate cells and MASLD mice. Summary/Conclusion : These results demonstrate for the first time that GDEVs can prevent MASLD‐ and ALD‐mediated intestinal hyperpermeability and liver fibrosis through the gut‐liver axis via restoration of gut microbiome and attenuating oxidative stress and fibrosis marker proteins. Funding : This work was supported by National Research Foundation of Korea (NRF) grant funded by Korea government (MSIT) (No.2021R1C1C1008117, 2023‐00210162, 2022R1C1C100633411).

Translation

Presenter: Dóra Kapui Semmelweis University, Budapest, Hungary Introduction : To implement extracellular vesicle (EV)‐based biomarker analyses for diagnostics and prognostics, isolation protocol resulting in high purity EV samples is needed. An established method based on iodixanol density gradient ultracentrifugation (DGUC) followed by CaptoCore700 bind‐elute size exclusion chromatography (BE‐SEC) was successfully applied to identify EV‐based metabolic alterations correlating with hypercholesteremia‐induced cardiotoxicity in rats. However, this workflow has not been tested for human samples. Thus, our aim was to apply this technology for human blood plasma samples. Methods : EVs were isolated from human platelet‐free plasma by iodixanol DGUC, qEV and Exo‐spin SEC columns, and iodixanol DGUC followed by Sepharose CL‐2B SEC column. EV isolates were characterized by Western blot (WB), nanoparticle tracking analysis, immune electron microscopy, and bicinchoninic acid protein assay. Results : WB analysis of human plasma DGUC isolates showed a significant amount of contaminants previously not detected in rat plasma, which made EV markers undetectable by WB. These results made it impossible to directly assess which DGUC fractions contain human EVs. By isolating EVs with qEV or Exo‐spin column, EV markers could be detected by WB, however, significant lipoprotein contaminants were present. Therefore, to eliminate the lipoprotein contaminants, DGUC was performed, and lipoprotein‐poor, particle‐rich fractions were further purified with a large‐scale Sepharose CL‐2B SEC column, which has similar separation characteristics to qEV columns. This resulted in the isolation of EVs positive for CD81, Tsg101 and HSP70 with lipoprotein contaminants below detection limits. Summary/Conclusion : Using Sepharose CL‐2B SEC column, a methodology similar to earlier used technology, resulted in EVs with lipoprotein contamination below detection limit. The applied method could be used to test the diagnostic potential of circulating EV metabolites in cardiovascular diseases. Further studies to test the effect of the difference in Sepharose CL‐2B versus CaptoCore700 SEC is needed. Funding : The Ministry for Innovation and Technology of Hungary from the source of National Research, Development and Innovation Fund supported CK (EKÖP‐2024‐226 New National Excellence Program), ZG (K139105) and the project (2020‐1.1.5‐GYORSÍTÓSÁV‐2021‐00011). The project was further supported by the European Union (RRF‐2.3.1‐21‐2022‐00003).

Unravelling

Presenter: Maureen Cambier Molecular Angiogenesis Laboratory, Leuven, Belgium Introduction : Immunotherapy has revolutionized cancer treatment, but understanding the resistance to this treatment is crucial for improving outcomes. In this study, we investigate the role of extracellular vesicles (EVs) in immune escape through immune checkpoint protein (ICP) expression, focusing on the PVR/CD155 axis. We developed a new methodology that identifies several ICPs on the surface of EVs in single‐liquid biopsies. Methods : We analysed the expression of several ICPs identified through literature screening and TCGA data analysis. Plasma samples from healthy individuals ( n = 24) and cancer patients ( n = 25) undergoing immunotherapy were collected. EVs were isolated and characterized using differential ultracentrifugation. A longitudinal study with responders ( n = 3) and non‐responders ( n = 3) was also performed before treatment and after 3 and 6 months. ICPs on EV surfaces were identified via MAGPIX (Luminex) analysis. Knockdowns were generated with siRNA and CRISPR‐Cas9 and studied with functional assays. Results : Analysis of TCGA data revealed high levels of PVR/CD155 in lung cancer. EV‐associated PVR/CD155 levels were elevated in patient samples. Furthermore, its expression was even more abundant during immunotherapy in non‐responders, suggesting an emergence of resistance. We studied ICP expression in several human and mouse models and discovered an enrichment of PVR in EVs. Knockdown of PVR/CD155 cells presented reduced migration and proliferation rates in human and mouse models. Summary/Conclusion : Our study introduces a novel method for characterizing circulating ICPs‐EVs in lung cancer patients, with implications for patient monitoring and therapeutic target discovery. Further investigation of PVR/CD155+EV in coculture systems with immune cells will deepen understanding of its role in immunotherapy resistance. Funding : This work is supported by ULiège, the Léon Fredericq Fundation, and the Walloon Region.

Visualizing

Dalya Gulseren 1,2 , Christian G. Specht 2 , Mehdi Kabani 1 1 Université Paris‐Saclay, CEA, CNRS, Labouratoire des Maladies Neurodégénératives (UMR 9199), Fontenay‐aux‐Roses, France; 2 Université Paris‐Saclay, INSERM, Maladies et Hormones du Système Nerveux (U1195), Le KremLin‐Bicêtre, France Christian Specht and Mehdi Kabani are co‐senior authors . Introduction : Misfolding and aggregation of tau into highly ordered amyloid filaments is characteristic of Alzheimer's disease (AD) and other tauopathies. Tau lesions progressively invade the brain by a prion‐like mechanism of self‐replication and spreading of diffusible tau species or ‘seeds.’ Extracellular vesicles (EVs) are major vehicles for tau dissemination, but the ultrastructural and molecular properties of Tau‐containing EVs are scarcely known. Here, we aim at characterizing tau‐containing EVs isolated from AD brains at single‐vesicle resolution using single‐molecule localization microscopy (SMLM). Methods : Post‐mortem human brain tissue samples (parietal and frontal cortex) from AD (Braak V/VI) and non‐demented age‐matched control subjects were obtained from the NeuroCEB brain bank, in accordance with French bioethics laws. After gentle dissociation of the tissues with collagenase, EVs were isolated by ultrafiltration and size‐exclusion chromatography and characterized by immunoblotting and negative staining transmission electron microscopy (TEM). For SMLM, EVs were selectively captured on multichannel coverslips, and then fixed, permeabilized and labelled with specific antibodies against EV marker proteins and their pathogenic cargo. Results : We were able to visualize pathological tau seeds within brain‐derived EVs from AD patients. The simultaneous detection of thousands of immobilized EVs per recording generates comprehensive datasets for categorizing and quantifying tau‐containing EV subpopulations. Summary/Conclusion : Due to its high spatial precision and outstanding sensitivity, SMLM is ideally suited to provide both detailed ultrastructural information as well as quantitative molecular information about the association of Tau with EVs. We have thus far succeeded in detecting tau in AD brain‐derived EVs. We will now apply this methodology for a more systematic analysis of the ultrastructure and molecular composition of EVs in neurodegenerative diseases. Funding : This study was funded by grants from France Alzheimer (M.K.) and the Graduate School of Life Science and Health of University Paris‐Saclay (M.K., C.S.).

Advancements

Presenter: Boi Hoa San Beckman Coulter Life Sciences, Indianapolis, Indiana, USA Introduction : The quantification of fluorescence intensity in flow cytometry is important for instrument sensitivity characterization and standardization of results. One of the key tools in this regard is Molecules of Equivalent Soluble Fluorochrome (MESF) beads. These beads are essential for standardizing fluorescence measurements and ensuring consistency across different experiments and instruments. Methods : We present here our ongoing research and latest advancements in the study of MESF beads. By leverageing new chemistry to incorporate a variety of fluorescent dyes (e.g., Pacific Blue*, SuperNova polymer dyes, FITC, APC, PE), we aim to provide a reliable method for quantifying the number of dye molecules per bead. *Pacific Blue is a registered trademark of Thermo Fisher Scientific Inc. Results : The nano‐sized MESF beads have low fluorescence background and a low known amount of incorporated dye molecules, thus making them particularly suitable for small particle flow cytometry applications. Preliminary evaluation using the CytoFLEX nano flow cytometer has confirmed the potential utility of the new nano‐sized MESF beads. Summary/Conclusion : Once fully developed, these beads are anticipated to become an essential tool for MESF characterization and for standardizing instruments and assays in the growing field of small particle flow cytometry.

Bdellovibrio

Kentaro Jingushi, Atsunari Kawashima, Takuro Saito, Akinaru Yamamoto, Toshihiro Uemura, Sassi Nesrine, Yu Ishizuya, Yoshiyuki Yamamoto, Hiroaki Hase, Norio Nonomura, Kazutake Tsujikawa University of Osaka, Japan Introduction : The gut microbiota characteristic of ICI (immune checkpoint inhibitor) responders has been reported, but how it affects intratumour immune activity in distant tumours is unknown. We have found multiple BEV (bacterial EV) common in the blood of ICI responders of urologic cancer patients and hypothesized that BEV may act as a link between the gut‐tumour axis. In this study, we tested whether the BEV identified in the blood of ICI responders could enhance the therapeutic effect of ICI treatment. Methods : BEVs isolation—Bdellovibrio bacteriovorus Stolp and Starr were grown predatorily on stationary‐phase E. coli prey (16 h, 29°C). Cultured media were centrifuged at 2000 ×  g for 30 min and filtered with a 0.2 um syringe filter. The filtered supernatant was centrifuged at 100,000 ×  g for 90 min to obtain BEVs pellets (Bdello EV). BEVs were subjected to nanoparticle analysis, western blot, and TEM. In vivo experiments: All animal experiments were approved by the Animal Experimentation Committee of the Graduate School of Pharmaceutical Science at Osaka University. Mice allografted with the murine renal carcinoma cell line RenCa received BEV intraperitoneally daily for 2 weeks (1 × 10 9 EV/mouse). In addition, PD‐1 antibody alone or in combination with PD‐1 antibody and BEV was administered along with antibiotics in drinking water. Results : Administration of the Bdello EV to a renal cancer allograft mouse model resulted in antitumour effects. We also found that administration of Bdello EV markedly increased the activity of intratumoural T cells. Furthermore, antibiotic administration decreased gut bacteria and suppressed the antitumour effect of the PD‐1 antibody, whereas concomitant administration of Bdello EVs resulted in antitumour effect of mouse PD‐1 antibody. Summary/Conclusion : Bdello EV found in ICI responder blood had the potential to enhance the therapeutic effect of ICI. And bacterial EVs may play a role in the gut‐tumour axis.

Bead‐Based

Presenter: Erez Eitan NeuroDex Inc., Natick, Massachusetts, USA Introduction : All cell types release extracellular vesicles (EVs), some entering the circulation. Typically, EVs are characterized after isolation via nanoparticle tracking analysis, though this approach can introduce variability due to isolation procedures and contamination from non‐EV particles. In this study, we applied a sandwich immunoassay with antibodies targeting two distinct proteins to directly measure CD63‐, CD9‐, and CD81‐positive EVs in plasma. Methods : We utilized a bead‐based multiplex immunoassay based on the Luminex system. EV analysis was validated following the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines and immunoassay qualification protocols to ensure specificity, sensitivity, linearity, and precision. Results : Both singleplex and multiplex assays produced comparable signals. Specific antibodies generated at least threefold higher signals than non‐specific antibodies, and EV depletion eliminated the signal, confirming specificity. Variability between wells and plates was below 15%, and parallelism was demonstrated across 16‐fold dilutions. Using this method, we assessed CD63‐, CD81‐, and CD9‐positive EV levels in 205 individual plasma samples. CD9‐positive EVs were significantly more abundant than CD63 and CD81. In healthy individuals ( N = 80), plasma EVs had coefficients of variance of 22.4% for CD9, 65.76% for CD63, and 71.7% for CD81. Neurodegenerative patients ( N = 78) showed significantly higher CD9‐positive EVs (+10%, p = 0.002) and significantly lower CD63 and CD81‐positive EVs (−27%, p = 0.002 for CD63; −28%, p = 0.03 for CD81). Post‐COVID patients ( N = 47, 2–5 months post‐infection) showed no significant EV differences. In 17 matched plasma and serum samples, EV levels (regardless of tetraspanin) were ∼1.8‐fold higher in plasma, with a strong plasma‐serum correlation ( R = 0.71). A slight age‐related EV decrease was observed, with CD63 showing significance ( R = 0.49, p = 0.02). Analysis is ongoing in a cohort of 1200 individuals. Summary/Conclusion : This method enables semi‐high‐throughput measurement of EV surface proteins in unprocessed plasma samples in a 96‐well format, supporting population‐level EV analysis and insights into factors driving EV variability. It is adaptable to other EV surface proteins, facilitating biomarker discovery and normalization efforts.

Biotinylated

Perla Elena 1 , Biagiotti Sara 1 , Tiboni Mattia 1 , Agostini Rachele 1 , Nozza Antonio 1 , Canonico Barbara 1 , Guescini Michele 1 , Rossi Luigia 1 , Magnani Mauro 1 1 Department of Biomolecular Sciences, University of Urbino, Italy Introduction : Red blood cells‐derived extracellular vesicles (RBCEVs) have recently emerged as promising tools for targeted drug and biologics delivery. Cargo‐loaded RBCEVs can be efficiently produced starting from preloaded RBCs using the innovative “soft extrusion” technique. To date, the produced RBCEVs have been employed in both in vitro and in vivo studies, leverageing their inherent uptake and biodistribution characteristics. Additionally, biotinylation is a fruitful strategy for attaching various peptides and antibodies to cell or EV surfaces, enabling specific targeting of particular cell types or tissues. This method is fully biocompatible and safe, so it has already found applications in clinical settings. The present work investigates the biotinylation of RBCEVs as a method for membrane engineering and targeted delivery, focusing on enhancing specificity for cardiomyocytes. Methods : Two biotin derivatives, NSH‐biotin and NSH‐LC‐biotin, were employed to biotinylate RBCEVs. Both the efficiency of biotinylation and cell recovery were assessed. Furthermore, flow cytometry analyses with Alexa Fluor‐streptavidin were conducted to quantify the degree of biotin incorporation. A three‐step delivery system was employed to target cardiomyocytes. It consists in the administration of the biotin‐Cx43 Ab followed by the introduction of avidin, which forms a stable complex with the Ab. Finally, biotinylated RBCEVs were decorated with the specific antibody biotin‐Cx43 using an avidin bridge and administered to allow them to attach via avidin's biotin‐binding sites. Results : Both biotinylation strategies demonstrated high efficiency and satisfying cell recovery. Flow cytometry analysis confirmed successful biotin incorporation on RBCEVs, facilitating specific targeting of cardiomyocytes via the biotin Cx43 Ab‐avidin bridge. The functionalization of RBCEVs with the antibody resulted in enhanced binding to target cells, highlighting the effectiveness of this approach for directed delivery. Summary/Conclusion : Both biotinylation strategies demonstrated high efficiency and satisfying cell recovery rates. Flow cytometry analysis confirmed successful biotin incorporation on RBCEVs, facilitating specific targeting of cardiomyocytes via the biotin Cx43 Ab‐avidin bridge. The functionalization of RBCEVs with the antibody resulted in enhanced binding to target cells, highlighting the effectiveness of this approach for directed delivery.

Caveolin‐1

Rachele Agostini 1 , Emanuela Polidori 1 , Paola Ceccaroli 1 , Laura Graciotti 2 , Stephanie Fondi 1 , Michela Battistelli 1 , Francesca Luchetti 1 , Silvia Codenotti 3 , Gabriella Pocsfalvi 4 , Massimiliano Bonafè 5 , Joao Ferreira 6 , Alessandro Fanzani 3 , Vilberto Stocchi 1 , Michele Guescini 1 1 Department of Biomolecular Sciences (DISB), Università degli Studi di Urbino Carlo Bo, Urbino, Italy; 2 Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy; 3 Department of Molecular and Translational Medicine (DMMT), Università degli Studi di Brescia, Brescia, Italy; 4 National Research Council (CNR), Napoli, Italy; 5 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Università di Bologna, Bologna, Italy; 6 Department of Proteostasis and Intercellular Communication, NOVA Medical School, Lisbon, Portugal Introduction : Caveolin‐1 (CAV1) is a transmembrane protein enriched in caveolae and lipid rafts, which are involved in signal transduction and intracellular trafficking processes. In rhabdomyosarcoma (RD) CAV1‐overexpression promotes tumour growth and metastatic dissemination. Since extracellular vesicles (EVs), lipid‐bound vesicles secreted by all cells, have a well‐established role in cancer disease, the present work aims to investigate if CAV1‐overexpression impacts the EV machinery and if these EVs can contribute to their increased aggressiveness. Methods : Three RD cell lines were employed for the study: RD‐Mock (transfected with an empty vector), RD‐CAV1‐F0 engineered for CAV1 overexpression, and RD‐CAV1‐F2 derived from the second generation of lung metastases after RD‐CAV1‐F0 injection in mice. EVs were isolated from conditioned media by sequential ultracentrifugation, and small‐EVs (sEVs) were further purified by density gradient centrifugation. For EV characterization, nanoparticle tracking analysis (NTA), western blot (WB), flow cytometry (FC) and proteomic analysis were employed. Intracellular trafficking was evaluated by immunofluorescence staining, WB analysis, endosome isolation and live cell imaging. Migration and proliferation assays were conducted on HUVECs. Results: The obtained data showed that RD‐CAV1 cells release more EVs compared to RD‐Mock. WB and FC analyses revealed that RD‐CAV1 sEVs exhibit TSG‐101 and Alix, but the expression of other typical exosomal markers CD63, CD81, and CD9 is much lower than in RD‐Mock. Proteomic analysis extended this alteration to other proteins, showing an overall reduction in protein loading and expression. These findings are combined with an increased intracellular trafficking in RD‐CAV1 cells, suggesting that CAV1‐overexpression induces an alteration of EV machinery and release. Moreover, RD‐CAV1 sEVs significantly increased HUVEC proliferation and migration compared to the control. Summary/Conclusion : Taken together, these data demonstrate that CAV1‐overexpression critically affects RD‐intracellular trafficking and EV cargo and release, which might contribute to RD‐CAV1 increased aggressiveness. Future studies will focus on the characterization of RD‐EV lipid‐ and miRNA‐loading and on the evaluation of RD‐EV effects in other cell types typical of tumour niche.

Cell‐Based

Invited Speaker: Steffen Goletz Technical University of Denmark, Lyngby, Denmark

Chemotherapy

M. Vecchitto 1 , M. Norimoto 1 , K. Funk 1 , Z. Wang 1 , T. Arai 1 , S. Martellucci 1 , S. L. Gonias 2 , W. M. Campana 1,3 1 Department of Anesthesiology, University of California, San Diego, La Jolla, California, USA; 2 Department of Pathology, University of California, San Diego, La Jolla, California, USA; 3 San Diego VA HealthCare System 3 , San Diego, California, USA Introduction : Painful peripheral neuropathy (PPN) is a severe side effect of chemotherapy, manifested as nerve toxicity and neuropathic pain. In cancer patients with PPN, treatment adherence is often compromised, resulting in suboptimal cancer therapy. Treatment options for PPN are limited. We hypothesized that extracellular vesicles (EVs), which participate in cell‐to‐cell communication, may regulate PPN and thus, represent a therapeutic target. Methods : We describe a novel method for capturing peripheral nerve‐derived EVs (pn‐EV) from extracellular spaces of the sciatic nerve. We applied this method to inducible turboGFP‐EV reporter (TIGER)‐P0‐Cre mice, which express tGFP only in Schwann cells (SCs). Pn‐EVs were isolated by centrifugation and size exclusion chromatography. nanoparticle tracking analysis (NTA) and electron microscopy revealed a concentration of ∼8.0 × 10 10 particles/mL, a modal diameter of 147 nm and crescent morphology. Pn‐EVs contain EV biomarkers, flotillin and TSG101, and glial biomarkers, CD9 and P0. Cellular proteins GM130, β‐actin and βIII‐tubulin were not detected. SC EVs were identified by tGFP using a ZetaView QUATT and super resolution microscopy with direct stochastic optical reconstruction (dSTORM). Results : Fluorescence NTA revealed that > 30% of pn‐EVs were derived from SC EVs with a size of 109 nm. When pn‐EVs were injected into mouse hind‐paws, no pain‐related behaviours were identified. However, when pn‐EVs were injected prior to inducing acute pain with capsaicin, pain‐related behaviours increased ( p < 0.05), suggesting that pn‐EVs exacerbate pain states. Next, we treated mice with the chemotherapeutic, paclitaxel (PTX), which induces mechanical and cold hypersensitivity. Pn‐EVs isolated after PTX treatment demonstrated no change in size or concentration. However, dSTORM and fluorescence NTA demonstrated a decrease ( p < 0.05) in pn‐EV fluorescence following PTX, indicating a decrease in the fraction of pn‐EVs derived from SC EVs. The modal size of PTX‐treated SC EVs was larger ( p < 0.05). Naïve and PTX‐treated pn‐EVs showed differential Akt/ERK1/2 signalling in primary sensory neuronees, suggesting distinct biological properties. Summary/Conclusion : Collectively, these studies establish a novel method for isolating peripheral nerve EVs in vivo, show that SCs are major contributors to the pn‐EV population in the peripheral nerve, and demonstrate that the cellular source and activities of pn‐EVs may change in PPN. Funding : This study was supported by Veterans Administration I101RX003363 to WM Campana.

Construction

Presenter: Yicong Xue Southern Medical University, Guangzhou, People's Republic of China Introduction : Colorectal cancer (CRC) is a major health challenge, ranking third in incidence and second in cancer‐related mortality. Evidence highlights the interplay between CRC and the gut microbiome. Bacterial extracellular vesicles (BEVs), originating from bacteria, contribute to inter‐bacterial and bacteria‐host communication by transporting proteins, lipids, and nucleic acids. BEVs from gut bacteria may play a critical role in CRC pathology. Investigating BEVs from human faeces is essential for understanding gut BEV heterogeneity in CRC, emphasizing the need for improved isolation and purification methods. This study optimized BEV isolation and applied it to assess BEVs’ diagnostic potential in CRC. Methods : BEVs from gram‐positive and gram‐negative bacteria, as well as from human faeces, were analysed to evaluate their distribution in density gradient centrifugation (DGC) using both top‐down and bottom‐up methods, along with size exclusion chromatography (SEC). These methods were optimized individually and in combination to establish the most effective separation strategy. Evaluation metrics included particle morphology, size, concentration, and specific markers (LPS, OmpA, LTA for BEVs). These were analysed using TEM, NTA, NanoFCM, and WB. DNA from fecal bacteria and fecal BEVs in both healthy individuals and CRC patients was amplified (16S rRNA gene v3‐v4) and sequenced using Illumina MiSeq. A random forest model was developed to assess diagnostic performance through ROC analysis. Results : A concentration of BEVs was observed in DGC fractions 6‐8 (F6‐8), with F5 being rich in eukaryotic EVs, and F4‐7 for SEC. The combination of top‐down DGC and SEC was identified as the optimal approach for isolating fecal BEVs from clinical samples, yielding high purity and more protein types regarding faecal BEVs, with a faecal BEVs concentration of 10 8 /mg. Sequencing results showed marked compositional differences between gut bacteria and fecal BEVs, notably between healthy and CRC individuals, both at the phylum and genus levels. Compared to whole bacteria, faecal BEVs demonstrated equally important diagnostic potential with fewer genus‐level types. Summary/Conclusion : The optimized method enables standardized BEV isolation, providing a robust foundation for multi‐omics and functional analyses. Significant changes in the types and abundance of fecal BEVs in CRC underscore their potential as diagnostic markers. Funding : This study was supported by the National Science Fund for Distinguished Young Scholars (82025024).

Contribution

Vásquez‐Pérez Jorge Manuel 1,2 , Flores‐Ramos Mónica 1 , Avendaño‐Estrada Arturo 2 , Ruiz‐May Eliel 3 , Ramos‐Godínez María del Pilar 4 , Ramírez‐Rodríguez Gerardo Bernabé 1 1 National Institute of Psychiatry Ramón de la Fuente Muñiz, Mexico City, Mexico; 2 National Autonomous University of Mexico, Mexico City, Mexico; 3 National Institute of Ecology, Mexico City, Mexico; 4 National Institute of Cancerology, Mexico City, Mexico Introduction : Major depressive disorder (MDD) is one of the most common and disabling psychiatric disorders worldwide. It is classified as an affective disorder, as described in the DSM‐V. It is considered a multifactorial disorder, with influence on the central nervous system (CNS) and endocrine and immunological systems, impacting different neurochemical and inflammatory pathways through common signalling mechanisms such as metabolites, soluble factors, and even extracellular vesicles, such as exosomes. Methods : Four groups of samples were included: healthy female controls ( n = 12), women diagnosed with depression ( n = 12), healthy female Balb/C control mice ( n = 10), and Balb/C mice under the mild chronic unpredictable mild stress (CUMS) protocol ( n = 10). EVs were isolated from human and murine blood serum using size exclusion chromatography (qEV1 70 nm, Izon Science). We characterized the exosomes by transmission electron microscopy, Exo‐Check Antibody Array (Neuro) Standard Kit and ExoELISA‐ULTRA CD63 assay (SBI, System Biosciences). We used human and murine (AAM‐CYT‐1000‐8, RayBiotech) arrays to analyse their protein content. Human CTRL and MDD exosomes were administered using robotic stereotactic surgery, and we performed a positron emission tomography (PET) functional analysis to identify CNS metabolic changes and depressive‐like behaviour associated with the exosomes that were administered. Results : Three statistically different protein sets were identified ( p < 0.05): 37 proteins from human exosomes (CTRL vs. MDD), 18 proteins in murine exosomes (CTRL vs CUMS), and 12 proteins from the groups of human and murine exosomes. We used STRING and UniProt to identify protein‐protein interaction (PPI), and gene ontology functional bioinformatics analysis was performed using WEBGESTALT and the KEEG database. Signalling pathways associated with MDD and chronic stress pathophysiology were identified: cytokine‐cytokine receptor interaction, JAK‐STAT signalling, and chemokine signalling. In addition, the exosomes administered caused depressive‐like behaviours in the mice in the forced swim and tail suspension tests. Summary/Conclusion : Our findings suggest the importance of exosomes in MDD, highlighting in part the validity and homology of murine models and their correlation with clinical observations, highlighting CUMS as an efficient model for the development and experimental modelling of subjects that replicate depressive‐like behavioural aspects with apparently similar signalling pathways. Funding : S.I.C.‐2000, Fondo Sectorial de Salud‐CONACYT 262307; Ciencia de Frontera 2023, CONAHCYT, Mexico (Proyecto CF‐2023‐I‐2651).

Customizable

Isabella Pesce 1 , Marinella Pinelli 2 , Lorenzo Lunelli 3,4 , Cristina Potrich 3,4 , Martin M. Hanczyc 2 1,2 University of Trento, Trento, Italy; 3 Fondazione Bruno Kessler‐Trento, Trento, Italy; 4 Italian National Research Council‐Trento, Trento, Italy Introduction : Characterization of extracellular vesicles (EVs) by optical methods is challenging due to their small size, high polydispersity and low refractive index. Their limited abundance in biological fluids and low antigen density make their analysis by flow cytometry even more complex. Flow cytometers with improved sensitivity and detection limits and suitable standardized methods can greatly contribute to EV characterization. By taking advantage of both the ImageStreamX MkII imaging flow cytometer and the BD‐FACSymphony A1 flow cytometer equipped with the small‐particle detector, we aim at setting up suitable reference nanoparticles based on biotinylated liposomes for small particle analysis. Biotin is exploited for the binding of fluorophores conjugated with streptavidin, obtaining useful and flexible tools for the optimization of flow cytometer settings and protocols. Methods : Biotinylated liposomes of different lipid compositions are prepared both as giant unilamellar vesicles obtained by droplet transfer followed by extrusion and as large unilamellar vesicles obtained by direct extrusion of multilamellar vesicles. Liposomes are characterized for size and concentration with NanoSight NS300. Monovalent and tetravalent streptavidins conjugated with various fluorophores are used to label biotinylated liposomes, and streptavidin fluorescence is quantified by using commercially available molecules of equivalent soluble fluorochrome (MESF) calibration beads. Results : Biotinylated liposomes were prepared by two standard methods and then varied by diameter, lipid composition, concentration and amount of added fluorescent streptavidin to establish the best protocol for the preparation of standard fluorescent calibrators for flow cytometry. The stability of biotinylated liposomes as size and concentration was evaluated over a 2‐month period, resulting in small variations. Biotinylated liposomes stained with different combinations of fluorescent streptavidin gave efficient binding and fluorescent signals after 30 min, showing the versatility of this tool for the different applications. Preliminary tests were performed to assess the potential of biotinylated liposomes as calibration nanoparticles for EV analysis. Summary/Conclusion : We successfully developed new versatile nanoparticles as fluorescent calibration nanoparticles for small particle analysis by flow cytometry. The proposed biotinylated liposomes have the huge advantage of being more similar to natural EVs with respect to standard polymeric calibration beads.

Differential

Presenter: Chul Won Seo Soonchunhyang University, Asan, Chungcheongnam‐do, Republic of Korea Introduction : Mutations in RAS and RAF play critical roles in the pathogenesis of various cancers, including colorectal and lung cancer, influencing their progression, diagnosis, and treatment strategies. Importantly, these mutations alter the release of extracellular vesicles (EVs), affecting their uptake efficiency, systemic circulation, and molecular heterogeneity. In this study, we applied nano‐flow cytometry, quantitative proteomics, high‐throughput uptake screening, and in vivo tracking to analyse EVs released by cancer cells with KRAS and BRAF mutations. Methods : EVs were isolated by size exclusion chromatography from wild‐type (Caco2, H292, H1703), BRAF‐mutant (HT29, WiDr), and KRAS‐mutant (HCT116, LoVo, A549, H358) colorectal and lung cancer cells. EV uptake and their subcellular trafficking (endoplasmic reticulum, nucleus, lysosome) were measured using automated microscopy. The biodistribution of EVs in mice was measured using IVIS imaging. EV proteomes were analysed with an Orbitrap mass spectrometer and processed with MaxQuant. For nano‐flow cytometry, EVs were labelled with fluorescently tagged antibodies (CD147, CD44, CD63, CD9, CD81) to analyse their subtypes. Results : High‐throughput uptake screening with fluorescent‐labelled EVs in A549 cells showed that HT29 and LoVo EVs had unique trafficking efficiency to the endoplasmic reticulum compared to other EVs. However, in HEK293T cells, these EVs were primarily trafficked to lysosomes, suggesting that EV uptake and cellular utilization may depend on both EV and cell types. Additionally, IVIS imaging showed that colorectal cancer HT29 EVs preferentially trafficked to the caecum and intestines, while lung cancer H292 EVs primarily targeted the lungs. Proteomic analysis revealed unique surface proteomes influenced by RAS and RAF mutations. Specifically, ECM‐receptor interaction, focal adhesion, and junction proteins were regulated by these mutations. Notably, surface proteins such as CSPG4, LSR, CD44, CD133, NRP‐1, and various integrins were increased in EVs with these oncogenic mutations. Summary/Conclusion : In summary, this research on EV heterogeneity and preferential uptake advances our understanding of intercellular communication by EVs in the context of oncogenic transformation. By identifying targeting proteins on EVs and their specific trafficking to subcellular locations, this study would unlock new avenues for developing novel drug delivery platforms that enable the efficient transfer of therapeutic cargo. Funding : RS‐2024‐00348103 and RS‐2023‐00219563.

Establishing

Clara Bernardelli, Eduardo Sommella, Piera Selvaggio, Vicky Caponigro, Valentina Citro, Domenica Giannandrea, Ehsan Soleymaninejadian, Carla Martinelli, Monica Miozzo, Pietro Campiglia, Raffaella Chiaramonte, Elena Lesma University of Milan, Milan, Italy Raffaella Chiaramonte and Elena Lesma are co‐senior authors . Introduction : Saliva is a complex oral biofluid that has gained considerable attention in biomarker identification due to its non‐invasive and simple collection. Thus, saliva analysis represents a potentially cost‐effective approach for large population screening and follow‐up studies. Among particles secreted in saliva, extracellular vesicles (EVs) are emerging as potential diagnostic and predictive tools. Here we present an effective isolation protocol and proteomic analysis of salivary EVs that could prospectively be used to identify EV‐related biomarkers to monitor autoimmune diseases such as type 1 diabetes (T1D) and multiple sclerosis (MS). Methods : Saliva specimens were collected with lollipops and saliva rolls from different groups of healthy donors ( n = 30) matched for gender and age ( 45 years). Saliva samples were collected in the morning, 1 h after the mouthwash and fasting from food and water, and were cleared by differential centrifugations, with the last at 100,000 ×  g . Ten micrograms of intact EVs were used for proteomic analysis with nLC‐HRMS/MS and subsequent statistical and bioinformatic analyses, including PCA and Gene Ontology, and literature review to identify potential EV‐related disease biomarkers. Results : Saliva rolls allowed the collection of 3 to 5 mL. Post‐isolation EVs integrity was demonstrated by TEM microscopy. Saliva EVs are positive for CD81 and TSG101. Nanoparticle tracking analysis (NTA) showed no differences in EVs size and number between male and female and across the different ages. Proteomics analysis in label‐free quantification yielded 1225 annotable features in data‐dependent acquisition, while > 2000 annotable and quantifiable proteins were obtained with data‐independent acquisition. The PCA shows a perfect separation between groups and the greatest impact of the age factor on dataset clusterization. Gene ontology revealed the enrichment in components related to EVs and biological functions associated with inflammation and immune response. Accordingly, the literature review showed the presence of potential EV‐associated biomarkers for T1D and MS related to inflammation, complement, lipid metabolism and beta‐cell function regulation. Summary/Conclusion : Our protocol provides an optimal EVs recovery and a high‐throughput proteomic analysis which promises to be useful for the identification of salivary EV biomarkers to monitor T1D and MS. Funding : This study was supported by the Project PNC 0000001 D3 4 Health, CUP B53C22006080001, and funded by NextGenerationEU.

Fluorescence

Presenter: Hyeonseung Lee Korea Basic Science Institute, Daejeon, Republic of Korea Introduction : Extracellular vesicles (EVs) have emerged as promising therapeutic targets, and understanding their biodistribution is essential for evaluating potential effects. This study presents a novel quantitative polymerase chain reaction (qPCR) assay to detect unmodified EVs by targeting mitochondrial DNA (mtDNA) unique to human EVs. Methods : We focused on specific mtDNA regions with distinct variations from rodent mtDNA to enhance specificity. We developed primers and probes for both human and rodent mtDNA, resulting in a sensitive and specific qPCR method. We also found that the quantification of EVs varied based on the cell source and manufacturer. Results : To assess biodistribution, we administered IRDye 800CW‐labelled Expi293F EV mimetics to animals via tail vein injection and compared imaging results with mtDNA‐qPCR data. Summary/Conclusion : This study established a novel qPCR‐based molecular platform that can analyse EVs without manipulation using certain mtDNA primers and probes. The method demonstrates high sensitivity and allows quantitative assessment of EV particles at the level of individual particles in the body. The technique also facilitates the analysis of EVs present in blood samples that are difficult to visualize using an imaging approach. In addition to mtDNA, additional nucleic acid targets that can distinguish EVs should be identified, and optimal quantification methods tailored to each EV should be devised.

High‐Yield

Carlos Jesus 1,2,3 , Miguel Lino 2,3 , Hugo Fernandes 1,2,3,4 , Lino Ferreira 1,2,3 1 Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 2 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 3 Centre for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal; 4 Multidisciplinary Institute of Aging (MIA‐Portugal), University of Coimbra, Coimbra, Portugal Introduction : The full regenerative/therapeutic potential of extracellular vesicles (EVs) isolated from mesenchymal stromal/stem cells has been demonstrated in the setting of several diseases, including cardiovascular diseases. However, the limited scalability of production of therapeutically relevant EVs hinders its clinical translation. Here, we used a hollow fibre bioreactor to improve the yield of native EVs from MSC whilst maintaining their therapeutic potential. Methods : We used human Wharton's Jelly MSC (WJ‐MSC)—upon informed consent—due to its easy isolation from the umbilical cord, retention of primitive stem cell characteristics and well‐known therapeutic capacity. A minimum of 100 million WJ‐MSC were inoculated, and conditioned media was collected every day. EV isolation was done by differential ultracentrifugation followed by purification by size exclusion chromatography. EVs were characterized according to their concentration, size, protein amount, surface charge and tetraspanins expression. We further validated EVs bioactivity in a scratch assay using endothelial cells. Results : Compared to EVs secreted by cells cultured in 2D systems (T‐175 flasks), our results showed that EVs produced by the bioreactor are slightly bigger (203.2 vs. 161.9 nm), showed high expression of CD63 (93.0% vs. 97.8%) and similar surface charge (−24.3 vs. −20.9 mV). In addition, we showed a 3.3‐fold increase of EV collection per day, as compared to a single isolation using 10–12 T‐175 flasks. We were able to isolate over 2.0 × 10 12 EVs in total (after purification) in a 15‐day period. Our results showed that the bioactivity of EVs generated in a bioreactor is stronger (73.4% vs. 40.8%) compared to EV from 2D systems. Summary/Conclusion : Overall, hollow fibre bioreactors are a suitable method for scalable production of EVs, maintaining their therapeutic properties. Funding : The authors would like to acknowledge the funding by the FCT PhD Studentship (SFRH/BD/144092/2019), Programa Operacional Competividade e Internacionalização (POCI) na sua componente FEDER e pelo orçamento da Fundação para a Ciência e a Tecnologia na sua componente OE (Project 2022.03308.PTDC; 2022.07615.PTDC; 2022.02803.PTDC); EC projects REBORN (Ref. 101091852); PRR project HfPT—Health from Portugal (Ref: 02/C05‐i01.01/2022.PC644937233‐00000047); and ‘Project RESET_BONE_AGING_2’ (Ref: COMPETE2030‐FEDER‐01175000). MIA‐Portugal (European Union's Horizon 2020 No. 857524).

Horizontally

Cong Hu 1 , Tianyang Wu 1 , Yinjie Zhu 1 , Xinxing Du 1 , Xinrui Wu 1 , Yanhao Dong 1 , Zehong Peng 1 , Kenneth J. Pienta 2 , Jiahua Pan 1 , Liang Dong 1 , Wei Xue 1 Kenneth J. Pienta, Jiahua Pan, Liang Dong, and Wei Xue are co‐senior authors. 1 Department of Urology, Ren Ji Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 The Cancer Ecology Center, The Brady Urological Institute, Johns Hopkins School of Medicine, Baltimore, USA Introduction : Prostate cancer (PCa) incidence is increasing rapidly, and metastatic PCa remains incurable. The tumour microenvironment exerts a multifaceted and important influence on tumour metastasis. Tumour‐associated macrophages (TAM) are important components of the tumour microenvironment, most of which favour the M2 phenotype that facilitates PCa metastasis. However, the precise mechanisms underlying this process remain inadequately understood. Extracellular vesicles (EVs) are pivotal mediators of intercellular communication, with the capability to transmit molecular signals; however, their involvement in TAM‐mediated PCa metastasis remains to be elucidated. Methods : Single‐cell RNA sequencing was employed to investigate the infiltration of M2 macrophages in metastatic and non‐metastatic PCa (mPCa, nmPCa) primary foci. M2 macrophages were induced in vitro from THP1. Collected cell culture medium (CCM) from M2 macrophages CCM‐isolated EVs were employed for co‐incubation with PCa, and the effects of M2 macrophage CCM and the EVs in it on PCa metastasis were explored by transwell and scratch assays. To further investigate what components of EVs could mediate the above effects, we focused on co‐altered mRNA molecules after treatment of PCa cell lines with M2 EVs by RNA sequencing. The horizontal cell‐to‐cell delivery of mRNAs was further explored by detecting constructed fluorescent‐tagged mRNAs in EVs with nanoflow cytometry. Results : Compared with nmPCa, mPCa primary foci exhibited greater infiltration of M2 macrophages, and M2 macrophage‐derived EVs accounted for a higher proportion of total tissue EVs. M2 EVs were able to alter the mesenchymal phenotype of PCa cells and promote metastasis of PCa. We found that TXNDC5 mRNA was co‐elevated in two DU145 and PC3 cell lines after treatment with M2 EVs. We subsequently further demonstrated that TXNDC5, as well as TXNDC5 in M2 EVs, promotes PCa metastasis. By exogenously transfecting Cy3‐tagged TXNDC5‐Flag fused mRNA into M2 macrophages, we demonstrated the presence of TXNDC5 mRNA in M2 EVs by nanoflow cytometry and confocal microscopy to validate cellular horizontal transfer. Summary/Conclusion : We demonstrated for the first time that horizontally intercellular delivery of TXNDC5 mRNA‐containing small extracellular vesicles contributes to TAM‐mediated PCa metastasis.

Interkingdom

Zóra Szilovics 1 , Éva Veres 1 , Krisztina Buzás 2 , Attila Gácser 1 1 University of Szeged, Hungary. 2 Biological Research Centre, Hungary. Introduction : The human oral cavity is colonized by more than 700 microbes, such as bacteria, viruses, fungi, known as the oral microbiota. As a result of environmental effects, such as smoking or infections, the microbial composition may change, which can result in dysbiosis that may lead to diseases, such as oral candidiasis. Oral candidiasis is most commonly caused by Candida albicans, which can alter the bacterial diversity. To examine the nature of such fungal‐bacterial interactions, we aim to investigate the interaction between Candida species‐ and oral pathogenic bacteria at the level of extracellular vesicles (EV). Methods : For our experiments we used the C. albicans SC5314 and C. parapsilosis CLIB214 strains, along with Staphylococcus aureus as pathogenic bacterial counterpart. We optimized the fungal and bacterial EV isolation protocol from solid media. The characterisation of the EVs by transmission electron microscopy and NanoSight showed round shaped particles with diameters between 50 and 250 nm. We examined the effects of EVs released by C. parapsilosis and the yeast and hyphae form of C. albicans on the growth and biofilm formation efficiency of S. aureus and vica versa. Results : As a results, we found that EVs from C. albicans and C. parapsilosis had different effects on the growth and biofilm formation efficiency of S. aureus. Regarding the effect of bacteria, the S.aureus EV treatment can induce the hyphae formation of C. albicans cells. Summary/Conclusion : Altogether these results suggest the presence of an active interaction between fungal and bacterial cells at the level of EVs.

Isolation‐

Presenter: Pascal Rüedi Institution ETH Zurich, Zurich, Switzerland Introduction : Understanding how extracellular vesicle (EV) subpopulations relate to specific biological states and functions remains a long‐sought goal. However, EV heterogeneity poses significant challenges for their isolation, detection, and characterization. While single EV characterization techniques have advanced, they rely heavily on the quality of the input samples, making them susceptible to biases introduced during isolation and purification. Here we introduce an optofluidic platform that characterizes EV subpopulations with single particle resolution directly in complex sample matrices without the need for isolation steps, at high‐throughput and in a label‐free manner. Methods : The optofluidic platform features a PDMS chip with parallelized single‐input, single‐output channels for multiplexing and rapid screening. As a key‐enabler for isolation‐free analysis, we developed a novel rapid immunoaffinity functionalization method (< 5 min of hands‐on time), ensuring surface passivation against non‐specific binding while selectively capturing EV subpopulations based on pan‐EV markers (CD9, CD63, CD81). The surface combines biomimetic membranes, and bio‐orthogonal antibody conjugation compatible with label‐free optical measurements. EVs from lung cancer cells (H358) were profiled, either isolated via size exclusion chromatography or directly from unpurified cell culture supernatant. Imaging was performed with a custom digital in‐line holography microscope, enabling single‐EV sensitivity and continuous monitoring of binding kinetics. The platform requires only 50 µL of sample and a 10‐min assay per channel for high‐throughput analysis. Results : Following validation with purified EV samples and functionalized liposomes, the platform successfully characterized EVs from unpurified cell culture supernatant while maintaining near‐zero non‐specific binding, even in the presence of high concentrations of interfering molecules from other secretomes and serum, paving the way towards applications with clinical samples. Comparisons between EVs isolated via size exclusion chromatography and those directly from supernatant revealed differences in EV size and composition, demonstrating purification‐induced biases. This approach allows estimation of EV concentrations and compositions directly from large‐scale cell cultures. Summary/Conclusion : This novel platform enables label‐free, high‐throughput single‐EV analysis without isolation or purification, advancing unbiased EV heterogeneity studies. Applications include real‐time EV secretion monitoring for quality control in therapeutics and profiling EV subpopulations from complex samples for diagnostics or treatment monitoring. Funding : Swiss National Science Foundation, grant no. 207485.

Label‐Free

Presenter: Evgenia Kim Beckman Coulter Life Sciences, Indianapolis, Indiana, USA Introduction : Flow cytometry is a powerful method to analyse heterogeneous particle populations based on single particle detection and is widely used for cellular analysis. However, the sensitivity of most current conventional cytometers has limitations to detect nanoparticles with sizes below 200 nm, creating a challenge for extracellular vesicle analysis. Methods : This work aims to demonstrate the performance of the CytoFLEX nano Flow Cytometer as a sensitive label‐free tool to characterize nanoparticles by leverageing its capability to collect and analyse scatter parameters at multiple wavelengths. Results : In this study, we will demonstrate the instrument's performance using commercially available samples, such as platelet EVs, RBC EVs and liposomes. Summary/Conclusion : As a part of post‐processing step, we will demonstrate capabilities of calibration techniques for size and refractive index based on NIST control beads with known size and refractive indices and biological controls of murine leukaemia virus (MLV), Herpes simplex virus (HSV), adenovirus, and vaccinia virus.

Longitudinal

Presenter: Zhen Zhang University of Queensland, Brisbane, Australia Introduction : Assessing therapeutic response in glioblastoma (GBM) is limited by the inability to perform serial biopsies due to the tumour's intracranial location. This challenge restricts longitudinal monitoring of the tumour's molecular profile and hinders the development of effective treatment strategies. Liquid biopsy techniques, particularly small extracellular vesicle (sEV) analysis, offer promising opportunities by providing real‐time insights into tumour molecular profiles using peripheral blood. To address this, we developed the GBM Extracellular Vesicle Monitoring Phenotypic Analyzer Chip (GEMPAC), which profiles a novel central nervous system (CNS) and glioma stem cell (GSC) biomarker panel (ATP1B2, EAAT2, CD24, CD44, CD133, and EGFR) on circulating sEVs. Methods : GEMPAC identifies a unique GBM signature in sEVs by targeting CNS‐specific markers ATP1B2 and EAAT2. Using a nanoshearing multiplex surface‐enhanced Raman spectroscopy (SERS) approach, the GEMPAC platform simultaneously tracks GSC subpopulations (CD24 in neural progenitor cell‐like, CD44 in mesenchymal cell‐like, CD133 in oligodendrocyte progenitor cell‐like, and EGFR in astrocyte‐like) to monitor tumour progression and therapeutic resistance. We applied this platform to 36 GBM clinical samples, including 12 longitudinal samples with five time points, to detect tumour recurrence and track therapeutic response. Results : GEMPAC successfully captured and analysed GBM‐specific sEVs, enabling real‐time monitoring of GSC dynamics on sEVs during treatment. Key findings included elevated CD44 levels, indicating mesenchymal‐like (MES‐like) GSCs associated with disease progression and recurrence. Additionally, CD133+ GSCs emerged in a subset of patients, suggesting resistance to treatment. While EGFR and CD24 markers did not show a strong link to recurrence, a shift to a MES‐like state was observed with downregulated EGFR and elevated CD44 during therapy. These findings highlight the platform's potential to provide real‐time insights into tumour heterogeneity and therapeutic resistance through sEV analysis. Summary/Conclusion : The GEMPAC platform offers a non‐invasive, real‐time method for monitoring tumour evolution in GBM patients, with significant potential to improve treatment outcomes. It provides valuable insights into tumour heterogeneity and can detect therapy‐resistant GSC subpopulations, creating opportunities for targeted interventions. With further validation in larger clinical cohorts, GEMPAC could become a tool in personalized GBM management, enhancing therapeutic strategies and improving patient survival.

Manipulation

Vincenzo Verdi 1 , Maribel Lara Corona 1 , Pierre‐Michaël Coly 1 , Wendy van Straaten 2 , Daniele D'Arrigo 3 , Amandine Robac 1 , Erwan Boëdec 1 , Alessia Di Maggio 2 , Frederik J. Verweij 1,2 and Guillaume van Niel 1,4 Presented by Ela Babursah 1,4 1 Institut de Psychiatrie et Neurosciences de Paris, France. 2 Universiteit Utrecht, The Netherlands. 3 Abbelight Biotech Research Company, France. 4 Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers (CRCI 2 NA) France Introduction : Tumour‐derived EVs are acknowledged as important cellular mediators that are involved in several hallmarks of cancer progression, such as tumour proliferation, immune system modulation, angiogenesis induction and the development of pre‐metastatic niches. However, there are still a lot of unanswered questions in the field about the function of EVs in vivo. A number of methods, most notably in the zebrafish embryo, have been developed to live‐track the dynamics and fate of tumour‐derived EVs at a single particle scale in vivo. Nonetheless, there are currently insufficient methods available to precisely direct and control the dispersal and targeting of tumour EVs in vivo, to either better understand intercellular communication during pathological development or improve EV‐based therapeutic strategies. To fill this gap, we present here a novel tool, the EV‐trap, for selectively capturing and locally accumulating circulating tumour EVs towards engrafted trapping cells or endogenous tissues. Methods : We have generated, on one hand, stable lines of prostate cancer cells (PCa) expressing the pHluorin tag associated to tetraspanins C63 or CD9 and, on the other hand, cells and a zebrafish strain expressing an “EV‐trap”, consisting of a transmembrane domain exposing specific nanobodies at the cell surface. After full characterization of the PCa‐derived EVs, we have used imaging approaches to evaluate the trapping of this tool in vitro and in vivo using zebrafish embryos. Results : We show here that modified PCa produce large amounts of EVs carrying pHluorin moieties. The EV‐trap expressing cells can selectively and specifically capture EVs within min, up to 20,000 EVs per cell without saturation. This capture is efficient in vivo for exogenous and endogenous EVs and redirects EVs from their intended destinations. Summary/Conclusion : We have developed an ‘EV‐trap’ tool able to capture and redirect EVs thanks to the expression of a chimeric receptor. This tool will provide new insights into the function of EVs and possible new therapy avenues in a number of physiopathological processes. We are now adapting this tool to natural tumour antigens, such as PSMA, to gain a better understanding of the biological activities of tumour EVs, such as in the creation of pre‐metastatic niches. Funding : Marie Curie Actions, proEVLifeCycle, Fondation ARC.

Mechanically

Carolina S. Martins 1,2 , Mimma Maggio 1,2, , Mathieu Y. Brunet 1,2 , Cansu Gorgun 1,2,3 , Rawan Almasri 4 , Lorraine O'Driscoll 4 , David A. Hoey 1,2,5 1 Trinity Centre for Biomedical Engineering, Ireland, 2 Dept. of Mechanical, Manufacturing, and Biomedical Engineering, School of Engineering, Trinity College Dublin, Ireland, 3 School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Ireland, 4 School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute & Trinity St James's Cancer Institute, Trinity College Dublin, Ireland, 5 Advanced Materials and Bioengineering Research Centre, Trinity College Dublin & RCSI, Ireland Introduction : The formation of new blood vessels, angiogenesis, plays a critical role in bone repair. We have previously highlighted the significance of bone cell‐derived extracellular vesicles (EVs) in mediating angiogenesis. However, the impact of mechanical stimulation, a potent regulator of bone regeneration, on bone cell‐derived EVs remains uncertain. Therefore, this study aimed to determine the role of EVs derived from cells at different stages of the osteogenic lineage in regulating angiogenesis, and how this is influenced by mechanics. Methods : Fluid shear was applied to human MSCs, human osteoblasts (OBs), or osteocyte‐like (OCY) cells at 1 Pa, 1 Hz for 2 h. Following 24 h in culture, the conditioned media was collected. EVs were collected by ultracentrifugation at 110,000 ×  g for 75 min at 4°C, using a 70Ti fixed‐angle rotor. NTA, flow cytometry and TEM were used for characterization. Human umbilical vein endothelial cells (HUVECs) were used to assess tube formation or fixed for immunofluorescent imaging (CD31 + ). Proliferation was assessed through BrDU incorporation and migration was evaluated by the Transwell assay. Results : The treatment of HUVECs with EVs collected from statically cultured MSCs (st‐EV) inhibited junction formation, although this affect was rescued following mechanical stimulation of the parent cell. EVs collected from mechanically activated OBs (maEVs) further enhanced the number of junctions compared to the negative control, which was further augmented with OCY‐derived EVs. In particular, OCY‐derived maEVs led to a significant increase in tube formation with thicker vessels present, comparable to the +VEGF control, and also increased HUVEC migration and proliferation. Remarkably, treatment with OCY‐derived maEVs leads to a substantial increase in CD31 + expression. Summary/Conclusion : We demonstrate the importance of the stage of differentiation, as well as mechanical stimulation on the angiogenic effect of EVs released by bone cells, highlighting OCY maEVs as a possible therapy for bone repair.

Microcarrier

Presenter: Andrea Haid FH Campus Wien Biomedical Science, USA Introduction : Producing extracellular vesicles (EVs) from different cells is relevant because they possess unique compositions and functions reflecting their cell of origin, and producing EVs from various adherent cells allows researchers to study cell‐specific intercellular communication mechanisms and explore their potential therapeutic applications. Methods : By using Cytodex 1 microcarrier, we developed a platform for adherent cells to produce EVs in middle scale (up to 50 L). Different cell types can be grown on Cytodex 1, like human dermal fibroblast cells, CACO‐2 and others. EV extraction was performed with trans flow filtration (TFF) or, in lower volumes, by Centricons (100 kDa cut‐off), followed by size exclusion chromatography with Sephacryl S‐500 HR). Identity of EVs was determined by western blot (CD63, CD9), integrity was tested by atomic force microscopy (AFM), as well as concentration determined by total protein and RNA measurements and dPCR. Results : We were able to grow different cells on Cytodex 1 microcarriers. With one cell line we scaled the process up to a 10 L bioreactor. A Design of Experiments (DOE)‐based optimizing approach led to optimal process conditions for EV production of this cell line. Summary/Conclusion : The ability to cultivate diverse cell types, including human dermal fibroblasts and CACO‐2 cells, in stirred‐tank bioreactors represents a significant advancement, particularly for cell lines previously unadapted to such systems. The use of TFF and size‐exclusion chromatography ensured high‐purity EV isolates, validated by CD63/CD9 markers and structural integrity via AFM. Implementing a DOE approach to optimize EV production underscores the potential for reproducible, large‐scale manufacturing, which is essential for therapeutic applications. This work establishes a robust, mid‐scale bioreactor platform for EV production from adherent cells, overcoming scalability challenges while maintaining EV integrity and functionality. Funding : This work was funded by Anschubfinanzierung ASF—Etablierung von Produktion und Charakterisierung von extrazellulären Vesikeln “Pro‐CHAR*EV.”

Microfluidic

Sara Cavallaro 1,2 , Sahbra Eldosougi 1 , Uma Paithankar 1 , Sara I. Veiga 1,2 , Raheel Ahmad 1,2 , Cecile Riviere‐Cazaux 3 , Terence C. Burns 3 , Brian V. Nahed 1,2 , Shannon L. Stott 1,2 . 1 Massachusetts General Hospital, Boston, Massachusetts, USA; 2 Harvard Medical School, Boston, Massachusetts, USA; 3 Mayo Clinic Rochester, Rochester, Minnesota, USA Introduction : Liquid biopsies represent a revolutionary approach in brain cancer management, being significantly less invasive than brain biopsies while also enabling real‐time tumour monitoring. However, a blood test for brain tumours has not translated into clinical practice due to the low abundance of circulating tumour biomarkers. While less accessible than blood or other biofluids, cerebrospinal fluid (CSF) is viewed as one of the most valuable sources of tumour‐specific biomarkers, particularly for glioblastoma (GBM), due to its proximity to the brain and direct access to molecules that cannot easily cross the blood‐brain barrier. Several studies have successfully identified GBM biomarkers in CSF, including extracellular vesicles (EVs). Microfluidic devices are especially well‐suited for analysing CSF, as they can handle incredibly small volumes of fluid (10 µL to 1 mL) with exquisite precision and sensitivity and relatively low cost. Methods : In this work, we present a microfluidic technology that can isolate cancer‐specific EVs from the CSF of brain cancer patients. Inspired by the ‘backbone’ of our EV isolation technology, the EV‐HB‐Chip, our device will use herringbone patterns and immunoaffinity capture to isolate brain cancer‐specific EVs. The developed platform will be specifically designed for CSF and optimized for small biofluid volumes (∼50 µL–1 mL). Following isolation, the EV RNA content will be analysed using ddPCR. Results : To demonstrate the feasibility of our approach, we processed 500 µL of CSF obtained from adult brain tumour patients (one of which was serially sampled at three time points). For these experiments, EVs were captured into the EV‐HB‐Chips using a cocktail of antibodies targeting EGFR, podoplanin, PDGF and Axl. The results showed successful detection of some of the brain tumour specific genes (EGFR, MYCN) using ddPCR following EV capture. For the patient serially monitored, the data showed high signals of GFAP gene expression that decreased over time, mimicking the patient's clinical response (decreasing tumour burden). Summary/Conclusion : The preliminary data suggest the feasibility of the proposed technology, while also indicating areas for further optimization. Funding : National Cancer Institute under Grant R01‐ CA226871 (S.L.S.); National Cancer Institute under Grant F32‐ CA236417 (D.C.R.); American Cancer Society under Grants 132030‐RSG‐18‐108‐01‐TBG (S.L.S.) and PF‐23‐1151433‐01‐CCB (S.C.); V Foundation (S.L.S.).

Miniaturized

Presenter: Andrea Capuano Leiden Universiteit, Leiden, The Netherlands Introduction : Placenta‐derived extracellular vesicles (EVs) play key roles in fetomaternal communication, modulating immune responses and cellular functions. Efficient isolation of EVs is crucial to enable accurate analysis for downstream applications. Using depletion zone isotachophoresis (dzITP), we achieve high‐purity EV separation and enrichment, enhancing analytical sensitivity and reproducibility. This method supports EV characterization, advancing research on pregnancy‐related pathologies. Methods : Miniaturized silicon‐glass devices based on dzITP were used to concentrate EVs and separate them from contaminants. Optimal electrical parameters were established with JEG‐3‐derived EVs (from cell cultures), followed by the analysis of EVs from human placenta perfusate—both pre‐purified (clean) and unprocessed (raw). These samples were collected and characterized (NTA, dot blot) by the Placenta Lab at Jena University Hospital (Germany). Sample collection from the microfluidic chip was optimized for downstream analysis. Results : Experiments showed how different pre‐treatments of JEG‐3‐derived EVs affected dzITP fractionation. Small EVs, isolated via serial ultracentrifugation steps, and larger, more contaminated EVs obtained with less refined ultracentrifugation processing showed distinct patterns—more complex samples yielded more fractions. Mixed EV samples displayed cumulative electropherograms and retained both profiles. Under the same dzITP conditions, EVs from raw and clean placenta perfusate were fractionated, reflecting their complexities. An electroosmotic flow‐based method enabled selective retrieval of fractions for downstream analysis. Summary/Conclusion : dzITP enables efficient, precise electrical‐based fractionation of placenta‐derived EVs, reflecting sample complexity. Its ability to concentrate EVs, separate them from contaminants, and retrieve the different fractions will potentially enhance downstream analyses, supporting research on EV roles in pregnancy‐related conditions. Funding : This study was supported by HORIZON EUROPE Marie Skłodowska‐Curie Actions (Grant No. 101111215).

Multi‐Omic

Presenter: Catherine R. Taylor Atlantic Cancer Research Institute, Canada Introduction : Immune‐oncology treatment has revolutionized cancer treatment, but not all patients benefit due to complexities in the interplay between cancer and the immune system. Extracellular vesicles (EVs) are particles secreted by both tumour and immune cells that reflect the state of the cell of origin and participate in the interplay between cancer and immune cells. Our study used RNA sequencing and proteomic profiling of circulating EVs isolated from the plasma of NSCLC patients treated with anti‐PD1 therapy to identify novel biomarkers of response and EV‐associated immune evasion mechanisms. Since the cargo of circulating EVs reflects the state of the cell of origin, we hope to identify predictive biomarkers of response to anti‐PD1 therapy as well as targetable pathways of treatment resistance. Methods : Plasma from NSCLC patients treated with anti‐PD1 therapy was obtained with informed consent. EVs were isolated using peptide‐affinity purification with Vn96 peptide to precipitate EVs from plasma for RNA extraction or protein lysate. RNA was sequenced using the ComboSeq mRNA/miRNA kit on a NovaSeq 6000. Proximal Extension Assay (Olink Inc.) was used to profile ∼3000 proteins in the EV protein lysate. The Bioconductor package edgeR and Olink Insight were used to identify differentially expressed RNAs and proteins between responders and non‐responders, respectively. Cox regression analysis was performed in the R environment to identify RNAs and EV proteins that correlate to progression‐free survival (PFS). Results : EV RNA analysis revealed enrichment of neutrophil recruitment, VEGF signalling, and CXC chemokine receptor signalling associated with poor survival. Proteomics analysis identified EV proteins associated with shorter PFS, including TRAIL‐R2 and VMO1, and pathway analysis revealed enrichment of VEGF signalling, chemotaxis, and macrophage polarization associated with poor progression‐free survival. Summary/Conclusion : Profiling circulating EVs has unveiled EV proteins and de‐regulated pathways that may improve our understanding of how EVs participate in immune evasion during immunotherapy treatment and may point to new treatment modalities that could improve responses to immune checkpoint blockade in lung cancer patients. Furthermore, the data suggests that circulating EVs provide RNA and protein signatures that reflect the tumour immune microenvironment and are predictive of anti‐PD1 response. Funding : Canadian Cancer Society J. D. Iriving Ltd Excellence in Cancer Research Fund

Nanophotonic

Presenter: Wei‐Chuan Shih University of Houston, USA Introduction : Recently, our research group has developed PlAsmonic NanO‐apeRture lAbel‐free iMAging (PANORAMA), a technique to detect individual nanoparticles as small as 25 nm using an array of gold nanodisks on an invisible substrate (AGNIS). (1) PANORAMA enables the counting, sizing, and localization of individual sEVs in both healthy individuals and cancer patients across various stages and types by utilizing AGNIS functionalized with antibodies against exosomal surface proteins CD9, CD63, and CD81. (2) By quantifying a portion of sEVs from 20 µL blood plasma, PANORAMA can differentiate 205 cancer patients and 106 healthy individuals with a sensitivity of 99.5% and a specificity of 97.3%. (3) In this study, we report new results towards a hepatocellular carcinoma (HCC) test. Methods : We have analysed samples of three donor types: healthy plasma donors (HPD), non‐cancer disease patients (NCD) (including cirrhosis, fatty liver, etc.), and cancer patients (CP) (including liver cancer and cholangiocarcinoma). A test set of 76 samples was analysed, comprising 13 HPD, 21 NCD, and 42 CP. Among them, the sEV counts are 156±55 (HPD), 370 ± 130 (NCD), and 458 ± 100 (CP). Using the previously obtained sEV count threshold value of 280 results in a cancer detection sensitivity of 100% and a specificity of 55%. The moderate specificity is a direct consequence of 15 false positives from the NCD samples. Results : To enhance specificity, we have next explored known molecular biomarkers such as cargo microRNAs (miRNAs): miR‐126‐3p, miR‐222‐3p, miR‐31, miR‐21, and miR‐221. Using a standard molecular beacon probe design (3′ BHQ2 quencher/5′ Cy3), we have analysed another sample set consisting of 80 samples (NCD and CP) in a blinded manner, yielding a sensitivity of 97%, a specificity of 95% (3 false positives and 1 false negative), a positive predictive value (PPV) of 91%, and a negative predictive value (NPV) of 98%. ROC analysis for the blinded set yielded an AUC of 0.94. Summary/Conclusion : Our study underscores the potential of PANORAMA in quantifying and profiling sEVs for HCC detection. The test runs on 20 µL of plasma without any additional sample preparation or isolation and thus is highly suitable for routine tests and point‐of‐care applications. Funding : National Institute of Health R01EB030623

Nanoplastics

Wei‐Hsuan Hsu 1 , Tang‐Long Shen 2 , Bao‐Hong Lee 3 1 Department of Food Safety / Hygiene and Risk Management, National Cheng Kung University, Tainan, Taiwan. 2 Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan.  3 Department of Horticulture, National Chiayi University, Chiayi, Taiwan. Introduction : Recent studies confirm that nanoplastics (NP) cause severe microbial imbalances in various ecosystems, significantly affecting microbial diversity and abundance. Hydroponic systems vital for lettuce production are increasingly threatened by NP contamination in irrigation water and this issue is gaining global attention. This study investigates microbial species in hydroponic irrigation water altered by NP exposure and their impact on lettuce growth. Methods : Lettuce (Lactuca sativa L.) was cultivated in two hydroponic models to assess the effects of NP treatments and Curvibacter fontanus‐derived extracellular vesicles (EVs). Model‐1 involved continuous water conditions, while Model‐2 incorporated water changes every four days, both using a nutrient solution with specific concentrations of minerals and NP. Over ten days, growth metrics such as leaf dimensions and root length were recorded. Microbial assays involved centrifuging water samples for DNA extraction and sequencing of the 16S rRNA gene. The uptake of NP by C. fontanus was visualized using confocal microscopy. EVs were isolated from cultured C. fontanus, characterized by size and morphology, and applied to hydroponically grown lettuce to evaluate their effects on oxidative stress, measured by enzyme activity and malondialdehyde levels. Results : While NP did not directly harm or accumulate in lettuce, significant changes in water parameters and microbial communities were observed, particularly an increase in Curvibacter fontanus abundance. Inoculation of sterile irrigation water with NP and C. fontanus led to lettuce mortality, suggesting C. fontanus as a critical mediator. Furthermore, extracellular vesicles (EVs) isolated from C. fontanus, treated with NP, were shown to suppress leaf development, growth, antioxidant defenses, and lettuce survival. Summary/Conclusion : This study concludes that NP‐induced microbial shifts, particularly involving C. fontanus EVs, indirectly harm hydroponic lettuce production. Funding : This research work and subsidiary spending were mainly supported by the National Science and Technology Council (NSTC) in Taiwan under Grant No. NSTC 112‐2628‐B‐415‐001‐MY3 (Young Scholar Fellowship Program).

Neurobiology

Invited Speaker: Roosmarijn E. Vandenbroucke Ghent University, Belgium

Neutralizing

Anjali Singh, Alok Kumar Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India. Introduction : Microparticles (MPs), annexin‐positive vesicles ranging from 100 to 1000 nm, are critical players in cellular communication, transferring active biomolecules that influence cellular functions. Recent studies highlight that disease states, including viral infections, can alter MPs composition, messageing, and function. This study examined the characteristics of MPs shed by Japanese Encephalitis Virus (JEV)‐infected murine macrophages (RAW 264.7) and their role in promoting infection and inflammation. Furthermore, we demonstrated that neutralizing MPs can control JEV replication, subsequent propagation of JEV copy numbers into macrophages, and inflammation. Methods : MPs were isolated from cell‐conditioned media using differential ultracentrifugation. MPs were characterized through scanning electron microscopy, flow cytometry, and nanoparticle tracking analysis (NTA). Viral copy numbers and cytokine levels were assessed using RT‐PCR, flow cytometry, and Western blotting. Furthermore, enriched MPs were neutralized, co‐cultured with macrophages, and used for viral propagation assays. Results : Macrophage activation following JEV infection was evidenced by cytopathic changes, cell death, increased viral copy numbers, and M1‐associated inflammation. NTA and flow cytometry analysis revealed a significant increase in the concentration of Annexin V+ MPs post‐infection. We further noted that JEV‐infected MPs exhibited unique protein profiles, with elevated levels of flotillin‐1 and Annexin V compared to control MPs, indicating altered biogenesis of the MPs. Additionally, we detected enriched JE viral RNA and elevated cytokine levels in the MPs after infection. Furthermore, we found that MPs from JEV‐infected cells carry infectious viruses and facilitate their spread to uninfected cells. Notably, MPs‐mediated infection was inhibited by neutralizing MPs with Annexin V antibody, heat inactivation, and PEG‐TB surface neutralization. Summary/Conclusion : Together, these results provide insight into the mechanisms of disseminating JEV infections. MPs containing JEV viral particles facilitate the seeding and spread of the infection, representing a potential therapeutic target for controlling JEV propagation. Funding : This study was supported by the Anusandhan National Research Foundation (ANRF) Science & Engineering Research Board (SERB) Core Research Grant (FILE NO. CRG/2023/006849) and SRF Fellowship (DBT/2020/SGPGIMS/1373) from DBT, Govt. of India to Anjali Singh.

Neutrophilic

Ákos M. Lőrincz 1,2 , Mátka Nagy 1 , Viktória Szeifert 1 , Ferenc Kolonics 1 , Nóra Borsos 1 , Roland Csépányi‐Kömi 1 , Erzsébet Ligeti 1 1 Semmelweis University, Hungary, 2 Szent György Hospital, Hungary Introduction : Neutrophils (PMNs) produce extracellular vesicles (EVs) predominantly ranging in diameter from 100 to 600 nm. We previously characterized three distinct EV populations: spEVs produced by resting PMNs, apoEVs generated during apoptosis, and ozEVs induced by opsonized zymosan particles. Only ozEVs exhibited antibacterial effects, and we have explored the specific signalling pathway leading to their production. This study investigates the roles of these EV populations in intercellular communication with other myeloid and endothelial cells. Methods : PMNs and monocytes were isolated from healthy adults in accordance with the Declaration of Helsinki and with ethics approval from ETT TUKEB (31937‐7/2020/EÜIG). Human PMNs were either stimulated with opsonized zymosan or left unstimulated. The EV fractions were isolated by differential centrifugation and filtration. These distinct EV fractions were then co‐incubated with resting neutrophils, monocytes, monocyte‐derived macrophages, and HUVEC cells. Phagocytosis was assessed by flow cytometry, ROS production via chemiluminescence, and IL‐8 and TGF‐beta production by ELISA. Cell surface markers were quantified by flow cytometry, and macrophage migration was monitored by microscopy. Results : PMN‐derived EVs did not influence phagocytosis in PMNs and only apoEV in monocytes. However, spEVs inhibited ROS production in both cell types, while ozEVs elevated ROS production in PMNs. SpEVs significantly increased anti‐inflammatory TGF‐beta production, whereas ozEVs increased pro‐inflammatory IL‐8 secretion in PMNs, monocytes, and HUVEC cells. SpEV‐treatment lowered CD11b amount on the monocyte cell surface. OzEV increased CD11b expression on monocytes and E‐selectin and VCAM‐1 on HUVEC cells. Additionally, apoEVs enhanced macrophage migration. Summary/Conclusion : According to these data, we hypothesized that the PMN EVs do not simply reflect the state of the mother cell but also deliver addressed information to the different partner cells. This selective EV‐mediated communication underscores the role of neutrophils in dynamic modulation of immune responses and highlights their possible therapeutic roles in immunomodulation. Funding : EFOP‐3.6.3‐VEKOP‐16‐2017‐00009, NKFIH FK 137770, TKP2021‐EGA‐24, János Bolyai Research Scholarship of the Hungarian Academy of Sciences

Non‐Coding

Presenter: Sarmeela Sharma LV Prasad Eye Institute, Hyderabad, India Introduction : Diabetic retinopathy (DR) is an ocular complication of diabetes with higher prevalence among the developing countries. Management of this disease only targets the end‐stage symptoms, thereby making the vision loss irreversible in DR subjects. Early diagnosis and better prognosis of this disease could help overcome this problem in the future. Methods : To understand the regulation of genes involved in DR, global expression profiling for regulatory molecules miRNA was performed in DR blood samples ( n = 3 each from NPDR, PDR and healthy controls) using microarray. The identified miRNAs were subjected to pathway analysis. Expression of significant miRNAs was further explored in extracellular vesicles (EVs) isolated from DR serum and vitreous samples. Results : A total of 1486 differentially expressed ncRNA genes were identified in this study. Among those, 890 genes were downregulated, while the remaining 596 were upregulated. The miRNAs found dysregulated were implicated in DR‐associated pathways: cytoskeleton remodelling, oxidative stress, angiogenesis, autophagy, apoptosis, lipid metabolism, inflammation, cellular adhesion, proliferation and vascular homeostasis. A concurrent negative regulation of the genes regulated by these miRNAs was also observed in DR blood samples. Among the significant pathways, extracellular vesicle related processes were enriched. The significant miRNAs were also found differentially expressed in the EVs derived from systemic (serum) and ocular fluids (vitreous). Summary/Conclusion : Vitreous/serum derived EV cargo packed significant information on the type of miRNAs involved in the DR pathogenesis. The specific signature of these EVs could also be explored as a predictive marker for DR development or progression among diabetic individuals. Funding : This study was supported by the ICMR.

Optimization

Presenter: Ons Ben Hadj Hassen Université de Caen‐Normandie, Caen, France Introduction : Ischaemic stroke remains a major health problem. Extracellular vesicles (EV), derived from mesenchymal stem cells (MSC), have been shown to mitigate brain damage and improve neurorepair and neurological outcome following ischaemic stroke. Though promising, EV production and their route of delivery remain a major challenge. Here, the effects of EV generated by an innovative turbulence approach were characterized on stroke‐induced brain lesion and neurological deficits in the rat. Methods : EV were isolated from human adipocyte tissue stromal cells (hASC) using a patented high‐yield turbulence bioproduction process. EV were quantified and characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), ExoView and proteomic analyses. Rats were subjected to a transient occlusion (1 h) of the middle cerebral artery (tMCAO) and treated with EV at reperfusion time (1010 EV/rat; I.A.) or 24 h later (1011 EV/rat; I.V.). MRI were performed at 1 and 14 days after tMCAO to quantify brain lesion volume. Behavioural tests were carried out up to 40 days post‐tMCAO. Results : NTA, TEM and proteomic analyses confirmed EV characteristics (size, shape, tetraspanin expressions). While I.V. EV administration showed limited therapeutic effects. I.A. EV delivery at reperfusion time reduced the lesion volume 24 h post‐tMCAo compared to the control group ( p < 0.05). Moreover, 7 days after tMCAO, rats treated with EV immediately after reperfusion tend to improve their neurological score ( p = 0.07) and showed a significantly better limb placing test performance ( p < 0.05). In addition, these rats were less lateralized compared to the control ones in the corner test and performed better in the adhesive test 30 days after tMCAO ( p < 0.01). In contrast, I.V. EV administration 24 h post‐tMCAO showed only a significant effect on spatial memory ( p < 0.05, t ‐test). Immuno‐histological analyses of brain tissues are ongoing to investigate post‐ischaemic cellular effects. Summary/Conclusion : These results suggest that (1) MSC‐derived EV produced by an original turbulence method are efficacious on stroke‐induced brain lesion and neurological deficits and (2) optimizing the administration route and timing enhances the efficacy of EV after stroke. Overall, the results showed that MSC‐derived EV produced by an original turbulence method are promising as an efficient therapeutic intervention in ischaemic stroke.

Osteosarcoma

Snehadri Sinha 1,2 , Virinder Kaur Sarhadi 1,2 , Md Mahamudul Alam 1,2 , Pia Siljander 1,3 , Riitta Seppänen‐Kaijansinkko 1.2 , Tuula Salo 1,2,4 1 University of Helsinki, Helsinki, Finland; 2 HUS Helsinki University Hospital, Helsinki, Finland; 3 Finnish Red Cross Blood Service, Finland, Helsinki, Finland; 4 Univeristy of Oulu and Oulu University Hospital, Oulu, Finland Introduction : Osteosarcoma (OS) is an aggressive form of bone cancer that primarily affects adolescents and older adults. Diagnosis typically involves imaging tests and biopsy procedures, which can be invasive and cause discomfort for patients. Currently, there are no established biomarkers for OS, although high levels of alkaline phosphatase and lactate dehydrogenase in blood may suggest an advanced stage of the disease. Liquid biopsies, such as plasma‐derived extracellular vesicles (EVs), offer a less invasive alternative for diagnosis. Here, we conducted a case‐control study using plasma from OS patients and controls, and screened plasma EVs for candidate biomarkers and evaluated their diagnostic utility. Additionally, we tested two common EV isolation methods to determine the optimal choice for proteomic analysis. Methods : Plasma samples from OS patients and control individuals were obtained from the Helsinki biobank, while OS cell lines were derived from patient biopsies or commercial sources. EVs were isolated from plasma and cell culture conditioned media using either ultracentrifugation (UC) or size exclusion chromatography (SEC), and the efficacy of each method was assessed based on EV purity and yield. EVs were characterised by nanoparticle tracking analysis, electron microscopy, and Western blotting for established EV markers. Next, the EVs will be analysed with a proximity extension assay to quantify selected cytokines and immune surveillance‐related molecules. Comparisons between plasma EVs and cell‐derived EVs will also be performed. Lastly, the expression of biomarkers of interest will be validated using additional protein assays. Results : Both UC and SEC methods isolated an adequate amount of EVs from plasma for proteomic analysis. However, UC was more effective than the commercial SEC columns at separating EVs from other plasma components, particularly lipoproteins. The UC‐isolated EVs were therefore considered more suitable for proteomic analysis. The results from proteomics are currently pending. Summary/Conclusion : The proteomics data will allow us to identify biomarkers of interest and to evaluate the suitability of small plasma volumes for EV‐based diagnostics. UC allows better separation of EVs from plasma components than SEC. This study advances the understanding of EVs as potential non‐invasive biomarkers, with implications for improving OS diagnostic accuracy and patient outcomes.

Preoperative

Dapi Menglin Chiang 1,2,5 , Susanne I. Wudy 3 , Christina Ludwig 3 , Marlene Reithmair 2 , Laura Benecke 4,5 , Yannik da Silva 4,5 , Laurent Muller 4,5 and Michael W. Pfaffl 1 1 Division of Animal Physiology and Immunology, School of Life Sciences, Technical University of Munich, Freising Weihenstephan, Germany; 2 Institute of Human Genetics, University Hospital, LMU Munich, Munich, Germany; 3 Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich (TUM), Freising Weihenstephan, Germany; 4 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Basel, Switzerland; 5 Department of Biomedicine, University of Basel, Switzerland Introduction : Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with more than 650,000 new cases and 330,000 deaths annually. Despite advancements in treatment, the prognosis remains poor, particularly for patients with recurrent or metastatic disease. Surgery is the primary treatment, especially for oral cavity tumours. Identifying new tumour‐related and pre‐operative biomarkers in tissues or circulation is crucial for improving diagnosis and prognosis. Liquid biopsy is a promising method for biomarker discovery, and especially extracellular vesicles (EVs) carry tumour‐derived proteins and genetic material, offering insights into cancer progression and treatment response. Methods : This study examines the protein content of EVs before and after surgery in HNSCC patients' plasma ( n = 11). Platelet‐poor plasma was collected peripherally before operation (BOPP) and 1 h after tumour removal (AOPP), alongside local tumour venous plasma before (BOTV) and after tumour removal (AOTV). We utilized galectin‐based glycan recognition particles for EV isolation, bead‐based flow cytometry, nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo‐EM) for EV characterization, and high‐resolution liquid chromatography‐mass spectrometry (HRLC‐MS/MS) for proteomics. Results : NTA and cryo‐EM showed comparable EV sizes, concentrations and morphology across all groups. HRLC‐MS/MS identified 14 proteins, including platelet‐derived growth factor subunit B (PDGFB) and tissue inhibitor of metalloproteinases 3 (TIMP3), which were significantly elevated in BOPP compared to AOPP. In addition, PDGFB levels were also higher in BOTV than in AOTV. Gene ontology analysis linked these proteins to cancer pathways and migration. Treatment with BOPP and BOTV plasma EVs significantly increased cell proliferation in the HNSCC cell line UPCI‐SCC‐016; hence, high expression of PDGFB and TIMP3 in solid tissue correlates with poor prognosis. Summary/Conclusion : These results suggest that cancer surgery removes most tumour‐related EVs in peripheral and local biopsies measurable during surgery. Increased PDGFB and TIMP3 levels in BOPP plasma indicate elevated tumour growth and their decrease post‐surgery points to effective oncological treatment, suggesting their potential as powerful preoperative biomarkers during cancer ablation.

Purification

Mariana Trigo‐Chapou 1 , Alicia Viloria‐Petit 1, Huiyan Li 2 , Geoffrey Wood 3 , Danny Carreira 1 , Aaron Dhillon 1 1 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada; 2 School of Engineering, College of Engineering and Physical Sciences, University of Guelph, Guelph, Ontario, Canada; 3 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada Introduction : Osteosarcoma (OS) is the most common primary bone cancer in dogs. Metastasis to the lungs is the main cause of death in canine OS patients, but there are no biomarkers to detect early metastatic disease. We previously identified OS‐enriched protein cargo with prognostic value via proteomics analysis of EVs released by canine tissue explants. For future clinical application of our findings, a quick and reliable method to purify EVs from dog plasma is necessary. This study tested the SmartSEC DeLipo protocol (SBI) for such purpose. Methods : Canine blood was collected and processed by the OVC Tumour Bank following standard protocols. To obtain plasma, blood‐containing EDTA tubes were centrifuged at 1500 ×  g for 10 min. Human plasma and serum were obtained from a commercial source. All samples were stored at −80°C. Three canine plasma samples were first subjected to the SmartSEC DeLipo and to a SEC protocol (qEV 70, IZON), following the manufacturer's instructions. Next, only the SmartSEC DeLipo protocol was used to compare plasma and serum from canine OS patients ( n = 2) to plasma and serum from healthy canine controls ( n = 2) and a healthy human control ( n = 1). EVs were characterized by DLS, western blotting (WB) and TEM. Two previously identified canine OS biomarkers, PSMD14 and PSMA7, were assessed in EV lysates by WB. Results : The SmartSEC DeLipo protocol resulted in a 6× reduction in 10 nm particles and a 6× enrichment of EV‐compatible particles (> 100 nm) when compared to SEC. EVs obtained with the former method have similar size profiles in plasma versus serum, but different size profiles when comparing serum/plasma from dogs versus human or from dog OS patients versus healthy dogs. EVs were enriched in plasma as compared to serum. Both PSMD14 and PSMA7 were enriched in the plasma of dogs with OS as compared to that of healthy dogs. Summary/Conclusion : The SmartSEC DeLipo is an effective protocol for the purification of EVs from dog plasma/serum, but plasma improves the EV yield. PSMD14 and PSMA7 can be detected in EV lysates from dog plasma and at higher levels in OS versus healthy dogs. Further studies with larger sample sizes are guaranteed.

Quantitative

Maria Jaritsch 1 , Tanja Plank 1 , Melanie Schürz 1,2 , Joachim Danmayr 1 , Cristian T. Matea 1 , Vesna Stanojlovic 1,2 , Eva Klinglmayr 1,2 , Patricia Hrasnova 1,3 , Heloisa Melo Benirschke 1 , Sarah Schönleitner 1 , Anna Müller 1 , Jana Kiefer 1 , Laurens Kober 1 , Petra Reinthaler 1 , Angelika Sales 1 , Martin Wolf 3,4 , Dirk Strunk 3,4 , Mario Gimona 2,3 , Eva Rohde 2,3,5 , Irma Schabussova 6 , Valentina Perova 1 , Gunda Üblagger 1 , Theresa Neuper 1 , Silja Wessler 1 , Wolf Heusermann 7 , Martin Hintersteiner 7 , Nicole Meisner‐Kober 1,2 1 Paris Lodron University of Salzburg, Austria;  2 Ludwig Boltzmann Institute for Nanovesicular Precision Medicine at PLUS, Austria; 3 Paracelsus Medical University Salzburg, Austria; 4 Austrian Red Cross Research, Austria;  5 University Hospital of Salzburg, Austria; 6 Medical University of Vienna, Austria;  7 EvoBiotiX SA, Switzerland; Introduction : Interest in extracellular vesicles (EVs) as therapeutics or for drug delivery is steadily increasing, but data on quantitative in vivo pharmacokinetics/dynamics, bioavailability and biodistribution—especially a systematic comparison between EVs isolated from different sources—are still limited. Since EVs are heterogeneous in size and composition, normalization of the labelling and detection strategies are challenging. This study presents a strategy for normalization and a side‐by‐side biodistribution map of vesicles from different sources. Methods : We investigated EVs from different sources (bovine, goat and human milk, umbilical cord MSCs, HEK293 cells, H.pylori and E.coli) by labelling with FRET‐pair fluorophores in the NIRF‐range, quantifying endotoxin levels and administering them intravenously into mice. To quantify EVs directly in serum and PFA‐fixed tissue cryosections, we developed a high‐content imaging workflow using automated high‐resolution widefield imaging and EVAnalyzer2. For normalisation of whole organ fluorescence, we investigated cow milk EVs labelled at different EV‐to‐dye stoichiometries and developed a normalisation strategy using single vesicle imaging. Results : Covalent labelling of EVs with NIRF‐dyes allowed the detection of single vesicles and provided the best signal‐to‐noise in serum and tissue, but we observed that for different EV‐sources the same labelling stoichiometry leads to different brightness and number of labelled vesicles. To overcome this heterogeneity, we have developed a normalisation strategy that enables a cross‐comparison of EV‐sources. In general, we observed a uniformLy short serum half‐life (< 5 min) of all EV‐sources in mice. This suggests rapid extravasation into tissue which was confirmed by cryosections of selected organs. In the liver, high EV signals were observed within cells after 5 min, followed by a decrease and redistribution to other organs at later timepoints. In addition, we observed differences in organ distribution between the EV‐sources, supporting the concept of a differential tropism linked to the EV‐source. Summary/Conclusion : We successfully established a workflow for quantifying EVs from diverse sources in serum and cryosections using NIRF‐dyes and automated high‐resolution widefield imaging. In addition, by normalizing the whole organ fluorescence data based on single vesicle imaging, we were able to compensate for the different labelling of the EV‐sources thereby allowing to link pharmacokinetics, biodistribution and spatial biology of EVs in vivo.

Regenerative

Presenter: Ayelet Lotan Technion—Israel Institute of Technology, Haifa, Israel Abstract unavailable

Single‐Pot

Presenter: Tyler T. Cooper Queen's University, Kingston, Ontario, Canada Introduction : High‐grade serous carcinoma (HGSC) is the most common and lethal form of epithelial ovarian cancer, largely due to the lack of effective biomarkers for early detection. Ovarian cancer is often diagnosed at an advanced stage when treatment options are limited, and prognosis is poor. Current biomarkers, such as CA‐125, are insufficient for early‐stage detection, as they lack the sensitivity and specificity to identify the disease at its onset. Therefore, there is an urgent need for new, reliable biomarkers that can detect ovarian cancer at an earlier stage, improving patient outcomes and enabling timely intervention. Therefore, developing technologies that can provide deep, insightful analysis to support biomarker discovery is crucial. Methods : In this study, we developed and optimized a workflow for the isolation and multi‐omics analysis of extracellular vesicles (EVs) from different biofluids, specifically plasma and ascites. We took this approach because EVs are known to carry a rich array of molecular information reflective of their cell of origin, making them ideal candidates for biomarker discovery. Using strong anion exchange (SAX) chemistry on magnetic beads, we successfully isolated EVs and applied a single‐pot multi‐omics approach to analyse their metabolomic, lipidomic, proteomic, and surface‐enhanced Raman (SERS) profiles. EV isolations were validated by mass spectrometry, atomic force microscopy, and nanoparticle tracking analysis. Results : Our results demonstrate the feasibility of obtaining comprehensive proteomic and Raman data from as little as 10 to 100 µL of ascites. Likewise, we were able to obtain a metabolic, lipidomic and proteomic profile from 100 µL plasma. Notably, SAX capture of EVs was able to deplete lipoproteins, TF, and albumin orders of magnitude. We also found that the use of dodecyl‐β‐D‐maltoside (DDM) significantly enhances peptide identification up to 30%, contributing to a more in‐depth characterization of EV proteomes. The workflow was initially piloted on umbilical cord plasma (human) and murine (ID8) ascites, and we are now extending the application to plasma and ascites from human patients with HGSC. Summary/Conclusion : This work offers a promising approach for the detailed molecular characterization of EVs and may contribute to advancing biomarker discovery for ovarian cancer detection and monitoring. Funding : Cancer Research Society and Canadian Institute of Health Research.

Size‐Based

Ivy Bhattacharya, Marzia Iarossi, Navneet Chandra Verma, Diana Huttner, Amit Meller Technion—Israel Institute of Technology, Haifa, Israel Introduction : Extracellular vesicles (EVs) are increasingly being accepted as circulating biomarkers accessible to liquid biopsies. However, the heterogeneity in the size profile of EVs originating from different sources, even when identified as small EVs (sEVs), and the contrasting dearth of evidence directly correlating EV size to its biophysical characteristics and, in turn, to its diagnostic potential, will likely mandate the development of novel, precise and democratic technologies for EV detection and sizing. Recently, the ability to fabricate sub‐wavelength solid‐state nanochannels and load them with biomolecules of interest has opened new avenues to achieve far more sensitive quantification of both concentration and single‐EV trajectory dynamics over time. Methods : To fabricate our nanochannel device in‐house, we used a combination of chemical dry etching and anodic bonding. To minimize pre‐processing of an HCT‐116 derived EV sample so that we retain accessibility of the device, we first fluorescently labelled the lipid membrane with DiD using the salt‐change method to avoid conflation of dye aggregates and EVs. To further ensure sample purity, we labelled the particles with a dye‐antibody complex specific to CD63 and assured colocalization of the dyes on the particles identified as EVs on our simple widefield optical setup. Results : We develop a generic 4‐dimensional single EV analysis method that allows not only highly sensitive EV detection but also size recognition based on single particle tracking of individual EVs during their electromigration. Tracking of the EVs provides rich multi‐dimensional information, which includes their migration velocity profiles and their diffusion velocity profiles, as well as their available lipid surface area. This information is then processed by a neural network trained on particles of known radii to output a most probable radius value for a specific EV, with an accuracy up to ±4 nm. Summary/Conclusion : We build an experimental and analytical framework that raises the limit of detection of EVs in a sample by several orders of magnitude compared to conventional techniques of EV counting and sizing. Importantly, our method does not rely on specific antibodies for EV sensing and is non‐destructive in the sense that EVs retain their biophysical characteristics and membranal integrity.

Standardized

Presenter: Cristina Zivko University of Bern, Switzerland Introduction : Extracellular Vesicles (EVs) are released by cells across all biological domains, playing crucial roles in many intercellular interactions under physiological and pathological conditions. They are thus increasingly studied for diagnostic and therapeutic purposes, some already reaching clinical trials. Methods : EVs were isolated from five distinct human or bovine model tissues (hepatic, ureteral, intervertebral disc, umbilical cord mesenchymal, and induced pluripotent stem cells). Isolation was performed by standardized ultracentrifugation followed by size exclusion chromatography to ensure high purity and minimal protein aggregation. The resulting EV populations were thoroughly characterized in terms of yield, size distribution, and surface charge. For all samples, membrane blotting of the purified EVs was performed to recover proteins for untargeted shotgun proteomics analysis. Proteins were also isolated from the cells that shed them. Results : EV isolation yielded 10⁸–10 11 particles per sample depending on the origin, indicating successful recovery but also differences across tissue types. Nanoparticle tracking analysis confirmed heterogeneous but consistent vesicles’ size distribution profiles, averageing 180±100 nm. Measurements of zeta potential indicated a mean surface charge of ‐29±11 mV, suggesting colloidal stability and a negatively charged membrane, which aligns with typical EV properties. Proteomics analysis revealed a highly distinctive yet reproducible protein signature for each tissue‐derived EV population. It was powerful enough to also discriminate between different treatment conditions within the tissue of interest. Cells proteomics were clearly discriminated from EVs. Comparative analysis between EVs and their respective parent cells uncovered selective protein enrichment patterns, supporting the hypothesis that EVs actively package specific biomolecules rather than merely reflecting cellular protein content. Summary/Conclusion : We provide reproducible evidence that EVs are not passive cellular byproducts but functionally specialized particles. Results were consistent across 5 tissues of origin, contributing to the growing body of knowledge on EV heterogeneity, highlighting the potential of tissue‐specific EVs for translational applications. By establishing a solid foundation for future clinical/biotechnological applications, our study advances the standardized understanding of EV biology and paves the way for their integration into therapeutic strategies. Funding : Center for Extracellular Vesicles Research (EVR) at the University of Bern, the Proteomics and Mass Spectrometry, the Flow Cytometry and Cell Sorting Core Facilities.

Amphiectosome

Tamás Visnovitz 1 , Dorina Lenzinger 1 , Anna Koncz 1,2 , Lilla Lankovics 1 , István Dudás 1 , Tünde Bárkai 1 , Krisztina V Vukman 1 , Kelsey Fletcher 1 , Csaba Cserép 3 , Ádám Dénes 3 , Péter Lőrincz 4 , Martin Krátký 5 , Szilvia Bősze 1,6 , Gábor Valcz 1 , Edit I Buzás 1,2,7 1 Semmelweis University, Department of Genetics, Cell‐ and Immunobiology, Budapest, Hungary; 2 HUN‐REN‐SU Translational Extracellular Vesicle Research Group, Budapest, Hungary; 3 Laboratory of Neuroimmunology, HUN‐REN Institute of Experimental Medicine, Budapest, Hungary; 4 ELTE Eötvös Loránd University, Department of Anatomy, Cell and Developmental Biology, Budapest, Hungary; 5 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, Czech Republic; 6 HUN‐REN–ELTE Research Group of Peptide Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 7 HCEMM‐SU Extracellular Vesicle Research Group, Budapest, Hungary Introduction : Small extracellular vesicle (sEV) biogenesis remains partially understood. Our recent findings show that all tested cell types, including cell lines and cells in their tissue environment, release multivesicular large extracellular vesicles (MV‐lEVs) that enclose intraluminal vesicles (ILVs). When the external membrane of these MV‐lEVs ruptures spontaneously, ILVs are released into the extracellular space through a ‘torn bag’ mechanism, a process distinct from multivesicular body (MVB) exocytosis and plasma membrane shedding of small ectosomes. Methods : Here we investigated this unique MV‐lEV‐mediated sEV release pathway. We employed high‐resolution confocal microscopy, super‐resolution nanoscopy (Lattice SIM2 and STED) on both fixed and live cells, and transmission electron microscopy (TEM) with or without immunogold labelling. ATG5 and Rab27a genes were silenced using siRNAs, and protein analysis was conducted by Western blotting. We modulated various cellular pathways with chemicals (e.g., chloroquine, bafilomycin, rapamycin, cytochalasin B and colchicine) as well as drug candidates (e.g., niclosamide and salicylanilide). Additionally, we examined the effects of the A23187 Ca 2+ ionophore on sEV release using STED nanoscopy, TEM and high‐resolution flow cytometry. The subcellular origin of the external membrane of MV‐lEVs was tracked with PalmGFP and fluorescently labelled wheat germ agglutinin. Results : We identified the observed MV‐lEVs as secreted amphisomes containing ILVs from both MVBs and autophagosomes. The external membrane of the budding MV‐lEVs is clearly derived from the plasma membrane; therefore, we termed the released MV‐lEVs ‘amphiectosomes.’ By super‐resolution nanoscopy, we captured consecutive stages of the intracellular amphisome formation, including fragmentation of the LC3‐positive autophagosome‐derived membrane and ILV formation. Amphiectosome secretion was found to be an active process, distinct from any other known EV release pathways. Surprisingly, under steady‐state conditions, exocytosis‐dependent exosome release was not observed by TEM; however, it was significantly induced by A23187. Summary/Conclusion : Our findings suggest that sEV release by the ‘torn bag’ mechanism of amphiectosomes is a general, essential sEV secretion pathway in non‐stressed, steady‐state mammalian cells. It is a novel EV secretion mechanism which is substantially distinct from the known exocytosis of exosomes. Funding : This study was supported by the NVKP_16‐1‐2016‐0004; OTKA K120237; FK138851 ); EKA2022/045‐P101; VEKOP‐2.3.2‐162016‐00002; VEKOP‐2.3.3‐15‐2017‐00016; TKP2021‐EGA‐23; RRF‐2.3.121‐2022‐00003; 2019‐2.1.7‐ERA‐NET‐2021‐00015; EU's Horizon 2020 No.739593; NAP2022‐I‐1/2022; János Bolyai Research Scholarship; LP2022‐13/2022; LP2022‐5/2022.

Anti‐Cancer

Nil Kılıç 1 , Eylül Gülşen Yılmaz 1,2 , Fatih Inci 1,2 1 UNAM‐National Nanotechnology Research Center, Bilkent University, Ankara, Turkey; 2 Institute of Materials Science and Nanotechnology, Bilkent University Ankara, Turkey Introduction : Natural killer (NK) cells and their anti‐cancer effects have been the focus of many researchers in cancer immunotherapy. Extracellular vesicles (EVs) play significant roles in functions such as cell differentiation, and cell communication by regulating gene expression in recipient cells. Research on cancer treatment using EVs has also become one of the focal points of today. In this study, the anti‐cancer effect of EVs from IL‐21‐activated and non‐activated NK‐92 cells on breast cancer cells was investigated. Methods : After activation with IL‐21 cytokine, NK‐EVs isolation was performed using microfluidic chip‐based methods. The characterisation of EVs was conducted using three analytical techniques: nanoparticle tracking analysis (NTA), western blot and SEM imaging. The most effective concentration and time for breast cancer cells incubated with NK‐EVs were identified using a WST‐1 cytotoxicity assay. The expression of apoptotic genes BAX, BCL‐2, CASPASE3, CASPASE8 and CASPASE9 in breast cancer cells was analysed using qRT‐PCR. Additionally, the synthesis of proteins associated with apoptosis (BAX, CASPASE3, CASPASE8 and CASPASE9) in breast cancer cells was examined using western blot. Results : EVs characterization was conducted on both IL‐21‐activated and non‐activated NK‐92 cell lines. It was established that the concentration of EVs was higher in the group of cells that had been activated. The results showed that EVs from IL‐21‐activated NK‐92 cells significantly increased the apoptotic effect compared to EVs from IL‐21 non‐activated cells. The expression and synthesis of BAX, BCL‐2, CASPASE3, CASPASE8 and CASPASE9 apoptotic genes and proteins were observed to be elevated in comparison to the control group that had not been stimulated by IL‐21. Summary/Conclusion : EVs obtained from the activated NK cell group showed more cytotoxic effect. In the future, miRNA profiles of NK‐EVs obtained from two different groups will be analysed. Furthermore, miRNA profiles of NK‐derived EVs with apoptotic effect against breast cancer cells will be revealed. Funding : This study is supported by the 2218 National Postdoctoral Research Fellowship Program from The Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project No: 123C139).

Bioengineered

Presenter: Meenakshi Mendiratta All India Institute of Medical Sciences, New Delhi, India Introduction : Mesenchymal stem cell‐derived small extracellular vesicles (MSCs‐sEV) are a promising candidate for cell‐free therapeutics in regenerative medicine. However, there are several challenges with the utility of sEV, including non‐specific packageing of cargo, loss in circulation, and target specificity. The current study employs surface and cargo modification strategies to overcome these challenges to enhance sEV homing, target specificity, and specific cargo enrichment for liver diseases. This research explores dual modification strategies to improve targeting precision and therapeutic efficacy of mesenchymal stem cell‐derived small extracellular vesicles, specifically emphasizing hepatic cell targeting. Methods : Tissue‐specific MSCs (bone marrow, Wharton's jelly) were isolated with due patient consent ((IC‐SCR/140/23 (O)) and characterized as per ISCT guidelines. MSCs were cultured in serum‐free media for isolation of sEV via ultracentrifugation. The plasma‐derived sEV were isolated from healthy control, ACLF, and CLD patients and further evaluated for their content by small RNA sequencing, with a primary focus on hepatoprotective miRNA distribution. The surface of tissue‐specific sEV was bioengineered with hepato‐specific ligands using chemical conjugation methods, while the cargo of sEV was enriched with hepatoprotective miRNA using lipofectamine RNAiMAX. Naive and BioEn‐sEV were characterized as per MISEV 2023 guidelines. Surface modification was validated via the lectin‐induced aggregation method. Cargo enrichment was validated via qPCR, and hepatoprotective functionality was validated via ROS, macrophage polarization, T cell proliferation in an in vitro ACLF model. Results : It was observed that BioEn‐sEV and Naïve sEV demonstrated a cup‐shaped morphology through TEM. Additionally, BioEn‐sEV demonstrated an increased diameter and became a positive charge compared to Naïve sEV. Furthermore, cellular uptake efficiency of BioEn‐sEV was notably higher in hepatic cells compared to non‐hepatic cells, surpassing that of Naïve sEV. BioEn‐sEV significantly reduced ROS production and T‐cell proliferation and enhanced polarization of macrophages towards M2 macrophages and Treg induction as compared to Naïve sEV. The significant hepatoregenerative capacity of miRNA (identified from CLD and ACLF patients) of BioEn‐sEV in hepatic cells further underscores their therapeutic promise. Summary/Conclusion : This study has demonstrated precise sEV targeting through surface modification, enhancing anti‐inflammatory effects via cargo modification. Bioengineered sEVs emerge as a promising avenue for targeted therapeutic interventions in diseases, prompting further clinical exploration in this innovative field.

Cerebrospinal

Presenter: Jamal Ghanam University Hospital Essen, Essen, Germany Introduction : Understanding the properties and dynamics of medulloblastoma (MB) progression remains elusive due to a lack of established minimally invasive monitoring techniques. Extracellular vesicles (EVs) carrying disease‐specific signatures have emerged as promising analytes for repetitive liquid biopsies. However, this is hindered by the absence of standardized EV isolation methods and the difficulty of targeting cancer‐specific EVs in body fluids. Here, we evaluate the clinical utility of blood plasma (BP) and cerebrospinal fluid (CSF) small EVs (sEVs), which remains unexplored in MB. Methods : We employed ultrafiltration and size exclusion chromatography (UF‐SEC) to isolate sEVs from BP and CSF of children with MB. sEVs were characterized according to MISEV2023 guidelines and subjected to liquid chromatography‐tandem mass spectrometry. Gene Ontology analysis and Cytoscape pathway analysis were conducted to investigate associated functions and further identify proteins with datasets from MB tissue, CSF, and common EV markers. Results : UF‐SEC was optimized to separate sEVs from low volumes of BP and CSF with high recovery rates. We observed a shift in particle number, protein concentration, and CD81‐positive sEVs towards later fractions in CSF‐derived EV preparations, indicating that CSF‐sEVs are notably smaller in size compared to BP‐derived EVs. This was further confirmed by TEM imaging. We analysed the EV proteome to investigate whether the disparities in size and bodily fluid type have a measurable impact on the quantity and quality of EV protein cargo. 210 proteins were identified and grouped into 10 clusters by K‐means clustering, revealing a body‐fluid‐specific protein ontology enrichment. Proteins associated with the extracellular matrix (ECM) and integrin functions were significantly upregulated in CSF‐sEVs. Notably, analysis of MB tissue datasets revealed that ECM and integrin proteins such as ANXA6 were exclusively enriched in CSF‐sEVs. Summary/Conclusion : This study underscores the importance of tailoring EV isolation methods to specific body fluids and highlights the potential of CSF‐sEVs over BP‐sEVs for MB diagnosis and monitoring. Further research in a larger cohort is needed to confirm these findings and assess their contribution to predicting recurrence, metastasis, outcome, and prognosis.

Cll‐Derived

Presenter: Orit Uziel Tel Aviv University, Tel Aviv, Israel Introduction : We studied here whether CLL cells communicate with their microenvironmental cells (endothelial and B cells) to acquire a survival advantage. We hypothesized that both interactions of CLL cells, either with the endothelial cells or with the normal B cells are mediated by CLL‐derived EVs for their benefit. Methods : CLL derived EVs were isolated from CLL cells of several untreated patients. EVs were harvested by ultracentrifugation and characterized by NTA, Western blotting and electron microscopy. The engulfment of stained EVs was followed by flow cytometry. Endothelial cells: (1) Underwent phosphoproteome analysis; (2) results were validated by western blotting; (3) secretion of IL‐6 was assessed by ELISA; (4) apoptosis was measured by flow cytometry; (5) CTNNB1 was ectopically expressed; (6) Interaction of all phosphoproteins and CTTNB1 was done by ANAT; (7) Binding of transcription factors was measured by ChIP. B cells: (1) RNA‐seq was performed. (2) Validation of the results: by Q‐RT PCR. (3) SMAD6 was ectopically expressed, and apoptosis was assessed as above. Results : CLL‐derived EVs were shown to possess the expected 30–150 nm diameter size and shape. These EVs were engulfed by either endothelial or B cells in a dose‐ and time‐dependent manner. The endothelial exposed cells (1) became into IL‐6 producing cells. (2). The IL‐6 enriched medium promoted the phosphorylation of STAT‐3 in fresh CLL cells, decreasing their apoptosis. (3) Twenty‐three proteins were phosphorylated in response to CLL‐ EVs. (4) Network analysis unravelled the central role of phosphor‐β‐catenin. (5) Ectopic expression of β‐catenin increased IL‐6 levels in the culture medium. (6) ChIP analysis revealed that all 3 transcription factors possessed an increased binding to the IL‐6 promoter. In contrast, exposed B cells (1) underwent apoptosis; (2) 24 transcripts were differentially expressed; (3) ectopic expression of SMAD6 in B cells induced apoptosis. Summary/Conclusion : CLL cells differentially communicate with endothelial and B cells through secreting EVs. Once they are taken up by endothelial cells, they turn them into IL‐6‐producing cells, thus providing the neoplastic cells with a survival advantage. However, B cells get a ‘death message’ sent by the neoplastic EVs to eliminate their potential competitors.

Combinational

Haroon Khan 1 , Jiannan Xiao 1 , Ningqiang Gong 2 , Zhaoting Li 1 , Chuancheng Ren 1 1 The Chinese University of Hong Kong Shenzhen, Shenzhen, People's Republic of China; 2 University of Science and Technology, Hefei, People's Republic of China Introduction : Ischaemic stroke is the leading cause of death worldwide, caused by blockage of cerebral blood flow, leading to neuronal death and disability. Fibrinolytic S100A10‐EVs, along with plasmin resistant D‐tat‐EE3 could be delivered locally by encapsulating them in thermosensitive Pluronic F127 hydrogel to mitigate both primary and secondary damage mechanisms of stroke. Methods : S100A10 lentivirus transfected MSC‐derived EVs are isolated and characterized. Plasmin generation, clot lysis and thrombolytic assays are performed to evaluate the fibrinolytic efficiency of S100A10‐EV. Cellular uptake and in vitro ischaemic stroke mitigation efficiency are evaluated for the EVs and peptide. For animal studies, the transient middle cerebral artery occlusion model is used, and the results were confirmed through Nissl staining to examine the infarct size, and neurobehavioural tests are performed to evaluate recovery in rodents. Laser speckle imaging and immunostainings are used to examine cerebral blood flow and neuroprotection. Results : Lentiviral transfection significantly increased S100A10 expression both in MSCs and isolated EVs, as confirmed by western blotting, qPCR, and fluorescent microscopy. In vitro studies demonstrate the fibrinolytic, neuroprotective, and anti‐reactive oxygen species capabilities of EVs and D‐tat‐EE3. Localized injection of Pluronic F127 hydrogel encapsulating S100A10‐EVs and D‐tat‐EE3 exerted distinct therapeutic effects in vivo, including enhanced cerebral blood flow and reduced neuronal death. Mice receiving the loaded hydrogel exhibit reduced infarct volumes and improved functional and behavioural outcomes. Summary/Conclusion : This combinational therapy offers a novel and promising multi‐target approach to address the primary and secondary damage mechanisms of ischaemic stroke. Funding : The work is supported by Guangdong Natural Science Foundation (JCYJ20240813113550064); the University Development Research Start‐up Fund (UDF01003417); the Pengcheng Peacock Project Special Fund (2023TC0190); National Natural Science Foundation of China Youth Science Foundation Project (82404527).

Comprehensive

Presenter: Marco Falasca University of Parma, Parma, Italy Introduction : Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers, with only 12% of patients surviving 5 years post‐diagnosis. Effective early detection remains a critical unmet need in PDAC management. Our study explores the role of small extracellular vesicles (sEVs) in PDAC, as they promise to deliver tumour‐specific biomarkers and therapeutic targets. Tumour‐derived sEVs can carry oncogenic proteins that drive tumour progression, metastasis, immune evasion, and invasion. Identifying specific molecular signatures in these vesicles could yield novel PDAC diagnosis and treatment avenues. Methods : We examined sEVs from three groups: healthy individuals ( n = 10), PDAC patients ( n = 19), and chronic pancreatitis patients ( n = 3), using the latest MISEV 2023 guidelines. sEVs were isolated via ultracentrifugation and EV‐affinity techniques and characterized by scanning electron microscopy, nanoparticle tracking analysis, immunoblotting, and quantitative proteomics. For proteomic profiling, we utilized data‐independent acquisition mass spectrometry with an orbitrap eclipse tribrid mass spectrometer, followed by nano‐HPLC separation and peptide quantification with DIA‐NN software. Ethical approval was obtained from Royal Perth Hospital and Curtin University ethics committees. Results : Proteomic analysis identified a distinct panel of 12 proteins in PDAC and chronic pancreatitis sEVs. Among these, SAA2 was exclusively found in PDAC‐derived sEVs, suggesting its potential as a PDAC‐specific biomarker linked to inflammatory processes in the tumour microenvironment. Additionally, detoxification and metabolic enzymes such as GGT1 and ALDH1A1, uniquely enriched in PDAC sEVs, could reflect metabolic adaptations of tumour cells. This protein profile highlights specific pathways in PDAC pathology that are encapsulated within sEVs and may aid in non‐invasive PDAC detection. Summary/Conclusion : Our findings underscore the promise of sEVs in advancing the field of PDAC diagnostics and therapeutics. The unique molecular profile of PDAC‐derived sEVs, particularly the presence of SAA2 and metabolically active enzymes, presents new possibilities for biomarker development and mechanistic insights into PDAC progression. These insights could inform novel strategies for early detection and intervention, positioning sEVs as a valuable tool in the fight against PDAC. Funding : This research was funded by a grant from PanKind, The Australian Pancreatic Cancer Foundation.

Concentrating

Markus Bergqvist 1 , Cecilia Lässer 1 , Rossella Crescitelli 2 , Kyong‐Su Park 1 , Jan Lötvall 1 1 Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 2 Department of Surgery, Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Introduction : Extracellular vesicles (EVs) are enriched from in vitro cell culture supernatants and various biofluids by multiple types of methods, including ultracentrifugation, which, however, has significant limitations, especially from the perspective of scalability. We have aimed to develop a non‐centrifugation‐based technique, removing non‐EV components of harvested cell culture medium using superabsorbent polymer (SAP), followed by size exclusion chromatography (SEC), with the goal to achieve similar yield and purity as ultracentrifugation (UC) followed by iodixanol density cushion (IDC). Methods : HEK293 cells expressing CD63 coupled with Thermo Luciferase (T‐Luc) were used as a starting material to help quantify the yield and purity of EVs. After removal of cells and large debris from a 0.5 L cell culture by slow centrifugation, the cell culture supernatant was concentrated by pressure ultrafiltration as a primary step (Sartorius Vivaflow 200). EVs were then concentrated by either UC, centrifugal filter units (MERCK 10 kDa cutoff, CFU) or SAP within a dialysis membrane with a 3.5 kDa cutoff. After concentration of the EVs, further purification by either IDC or SEC using either Sepharose CL‐2B or Sepharose 6B, in either 10 or 20 mL columns (2B‐10, 2B‐20, 6B‐10 or 6B‐20). Results : The pressure ultrafiltration concentrated the supernatant from the cell cultures from 500 mL down to 20–30 mL. All the subsequent concentration methods, including UC, SAP or CFU, resulted in a final volume of approximately 1 mL. The highest yield was with UC (70%–80%), followed by SAP (60%–70%) and CFU (50%–60%). For evaluation of the four SEC methods and the IDC, the yield was 60%, 50%, 50%, 40% and 60%, respectively, for 2B‐10, 2B‐20, 6B‐10 or 6B‐20 and IDC. Purity was evaluated as the relationship between luciferase signal vs protein concentration measured by BCA. Purity was 26, 25, 15, 15 and 38, respectively, for 2B‐10, 2B‐20, 6B‐10 or 6B‐20 and IDC (arbitrary numbers). Summary/Conclusion : In summary, UC combined with IDC resulted in the highest purity and highest yield. An alternative approach also achieving high yield and purity is SAP combined with 2B‐10 or 2B‐20. Thus, utilization of SAP followed by SEC is an alternative efficient way to enrich EVs from cell culture.

Dual‐Ligand

Presenter: Lingyan Yang Guangzhou National Laboratory, Guangzhou, People's Republic of China Introduction : WNT signalling is an essential pathway regulating tissue morphogenesis and regeneration across multiple organs through a large family of ligands. However, harnessing the WNT signalling pathway for regenerative medicine has been challenging due to the lack of approaches to identify specific WNT ligands and deliver them for tissue‐specific regeneration. Methods : Herein, we developed a panel of WNT signalling‐inducing exosomes as a protein delivery system and uncovered their therapeutic potential. Results : We showed that WNT signalling‐activating ligands, including the WNT and R‐spondin (RSPO) proteins, could be transported on engineered exosomes and synergistically achieve efficient pathway activation. Through systematic screening of human liver progenitor cell cultures, we identified a specific combination (WNT3A and RSPO1) that could effectively regulate hepatic cell fate and uncovered potential functional crosstalk with the PPARα signalling pathway. We also observed that dual ligands loaded on a single exosome (exoWNT3A/RSPO1) hyperactivate the WNT pathway and promote efficient adult liver organoid growth compared to the small molecule inhibitor CHIR99021. After exoWNT3A/RSPO1 administration, we confirmed the activation of WNT signalling in the liver using a transgenic Axin2‐mGFP mice model. Importantly, exoWNT3A/RSPO1 could accelerate liver repair and regeneration under various conditions, including acute and chronic injuries and aged‐associated phenotypes. Summary/Conclusion : Collectively, our work revealed the broad therapeutic effects of WNT signalling activation in the liver through the dual‐ligand‐carrying exosomes, which activate PPAR signalling for hepatic lineage maintenance and proliferation.

Early‐Stage

Presenter: On Shim EXoPERT, Seoul, Republic of Korea Introduction : Lung cancer, with one of the highest mortality rates among cancers, remains an area where liquid biopsy techniques are underutilized. While extracellular vesicles; EVs in blood have distinctive features across various tumours, including lung cancer, utilizing these features for diagnosis poses challenges due to biomarker variability and tumour heterogeneity. Here, we present a lung cancer liquid biopsy system based on AI analysis of Raman spectroscopic signals of plasma EVs, validated through a clinical trial using prospective and retrospective multi‐ethnic samples. Methods : EVs were isolated from plasma using the dual‐size exclusion chromatography (SEC) method. Raman signals were detected through the surface‐enhanced Raman spectroscopy (SERS) technique. To reflect the heterogenicity of EVs, signals were detected at 100 different spots per sample. The EV isolation and detection were implemented using our automated isolation and detection equipment and rigorously performed through an external lab. Blood samples were retrospectively and prospectively collected from 11 institutions in Korea and 4 institutions in the United States. Our CNN‐based algorithm has three parts: EV signal filtering, cancer signal classification, and lung cancer progression score refinement. The final cancer score integrates the results from the 100 signals. To train and validate the algorithm, we used over 690k SERS signals from 1853 Korean subjects and 244 subjects from the US clinical study. Finally, we validated the clinical performance. Results : The algorithm prediction result of our 135 hold‐out test set achieved a sensitivity of 75% and a specificity of 95%. The sensitivity by cancer stage reaches 69% and 82% for early‐stage and advanced stage, respectively. The false‐positive rate for other cancer types—prostate, ovarian, and liver cancer, is 20%. Summary/Conclusion : We implemented and validated a novel liquid biopsy system for lung cancer, encompassing a series of processes from EV isolation to signal detection. We evaluated clinical performance on multi‐ethnic samples, demonstrating high predictive accuracy for early lung cancer detection. These results highlight the potential for this system as a new screening approach. Funding : This work was supported by the Korea Medical Device Development Fund grant funded by the Korean government (Project Number: 1711195429, RS‐2020‐ KD000094 ).

Elisa‐Based

Tatsuya Nishimura, Keiichi Takizawa, Yousuke Hirakawa, Yuko Kajiho, Shoichiro Kanda, Yutaka Harita The University of Tokyo Hospital, Japan Introduction : Chronic kidney disease (CKD) has emerged as a global health crisis, posing significant challenges to healthcare systems worldwide. The progressive loss of functional nephrons is a key factor in the development and progression of CKD. Early detection of CKD is crucial, as it allows for timely intervention that can significantly slow disease progression, reduce complications, and improve patient outcomes. Our previous work highlighted the potential of expression levels of mucin 1 (MUC1) on urinary EVs (uEVs) as an indicator for childhood CKD (iScience 2022), and we developed an ELISA‐based method for the quantitative assessment of uEVs (STAR Protocols, 2023). In this study, we optimized the methodology and examined its utility in a broad range of patients, including adults. Methods : We generated monoclonal antibodies against MUC1 and evaluated their performance across two uEVs purification methods (Tim4 and CD9 antibodies) on ELISA plates using urine samples from 88 children and 160 adults with varying kidney functions who are receiving outpatient care. The MUC1‐uEVs values were standardized with urinary creatinine. This study was approved by the ethics board of the University of Tokyo. The institutional review board waived the requirement for individual informed consent. We provided comprehensive information about the study to potential participants and offered a clear opt‐out mechanism. Results : Among the developed monoclonal antibodies, the most sensitive one, in conjunction with both Tim4 and CD9 antibodies for uEVs purification on ELISA plates, effectively captured variations in MUC1 levels on uEVs. The CD9 plate method more accurately mirrored decreased kidney function outcomes than Tim4. The area under the curve (AUC) values from receiver operating characteristic (ROC) curve analysis for predicting the presence of moderate CKD (eGFR < 60) in adults was 0.912. This predictive accuracy was consistent across gender‐specific analyses. Similar results were obtained in paediatric patients. The samples stored at 4°C or −80°C are stable for uEVs‐MUC1 quantification, allowing for future batch analysis. Summary/Conclusion : This system demonstrates significant potential as a clinical biomarker for CKD, offering a novel, non‐invasive testing method. Its cost‐effectiveness makes it feasible for widespread implementation in clinical settings.

Encapsulation

Yaoyao Lu, Gege Zhao, Nicolas Soucy, Joel Rousseau, Jacques P.Tremblay Centre de Recherche du CHU de Québec – Université Laval, Québec, Canada Introduction : Prime editing (PE) is a precise genome manipulation technology based on the CRISPR‐Cas9 system, also known as the “search and replace” approach, which does not require exogenous donor DNA or DNA double‐strand breaks (DSBs) to repair the mutated gene. PE provides advantages over other nuclease gene editing technologies, including greater precision and the ability to edit different types of mutations. Nevertheless, due to its coding sequence of 6.3 kilobases, there is no effective method to deliver PE systems for in vivo treatment, which restricts its use in fundamental research and agriculture. Extracellular vesicles (EVs) are a diverse set of lipid‐ and protein‐delimited vesicles released from cells to the extracellular environment to transport nucleic acids, proteins, and metabolites between cells. Palmitoylation is an important post‐translational modification that regulates multiple aspects of protein function, including the localization to membranes, intracellular trafficking, protein interaction, protein stability, and protein conformation. Therefore, we encapsulated the components of the PE system into extracellular vesicles (EVs) through their biogenesis by using S‐palmitoylation to correct the DMD gene mutation. Methods : To reach this goal, we incorporated a palmitoylation peptide derived from growth‐associated protein 43 (GAP43) into the N‐terminal of SpCas9 nickase and the N‐terminal of reverse transcriptase (RT). The palmitoylation peptide‐modified Cas9 nickase and RT were transfected into cells using polyethyleneimine (PEI). Afterward, the medium was changed to an EV‐depleted FBS medium. The EVs were subsequently purified by ultracentrifugation and added to EV recipient cells already containing an epegRNA permitting the correction of the G to C mutation in the EMX1 gene. The correction of this point mutation in the EVs recipient cells was analysed by Sanger sequencing. Results : The palmitoylation peptide from GAP43 permitted the successful loading of the SpCas9 nickase and the RT into EVs. The addition of these EVs to the culture medium of EV recipient cells, permitted the correction of the G to C mutation in 10% of the EMX1 gene. Summary/Conclusion : The incorporation of palmitoylated Cas9 nickase and reverse transcriptase proteins into EVs creates a new avenue for the safe and effective delivery of the PE components to treat hereditary diseases.

Endometriotic

Jaelis P. Holmes, Katherine B. Zutautas, Danielle J. Sisnett, Chandrakant Tayade Department of Biomedical and Molecular Sciences, Queen's University, Canada. Introduction : Endometriosis (EM) is a chronic, estrogen‐dependent inflammatory disease with high heterogeneity in phenotype and symptom severity, resulting in diagnostic delays of 4–11 years. Small extracellular vesicles (sEVs), contain bioactive molecules and show distinct expression profiles between EM patients and controls, making them potential candidates for disease biomarkers. This study aims to profile sEVs in mild and severe EM cases using multi‐omics data, to unravel biomarkers associated with EM. Methods : sEVs were isolated from ectopic lesions, matched eutopic endometrium, plasma, and peritoneal fluid samples from 24 patients (ASRM Stages I‐II, n = 8; Stages III‐IV, n = 8; controls, n = 8). Isolation involved combination of ultracentrifugation and Norgen Biotek resin technology, followed by characterization using NTA, TEM, and the MACsPlex EV kit, following MISEV2023 guidelines. sEV molecular content (miRNAs, lncRNAs, proteins, lipids) was assessed via transcriptomic, proteomic, and lipidomic analyses to identify conserved differential expression patterns relative to controls. All suppliers were ISO 0991 and ISO 13485 certified. Results : Nanoparticle tracking analysis confirmed sEV size distribution (50‐250 nm), while TEM showed double membrane vesicles characteristic of sEVs. Preliminary MACsPlex analysis identified high expression of tetraspanins (CD9, CD63, CD83) and surface markers associated with B cells (CD24), cell adhesion/integrins (CD29, CD41b, CD42a), and platelets/endothelium (CD42a). Lipidomic analysis revealed significantly elevated saturated fatty acids and triacylglycerides in EM patient plasma and ectopic lesions compared to controls, shedding insights into EM‐associated metabolic pathways. Proteomic and transcriptomic analyses are in progress. Summary/Conclusion : This study uniquely integrates nucleic acids, proteins, and lipids in sEVs from matched patient samples. Our results will offer a comprehensive profile of sEV signatures, and findings will aim to clarify sEV contributions to EM pathology and aid in identifying potential biomarkers for diagnostic applications. Funding : This work is supported by the Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council (NSERC).

Escrt‐0/Hrs

Presenter: Beike Wang Wuhan University, China Introduction : The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery plays a pivotal role in the biogenesis and cargo sorting of small extracellular vesicles (sEVs). While it is established that ESCRT machinery is crucial for the selective inclusion of proteins in sEVs, its impact on the lipid composition of sEVs remains unexplored. This study investigates the role of ESCRT‐0/HRS in regulating the lipid content of sEVs derived from tumours. Methods : We generated HRS knockdown (KD) and knockout (KO) cell lines using human head and neck squamous cell carcinoma Cal27. Tumour‐derived sEVs were characterized using Nanoparticle Tracking Analysis, transmission electron microscopy (TEM), and western blotting. Lipidomics profiling of both tumour cells and their sEVs was performed using Liquid Chromatography‐Mass Spectrometry (LC‐MS). Results : Significant differences in lipid composition were observed between tumour cells and their derived sEVs. Cal27 cells predominantly contained phosphatidylethanolamine (PE) and triacylglycerols (TAGs), whereas Cal27‐derived sEVs were rich in phosphatidylcholine (PC) and PE. In HRS KD Cal27 cells‐derived sEVs, there was a notable increase in ceramide (CE), lysophosphatidylcholine (LPC), and phosphatidic acid (PA). Enrichment analysis indicated activation of glycerophospholipid and glycerolipid metabolic pathways. Lipid droplet accumulation in HRS KD cells, as evidenced by Oil Red and BODIPY staining, suggested impaired lipid metabolism. The lipolysis rate‐limiting enzyme, ATGL, was significantly downregulated in HRS KD cells, potentially contributing to the observed lipidomic alterations in sEVs. Summary/Conclusion : Significant differences in lipid composition were observed between tumour cells and their derived sEVs. Cal27 cells predominantly contained phosphatidylethanolamine (PE) and triacylglycerols (TAGs), whereas Cal27‐derived sEVs were rich in phosphatidylcholine (PC) and PE. In HRS KD Cal27 cells‐derived sEVs, there was a notable increase in ceramide (CE), lysophosphatidylcholine (LPC), and phosphatidic acid (PA). Enrichment analysis indicated activation of glycerophospholipid and glycerolipid metabolic pathways. Lipid droplet accumulation in HRS KD cells, as evidenced by Oil Red and BODIPY staining, suggested impaired lipid metabolism. The lipolysis rate‐limiting enzyme, ATGL, was significantly downregulated in HRS KD cells, potentially contributing to the observed lipidomic alterations in sEVs.

Establishment

Tomofumi Yamamoto, Hirotaka Nishimura, Noritaka Hashii, Akiko Ishii‐Watabe National Institute of Health Sciences, Japan Introduction : In recent years, extracellular vesicles (EVs) have attracted attention as a new drug modality and are being developed worldwide. Mesenchymal stem cell (MSC)‐derived EVs are expected to have various effects on immune diseases and wound, and their application to humans is progressing. However, because EVs have heterogeneity, it is difficult to homogeneous EV preparations, the establishment of appropriate potency assay is the key issue for quality control of therapeutic EV products to ensure their safety and efficacy. Methods : We used a cell‐based assay in which LPS stimulation of Raw 264.7 mouse macrophage cells induces an inflammatory response in order to establish a method for quality evaluation and control of MSC‐derived EV preparations. In this assay, the anti‐inflammatory effect of EV administration was evaluated by change in TNFα mRNA expression level. hTERT‐adipose tissue MSC was used in this study. MSC‐derived EV was isolated by ultracentrifugation method. Results : MSC‐derived EVs showed anti‐inflammatory activity dependent on the number of particles added. Next, we checked whether the anti‐inflammatory effect was induced by components other than EVs in the supernatant of the EV samples used. No anti‐inflammatory activity was detected in the supernatant without EVs. Next, we prepared samples of degraded EVs that were subjected to repeated freeze‐thaw cycles. After several freeze‐thaw cycles, the samples showed changes in several physical properties, such as an increase in particle size and a decrease in particle concentration. However, there was no significant change in anti‐inflammatory activity, suggesting that the evaluation of biological activity using TNFα does not correlate with changes in the physical properties of EVs. Therefore, we comprehensively measured the expression levels of chemokines and cytokines after LPS stimulation to search for novel markers that correlate more sensitively with changes in physical properties. The results indicate that CXCL10 may be a more sensitive indicator of EV bioactivity than TNFα. Summary/Conclusion : We found that CXCL10 expression level is more suitable marker in the potency assay for MSC‐derived EV preparations. It is necessary to clarify whether the change in expression level of CXCL10 reflects the mechanism of action of EVs in vivo. Funding : AMED JP22mk0101218, Grant‐in‐Aid for Early‐Career Scientists 24K18318

Ev‐Mediated

Baoye He 1 , Brisa Davila 1 , Qiang Cai 2 , Shumei Wang 3 , Hailing Jin 3 1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, USA; 2 State Key Laboratory of Hybrid Rice, College of Life Science, Wuhan University, Wuhan, China; 3 Department of Microbiology and Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, California, USA Introduction : Cross‐kingdom RNA trafficking between plant hosts and microbes is a crucial process that regulates gene expression in both interacting partners during infection. Despite its significance, the mechanism of RNA transport between plants and microbes has remained elusive. Here, we demonstrate that extracellular vesicles (EVs) serve as a means for both plants and fungal pathogens to deliver RNA to their respective interacting partners. Methods : Arabidopsis EVs were isolated from the apoplastic washing fluid of Botrytis cinerea‐infected plants by sequential ultracentrifugation at 100,000 × g followed by sucrose gradient fractionations or immunoisolation. Fungal EVs were isolated from the liquid culture by sequential ultracentrifugation at 100,000 × g , followed by sucrose gradient fractionations. Both EVs were characterized by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Results : We performed RNA‐seq analysis and revealed that both small RNA (sRNA) and messenger RNA (mRNA) were found in plant EVs. These sRNAs can be transferred to the interacting fungal pathogen B. cinerea , where they can suppress fungal virulence‐related gene expression. Meanwhile, the mRNAs can also be transported to fungal cells, where they can be translated to modulate fungal virulence. Plant tetraspanin proteins, for example, TET8, are enriched in plant EVs and can be used as a plant EV biomarker. The fungal pathogen B. cinerea can also secrete EVs to deliver fungal sRNAs into plant cells to suppress plant immune‐related gene expression and facilitate its infection. B. cinere a EVs can be internalized by plants via clathrin‐mediated endocytosis. B. cinerea tetraspanin protein BcPLS1 serves as a biomarker for B. cinerea EVs and plays an essential role in fungal pathogenicity. Summary/Conclusion : EVs are identified as a mechanism used by both plants and fungal pathogens to deliver RNAs to the interacting sides to modulate the plant‐pathogen interaction. These findings could have significant implications for the development of novel plant protection solutions. Funding : NIH (R35GM136379), NSF (IOS 2020731), USDA (2021‐67013‐34258), USDA (2019‐70016‐29067) to H.J., and the Texas A&M startup funding to B.H.

Extracellular

Presenter: Savvas Ioannou University of York, York, UK Introduction : Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) demonstrate potential as a therapeutic for numerous disease conditions. However, the use of primary MSCs can lead to variation in the therapeutic effect due to differences in the donor source and inherent MSC heterogeneity. Here we used immortalised clonal MSC lines, termed Y201 and Y202, to examine how MSC phenotype influences EV character and function. Methods : EVs were isolated by ultracentrifugation and characterised according to recommended MISEV guidelines, including nanoparticle tracking analysis, protein and miRNA analysis and transmission electron microscopy. In vitro analysis of the functional properties of these EVs included chondrocyte proliferation assays, activated T‐cell suppression assays, and cell signalling analyses. The in vivo efficacy of Y201‐EVs was examined using two independent models of arthritis, an antigen‐induced arthritis (AIA) model and a collagen‐induced arthritis (CIA) model. Results : Both EV subtypes were morphologically similar; however, Y201‐EVs were more abundant in EV markers (CD63 & CD81) with an enhanced miRNA and proteomic content, predicted to contribute to an elabourate EV corona, particularly abundant in RGD‐containing proteins fibronectin and MFG‐E8. EVs from both cell subtypes could significantly suppress proliferation of activated T‐cells, however, only Y201‐EVs could significantly stimulate chondrogenesis and proliferation of primary human articular chondrocytes, mechanistically through an RGD (integrin)‐FAK‐ERK1/2 signalling axis. Considering the apparent anti‐inflammatory and pro‐regenerative properties of Y201‐EVs, they were tested in two mechanistically different in vivo models of arthritis (AIA and CIA), performed 2 years apart, to determine therapeutic scalability using the same EV‐producer cell. Y201‐EVs significantly reduced all measures of disease activity compared to vehicle controls, including inflammation, synovial infiltration, joint exudate, pannus formation, cartilage lesions and bone resorption. The in vivo immunomodulatory effect of Y201‐EVs was at least in part mediated by promoting T‐cell polarisation towards a pro‐resolving Treg phenotype. Summary/Conclusion : Comparing EVs derived from two different MSC clonal lines demonstrates that EV therapeutic potential is significantly affected by cell subtype. EVs derived from immortalised Y201 MSCs have immunomodulatory and pro‐regenerative properties, with in vivo efficacy in preclinical models of inflammatory arthritis over an extended timeframe, demonstrating suitability as a scalable EV therapeutic. Funding : This study was supported by BBSRC, Versus Arthritis, Northern Accelerator, and Mesenbio.

Full‐Length

Chantal Scheepbouwer 1,2,3,4 , Ernesto Aparicio 5 , Cristina Gómez‐Martin 1,4 , Monique A. J. van Eijndhoven 1,4 , Esther E. E. Drees 1,4 , Leontien Bosch 1,4 , Daphne de Jong 1 , Thomas Wurdinger 3 , Josée Zijlstra 6 , Michael Hackenberg 5 , Alan Gerber 2,3* and D. Michiel Pegtel 1,4* 1 Department of Pathology, Amsterdam UMC, VU University, Amsterdam, The Netherlands; 2 Cancer Center Amsterdam, Cancer Biology, the Netherlands; 3 Department of Neurosurgery, Amsterdam UMC, VU University, Amsterdam, The Netherlands; 4 Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, the Netherlands; 5 Faculty of Science, Department of Genetics, University of Granada, Granada, Spain; 6 Department of Hematology, Cancer Center Amsterdam, Amsterdam UMC, VU University, Amsterdam, The Netherlands Alan Gerber and D. Michiel Pegtel contributed equally to this study . Introduction : High‐throughput sequencing has advanced our understanding of nucleic acid content in extracellular vesicles (EVs), mainly focusing on short non‐coding RNAs (ncRNAs) under 50 nucleotides. However, the composition and abundance of longer ncRNAs in EVs are less well characterized. To address this gap, tailored next‐generation sequencing (NGS) protocols have been developed to minimize length bias from conventional RNA sequencing. Our study used the ALL‐tRNAseq approach, allowing the detection of a diversity of structured full‐length RNA species, including transfer tRNA (tRNA), small nucleolar RNA (snoRNA), and Y RNA. Methods : Small RNA in the 18–120 nt size range from four cell lines, including cellular, EV‐enriched, and non‐vesicular extracellular particle (NVEP) fractions, was sequenced using an adapted ALL‐tRNAseq approach. Data processing was performed using sRNAbench with normalization via the NormSeq web server. Results : Our analysis confirmed a predominance of tRNA molecules, constituting 60%–85% of assigned reads across all samples. Sequencing and northern blotting confirmed the presence of mature, full‐length tRNAs in both cells and in the lumen of EVs. The tRNA modification profiles showed similar misincorporation signatures in cells and EVs, with no significant differences impacting EV secretion, suggesting that EV‐tRNAs primarily derive from the mature tRNA pool. We assessed 3′ CCA tails of tRNAs, finding that 98% of cellular tRNAs contained the essential CCA sequence, while EV‐tRNAs exhibited substantial truncation. We also analysed plasma EVs (pEVs), revealing that CCA‐truncated tRNAs remained the predominant component in both in vitro and in vivo. Summary/Conclusion : Our findings challenge previous assumptions by demonstrating that tRNAs in EVs are primarily non‐functional full‐length molecules lacking the essential CCA tails required for aminoacylation. Despite their inability to participate in translation, these fully modified tRNAs resemble cellular counterparts and likely originate from previously functional cytoplasmic tRNAs. The consistent CCA truncation pattern across all tRNA types in EVs suggests a potential sorting mechanism for mature tRNAs. Altogether, this study highlights an important and underappreciated aspect of small RNA composition in EVs. Funding : Stichting CCA, MRD Hodgkin Lymphoma, and AQrate TKI.

Genome‐Wide

Miguel Palma‐Cobo 1 , Victor Toribio 1 , Carlos Enrich Bastús 2 , Albert Lu 2 , María Yáñez‐Mó 1 1 Department of Biología Molecular, Universidad Autónoma de Madrid, IUBM, Centro de Biología Molecular Severo Ochoa, IIS‐IP, Spain; 2 Universidad de Barcelona, Facultad de Medicina, IDIBAPS, Spain Introduction : Extracellular vesicles (EVs) hold immense potential in therapeutic delivery, warranting a comprehensive investigation of the molecular mechanisms that regulate their uptake by target cells. To identify key molecular regulators of EV internalization, we conducted a genome‐wide CRISPR (GWC) screen utilizing flow cytometry sorting aimed to pinpoint candidate genes that influence EV uptake. Methods : We employed a GWC library in K562 cells, which included 10 single‐guide RNAs (sgRNAs) per gene spanning the entire human genome, along with appropriate negative controls, for a total of 250,000 individual sgRNAs. EVs were isolated from the SKMEL147 human melanoma cell line using serial ultracentrifugation and were characterized through nanoparticle tracking analysis (NTA), electron microscopy, western blotting, and confocal microscopy. For the GWC screen, 3.6×10 1 2 EVs were labelled with Alexa633‐C5‐Maleimide, a fluorescent dye that covalently binds to sulfhydryl residues on surface proteins, and incubated for 2 h with 500 × 10⁶ K562 cells, providing a 2000× coverage of the library. The top 5% of high and low fluorescence populations were sorted. Genomic DNA was extracted, and sgRNA sequences were amplified by PCR with Illumina adaptors added as overhangs. Next‐generation sequencing (NGS) was performed to quantify sgRNA enrichment in the sorted populations compared to the unsorted control. Results : Remarkably, several members of the COMMANDER complex emerged as significant hits in our screen. To validate these findings, we employed quantitative EV uptake assays developed in our Laboratory, utilizing luciferase or maleimide‐based readouts. We conducted validations in knockout (KO) cell lines of both K562 and HeLa cells using EVs derived either from melanoma or breast cancer cell lines and kinetic follow‐up of EV cargo, including surface proteins, luminal proteins, and RNA. Summary/Conclusion : Our data suggest that the COMMANDER complex plays a pivotal role in the early stages of EV uptake and in the recycling of transmembrane EV components.

Glycosylation

Presenter: Alicja Głuszko Medical University of Warsaw, Warsaw, Poland Introduction : Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer‐related morbidity and mortality, with limited early detection methods and prognostic biomarkers. Small extracellular vesicles (sEVs), including exosomes, are key mediators of intercellular communication and have emerged as potential sources of biomarkers. Glycosylation, a post‐translational modification, plays a critical role in cancer progression and metastasis. In this study, we explore the glycosylation profiles of sEVs derived from HNSCC to identify potential biomarkers for diagnosis and prognosis. Methods : Three HNSCC cell lines (FaDu, PCI‐30, and SCC‐25) and normal keratinocytes (HaCaT) were cultured under normoxic (21% O 2 ) through 72 h. sEVs were isolated from supernatants of those cells as well as from HNSCC patient plasma ( n = 10) and healthy donors ( n = 10), using size exclusion chromatography. sEVs were characterized by western blot, electron microscopy and nanoparticle tracking analysis. Flow cytometry was used to assess the sEV‐associated glycan pattern. Mass spectrometry and TCGA‐based database analysis were used to assess modifications in glycosylation‐associated proteome and transcriptome. For correlation analysis, vesiculation‐associated gene sets were used. Results : sEVs range in size from 85 to135 nm, carry CD63, CD9, TSG101, and Alix, but not Calnexin nor Grp94. Bulk RNAseq analysis revealed significant correlation with patient survival for LG3BP ( p = 0.042), LEG1 ( p = 0.014), OST48 ( p = 0.042), DAD1 ( p = 0.017), B4GALNT1 ( p = 0.039), PYGL ( p = 0.023), ALG3 ( p = 0.017), EXT2 ( p = 0.0069), and KDELC1 ( p = 0.0061). Compared to HPV‐positive tumours significantly higher expression of LG3BP, LEG1, B4GALNT1, PYGL, EXT2, and KDELC1 was observed in HPV(−) tumours. These genes correlated significantly with tumour stage and with the vesiculation‐associated gene set. Single‐cell RNA‐seq revealed upregulation of LEG1 in immune cells subsets. MS‐based comparative analysis between tumour and normal cells revealed upregulation of mannose receptor (MPRD) and downregulation of oligosaccharyl transferase (OST48) and dolichyl‐diphosphooligosaccharide‐protein glycosyltransferase (RPN1) in tumour cells, with galectin‐3 binding protein (LG3BP) enriched in sEVs. HNSCC patient sEV glycan profiling revealed significant downregulation of ConA lectin binding compared to normal controls. Summary/Conclusion : In conclusion, the glycosylation profiles of small extracellular vesicles in head and neck squamous cell carcinoma hold promise as potential biomarkers for early detection and prognosis. Further clinical validation is needed to establish their role in improving patient outcomes. Funding : This study was supported by Medical University of Warsaw subvention for science.

Hematopoietic

Presenter: Danielle Kobulsky Children's Hospital of Philadelphia, USA Introduction : Crosstalk between hematopoietic stem and progenitor cells (HSPCs) and specialized bone marrow (BM) niche cells is critical for engraftment after therapeutic HSPC transplantation. Emerging evidence supports a dynamic role for HSPCs in shaping their niche to promote BM occupancy. However, key signalling events originating from HSPCs that govern these processes, including through secreted extracellular vesicles (EVs), are not well understood. Methods : To better understand the composition and functions of secreted HSPC vesicles, small EVs were isolated from brief ex vivo cultures of human CD34+ progenitors and donor‐matched plasma from mobilized leukapheresis products. Further density‐gradient enrichment was performed for quantitative proteomic analysis, and characterization of vesicles was performed according to MISEV2023 guidelines. Key differentially expressed proteins were validated in murine HSPC EVs for downstream functional studies. Results : HSPC EVs are enriched in integrin α4β7, a receptor for vascular cell adhesion molecule‐1 (VCAM‐1), compared to plasma EVs in both murine and human model systems (6.5‐fold, p < 0.05). Using a well‐established model of primary murine BM endothelial cells (ECs), we demonstrate robust uptake of HSPC EVs into ECs that is reduced after pharmacologic and genetic disruption of α4β7/VCAM‐1 interactions. Upon uptake into ECs, HSPC EVs induce canonical NF‐κB activation and downstream transcriptional upregulation of CC motif chemokine receptor 2 ligands (CCR2L; including Ccl2, Ccl7, and Ccl12) (> 8‐fold, p < 0.05). Small RNA sequencing studies of HSPC EVs revealed KLF4, a negative regulator of NF‐κB, to be an enriched cellular target of EV miRNA, and transcriptionally suppressed in ECs after EV uptake (0.7‐fold, p < 0.01). Functionally, recapitulation of vesicle‐mediated HSPC:EC signalling by intraosseus injection of EVs or CCR2L improved donor cell homing and engraftment in murine bone marrows after transplantation. Summary/Conclusion : Overall these findings support a novel, integrin‐dependent role for HSPC EVs in shaping the EC secretome to promote BM homing. Importantly, our study identifies a therapeutically accessible axis of HSPC:EC vesicle crosstalk to inform potential targeted conditioning strategies to improve HSPC transplantation outcomes. Funding : This work was supported by the National Institutes of Health grants R01HL164633 (P.K.), 4‐R38‐ HL143613 ‐04 (S.N.H.), and T32 HL‐07439 (S.N.H.).

Heteronuclear

Galisova A. 1,2 , Havlicek D. 1,2 , Zahradnik J. 2 , Tunca A. 2 , Androvic L. 3 , Sedlacek O. 2 , Laga R. 3 , Jirak D . 1,2 1 Institute for Clinical and Experimental Medicine,  Prague, Czech Republic; 2 Charles University,  Prague, Czech Republic; 3 Czech Academy of Science,  Prague, Czech Republic Introduction : Imaging of extracellular vesicles (EVs) is crucial for understanding their role in intercellular communication and disease mechanisms. We aim to develop an array of tracking strategies for genetically‐engineered EVs by magnetic resonance imaging (MRI). Here, we encapsulate synthetic biocompatible polymer probes composed of different MR‐active isotopes, as 19F and 31P, in EVs and then monitor the EV distribution in tissues in vivo. Such a heteronuclear approach allows specific tracking of labelled EVs compared to conventional 1 H MRI that provides only relative signals. Methods : HEK293 cells stably displaying a cancer‐targeting peptide (RGD) were used for EV isolation by a standard ultracentrifugation method (100,000 ×  g , 2 h) combined with ultrafiltration (100 kDa). RGD‐displaying EVs were characterized by western blot and NTA. Binding to cancer cells (4T1) was tested in an uptake experiment by flow cytometry. Isolated EVs were labelled with MR‐active macromolecular agents: fluoropolymers (TFEAM‐based) and phosphopolymers (pMPC‐based) using microporation (1000 V, 2 pulses, 20 ms) or extrusion (5 µm, 1 µm, 400 nm). Polymer‐labelled and unlabelled EVs were tested on a 7T MR scanner. Then, fluorescently and magnetically labelled EVs were intravenously injected into tumour‐bearing Balb/c mice and visualized by fluorescence and MR imaging. Results : Vesicles originated from the engineered cells contain the full‐length of the targeting peptide on the surface, as confirmed by western blot. Preferential uptake of engineered RGD‐EVs in 4T1 cells compared to unmodified EVs was proved by flow cytometry and in vivo fluorescence imaging in mice. Polymer‐labelled EVs in solution were specifically visualized by 19 F and 31 P MRI showing specific MR signals on a background 1 H MR image. Targeted 19F‐labelled EVs were visualized in a tumour by ex vivo MRI. Summary/Conclusion : We implemented protein engineering in combination with cutting‐edge MRI technology to create efficient EV‐based drug delivery and diagnostic systems for cancer therapy. MRI, as non‐invasive, three‐dimensional and clinically translatable imaging method, allowed visualization of magnetically labelled EVs in solutions and in tissue. Heteronuclear MRI (19F/31P) showed promise for specific in vivo detection of EVs, although the sensitivity needs to be tailored by improvement of chemical synthesis of probes or enhancement of labelling efficiency. Funding : This work was supported by the Czech Science Foundation (GACR)—Grant No. 22‐13334I.

Hypertonicity

Kristina Enge 1 , Dennis Gerloff 2 , Bayram Edemir 1,3 1 Department of Internal Medicine IV, Martin‐Luther‐University Halle‐Wittenberg Halle, Halle, Sachsen‐Anhalt, Germany; 2 Department of Dermatology and Venereology, Martin‐Luther‐University Halle‐Wittenberg Halle, Halle, Sachsen‐Anhalt, Germany; 3 Faculty of Health‐School of Medicine, Department of Physiology and Pathophysiology, Center of Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany Introduction : One of the key functions of the kidney is the generation of a concentrated urine. The osmotic gradient that is generated between the renal cortex and inner medulla is the driving force for water reabsorption in the renal collecting duct. The cells in the inner medulla are challenged with a hypertonic environment. The cells have developed mechanisms to adapt to this challenge by different mechanisms that include the induction of a specific gene expression pattern. In the recent study, we analysed how environmental osmolality affects the microRNA expression in extracellular vesicles (EV). Methods : We used primary cultivated murine inner medullary collecting duct (IMCD) cells. The cells were either cultivated at 300 or 600 mosmol/kg, and the EVs were isolated from cell culture supernatants. Total RNA was isolated using the QIAGEN miRNeasy Kit from the EVs and the cells. The RNA was sent for analysis by the company Novogene. Differentially expressed miRNAs were identified by the DESeq2 algorithm. Results : We detected 418 out of 944 microRNAs in the EV isolated from IMCD cell supernatant that was cultivated at 300 mosmol/kg and 468 out of 944 microRNAs in cells that were cultivated at 600 mosmol/kg. From these, 340 were detected in both groups. The analysis of differentially expressed microRNAs led to the identification of 112 differentially expressed microRNAs. The mmu‐miR‐9b‐5p showed the highest difference in expression in the 600 mosmol/kg group with a log2‐fold change of 6.6, and mmu‐miR‐134‐5p showed the highest expression level in the 300 mosmol/kg group with a log2‐fold change of 6.8. In the IMCD cells, we were able to detect 768 in the 600 and 771 in the 300 mosmol/kg group. A number of 652 microRNAs were common to both groups. A number of 94 showed differences in the expression level, with mmu‐miR‐1941‐5p showing the highest expression in the 600 and mmu‐miR‐199b‐5p showing the highest expression in the 300 mosmol/kg group. Summary/Conclusion : Taken together, we could show for the first time that environmental hypertonicity affects the microRNA expression profile in cells and also the composition of micro‐RNAs in EVs is affected. Similar differences in vivo might influence renal or systemic processes.

Immunological

Náthani Gabrielly Silva Negreiros, Nadjania Saraiva, Ana Claudia Torrecilhas Universidade Federal de São Paulo (UNIFESP); Instituto de Ciências Ambientais, Químicas e Farmacêuticas; Departamento de Ciências Farmacêuticas; Labouratório de Imunologia Celular e Bioquímica de Fungos e Protozoários. Introduction : Chagas disease (CD) caused by Trypanosoma cruzi (protozoa parasite), affecting more than 6‐7 million people worldwide. There are many factors are involved in the development the pathogenesis of CD; virulence, strains of parasite and host immune system. EVs shedding by T. cruzi trypomastigotes are involved in cell‐cell communication and can induce inflammation and activated host immune response. The goal was to evaluate the various additions of EVsTcruzi in macrophage to determine their pro‐ and anti‐inflammatory properties. Methods : Functional assays were conducted to determine the interaction of EVsT cruzi with macrophage. The THP‐1 cells differentiated into macrophages used 100 nM of phorbol 12‐myristate 13‐acetate (PMA) by 24 h. In parallel, we performed the interaction assay EVsTcruzi with macrophage was evaluated by confocal microscope and measure the number of EVs‐uptake during 1, 24, 48, and 72h. Cell viability was determined using the 7‐AAD and Annexin V stain by cytometry, and relative expression of cytokines (pro and anti‐inflammatory) mRNA and the levels of cytokines and cell culture supernatants were measured by RT‐PCR and Cytometric Bead Array‐CBA respectively. Results : Results showed that T. cruzi EVs interact with macrophages, and particles uptake by the cells over time, migrating to the nucleus after 1 h of incubation with EVs. After 72 h of EVs and macrophages interaction, the increase the number of particles and cell death via apoptosis. In addition, early exposure to EVsT.cruzi induce pro‐inflammatory cytokines (TNF‐alpha, IL‐6 and IL‐1) during 48, but after 72 h the profile were anti‐inflammatory response. T. cruzi‐secreted EVs interact with macrophages modulate the host immune response. Summary/Conclusion : T. cruzi‐secreted EVs interact with macrophages modulate the host immune response. These findings showed that internalization of T. cruzi EVs implications for immunological activation and control, opening the way for novel therapeutic targets and biomarkers in Chagas disease. Funding : Supported by CAPES, CNPq e FAPESP 2020/07870‐4.

Immuno‐Qpcr

Presenter: Lucia Ciglar Austrian Institute of Technology GmbH, Austria Introduction : The molecular cargo of EVs is reflective of their cellular origin, making them a promising source of biomarkers for various diseases. Traditionally, biomarker research has focused on nucleic acids rather than proteins, largely due to the limitations of existing methods for protein detection. Current methods often lack the sensitivity to quantify low‐abundance proteins in complex biological fluids, they are difficult to scale up, and they may require specialized equipment. To address these challenges, we developed an immuno‐qPCR method, which significantly enhances sensitivity while offering a cost‐effective and high‐throughput approach for detecting surface proteins on EVs. Methods : To adapt immuno‐qPCR for EV surface protein detection, we evaluated different protein immobilization strategies. The final optimized protocol employed magnetic beads to capture EVs, followed by surface protein detection using commercially available antibodies conjugated to DNA tags. These DNA tags were then amplified and quantified through qPCR. In a proof‐of‐concept study, we compared the performance of our immuno‐qPCR method to conventional flow cytometry‐based detection in clinical serum samples from patients with atopic dermatitis. Results : The use of magnetic beads, both general protein‐binding and exosome‐specific, allowed for efficient capture of EVs, with the exosome‐specific beads providing an additional advantage of increased specificity and faster workflow. When tested on a cohort of 16 atopic dermatitis patients and 16 healthy volunteers, our optimized immuno‐qPCR method simultaneously detected 10 different surface proteins and demonstrated superior sensitivity compared to conventional flow cytometry‐based detection. Notably, for one of the tested proteins, CD25, we detected a significant difference in levels between patients and healthy volunteers, which was not uncovered with flow cytometry. As CD25‐positive immune cells have been previously shown to be enriched in the blood of atopic dermatitis patients, this finding further validates our approach. Summary/Conclusion : The immuno‐qPCR method offers a highly sensitive, scalable, and versatile platform for the detection of EV surface proteins. This approach holds great promise for biomarker discovery and non‐invasive diagnostics, with potential applications extending beyond blood and atopic dermatitis. Funding : This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No. 853995 with EU Horizon 2020 and EFPIA support.

Intercellular

Hui Su Jeong, Ji Eun Lee SungKyunKwan University, Seoul, Republic of Korea Introduction : Chronic inflammation impairs the repair ability in the peripheral nervous system, including the neuromuscular junction (NMJ). Chronic inflammation inhibits Wallerian degeneration/regeneration of presynaptic structures and leads to NMJ disorders. Although Schwann cells (SCs) have been suggested as a key player in neuronee regeneration, their precise mechanism responsive to inflammation is poorly understood. In our study, we investigated the role of SCs in the NMJ and muscle regeneration in a state of inflammation. Methods : SCs in transverse abdominal and lumbrical muscles of young (4m) and old mice (22‐30m) were visualized via immunohistochemistry using NMJ and cilia markers. Cardiotoxin and LPS were used to induce tissue regeneration and inflammation. S16 SC lines were cultured for extracellular vesicle (EV) isolation that was characterized using nanoparticle trafficking analysis, western blotting, and proteomics analysis. Comparison of EVs from inflamed and non‐inflamed SCs revealed differences in size, number, and protein content. We treated EVs at damaged C2C12 myoblast cells to know their positive function in muscle regeneration. Results : NMJ defects accompanied by abnormal SC were observed in both aged and inflammation‐induced mice, suggesting SC involvement in NMJ abnormalities. While normal SCs exhibited dynamic changes after injury to regeneration, inflammation‐hyperinduced SCs lost this activity, indicating impaired molecular functions in the regenerative environment. Given NMJ's unique multicellular composition, we hypothesized that SCs would communicate through mediators, and we developed our experiments based on the hypothesis that these mediators were EVs. We performed a proteomic analysis of culture media from SCs and identified a cluster of proteins involved in both neuronee development and muscle growth factor pathways, including those regulating early myoblast differentiation. To validate the functional role of these proteins, we treated myoblasts with SC‐derived EVs and demonstrated their ability to accelerate myoblast differentiation following muscle injury. Summary/Conclusion : These findings reveal a novel mechanism whereby SCs regulate NMJ maintenance and muscle regeneration through EVs containing specific proteins, providing insight into potential therapeutic strategies for age‐ and inflammation‐related NMJ dysfunction.

Investigating

Presenter: Suzette Soerensen, Aalborg University, Denmark Introduction : Gut microbiota‐derived extracellular vesicles (GM‐EVs) are believed to play an important role in microbiome‐host communication in health and disease. The growing number of studies investigating GM‐EVs from clinical samples, often only rely on a single time point collection. The gut microbiota composition is known to be stable over time, it has, however, not been investigated if GM‐EV secretion is likewise stable. More knowledge on possible temporal fluctuations in GM‐EV secretion is therefore highly warranted. Methods : Fecal samples were collected from five healthy donors once every week for a period of five weeks. GM‐EVs were isolated by centrifugation followed by filtration and size exclusion chromatography. GM‐EV secretion was evaluated by particle number, particle size, and gram‐positive to gram‐negative EV ratio. Additionally, we investigated the bacterial origin of GM‐EVs by Oxford Nanopore sequencing of near full‐length 16S genes. Finally, the analysis of bacterial protein cargo of secreted GM‐EVs will be performed by mass spectrometry to assess for any changes in functional capacity. All participants gave informed consent. Results : Our 16S sequencing data revealed a clear distinction between the gut microbiota composition and the GM‐EV composition for all five donors. This includes a higher alpha‐diversity within the gut microbiota compared to the GM‐EVs. As for the beta‐diversity, the GM‐EV samples form a distinct cluster, which is separated from the gut microbiota samples. Notably, samples from the same donor cluster together within the GM‐EV group. Temporal fluctuations were smaller than inter‐donor differences, suggesting a stable GM‐EV composition over time. Summary/Conclusion : The GM‐EV composition appears to be stable over time, justifying single time point collections in gut microbiota EV studies. Funding : The Lundbeck Foundation

Investigation

Presenter: Yingchun Xie Sun Yat‐Sen University, Guangzhou, People's Republic of China Introduction : Extracellular vesicles (EVs) have emerged as promising therapeutic agents due to their ability to carry parental cell characteristics and potential for targeted modification and drug loading. Various EV labelling methods are often limited by spontaneous dye aggregation or low sensitivity. Achieving reliable in vivo tracking without altering physiological distribution remains a critical challenge for translating EV research into clinical applications. This study pioneered the use of metabolic glycan labelling and click chemistry to generate Cy5‐labelled EVs, and for the first time, investigated the plasma half‐life and biodistribution of umbilical cord mesenchymal stem cell‐derived small EVs (UC‐MSC‐sEVs) using this novel labelling approach in mice. Methods : UC‐MSC‐sEVs were isolated using anion exchange chromatography and characterized. The EVs were conjugated with Cy5 fluorophore through metabolic tagging technology and click chemistry. Azido membrane expression was verified by flow cytometry and fluorescence microscopy, while successful fluorophore conjugation was confirmed by nano‐flow cytometry. Following tail vein injection of 2 × 10 10 EVs, blood samples and organs were collected at predetermined time points (0 min–24 h). EV concentration in tissue homogenates was calculated based on Cy5 fluorescence intensity, and results were validated through ex vivo imaging. Results : UC‐MSC‐sEV‐Cy5 maintained characteristic EV properties with no significant changes in particle size or membrane potential. The labelled EVs were efficiently internalized by BEAS‐2B epithelial cells. Following intravenous administration, sEVs showed a rapid clearance with over 50% elimination within 5 min. Biodistribution analysis revealed predominant accumulation in the liver and spleen, moderate distribution in the kidneys and lungs, and minimal presence in the brain, heart, and intestines. Ex vivo imaging results correlated well with tissue homogenate data. Summary/Conclusion : We successfully generated Cy5‐labelled EVs without affecting their composition, size, or membrane potential based on metabolic glycoengineering and bio‐orthogonal click chemistry. And for the first time, investigated the plasma half‐life and biodistribution of umbilical cord mesenchymal stem cell‐derived small EVs (UC‐MSC‐sEVs) using this novel labelling approach in mice. UC‐MSC‐sEV‐Cy5 demonstrated rapid blood clearance with a half‐life of less than 5 min following intravenous administration. Funding : This work was supported by grants from National Natural Science Foundation of China (No. 82271144).

Lactobacillus

Soyeon Lee 1 , Haekang Yang 1 , Eunbi Jo 1 , Chul Won Seo 1 , Shinwon Chae 1 , Woocheol Jung 1 , Yoon‐Jin Lee 1,2 , Eun‐Young Lee 1,2 , and Dongsic Choi 1,2 1 Department of Biochemistry, 2 Research Center for Extracellular Particles, College of Medicine, Soonchunhyang University, Cheonan, Chungcheongnam‐do, Republic of Korea Introduction : Bacteria secrete the small extracellular vesicles (EVs) known as outer membrane vesicles from Gram negative bacteria and membrane vesicles from Gram positive bacteria. Theses bacterial EVs have emerged as key regulators in host immune system. Especially, Lactobacillus probiotics, widely used in dietary supplements, have showed the beneficial properties related in their anti‐inflammatory effect through the bacteria–host interactions possibly via EVs. To address the differential therapeutic effect of Lactobacillus species‐derived EVs, we investigated their anti‐inflammatory effect in a sepsis mouse model induced by cecal ligation and puncture (CLP). Methods : Bacterial culture supernatant from Limosilactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus crispatus, Limosilactobacillus fermentum, Lactiplantibacillus paraplantarum, and Lactobacillus acidophilus were concentrated with a 100‐kDa cut‐off. The concentrated supernatant was ultracentrifuged at 150,000 ×  g for EV sedimentation. Their size and concentration were measured by nanoparticle tracking analyses. Sepsis was induced in C57BL/6 mice through CLP, where the cecum was ligated and punctured with a 21‐gauge needle. EVs were injected by intravenous into mouse 1‐h after CLP operation. Therapeutic effects were measured by survival, cell counting by Diff‐Quick staining in bronchoalveolar lavage fluid (BALF), and expression of cytokines by PCR. Animal protocols were approved by the IACUCs (SCH24‐0053). Results : Isolated Lactobacillus EVs showed the spherical round shape under 100 nm size by electron microscope. In a sepsis model, most of Lactobacillus EVs by intravenous administration represented the increased survival with mild morbidity. Especially, Limosilactobacillus reuteri EVs showed significant therapeutic effect in about 80% survival. Also, these mice exhibited the decreased neutrophil infiltration in BALF, accompanying with increased anti‐inflammatory IL‐10 but decreased TNF‐α and IL‐6. Hematoxylin and eosin staining of lung tissue showed the reduced tissue damage, representing the suppression of inflammation by Limosilactobacillus reuteri EVs. Summary/Conclusion : In this study, we found the Lactobacillus EVs reduce the mortality and morbidity in a sepsis mouse model. These findings suggest that Lactobacillus EVs could be applicable in therapeutics for modulating excessive immune responses and improving survival in diverse inflammatory diseases. Funding : This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS‐2024‐00348103 and RS‐2023‐00219563).

Large‐Scale

Presenter: Emily C. Matchett Saint Louis University, St. Louis, Missouri, USA Introduction : New treatments for relapsed, refractory and rare cancers are needed. Glioblastoma (GBM) is an aggressive brain tumour that develops rapidly and lacks effective treatment. Patients have a median survival of 15 months and a 5‐year survival rate of 5.8%. They experience progressive and severe neurological impairment from the disease and from the treatments. Equally devastating is cognitive decline, personality changes and behavioural disorders that significantly diminish quality of life. Another cancer with a poor prognosis is acute myeloid leukaemia (AML), which is characterized by rapid clonal growth of myeloid lineage stem cells in the bone marrow. The 5‐year survival rate is ∼30% as patients become resistant to chemotherapy. We are investigating using EVs derived from natural killer (NK) cells as a new treatment for these diseases. To address the issue of efficient and large‐scale production, we immortalized the human NK cell line NK3.3 and developed an NK EV isolation protocol to maximize yield and activity. Methods : NK3.3 EVs were isolated from culture supernatants by differential high‐speed centrifugation, sterile filtration, followed by tangential flow filtration (TFF). EVs were characterized by protein concentration, nanoparticle tracking analysis, and enzyme linked immunosorbent assays. Cell counts, cytotoxicity assays, and flow cytometry were used to measure viability of GBM and AML cells after NK EV treatment. Tumour growth in vivo was monitored by bioluminescence imaging. Results : EVs isolated by TFF range in size from 100 to 200 nm, contain NK receptors and cytolytic proteins important for tumour recognition and killing and decrease tumour growth and viability both in vitro and in vivo. They kill drug‐resistant tumour cells but are nontoxic to healthy cells. Levels of the caspase activator granzyme B in NK EVs correlates with their anti‐tumour activity, making granzyme B a reliable and quantitative biomarker. Summary/Conclusion : EVs isolated from immortalized NK3.3 by TFF display strong anti‐tumour activity, with no toxicity to normal cells, making them a safe and effective treatment option for patients when current therapeutics fail, such as with AML, or for difficult to treat cancers, such as GBM.

Microvesicles

Presenter: Xiaoyan Tian Nanjing University, Nanjing, People's Republic of China Introduction : Enterovirus 71 (EV71) is a major etiologic pathogen for hand‐foot‐and‐mouth disease (HFMD) in young children. Severe cases of EV71 infection could lead to neurological complications and even death, while the mechanism inducing neurological complications remains poorly understood. Methods : Microvesicles (MVs) were isolated from HeLa cells infected or not infected with EV71 by differential centrifugation. The morphology and size of the separated MVs were examined by electron microscopy and nanoparticle tracking analysis (NTA). We investigated whether MVs contained viral proteins and/or virions, and a monolayer BBB model was established to detect the ability of MVs to cross BBB in vivo. EV71 or MVsEV71 was injected into mice via the intraperitoneal (ip) route, and the brains were prepared for subsequent analysis by H&E staining and QPCR assay. Subsequently, we investigated whether MVs contained damaged mitochondria using co‐immunoprecipitation assay and transmission electron microscopy (TEM). Results : We found that MVs could carry EV71 virions and mediate infection of target cells at a higher efficiency. MVs containing virions (MVsEV71) could cross the BBB with greater efficiency than EV71 alone both in vivo and in vitro. Meanwhile, MVsEV71 infection of animals induced more severe inflammatory infiltration in the brain compared to animals infected by EV71. Moreover, we elucidated that MVs crossed the BBB via disrupting tight junctions (TJs) and non‐clathrin‐dependent endocytosis pathways. Damaged mitochondria, as the ‘cargo’ of MVs, also facilitated MVsEV71 crossing the BBB. MVsEV71 crossing the BBB further induced mitochondrial damage and activated the NOX4‐derived ROS pathway in U251 cells. Summary/Conclusion : Our findings suggested that MVs transported EV71 virions across the BBB, while damaged mitochondria facilitated this process. MVsEV71 induced mitochondrial damage and activated the NOX4‐derived ROS pathway in U251 cells, leading to further brain injury. Funding : This study was supported by the National Natural Science Foundation of China.

Mitochondrial

Iris Potakowskyj 1,2 , Ee Lyn Lim 1 , Sneha Prabhakara Shastry 1,2 , Adrian Parrilla Mesas 1 , Gorjana Rackov 1 1 BioMed X Institute, Germany; 2 Heidelberg University, Germany Introduction : Mitochondrial dysfunction and immune cell senescence are well‐established hallmarks of ageing. T cells in systemic lupus erythematosus (SLE) show signs of premature senescence and are characterized by mitochondrial dysfunction and mitophagy defects. Recent studies showed that inefficient mitophagy may lead to increased mitochondrial content in EVs. Methods : Here, we aimed to characterize mitochondrial DNA and protein cargo in large and small EVs enriched from plasma of young vs. aged donors and SLE patients versus healthy controls. Large and small EVs were enriched from platelet‐depleted plasma using differential centrifugation coupled with size exclusion chromatography and ultrafiltration. In addition to basic EV characterization—including particle count, size distribution and protein content—mitochondrial protein content was assessed using liquid‐chromatography tandem mass spectrometry (LC‐MS/MS). MACSPlex flow cytometry array was used for assessment of EV surface markers. Results : Enrichment of EVs was confirmed by immunoblot and nanoparticle tracking analysis, showing distinct size distribution for large and small EVs. Our results showed that mitochondrial DNA and protein cargo can be detected both in large and in small EVs, designating them as MitoEVs. Although mitochondrial proteins were more abundant in large EVs, comparison to the MitoCarta database showed that only a subset of human mitochondrial proteins is detectable in EVs. The abundance of these proteins was altered in aged compared to young donors and in SLE patients compared to healthy controls. Summary/Conclusion : Our findings indicate that a specific subset of mitochondrial proteins is loaded in EVs. The altered levels of mitochondrial cargo in large and small EVs may serve as potential biomarkers in pathophysiological processes associated with mitochondrial dysfunction. Funding : This study is sponsored by Merck KGaA.

Msc‐Derived

Presenter: Christian Deininger Paracelsus Medical University, Salzburg, Austria Introduction : With the increasing incidence of osteoporosis, there is a critical need for innovative therapies to improve osteoporotic fracture repair. Effective fracture healing relies on enhanced bone regeneration alongside robust fracture fixation and implant osseointegration. Extracellular vesicles derived from mesenchymal stem cells have shown potential in promoting bone repair. Building on this, our study aimed to evaluate the effects of GMP‐grade human umbilical cord MSC‐sEVs in enhancing BMP‐2‐mediated bone repair and implant integration. This approach may not only improve therapeutic outcomes but also reduce safety concerns associated with high BMP‐2 doses applied in clinical settings. Methods : After inducing osteoporosis via bilateral ventral ovariectomy and a specialized diet, 64 rats underwent distal femoral metaphyseal osteotomy. Defects were stabilized with custom Y‐shaped mini‐locking plates and treated with one of four interventions: alginate alone, alginate with hUC‐MSC‐sEVs (2 × 10 9 particles), rBMP‐2 (1.5 µg), or a combination of sEVs and rBMP‐2. In a second study, drillhole defects were created in 42 rats and treated with alginate containing sEVs, rBMP‐2, or both to evaluate screw implant integration. Animals were euthanized 6 weeks post‐surgery, and femora were analysed by micro‐CT, histology, and biomechanical testing to assess bone healing and osseointegration. Results : Radiographs and µCT showed improved bone union in metaphyseal defects treated with hUC‐MSC‐sEVs and a low dose of BMP‐2 at 6 weeks, which was confirmed by histological analysis. Torsional testing indicated increased stiffness with rBMP‐2 alone or combined with sEVs, while torque was significantly enhanced only in the combination group. Interestingly, sEVs alone did not improve osseointegration, and only the localized application of 1.5 µg rBMP‐2 improved biomechanical outcomes. Further, no additive effect was observed when sEVs were combined with rBMP‐2. Summary/Conclusion : Combined rBMP‐2 and sEVs improved osteoporotic defect healing compared to either treatment alone but did not enhance implant osseointegration. sEVs alone slightly reduced bone formation around implants, and rBMP‐2 alone was the most effective for implant integration, with no added benefit from combined sEV treatment. Funding : This work was supported by the Lorenz Böhler Fonds (#1/20), Land Salzburg/IWB/EFRE 2014‐2020 (P1812596; “EV‐TT”), and a PMU‐RIF‐PRE fellowship (2023‐ PRE‐007).

Multi‐Omics

Chang Liu 1 , Li Sun 1,2 , Hannah Worden 3 , Justice Ene 1 , Sunghoon Jung 3 , Yan Li 1 1 Department of Chemical and Biomedical Engineering, FAMU‐FSU College of Engineering, Florida State University, Tallahassee, Florida, USA; 2 Department of Biomedical Sciences, Florida State UniversityTallahassee, Florida, USA; 3 PBS Biotech Inc, CA, Camarillo, California, USA Introduction : Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell‐free therapies in various diseases, including stroke. However, because of the significant amounts of EVs needed in preclinical and clinical trials, it is still challenging for EV application. Vertical‐wheel bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress. Methods : In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3‐D aggregates (Agg) and on synthemax II microcarriers (MC) in VWBRs was investigated compared to a static aggregate culture control. The spheroids were characterized by transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and western blot. The EV cargo was analysed by proteomics and miRNA‐sequencing. The in vitro oxygen and glucose deprived stroke model and proof of concept in vivo study were performed. Results : Human forebrain spheroids differentiated on microcarriers showed a higher growth rate than 3‐D aggregates. The VWBR culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes compared to the static culture. EVs from the three conditions had similar sizes, but the yields from high to low were microcarrier culture, dynamic aggregates, and static aggregates. The transcriptome of parent cells based on mRNA sequencing and EV proteomics data of Agg versus MC revealed a weak increasing trend of EV differentially expressed proteins (DEPs) with the parent cell differentially expressed genes (DEGs). The cargo is enriched with proteins (based on proteomics) and miRNAs (based on miRNA‐seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. The EVs demonstrated the ability to improve recovery in an in vitro stroke model and an in vivo rat model. Summary/Conclusion : Human forebrain spheroids differentiated in VWBR significantly increased EV yields (up to 240–750‐fold) and EV biogenesis compared to static control due to dynamic microenvironment and metabolism alteration. The biomanufactured EVs from VWBRs are enriched with therapeutic cargo and functional in vitro and are promising for in vivo study. Funding : This project is supported by NSF CAREER 1652992 and NIH R01NS125016 of the USA.

Mycobacterial

Han‐Gyu Choi 1 , Dahee Shim 2 , Sang Chul Park 2 , Hwa‐Jung Kim 1 1 Chungnam National University, Daejeon, South Korea; 2 Hallym University, Chuncheon, South Korea Introduction : Tuberculosis (TB) remains a global health concern, and there is an urgent need for effective vaccines. Extracellular vesicles (EVs) derived from mycobacteria have emerged as promising vaccine candidates. This study investigated the immunomodulatory properties and protective efficacy of EVs derived from different mycobacterial strains (BCG, Ra, and Rv). Methods : Mycobacterial strains were initially cultured in 7H9 medium (seed culture, OD600 = 1.0), followed by main culture in Sauton's medium (OD600 = 0.1) under static conditions for 3 weeks. EVs were isolated from culture supernatants using sequential filtration (TFF/UF/DF 300 kDa) and concentrated to 15 mL with PBS dialysis. EV characterization included NTA for size distribution and concentration analysis, and microBCA for protein quantification. Immunological properties were assessed by analysing DC surface markers and cytokine production upon EV treatment. T cell proliferation was evaluated using OT2 mouse splenocytes. Additionally, in vitro Mtb growth inhibition assays and in vivo prophylactic vaccination studies were performed. Results : Isolated EVs showed characteristic size distributions and protein content. EV treatment induced significant changes in DC surface markers and cytokine profiles, demonstrating their immunomodulatory potential. OT2 mouse splenocytes showed enhanced proliferation in response to EV treatment. Notably, EVs demonstrated significant inhibition of Mtb growth in vitro. In prophylactic vaccination studies, EV‐immunized mice showed reduced bacterial burden upon Mtb challenge. Summary/Conclusion : Our findings demonstrate that mycobacterial EVs possess immunomodulatory properties and provide protective immunity against Mtb infection, suggesting their potential as novel TB vaccine candidates. These results provide a foundation for further development of EV‐based TB vaccines. Funding : This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS‐2024‐00463203).

Neuroblastoma

Tal Manko 1 , Ishai Luz 1 , Meshi Zorsky 2 , Gad Vatine 2 , Tomer Cooks 1 1 Faculty of Health Sciences, The Shraga Segal Department of Microbiology, Immunology & Genetics, Ben‐Gurion University, Beer Sheva, Israel; 2 The Department of Physiology and Cell Biology, Ben‐Gurion University, Beer Sheva, Israel Introduction : Extracellular vesicles (EVs) play an important role in cell‐to‐cell communication, particularly when released by cancer cells, allowing them to interact with their tumour microenvironment. Neuroblastoma (NB) is a cancerous solid tumour commonly arising in paediatric children across the sympathetic nervous system, which causes multisite neuronal damage to children and might even lead to death. NB is considered an enigma with spontaneous remission, relapses, and primary tumour sites; despite extensive research of NB, there are still many gaps in our knowledge regarding its tumourigenesis, metastasis, and pathology. Methods : In this research, we sought to explore the effects of EVs shed by NB cells (NS20 and N2A, cell lines representing late and early onsets, respectively) upon endothelial cells (bEND3 cell line) of the blood‐brain barrier (BBB), employing a novel in vitro BBB model to study their interactions. Measuring their stability and permeability via Trans Epithelial Electrical Resistance (TEER) measurements and FITC penetrance. In addition, we also re‐educated NSC34 (non‐cancerous motor‐neuronees cells) via co‐culture NB cells or EVs and measured their ability to affect BBB permeability. Results : Our results indicate that NB EVs and educated NSC34 cells significantly impact bEND3 integrity. Mass spectrometry analysis of EVs from N2A EVs unravelled several proteins that affect tight junctions’ assembly, suggesting the first reported evidence of BBB manipulations via NB cancer cells. Higher levels of tight junctions’ proteins were found in the late‐stage NB EVs (NS20), including key proteins such as occludin, claudin and ZO‐2, matching with our TEER measurements showing higher degree of destabilization of the BBB. Summary/Conclusion : This study presents characterization of NB EVs and shows their capacity to promote disassembly of tight junctions, leading to destabilization of the BBB. Our results also suggest that EVs from NB sources can contain proteins involved in tight junction biology, leading to a new frontier of EV biomarkers and potentially enabling full intact tight junction formations that can interact with target sites.

Physiological

Presenter: Tomer Cooks Ben Gurion University, Beersheba, Israel Introduction : Extracellular vesicles (EVs) are essential mediators of intercellular communication. The method of EV administration in vivo significantly impacts their biodistribution and their effect on recipient tissues and cells. Current in vivo EV injection methods rely on direct pulses of large amounts of EVs, poorly mimicking the ‘real‐life’ chronic continuous release of EVs from cells and tumours. This study aims to compare the physiological EV secretion rates with common EV injection methodologies and assess the effectiveness of an osmotic slow‐release pump in delivering EVs continuously for substantial periods to create a chronic delivery mechanism faithfully reflecting physiological conditions. Methods : We employed two types of osmotic slow‐release pumps (Alzet pumps as well as iPRECIO pumps) to administer EVs at a controlled, sustained, programmable rate and compared them with the traditional injection methods (intraperitoneal [ip] and intravenous [iv]). Biodistribution and concentration of fluorescently labelled EVs were monitored in various organs, including the pancreas and liver, over multiple time points. The concentration of EVs was measured using NTA and TRPS, and EM was used to verify morphology. We used gold nanoparticles as markers for EV cargo deposited in the target tissues. Additionally, the study evaluated the negative side effects associated with high‐concentration EV injections. Results : The osmotic slow‐release pump demonstrated a more consistent and physiological distribution of EVs across the targeted tissues compared to traditional injection methods. Traditional injection methods led to a localized, short‐term, and intense cellular response with potential adverse effects, including cellular saturation and toxicity. The slow‐release method mitigated these issues, providing a more stable and prolonged delivery of EVs. When refillable pumps were used, we were able to load fresh batches of EVs and provide tissue targeting (pancreas and liver) spanning periods of weeks and months. Summary/Conclusion : The study highlights the limitations of robust single EV injections emphasizing the benefits of using an osmotic slow‐release pump for EV delivery. This method ensures a more physiological and sustained release of EVs, enhancing their therapeutic potential and reducing adverse effects. Future research should focus on optimizing the slow‐release delivery system for clinical applications and further exploring its impact on EV‐mediated therapies.

Polymicrobial

Lily Charpentier 1 , Roxanna Barnaby 1 , Fabrice Jean‐Pierre 2 , George A. O'Toole 1 , Douglas Taatjes 3 , Bruce A. Stanton 1 1 Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA; 2 Département de Microbiologie, Université de Sherbrooke, Sherbrooke, Québec, Canada; 3 Department of Pathology and Laboratory Medicine, The University of Vermont, Burlington, Vermont, USA Introduction : Despite highly effective modulator therapy (HEMT; ETI) that improves outcomes in people with cystic fibrosis (CF), HEMT does not eliminate antibiotic‐tolerant polymicrobial lung infections and inflammation, the major cause of morbidity and mortality in CF. Bacteria in the CF lung reside in mucus, and we have shown Pseudomonas aeruginosa communicates with human bronchial epithelial cells (HBEC) by secreting extracellular vesicles (bEVs) that induce inflammation. Since little is known about how polymicrobial bEVs, which are more representative of lung infections than a single genus, affect host‐cell immune responses, we developed a model containing four of the most common bacteria in CF to gain insight into how polymicrobial bEVs impact the immune response of HBEC. Methods : bEVs secreted by P. aeruginosa , Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown together in artificial sputum medium (ASM) were isolated by ultracentrifugation and Optiprep density gradient. bEVs were characterized by NTA, 16S rRNA sequencing, flow cytometry, and transmission electron microscopy according to MISEV 2023 recommendations. Wild‐type (WT), CF, and CF+ETI HBEC ( n = 5 donors/group) were treated with processed, uninoculated ASM (processed control; PC) or polymicrobial bEVs for 6 h. Gene expression was assessed by RNA‐Seq and cytokine secretion by the Millipore 48‐plex cytokine array. CFTR Cl‐ secretion was measured in Ussing chambers. Results : Characterization of bEVs revealed that all four bacteria secrete bEVs (∼30–100 nm) and induce a hyper‐inflammatory response by CF HBEC. CXCL3 secretion (which increases chemotaxis of monocytes and lymphocytes) is increased in CF HBEC compared to PC ( p = 0.02). TNFα secretion (which stimulates inflammation) also increases ( p = 0.001). bEVs increase inflammatory pathways, including TLR and MAPK signalling, in CF HBEC + ETI. bEVs reduce CFTR Cl‐ secretion by WT ( p = 0.002) and CF HBEC+ETI ( p < 0.0001). Summary/Conclusion : Polymicrobial bEVs elicit a robust inflammatory response in HBEC and reduce Cl‐ secretion by CF HBEC + ETI, effects that are predicted to increase recruitment of immune cells to CF lungs, which cause lung damage, and reduce the ability of ETI to clear polymicrobial infections. Studies in progress are investigating approaches to mitigate the adverse effects of bEVs on HBEC. Funding : CF Foundation (STANTO19R0) and NIH (P30‐ DK117469 , R01HL151385, T32AI007519, and F31HL172440).

Sev‐Pd‐L1

Presenter: Isen Naiken CHU Dijon, Dijon, France Introduction : There is a critical need for reliable biomarkers enable to monitor neuroendocrine tumours and refine therapeutic strategies. Chromogranin A (CgA) and other biomarkers used in current clinical practice lack specificity, making computed tomography (CT) and magnetic resonance imaging (MRI) the primary methods for disease monitoring. However, those methods can miss small pancreatic or gastrointestinal lesions and can be costly. New markers must be explored, among which immunosuppressive molecules the like of PD‐L1 and HSP70. Our study concentrates on markers found in liquid biopsies obtained through simple blood collection. Methods : In that prospect, we conducted a retrospective multicentric clinical study to determine whether the levels of PD‐L1 and HSP70 in sEV (sEV‐PD‐L1 and sEV‐HSP70) isolated from patients’ plasma could predict disease progression. Patients from the REMINET clinical studies were treated by placebo or lanreotide. Plasma samples were drawn at various stages of the disease, allowing results to be linked to disease progression. Plasma‐EVs where isolated using a precipitation kit. EV markers were tested by western blotting to control the isolation. Size and concertation were measured by nanoparticle tracking and transmission electron microscopy. ELISA assays were performed on isolated EVs and patient's plasma to quantify immunosuppressive molecules. Results : HSP70 and PD‐L1 were identified in DPNET‐patient's peripheral blood, as well as sEVs isolated from said blood samples. We show that molecules circulating in the blood or isolated from EVs are of different origins. The eventual linked between the variation of those molecules and disease progression was tested, revealing a significant correlation between levels of sEV‐PD‐L1 and disease progression in patients treated with lanreotide. Summary/Conclusion : Circulating sEVs where analysed and proved to be pertinent in the monitoring of disease progression in DPNET patients. A first promising circulating molecular marker was identified, strongly correlated to cancer progression. Further studies are needed to confirmed sensitivity and specificity of our marker and its potential use as a predictive marker of disease progression in DPNET. Those findings could also contribute to the advancement of rapid and non‐invasive diagnostic tests in DPNET.

Single‐Cell

Presenter: Patrick Pirrotte TGEN, Phoenix, Arizona, USA Introduction : Neural stem cell (NSC)‐based therapies offer significant potential for treating brain injuries by delivering neurotrophic factors and promoting endogenous brain repair. Despite promising evidence of the protective and restorative effects of NSCs and their extracellular vesicles (EVs), advancing these therapies to preclinical and clinical stages has been limited by inadequate molecular characterization of NSCs and their EV cargo. This study addresses this challenge by characterizing two human foetal NSC lines expressing the L‐MYC gene (LMNSC01 and LMNSC02) and their EVs through single‐cell RNA sequencing (scRNA‐seq) and proteomic analyses. Methods : LMNSC01 and LMNSC02 cells were cultured and analysed for differentiation potential into neuronees and glial cells using scRNA‐seq. Key NSC markers (SOX2, nestin) and differentiation markers (doublecortin [DCX], vimentin) were evaluated. Single extracellular vesicle nanoscopy was utilized to measure the size, shape, and tetraspanin content (CD9, CD63, CD81) of EVs from each line. Additionally, protein and RNA concentrations, particle numbers, and batch‐to‐batch consistency were assessed. Global proteomics was conducted to identify biological pathways in EV cargo, and neuroprotective effects were tested in a methotrexate‐induced brain organoid toxicity model. Results : scRNA‐seq revealed that both cell lines were homogeneous, expressing NSC and differentiation markers like DCX and vimentin. LMNSC01 was enriched in markers for astrocytes, excitatory neuronees, and glial cells, while LMNSC02 showed enrichment for inhibitory neuronees and oligodendrocyte progenitor cells (OPCs). LMNSC02 EVs had a higher tetraspanin content and larger size compared to LMNSC01. Proteomic analysis showed that LMNSC02 EVs were enriched in proteins involved in neuronee development and synaptic transmission, whereas LMNSC01 EVs were more linked to RNA processing. Both types of EVs demonstrated neuroprotective properties in the brain organoid model, suggesting potential in reducing chemotherapy‐induced brain damage. Summary/Conclusion : This study highlights the unique therapeutic potential of LMNSC01 and LMNSC02 and emphasizes the importance of detailed molecular characterization for guiding NSC‐based therapies for neurological conditions. Funding : This work was supported by National Institutes of Health grants P30CA033572, 1R01NS121037‐01 Kline/Gutova, and UG3/UH3 TR002878.

Standardizing

Presenter: Jen Bon Lui Beckman Coulter, Brea, California, USA Introduction : Traditional flow cytometry often falls short in sensitivity and resolution for extracellular vesicle (EV) analysis. By converting scatter parameters from arbitrary to standardized units, researchers can better compare results across different cytometers, enhancing study interpretation and reproducibility. Methods : The CytoFLEX nano flow cytometer paired with FCMPASS software offers a solution for the analysis EVs which are difficult to analyse due to their small size, heterogeneity, and low abundance. The CytoFLEX nano flow cytometer is equipped with a Violet Side Scatter (VSSC1) detector capable of detecting particles as small as 40 nm polystyrene NIST (National Institute of Standards and Technology) reference particles, and up to 1000 nm using the VSSC2 channel. This advanced detection capability is crucial for accurately analysing small particles like EVs. To facilitate cross‐platform comparisons, scatter data is calibrated to nanometres using Mie‐modelling software. Results : Here we show a workflow where VSSC data is converted from arbitrary units into standardized units (nanometres) for particle analysis, using the CytoFLEX nano flow cytometer and FCMPASS software. Standardizing this data into nanometres using a ready‐to‐use, NIST‐traceable size standard mixture, the nanoViS Low and High Sizing Standards, significantly enhances the value and comparability of small particle research data. Summary/Conclusion : This approach aligns with MISEV and MIFlowCyt‐EV guidelines, ensuring that research involving EVs and other submicron particles is repeatable, verifiable, and traceable.

Translocation

Shogo Tsubaki 1 , Juntaro Matsuzaki 2 , Rika Tanaka 3 , Yusuke Yoshioka 4 , Hitoshi Tsugawa 1,5 1 Transkingdom Signaling Research Unit, Division of Host Defense Mechanism, Tokai University School of Medicine, Isehara, Kanagawa, Japan; 2 Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Tokyo, Japan; 3 Division of Host Defense Mechanism, Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa, Japan; 4 Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan; 5 Institute of Medical Sciences, Tokai University, Isehara, Kanagawa, Japan Introduction : Klebsiella pneumoniae, a gut commensal bacterium, primarily causes pneumonia and liver abscesses in elderly individuals. However, the mechanisms behind the development of diseases in distant organs due to K. pneumoniae are not well understood. Recently, we identified bacterial extracellular vesicles produced by K. pneumoniae (Kp‐bEVs) using scanning electron microscopy. This study examined whether Kp‐bEVs are involved in diseases occurring in distant organs far from the K. pneumoniae ‐colonized gastrointestinal tract. Methods : Kp‐bEVs were collected using ultracentrifugation and characterized via transmission electron microscopy. DiR‐stained Kp‐bEVs were administered to mice, and their distribution was traced using NEWTON imaging analysis. Bone marrow‐derived macrophages (BMDMs) were cultured in DMEM with M‐CSF for 7 days, while osteoclasts were induced in DMEM with M‐CSF and RANKL for 7 days. Osteoblasts were induced by Osteoblast‐Inducer Reagent (TaKaRa Bio Co.) for 21 days. Results : To assess localization within BMDMs, GFP‐labelled Kp‐bEVs were constructed by fusing GFP to TufA, a protein localized in Kp‐bEVs. Kp‐bEVs did not co‐localize with organelles such as the Golgi, endoplasmic reticulum, or early endosome; instead, they distributed throughout the cytoplasm. These findings suggest that Kp‐bEVs exert biological activity on macrophages. Therefore, we next examined the polarization behaviour of Kp‐bEV‐treated BMDMs. Treatment with Kp‐bEVs to BMDMs significantly increased the population of CD206‐positive cells, an M2 phenotype marker. Moreover, Kp‐bEVs significantly inhibited nitric oxide (NO) production during K. pneumoniae infection. In vivo imaging analysis using Kp‐bEVs‐administered mice revealed that Kp‐bEVs accumulate in the liver, lungs, and vertebrae. In Kp‐bEVs‐treated osteoclasts, cell viabilities were significantly reduced, and tartrate‐resistant acid phosphatase (TRAP)‐staining positive cells were scarcely detected following treatment with Kp‐bEVs. Conversely, in osteoblasts, the number of alkaline phosphatase (ALP)‐positive cells significantly increased by the 21st day in the presence of Kp‐bEVs, and the intensity of Alizarin Red staining, indicating calcium deposition, was significantly enhanced by treatment with Kp‐bEVs. Summary/Conclusion : Kp‐bEVs attenuate the immunological activity against pathogens of macrophages. Moreover, in vertebral bone, Kp‐bEVs are thought to disrupt the balance of bone metabolism through their effects on osteoblasts and osteoclasts. Our findings suggest that Kp‐bEVs may contribute to disease development in the organs where they translocate and accumulate.

Understanding

Thea N. Golden 1 , Rita L. Leite 1 , Jerome F. Strauss III 1 , Samuel Parry 1 , and Rebecca A. Simmons 1,2 1 University of Pennsylvania, Philadelphia, Pennsylvania, USA, 2 Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA Introduction : Preterm birth (PTB) is a heterogeneous complication that affects over ten percent of pregnancies. To date there are no effective treatments nor the ability to predict who goes on to deliver preterm. The overarching goal of our research is to identify a biomarker obtained in early pregnancy to identify individuals at risk for delivering preterm. Circulating extracellular vesicle (EV) signalling is a key form of maternal‐foetal communication, and PTB is associated with changes in the EV profile. In this project we tested the hypothesis that the aetiology of PTB influences maternal circulating EVs. Methods : Subjects were enrolled at the time of delivery after obtaining informed consent and included uncomplicated term, spontaneous PTB (sPTB), and medically indicated PTB (mPTB). EVs were isolated from maternal plasma using serial centrifugation, and large (LEVs) were isolated by 15,000 × g and small (SEVs) were isolated by 100,000 × g . The number and size of EVs were quantified by nanoparticle tracking analysis. We quantified the percentage of EVs from trophoblasts (PLAP+), immune cells (CD45+), platelets (CD41a+), mature endothelial cells (CD31+CD34‐), and foetal endothelial cells (CD31+CD34+) using flow cytometry. We also measured the abundance of ND1 by qPCR to quantify mitochondrial DNA in EVs. Results : Compared to term, the number of LEVs was increased in mPTB, but there were fewer SEVs in sPTB. The size of LEVs was increased in sPTB but not mPTB, while the size of SEVs was unaffected. Also, in sPTB, we observed an increase in platelet and immune cell derived LEVs and maternal endothelial cell derived SEVs. In mPTB, there were more foetal endothelial cell LEVs and fewer mature endothelial cell SEVs. Finally, we observed an increase in the abundance of mitochondrial DNA in SEVs from PTB pregnancies regardless of the type. Summary/Conclusion : The changes observed at delivery reflect the underlying pathophysiology and identify different cell types affected in sPTB versus mPTB. However, increased mitochondrial DNA release was evident irrespective of the type of preterm birth. Our ongoing research, in a prospectively collected cohort, aims to identify when during pregnancy the size, cargo, and origin of circulating EVs are altered. Funding : This study was supported by the March of Dimes and NIH grants T32ES019851 and P30ES013508.

Visualization

Presenter: Venkatesh Kumar Chetty University Hospital Essen, Essen, Germany Introduction : Small extracellular vesicles (sEVs) are crucial players in intercellular communication in various diseases, including cancer, where they transport essential cargo molecules between cells. However, the limitations due to the lack of high‐resolution imaging techniques hindered a comprehensive understanding of sEVs, including their biogenesis and functional attributes. We addressed this knowledge gap by applying single‐molecule localization microscopy (SMLM), a powerful super‐resolution imaging technique, for the nanoscale imaging of sEVs within the recipient cells. Methods : sEVs were isolated using a combination of tangential flow filtration (TFF), size exclusion chromatography (SEC), and ultrafiltration (UF), collectively known as TSU. According to MISEV2023 guidelines, sEVs were characterized using NTA, TEM, and western blot. 5‐ethynyl‐2′‐deoxyuridine (EdU) was employed to label DNA cargo associated with sEVs (EV‐DNA) that are released by metabolically active donor cells. Following the sEV treatment in the recipient cells, boron dipyrromethene‐azide (BODIPY‐azide) was utilized to label EV‐DNA‐EdU via click chemistry, and Alexa647‐conjugated nanobody against GFP (Alexa647‐Nb) to label CD63‐eGFP+‐sEVs. Results : Following EV‐DNA labelling using BODIPY‐azide, we showed BODIPY's buffer‐independent blinking feature by performing EV‐DNA imaging in the recipient cells using SMLM. Next, the spatial distribution of GFP+‐sEVs in the recipient cells was visualized by performing immunostaining with Alexa647‐Nb. Using dual‐colour SMLM, we visualized the association of EV‐DNA cargo and CD63+‐sEVs in the recipient cells at nanoscale resolution. Furthermore, using dual‐colour SMLM imaging combined with cluster analysis, we revealed the intricate interactions between EV‐DNA and cytoplasmic DNA sensor cyclic GMP‐AMP synthase (cGAS) within the recipient cells. Summary/Conclusion : Altogether, our data illustrated that dual‐colour SMLM imaging has a promising scope to study how sEVs are being taken up along with their EV‐DNA cargo in the recipient cells and their molecular interaction with different cellular compartments, which would enable us to understand the biological function of sEVs in various diseases. Funding : This work was supported by grants to B.K.T. from the Stiftung Universitätsmedizin Essen, Deutsche Jose Carreras Leukämie‐Stiftung (DJCLS 14 R/2023), Deutsche Forschungsgemeinschaft (DFG TH 2012/1‐1), and Deutsche Kinderkrebsstiftung (DKS 2018.17). X.Z. and Q.Y. also acknowledge the support received from the Chinese Scholarship Council (CSC).

Yam‐Derived

Presenter: Do‐kyun Kim Jeonbuk National University, Republic of Korea Introduction : Several studies have reported that substances extracted from yam (diosgenin and dioscin) promoted osteoblast activation in osteogenic MC3T3‐E1 cells. Accordingly, we hypothesized that yam‐derived exosome‐like nanovesicles (YNVs) would play a pivotal role in osteoporosis preventive activity. In this study, an isolation of PENs to prepare YNVs containing RNAs, proteins, and lipids is optimized. Next, YNVs‐mediated osteogenic differentiation is demonstrated with a mechanism study on BMP‐2/p‐p38‐dependent Runx2 pathway in mouse primary osteoblasts and osteogenic MC3T3‐E1 cells. Finally, therapeutic potentials of YNVs are evaluated in ovariectomized (OVX)‐induced osteoporotic mice, in addition to the demonstration of biodistribution and biosafety in naive mice. Methods : 1) Isolation and purification of Yam‐derived exosome‐like nanovesicles 2) Nanoparticle tracking analysis 3) Negative‐stain transmission electron (TEM) microscopy and Cryo‐EM 4) OVX‐induced osteoporotic mouse model 5) Micro‐CT analysis 6) Bone histomorphometry. Results : # yam‐derived exosome‐like nanovesicles (YNVs) 1) YNVs are taken up by osteoblastic MC3T3‐E1‐cells, which stimulate osteoblast differentiation and mineralization 2) YNVs improves osteoblast proliferation, differentiation, and mineralization 3) YNVs show higher osteogenic activation than diosgenin and dioscin 4) YNVs modulate p38 MAP kinase pathway 5) YNVs promote osteogenesis in OVX‐induced mice. Summary/Conclusion : In summary, YNVs were successfully isolated, purified, and characterized, which osteogenic activity and mechanisms were first identified. In detail, MTT assay, immunoblot/RT‐qPCR, and ALP/von Kossa assays demonstrated that YNVs improved proliferation, differentiation, and biomineralization of osteoblast cells, respectively. HPLC analysis indicated that YNVs do not contain any diosgenin and dioscin that are well‐known major components of yam to allow osteoblast activation. Additional total RNA analysis using Volcano plot and DAVID program on KEGG pathway revealed that osteogenic activity of YNVs are associated with MAPK signalling pathways. In particular, immunoblotting assay and inhibitor‐assisted Alizarin Red staining assay confirmed that YNVs activate osteoblast via BMP‐2/p‐p38‐dependent Runx2 pathway rather than other MAPK pathways for ERK1/2 and JNK. YNVs administered orally were transported through GI tract and absorbed in the small intestine, followed by bloodstream and liver. The YNVs enhanced osteogenesis in OVX‐induced postmenopausal osteoporosis mice model in vivo. Therefore, YNV is a potential therapeutic agent for the treatment of osteoporosis with excellent biocompatibility and safety.

8Clc‐Derived

Ying Fu 1,2 , Qiang Guo 2,3 , Yunge Wang 2,3 , Mengwei Fu 2,4 , Yixin Yan 2 , Xuan Dong 2 1 Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China; 2 BGI Research, Hangzhou, China; 3 College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China; 4 College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China Introduction : Clinical management of psoriasis is often complicated by recurrence and challenges in maintaining long‐term efficacy. While exosomes derived from pluripotent stem cells have shown promising long‐term therapeutic effects, they are hampered by issues such as senescence and unclear effector molecules and mechanisms. Methods : In our previous research, we developed a system to induce pluripotent stem cells into 8C‐like cells (8CLCs), which exhibit characteristics similar to human 8‐cell embryos. These cells demonstrate totipotency and enhanced genomic stability. Results : Co‐culturing T cells with exosomes derived from 8CLCs (8CLC‐Exo) significantly reduces Th17 differentiation while promoting Treg generation. Topical application of 8CLC‐Exo alleviates psoriatic skin lesions and enhances Treg infiltration in an IMQ‐induced mouse model. Through integrated analysis of exosomal proteomics data and RNA sequencing data from psoriasis patients, we hypothesize that 8CLC‐Exo facilitates Treg differentiation and modulates immune homeostasis in psoriasis via the cellular retinoic acid‐binding protein 1 (CRABP1)‐dependent retinoic acid metabolism pathway. Summary/Conclusion : This project integrates disease models with multidimensional omics techniques to elucidate the precise molecular mechanisms and roles of 8CLC‐Exo in alleviating psoriasis, offering potential insights and innovative therapeutic strategies for its treatment. Funding : This work was supported by the General Program of the National Natural Science Foundation of China (32400746).

Acoustofluidic

Michael Gerlt and Thomas Laurell Lund University, Lund, Sweden Introduction : Blood plasma is a key medium for Extracellular vesicles (EV) research due to its accessibility but presents challenges for EV isolation, including high lipoprotein levels and other abundant proteins. Traditional ultracentrifugation methods are limited by low purity and high sample requirements, prompting interest in microfluidic technologies. This manuscript introduces ‘acoustofluidic chromatography,’ a method leveraging acoustic waves and micro‐capillaries for high‐throughput EV trapping with enhanced capacity and minimal sample volumes. Methods : The trapping device comprised a glass capillary (20 mm length, 4 mm width, 0.4 mm height, 0.25 mm wall thickness) attached to a piezoelectric element. The capillary was filled with polystyrene beads (100 µm diameter). Upon actuation of the piezoelectric element in the MHz range using a wave generator, ultrasonic waves are generated and scattered at the polystyrene beads enabling the trapping of nanometre sized particles (seed particle trapping). The setup consists of two syringe pumps and a fluorescent microscope enabling the analysis of trapping efficiency in flow, which we used for thorough device characterisation. The blood plasma samples were analysed using nanoparticle tracking analysis and micro BCA. Results : The device effectively traps particles over a broad frequency range (0.45–4 MHz) and supports high flow rates up to 100 µL/min, achieving trapping efficiencies of 82% ± 4% with electric power as low as 55 mW. Following comprehensive characterization, we performed blood plasma experiments using just 4 µL of triple‐centrifuged plasma, successfully isolating up to 2 × 10 9 particles/mL with the highest concentration of Particles with 50–60 nm diameter in only 8 min with a PBS wash of 360 µL. Protein contamination was minimal, with micro BCA measurements showing levels below detection (< 2 µg/mL). Our recent discovery of a frequency‐switching mode enhances trapping capacity by 100 times and throughput up to 4 times. Current efforts focus on isolating distinct EV subpopulations by size and density and confirming low lipoprotein contamination due to a repulsive acoustic force on these particles. Summary/Conclusion : Acoustofluidic chromatography is a promising method to enrich different EV subpopulations with high purity from minute plasma samples within min. Funding : This project is funded by the European Union—EIC transition.

Bioengineering

Presenter: Flurina Staubli University Medical Center Utrecht, The Netherlands Introduction : Extracellular matrix vesicles (MtVs) are essential mediators of endochondral ossification (EO), a pivotal process in bone development and repair. These nanovesicles, derived from hypertrophic chondrocytes, play critical roles in extracellular matrix remodelling, chondrocyte communication, and growth plate development. The manufacture of stem cell‐derived cartilage tissues in vitro is a promising approach to generate MtVs; however, obtaining MtVs which exhibit a hypertrophic phenotype has remained elusive. In this study, we aim to bioengineer hypertrophic stem cell‐derived cartilage microtissues in vitro to generate EO‐mimetic MtVs as a novel nanotherapeutic strategy for bone regeneration. Methods : Human bone marrow‐derived stromal cells (hBMSCs) microtissues were differentiated in chondrogenic medium with/without bone morphogenic protein 2 (BMP2) for 21 days. Following this, constructs were either maintained in chondrogenic medium or switched to hypertrophic medium till Day 28. The expression of chondrogenic and hypertrophic markers was assessed by biochemical and histological analysis. MtVs were isolated via enzymatic digestion, followed by ultracentrifugation. MtV size, morphology and concentration were assessed via nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and CD63 ELISA. MtVs therapeutic efficacy was assessed by evaluating their effects on hBMSCs recruitment, osteogenic differentiation, and human endothelial colony‐forming cells (hECFCs) angiogenesis. MtVs cytokine contents were determined via ELISA. Results : Our findings revealed that BMP2 and hypertrophic medium conditioning significantly upregulated microtissue chondrogenic (glycosaminoglycans, collagen type II) and hypertrophic (vascular endothelial growth factor (VEGF), alkaline phosphatase (ALP), type I‐ and type X collagen) marker expression. NTA and TEM confirmed the nano‐sized nature of the MtVs (∼50–200 nm). BMP2 and hypertrophic conditioning significantly increased MtV yield, ALP activity and calcium binding compared to MtVs from chondrogenic microtissues. Notably, MtVs derived from BMP2/hypertrophic conditioned microtissues substantially promoted recipient hBMSCs proliferation, migration and mineralization, in addition to improving the angiogenic tube formation of hECFCs. Cytokine profiling confirmed that MtVs obtained from hypertrophic microtissues were enriched with BMP2 and VEGF. Summary/Conclusion : Taken together, we showcase that synergistic BMP2 and hypertrophic conditioning significantly improve the yield and therapeutic potential of MtVs. These findings highlight the considerable potential of manufacturing EO‐mimetic MtVs as a nanotherapeutic approach for promoting bone regeneration.

Bone‐Derived

M. Maggio 1 , M. Y. Brunet 1 , C. Gorgun 1,2 , R. Almasri 1 , L. O'Driscoll 1 , D. A. Hoey 1,2,3 1 Trinity College Dublin, Ireland; 2 Royal College of Surgeons in Ireland, Ireland; 3 Advanced Materials and Bioengineering Centre (AMBER), Ireland Introduction : Matrix vesicles (MV) are membrane‐bound nanoparticles secreted by mature bone cells that are embedded in the extracellular matrix (ECM). As key mediators of biomineralization, they are essential for bone formation and repair. Moreover, there is emerging evidence for the presence and functional role of MV in ECM‐derived scaffold‐based tissue repair. MVs are also likely to be present in auto/allografts, which remain the gold standard for bone repair. Nevertheless, the multimodal activity of MVs, their mechanism of action, and their definition as an extracellular vesicle subpopulation remain unclear despite their initial discovery in the 1960s. Therefore, this study aimed to establish a reliable method to isolate MV from animal tissues and to investigate their pro‐regenerative potential for bone repair. Methods : Tissues from 5‐month‐old male C57BL/6J mice were dissected to obtain long‐bone (lb) and kidney (kd)‐derived MV. Tissues pieces were digested with collagenase type 2 for 3 h at 37°C prior to filtration (100 µm) and differential ultracentrifugation with a swinging rotor (TH660) at 110,000 ×  g for 90 min to collect MV. MVs were then characterized including NTA and alkaline phosphatase (ALP) activity. Finally, MV anabolic and anti‐catabolic potential was evaluated by adding increasing doses of MV to human osteoblastic cells and human CD14+ monocytes in the presence of osteogenic or osteoclastogenic factors, respectively. Results : Comparative MV characterisation revealed that lb‐MV were significantly smaller than kd‐MV ( p < 0.0001) with no yield variation observed upon g/tissue of origin. In terms of their intrinsic activity, all MV populations were found to express functional ALP, indicating their pro‐mineralising potential, with lb‐MV possessing significantly more ALP than kd‐MV ( p < 0.0001). Finally, lb‐MV displayed both anabolic and anti‐catabolic potential, eliciting increased mineralisation and decreased osteoclastogenesis in a dose‐dependent manner from 0.25 to 2.5 µg/mL. Summary/Conclusion : MV were successfully obtained and characterized from multiple adult mouse tissues, offering a physiologically relevant model to investigate MV's roles in mature bone beyond in vitro cultures. Importantly, for what we believe to be the first time, lb‐MV demonstrated both anabolic and anti‐catabolic effects for the first time, highlighting their role in bone physiology and their potency as nanotherapeutics for bone repair. Funding : SFI FFP Grant (19/FFP/6533).

Characterizing

Presenter: Victor U. Weiss TU Wien, Vienna, Austria Introduction : As recommended by the European Commission, nanoparticle characterization should be based on particle number concentrations (2011/696/EU from October 18th, 2011, updated in 2022). Gas‐phase electrophoresis on a nano electrospray gas‐phase electrophoretic mobility molecular analyser (nES GEMMA) allows such an approach. Single‐charged (bio‐)nanoparticles obtained after a nES process with subsequent surface‐drying and charge equilibration are separated based on their electrophoretic mobility diameter (EMD) corresponding to the particle diameter for spherical analytes. By voltage variation, particles of different EMD successively reach the instruments’ detector unit and are counted, resulting in a spectrum in the size range of several few up to several hundred nm EMD. Methods : EVs were either purified via ultracentrifugation or a combination of precipitation and centrifugation. Subsequently, the sample buffer was exchanged for ammonium acetate. EV monitoring was via nanoparticle tracking analysis (NTA, Particle Metrix, Inning am Ammersee, Germany), gas‐phase electrophoresis was on nES GEMMA instrumentation (TSI Inc., Shoreview, MN, USA). Results : In previous work, we succeeded in demonstrating the applicability of nES GEMMA for the analysis of liposomes, vesicles consisting of a lipid bilayer encapsulating an aqueous lumen. nES GEMMA enabled the characterization of vesicles in terms of size distribution, particle number concentration and the occurrence of smaller‐sized building blocks next to large vesicles. Also, offline hyphenation of gas‐phase electrophoresis with orthogonal analysis methods, for instance atomic force microscopy, MALDI mass spectrometry or spectroscopy was shown. In vivo, cell‐derived EVs, being part of cell/cell communication, are comparable to liposomes. They are envisioned as pharmaceutical cargo transporters, explaining the need for their characterization. We succeeded in porting gas‐phase electrophoresis of liposomes to the characterization of EVs, demonstrating protein co‐purification and loss of EV stability upon further polishing of vesicle preparations. Our nES GEMMA results were compared to data from NTA. Summary/Conclusion : We demonstrate gas‐phase electrophoresis relating additional characteristics for EV containing samples in comparison to prevailing techniques like, for example, NTA.

Cthrc1+Cxcl12+

Presenter: Chen Hongbo Sun Yat‐sen University, Guangzhou, People's Republic of China Introduction : Alveolar fibroblast subsets exhibit considerable heterogeneity and functional diversity after lung injury. However, the role of CTHRC1+ fibroblasts in responding to and transmitting damage signals caused by SARS‐CoV‐2 remains unclear. Here, utilizing publicly available data, we reveal that CTHRC1+ fibroblasts associated with epithelial cells and monocyte‐derived macrophages (MDMs). Further study demonstrates that these CTHRC1+ fibroblasts exhibit a pro‐inflammatory phenotype, primarily receiving damage signals from type 2 alveolar epithelial cells via exosomes uptake and subsequently secreting CXCL12 to recruit MDMs—a process closely related to ARDS progression. Based on these findings, we develop and preclinical test an inhalable, room‐temperature‐stable recombinant antibody‐exosome conjugated drug that, after ultrasonic nebulization, reduces inflammatory infiltrates and attenuate lung injury in mice. Methods : (1) scRNA‐seq and CellChat analysis identified fibroblast subsets involved in communication with MDMs. (2) TFA analysis and co‐culture assays were conducted to explore the mechanisms driving these fibroblast subsets to secrete elevated CXCL12 levels following SARS‐CoV‐2 infection. (3) miRNA‐seq identified regulatory miRNAs, with their effects confirmed via transfection and qRT‐PCR. (4) ALGGEN PROMO and TransmiR v2.0 predicted transcription factors potentially regulating the miRNAs. 5.Reverse chimeric‐Bebtelovimab was engineered and conjugated to MSCs‐Exo using a DSPE‐PEG linker. Results : (1) CTHRC1+ fibroblasts secrete elevated CXCL12 levels after infection, thereby recruiting excessive MDMs. (2) Exosome mediated communication between epithelia and fibroblasts. (3) EDEVs‐delivered miR‐126 downregulates CXCL12 mRNA levels in fibroblasts. (4) S protein inhibits the expression of miR‐126 by suppressing TFAP2A expression. (5) Fabrication and characterization of mBebtelovimab@MSCs‐Exo and its superior stability. (6) mBebtelovimab@MSCs‐Exo reduce inflammatory infiltrates and attenuate lung injury in vivo after nebulization. Summary/Conclusion : In conclusion, we demonstrated that the SARS‐CoV‐2 spike (S) protein, but not influenza virus, downregulated miR‐126 levels in epithelial cells by decreasing TFAP2A expression. This TFAP2A suppression reduced miR‐126 levels in exosomes derived from epithelial cells (EDEVs). Upon uptake of these EDEVs by CTHRC1+CXCL12+ fibroblasts, their capacity to downregulate CXCL12 expression was impaired, resulting in elevated CXCL12 secretion and increased MDMs recruitment. Based on these findings, we developed an aerosolized, S protein‐specific recombinant antibody‐conjugated MSC exosome therapy, which effectively reduced inflammatory cell infiltration and mitigated lung injury in a murine model.

Cutting‐Edge

Presenter: Jessica Gobbo INSERM1231, Dijon, France Introduction : Extracellular vesicles (EV) are emerging as powerful biomarkers in immunotherapy, carrying tumour‐derived signals and immunosuppressive factors. Their minimally invasive and dynamic nature makes them valuable for predicting treatment response, monitoring therapeutic efficacy, and analysing the tumour microenvironment. This study aims to define a multiparametric signature to predict responses to PD‐(L)1 inhibitors before treatment initiation and develop a rapid, robust, and clinically feasible biomarker analysis method requiring minimal biological samples. Methods : A total of 250 patients with metastatic melanoma, non‐small cell lung cancer, and lymphoma were included in this study ( NCT02662621 , NCT05744076 , NCT04417803 ). EVs were isolated from 500 µL of plasma using an optimized precipitation kit. Size and concentration were assessed via nanoparticle tracking analysis (NS300, Malvern) and transmission electron microscopy. EV markers (TSG101, CD9, CD63, CD81, Alix, and the negative control Grp94) were validated using capillary western blot (CWB). A multiplexed ELISA assay (MEA) was developed to establish a comprehensive protein signature, including PD‐L1. Results : We optimized EV characterization by refining antibody concentration, blocking reagents, and sample concentration for CWB and MEA. Key advantages include ultra‐sensitivity (pg/mL), minimizing blood collection needs, enhanced standardization via automation, Optimized processing: in 3 h (CWB) and 1.5 h (MEA, five targets in triplicate). Circulating PD‐L1‐EV were identified as dynamic biomarkers for anti‐PD‐(L)1 therapy response. The lack of correlation between EV‐associated and free PD‐L1 suggests distinct biological origins. We showed that PD‐L1‐EV analysis could predict tumour response before medical imaging evidence. We further demonstrated that PD‐L1‐EV are biologically active, mimicking cancer cells by inhibiting CD8⁺ T‐cells. This highlights their immunosuppression role in resistance mechanisms (local or at the tumour distance). Finally, we identified a protein signature capable of predicting anti‐PD‐(L)1 therapy responses before treatment initiation. Summary/Conclusion : We developed an ultra‐sensitive, rapid, and clinically adaptable EV biomarker detection method requiring minimal sample volume. This approach offers deeper insights into the tumour microenvironment from a simple blood test and could enhance patient selection in clinical practice. A national multicentre study is underway to validate this predictive signature in precision oncology.

Filter‐Aided

Presenter: Jarne Pauwels VIB‐Ugent, Ghent, Belgium Introduction : Cells secrete small extracellular vesicles (sEVs) with a diameter < 200 nm, which are critical mediators of intercellular communication and hold significant potential for disease diagnostics and therapeutics. Traditional methods for sEV isolation, such as ultracentrifugation (UC), are often not scalable and may result in variable yields and purities, especially for proteome studies. This study investigates a streamLined 96‐well format for EV enrichment for mass spectrometry‐based proteome analysis. Methods : We present a high‐throughput filter‐aided EV enrichment (FAEVEr) workflow for the enrichment of sEV from conditioned medium (CM) and biofluids with limited sample input (200–1000 µL). In short, CM is pre‐cleared using 96‐well 0.2 µm filtration plates, followed by sEV enrichment using ultrafiltration (UF, 300 kDa MWCO 96‐well plates) at low centrifugation speed (1000 × g for 10 min). Retained particles are purified by using relatively high percentages of the detergent TWEEN‐20 for consistent and near‐complete removal of non‐EV proteins. Purified sEVs are immediately lysed for optimal protein recovery and prepared for LC‐MS/MS analysis on a 96‐well S‐Trap plate. Results : Our results indicate that FAEVEr 96well effectively and consistently retains sEV particles and show that including TWEEN‐20 during wash steps results in consistent high sEV purity without compromising sEV integrity. Mass spectrometry analysis revealed high reproducibility of protein identifications with few missing values compared to UC, and that increasing the percentage of TWEEN‐20 in the wash buffer translates to a more efficient removal of non‐EV proteins and a higher abundance of membrane proteins and EV biogenesis‐related proteins. Summary/Conclusion : The FAEVEr 96‐well strategy enables the simultaneous processing of multiple samples, significantly reducing processing time yet improving the reproducibility, consistency and performance between replicates with overall fewer missing values, crucial for ensuring the validity and comparability between experimental results.

Identification

Anna Sophia Feix 1,2 , Bärbel Ruttkowski 1 , Anja Joachim 1 1 University of Veterinary Medicine Vienna, Austria, 2 Aarhus University, Denmark ABSTRACT UNAVAILABLE.

Investigations

Gülce Güralp 1,2 , Stuart James Lucas 1,2 1 Sabancı University, Türkiye; 2 Sabancı University Nanotechnology Research and Application Centre (SUNUM), Türkiye Introduction : Nitrogen is the most significant yield‐limiting nutrient for potato (Solanum tuberosum), which is the world's fourth most important crop for human nutrition. Nitrogen fixation is a significant process mediated by nitrogen‐fixing bacteria among beneficial soil microbes to convert nitrogen gas to ammonium, which is one of the most biologically available forms for plants. Thus, plant‐microbe interactions are important for plant nutrition, health and survival. Extracellular vesicles (EVs)‐released by microbes provide the communication between plants and microbes through their compositions of proteins, lipids, small RNAs (sRNAs) and nucleic acids. Several studies have demonstrated that EVs‐released from bacteria contribute to stress responses, quorum sensing, biofilm formation and symbiotic relationships with plants. The study purposes to explore roles of EVs in the effects of nitrogen‐fixing bacteria on potato plants, with the ultimate aim of developing biofertilizers from nitrogen‐fixing bacteria and their EVs. Methods : Endophytic bacteria have been isolated from surface‐sterilized tubers and roots of potato plants, collected from high‐yielding potato fields of Türkiye. Individual colonies sub‐cultured on fresh media have been evaluated for nitrogen fixation capacity by inoculating into Nfb medium and then will be identified by 16S rRNA and nifK gene sequencing. EV production from the nitrogen‐fixing bacteria into liquid medium will be optimized and characterized by nanoparticle tracking analysis, transmission electron microscopy and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and so forth, to reveal potential roles of EVs‐produced by them in nitrogen fixation. Beneficial nitrogen‐fixing bacteria and their EVs will be investigated for their effects on potato nutrition in controlled conditions. Results : Three promising isolates with nitrogen‐fixing ability have been selected, which will be identified by sequencing. We hypothesize that EVs‐released by nitrogen‐fixing bacteria may carry biomolecules or genes related to nitrogen fixation into potato plants, help to regulate interactions with other microbes in the soil, and mediate plant‐endophyte symbiosis. Summary/Conclusion : Characterization of the nitrogen‐fixing and EV‐producing bacteria identified in this study is expected to shed new light on communication mechanisms during plant‐endophyte symbiosis. Funding : This study has been supported by the Scientific and Technological Research Council of Turkey [TUBITAK, 124O159].

Ipsc‐Derived

Presenter: Pei‐Ling Chi Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (Republic of China) Introduction : Uncontrolled lung inflammation is a critical aspect of lung infection‐associated acute lung injury (ALI). Induced pluripotent stem cells (iPSCs) offer a scalable and effective source for therapeutic extracellular vesicles (iPSC‐EVs). However, their mechanisms of action and pathways of communication remain under exploration. This study examines the contents of iPSC‐EVs in reducing inflammation in a mouse model of septic lung injury induced by Vibrio vulnificus . Methods : Human iPSC‐EVs were administered intraperitoneally to 6‐week‐old male C3H/HeN mice 2 h before subcutaneous V. vulnificus infection. An in vitro model, using RAW 264.7 cells, was employed to investigate how iPSC‐EVs influence macrophage immune responses. Results : Results demonstrated that iPSC‐EV treatment attenuated V. vulnificus ‐mediated activation of the NLRP3 inflammasome in lung tissue. Additionally, miR‐223, miR‐18, and miR‐30 expression was elevated in the lung tissue of infected mice treated with iPSC‐EVs, relative to untreated controls. In RAW 264.7 cells, miR‐223 suppressed NLRP3 inflammasome expression in V. vulnificus ‐infected macrophages by inhibiting NLRP3 and ASC. Furthermore, miR‐18 and miR‐30 promoted M2 macrophage polarization. Notably, iPSC‐EVs contained higher levels of miR‐223, miR‐18, and miR‐30 than EVs derived from human umbilical cord mesenchymal stem cells (hUC‐MSC‐EVs), suggesting a superior protective effect of iPSC‐EVs against septic lung injury. Summary/Conclusion : In conclusion, iPSC‐EVs reduce V. vulnificus‐induced septic lung injury through the transfer of miR‐223, miR‐18, and miR‐30, leading to decreased NLRP3 inflammasome activity and an increase in anti‐inflammatory M2 macrophages, thereby supporting inflammation resolution. Funding : National Science and Technology Council 113‐2314‐B‐075B‐003‐MY2.

Lectin‐Based

Haruka Sei, Fumie Nakashima, Takahiro Shibata Nagoya University, Nagoya, Japan Introduction : Urinary extracellular vesicles (uEVs) are secreted into urine from a variety of cells in the kidneys and urinary tract and their contents reflect changes in the kidneys. Therefore, uEVs have been thought to be a potential source of biomarkers for kidney diseases. However, it is difficult to predict the original cells of uEVs because the original segment‐dependent sorting methods have not yet been developed well. In this presentation, since the surface sugar chain patterns differ among the nephron segments, we attempted to enrich nephron segment‐specific EVs from uEVs using biotinylated lectins. Methods : EVs were isolated by ultracentrifugation from rat urine. The lectin pull‐down assay of uEVs was performed to enrich nephron segment‐specific EVs. The flow‐through and pull‐down fractions were subjected to immunoblotting with antibodies to nephron segment‐specific proteins. Histochemical staining of the kidney sections was also performed using biotinylated lectins and nephron segment‐specific proteins. Results : The lectin pull‐down assay revealed that Lens culinaris lectin (LCA) interacted with uEVs derived from proximal tubular cells (PT‐uEVs) but not with uEVs derived from collecting duct cells (CD‐uEVs). Histochemical staining of kidney sections showed that LCA‐positive cells were colocalized with the proximal tubular marker‐positive cells in the cortex, but not with the collecting duct marker‐positive cells. Summary/Conclusion : Biotinylated LCA was a suitable lectin to enrich PT‐uEVs from rat uEVs. This method will be valuable in biomarker research involving rat uEVs.

Matrix‐Bound

Presenter: Genevieve Anghileri Loughborough University, Loughborough, UK Introduction : Matrix‐bound vesicles (MBVs) function as early sites of mineral formation in developing tissues such as cartilage and bone. However, therapeutic studies continue to prioritise media vesicles (MVs) isolated from suspension. Consequently, the therapeutic potential of MBVs remains largely undefined. This study sought to characterise MBVs and evaluate their therapeutic potential using in vitro and in vivo models. Methods : MBVs and MVs isolated from primary human bone marrow MSCs cultured under osteogenic conditions were profiled using ZetaView, western blotting, super resolution microscopy (ONI), and mass spectrometry. Pro‐mineralisation capacity and mineral composition were assessed using alkaline phosphatase (ALP) assay, phosphate hydrolysis assays, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). Association with collagen and decellularized/demineralised bovine bone hydrogels (dECM) was determined to identify an appropriate material delivery system. In vitro models assessed the pro‐mineralisation potential of MBV delivery, and in vivo subcutaneous rodent models were applied to quantify EV clearance (DiR labelled) and de novo mineralisation potential when administered with collagen sponges. Results : Significantly higher numbers of MBVs (2.56 × 10 12 ± 8.50 × 10 11 particles/mL) were liberated from the ECM compared to MVs recovered from conditioned medium (1.54 × 10 11 ± 2.73 × 10 10 particles/mL) ( p < 0.001). Phosphate hydrolysing enzymes and calcium channelling proteins required for mineralisation were elevated in MBVs, with confirmation of their localisation with tetraspanins confirmed at the single vesicle level by super‐resolution microscopy. MBVs rapidly hydrolysed polyphosphates (ATP) and significantly enhanced mineralisation in MSC cultures when compared with MVs as confirmed by turbidity assays and alizarin red staining ( p < 0.05). MBVs were comparatively enriched in electron dense material (TEM imaging), which was identified as hydroxyapatite by FTIR. In vitro and in vivo analysis confirmed MBVs exhibited a high binding affinity for collagen and dECM scaffolds (60%–70% binding in vitro), while MVs exhibited minimal binding (< 10%). DiR‐labelled MBVs were detectable on collagen sponges when implanted subcutaneously over a period of 4 weeks. Summary/Conclusion : MBVs represent a comparatively abundant and potent source of vesicles for hard tissue regeneration and are compatible with collagen‐based delivery systems for sustained delivery in vivo. Funding : This work was supported by the Academy of Medical Science, the EPSRC and the British Council.

Mesoangioblast

Presenter: Fabiana Geraci University of Palermo, Italy Introduction : Mouse mesoangioblasts (C57) are vessel‐associated multipotent progenitor stem cells, which are able to differentiate into different mesodermal cell types and are able to shed membrane vesicles (EVs) in the extracellular environment. EVs contain several molecules, such as MMP2/9 and HSP70, as transmembrane protein. The aim of our work was to determine the effect of C57 EVs on murine macrophages (Raw264.7), as they play a central role in all stages of the inflammatory response. In damaged tissue macrophages migrate to carry out their function of identifying and removing dead cells, debris, and foreign particles via phagocytosis. Methods : C57 EVs were collected from conditioned media by ultracentrifugation. Raw264.7 cells were cultured with or without C57 EVs to evaluate the effects on cell proliferation, cell migration, phagocytosis ability, and phenotype characterization. To this aim we performed FACS analysis, microscope observations and enzymatic activity quantification. Results : We have analysed the immunomodulatory effect of C57 EVs on Raw264.7 cells. We have first demonstrated that EVs are able to interact with Raw264.7 cells by negatively influencing their proliferation index and positively influencing their migratory capability. We also proved that this enhanced migration is mediated by an elevated expression and activity of MMP2/9. Moreover, in vitro phagocytosis index calculation highlighted that EV treatment is able to improve Raw264.7 phagocytic ability, which is important at early stages of tissue repair. The use of neutralizing antibodies (i.e., anti‐Hsp70, anti‐TLR2, and anti‐TLR4 antibodies) demonstrated that C57‐EVs are able to increase Raw264.7 phagocytosis through Hsp70 and its surface receptors. To point out whether EVs modulate macrophage phenotype, we explored the presence of M1/M2 markers (i.e., iNOS and arginase) and NO synthesis. At the initial stage after EV treatment, Raw264.7 synthesized both iNOS and NO, whereas no arginase mRNA was detected. These data suggest an M1 phenotype. After a recovery following EV treatment, an increased release of anti‐inflammatory cytokines, typical of M2 macrophages, was observed. Summary/Conclusion : These data suggest that C57‐EV could positively influence tissue repair through macrophage modulation. Funding : This research was supported by grants from the University of Palermo (FFR 2024 to FG).

Milk‐Derived

M. Brattini, M. Mosaico, E. Butturini, S. Mariotto Dept. of Neurosciences, Biomedicine and Movement Science, Biological Chemistry Section, University of Verona, Verona, Italy Introduction : One of the main attractive characteristics of extracellular vesicles (EVs) is the possibility of using them as drug delivery vehicles thanks to their natural origin and the intrinsic ability to carry molecular cargo and to cross biological barriers. However, the translation of EVs as delivery vehicles into clinical studies is hampered by the difficulties in obtaining them in an efficient and rapid way. Milk could represent a good EV source, offering an accessible and cost‐effective option with high EV yield through a relatively easy protocol. To demonstrate the potential of mEVs as a drug delivery system, myricetin, a natural polyphenol with anti‐inflammatory activity, was chosen as a model drug, while saponin was used as a loading agent. Methods : MEVs were isolated from whole pasteurized bovine milk through the differential ultracentrifugation method in combination with rennet pre‐treatment to remove casein micelles. Nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), transmission electron microscopy (TEM) and western blot analyses were carried out to characterize the isolated particles. Moreover, cellular uptake of MEVs was evaluated by analysing treated BV2 cells by confocal microscopy. Finally, myricetin was encapsulated through a saponin‐assisted method, and the encapsulation efficiency (EE%) was assessed using HPLC analysis after removing free myricetin by ultrafiltration. Results : NTA and DLS analysis revealed particles with a size in the range of 150 nm, a surface charge of −21.7 ± 2.67 mV and a concentration in the order of 10 13 particles/mL. TEM images displayed particles delimited by a lipid bilayer and with spherical morphology; moreover, western blot showed the presence of positive EVs markers CD81, Hsp70, and TSG101. MEVs were efficiently taken up by BV2 cells after 24 h, as proved by confocal images. Finally, HPLC analysis confirmed the optimization of myricetin encapsulation conditions with an EE% of 21% ± 8.5%. Summary/Conclusion : Our work demonstrates the effectiveness of the adopted isolation protocol, which enabled the efficient separation of MEVs from bovine milk. Moreover, this study highlights the potential of mEVs as a tool for optimizing the encapsulation parameters of myricetin in EVs. However, further analysis is needed to verify the cellular response to the treatment with myricetin‐loaded MEVs.

Overexpression

Presenter: Ravi Shankar Akundi South Asian University, New Delhi, India Introduction : Inflammation is the first line of the body's defence system, mediated by macrophages, which produce prostaglandin E2 (PGE2) in addition to cytokines and chemokines. Failure of resolution of inflammation leads to chronic inflammatory conditions such as rheumatoid arthritis, neurodegeneration, and/or cancer. We have previously identified extracellular ATP (eATP) as a synergistic enhancer of cyclooxygenase 2 (COX‐2)‐mediated inflammation in macrophages and found that activation of purinergic P2 receptors enhanced the synthesis of PGE2. Ectonucleotidases such as CD39 and CD73 sequentially dephosphorylate ATP to ADP and AMP and further to adenosine, respectively. Removal of ATP by ectonucleotidases, therefore, is an essential step for the resolution of inflammation. In addition, adenosine acts as an anti‐inflammatory molecule by binding to P1 receptors. In the case of tumours, we have shown that P2 receptor‐mediated increased expression of COX‐2 causes tumour progression and metastasis. Extracellular vesicles (EVs) released by activated macrophages or tumour cells are a potential mechanism through which pro‐inflammatory mediators such as COX‐2, cytokines, and PGE2 are transported to distant sites, thereby spreading metastasis in the case of cancer cells. In this work, we check whether ectonucleotidases reduce COX‐2 expression in macrophages and affect the packaging of EVs released by these cells. Methods : In this project, we cloned and overexpressed CD73 in J774A1 macrophages to isolate EVs. EVs were isolated and characterized using different conditioned media followed by differential ultracentrifugation and using various protein markers. The isolated EVs were used to stimulate naïve J744A1 macrophages to check for COX‐2 expression. Results : We show that overexpression of CD73, the rate‐limiting ectonucleotidase, in murine macrophages shows a reduction in inflammation and PGE2 release. Furthermore, we also show that EVs released from CD73‐transfected cells have reduced sequestration of COX‐2 mRNA and its expression. Summary/Conclusion : Modulation of inflammation through engineered EVs (EVs released from CD73 overexpressing cells) could be a potential alternative to non‐steroidal anti‐inflammatory drugs (NSAIDs) to combat chronic inflammatory conditions, autoimmune disorders, and neurodegeneration.

Preconditioned

Presenter: Polina Pikus National Academy of Sciences of Ukraine, Ukraine Introduction : Extracellular vesicles (EVs) are known to be promising tool in the treatment of various diseases. Of particular interest are EVs secreted from mesenchymal stem cells (MSCs) because they have a main role in modulating immune cells. Preconditioning of MSCs, which enhances their therapeutic efficacy, positively affects the immunomodulatory properties of their EVs. The aim of the study was to compare the effects of EVs secreted by native and preconditioned MSCs on intraperitoneal macrophages during resolution of inflammation in a mouse model of acute peritonitis. Methods : MSCs were isolated from umbilical cords of both sexes and characterized according to the minimal criteria for defining MSCs (ISCT). MSCs were preconditioned with H2O2 at a dose of 30 µM. EVs were isolated by ultracentrifugation. EVs were characterized by transmission electron microscopy, ZetaView Nanoparticle Tracking Analyzer and EV markers were identified by Western blot. A model of sterile acute inflammation of the abdominal cavity in mice was induced by intraperitoneal injection of 1 mL of 3% proteose peptone solution. Results : The maximum intensity of inflammation was 24 h after the injection of the proteose peptone solution, and at this time mice were injected with EVs in an amount equivalent to that obtained from 5 000 MSCs/mouse. 15 min after injection of MSCs at this dose led to sharp inflammation suppression, and the rate of inflammation suppression upon injection of preconditioned MSCs was even higher. Dynamics of inflammation suppression after the injection of EVs shows higher efficacy compared to MSCs. The level of IL‐10 expression in mononuclear cells obtained from the peritoneal cavity of mice after injection of EVs from native MSCs increases, suppressing inflammation compared to the positive control. But the level of IL‐10 expression is significantly higher after injection of EVs derived from H2O2‐preconditioned MSCs. Increased phagocytosis by intraperitoneal macrophages, observed during the inflammation suppression, is more intense after the injection of preconditioned MSCs. Summary/Conclusion : The injection of EVs derived from native and H2O2‐preconditioned HUC MSCs results in potent suppression of inflammation and macrophage polarization in the mouse peritoneal cavity, with both effects being more pronounced with injection of EVs from preconditioned MSCs.

Quantification

Liam Barry‐Carroll 1 , Céline Gounou 2 , Jean‐Christophe Delpech 1 , Stéphane Mornet 3 , Alain R. Brisson 2 1 INRAE UMR‐1286, Bordeaux, France; 2 University of Bordeaux, Bordeaux, France; 3 CNRS UMR‐ICMCB, Pessac, France Introduction : The quantification of extracellular vesicles (EV) constitutes a major challenge in the EV field. In this study, we compared two approaches of EV quantification by flow cytometry, namely the conventional approach of light‐scatter detection using a new‐generation flow cytometer dedicated to small particle detection (NanoAnalyzer, NanoFCM), and the alternative approach of fluorescence‐based detection, using a previous‐generation instrument (Gallios, Beckman‐Coulter). In addition, immuno‐gold cryo‐electron microscopy (immuno‐cryo‐EM) was used to determine the EV size and phenotype distributions. Methods : EV derived from red blood cell (RBC) and platelets (PLT) were prepared from blood bags, by osmotic lysis and activation with A23187 ionophore, respectively. EV derived from reticulocytes (RET) were isolated from blood samples obtained from sickle cell disease (SCD) patients, after written informed consent. EV samples were labelled with Annexin‐5 (A5) for identifying phosphatidylserine‐exposing EV, or with antibodies (Ab) specific of RBC (CD235), PLT (CD41) or RET (CD71), either fluorescently labelled for flow cytometry or conjugated to gold particles for immuno‐cryo‐EM. Results : Two types of EV samples were selected for this study: (1) EV derived from RBC and PLT, which are large EV (∼200 nm average size, as determined by cryo‐EM) and present a high density of membrane surface receptors, around 7000 CD235 (RBC) and 3000 CD41 (PLT) per µm 2 , respectively, and (2) RET‐EV expressing CD71, which are prototypical exosomes of small size (∼100 nm diameter). For RBC‐ and PLT‐EV, essentially identical numbers of EV were detected by the two flow cytometry approaches compared here, with all the markers used ‐A5, CD235 and CD41‐, over 20 independent experiments. As an example, the ratio between the numbers of A5‐labelled EV detected by light‐scattering vs. fluorescence was 1.4 ± 0.3 ( n = 11) for RBC‐ and PLT‐EV. In contrast, in the case of CD71‐expressing RET‐EV, we found that only a small amount (< 10%) of EV was detected by fluorescence triggering. This latter point is under further investigation. Summary/Conclusion : This study demonstrates, for the first time to our knowledge, that the fluorescence triggering approach allows an accurate and reliable quantification of EV, at least in the case of large EV expressing a high surface density of antigens. Funding : ANR grant

Rbcs‐Derived

Kulzhan Berikkhanova, Alexandr Gulyayev, Ernur Zakirov, Askhat Zhilkaidarov, Shynggys Sergazy Nazarbayev University, Kazakhstan Introduction : Patients with abdominal sepsis face toxemia and bacteremia, requiring continuous detoxification and liver support. Targeted drug delivery offers new ways to manage liver dysfunction. The hepatoprotector ademethionine distributes non‐selectively with low liver accumulation. Red blood cells (RBCs) are the primary vesicle‐secreting cells in blood circulation. RBCs‐derived carriers (RBCCs) have been created for targeted delivery of ademethionine to the liver. Ademethionine‐loaded RBCCs are recognized by liver macrophages as damaged RBCs, resulting in their capture and digestion. This process extends the duration of peak ademethionine concentration in the liver following phagocytosis of RBCCs. In this study, ademethionine‐loaded RBCCs were created, and in vitro release of ademethionine from RBCCs was described. Methods : This study was approved by institutional ethics committee. Blood samples were taken from volunteers after receiving signed informed consent. RBCCs loaded with 500 mg of ademethionine were developed using hypoosmotic hemolysis method, with our original modifications. To evaluate degree of ademethionine release from RBCCs, equilibrium dialysis with semipermeable membrane was performed. Samples were taken at intervals of 15 min, 30 min, 1 h, 3 h, 17 h, and 24 h. The concentration of ademethionine in these samples was determined using spectrophotometric analysis. Results : The release of ademethionine from RBCCs occurred gradually over a 24‐h period. The highest release was observed at 15 min (17.02 µg/mL ± 0.21), attributed to the release of both the internally deposited and superficially adsorbed drug. This was followed by gradual release of ademethionine over the next 3 h, maintaining levels of 5‐8 µg/mL. By 17 h, concentration of released ademethionine had decreased to 1.14 µg/mL ± 0.13 and reached zero by the 24th h. Summary/Conclusion : RBCCs demonstrated ability to encapsulate the ademethionine and release it gradually into the external environment over 24 h, achieving the highest concentration within first 17 h. These findings can be applied to study pharmacokinetics of ademethionine‐loaded RBCCs in animal models. This approach could provide promising therapeutic strategy for manageing liver disorders in abdominal sepsis. Funding : This research is funded by Science Committee of Ministry of Science and Higher Education of Kazakhstan, Grant №AP19676272 and by Nazarbayev University under Collabourative Research Program Grant № 211123CRP1614, A.G.

Tki‐Mediated

Jeannette Salsetta 1 , Elisa D'Angelo 1 , Gianpiero Lupoli 1 , Eriomina Shahaj 2 , Elisabetta Vergani 1 , Agata Cova 1 , Paola Squarcina 1 , Licia Rivoltini 1 , Elena Verzoni 3 , Veronica Huber 1 1 Translational Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2 Genetics and Genomic Medicine Research & Teaching Dept, UCL GOS Institute of Child Health, London, UK; 3 Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Introduction : CD49b (ITGA2), the alpha subunit of α2β1 integrin, is expressed by T and NK cells, fibroblasts and platelets and is involved in adhesion and cell activation. CD49b expression by renal cell carcinoma (RCC) is associated with poor prognosis. As a heterodimer with CD29, CD49b functions as a collagen receptor and is involved in cancer progression and angiogenesis. Pazopanib is a tyrosine kinase inhibitor (TKI) with anti‐angiogenic and immunomodulatory properties (doi: 10.1002/ctm2.434). Here we investigated CD49b expression by plasma extracellular vesicles (EVs) in the context of TKI‐induced immune modulation and antiangiogenic activity in metastatic (m)RCC patients. Methods : Peripheral blood samples from mRCC patients ( n = 8, INT146/14) were collected at baseline and at 3 and 6 months on‐therapy with Pazopanib. EVs, isolated by differential centrifugation, were evaluated by NTA, microsphere‐assisted flow cytometry (MACSplex) and ELISA for CD49b expression. Statistical analysis was performed with GraphPad Prism. Results : Circulating CD49b+ EVs originated primarily from T regulatory cells and platelets, as indicated by correlation analysis with cellular immune profiles and blood counts. After 3 months of therapy, we recorded a decrease of plasma EV concentration and of CD49b+EVs with a concomitant decrease of immune suppression‐associated EV markers of Treg (CD2, CD44, CD25, CD44, CD29, CD45, HLA‐DR). This effect was accompanied by increased EVs expressing NK (CD56, CD69, HLA‐DR), T cell (CD2, CD3, CD4, CD8, CD25, CD69) and myeloid (CD14, CD1c, CD11c, CD86, HLA‐DR) markers, suggesting a reactivation of antitumour immunity detectable at the plasma EV level. CD49b+ EV levels correlated with platelet counts, Treg cells and PD‐L1+ monocytes. All of these showed a significant decrease in peripheral blood of patients after 3 months therapy while at the 6‐month time point we measured a recovery close to baseline levels. Summary/Conclusion : Our findings suggest TKI‐induced decrease of CD49b+ EVs is associated with a reduction of platelets and the immune suppressive components Tregs and PD‐L1+ monocytes, resulting in a systemic immune reactivation. Inhibition of CD49b+ EVs by TKI could affect the local immune response and function of T cells, including Treg. We hypothesize the TKI‐mediated reduction of CD49b+ EVs coincides with the anti‐angiogenic and anti‐tumour activity of TKI via disruption of the CD49b/CD29 axis. Funding : Italian Association for Cancer Research, AIRC, IG25078.

Ultrasensitive

Pablo Sánchez‐Martín 1 , John Atanga 2 , Irina Nazarenko 2 , Tobias Groß 1 1 Actome GmbH, Witten, Germany; 2 Institute for Infection Prevention and Hospital Epidemiology, University Hospital Münster, Münster, Germany Introduction : Research on extracellular vesicles (EVs) is currently limited by the lack of suitable tools to analyse and quantify the heterogeneous populations of these interesting biomarkers. Based on the PICO digital immunoassay, we developed a novel assay to quantify and characterize EVs. Methods : DNA‐labelled antibodies against different surface or intravesicular proteins were incubated overnight with intact or lysed purified EVs. In the case of intact EVs, the antibodies were selected to prevent the detection of individual solubilized proteins. The binding of at least two antibodies to their protein targets forms the detection unit of the PICO assay, named Couplex. Samples were subsequently diluted and analysed by digital PCR, which provides absolute quantification down to single EV level. Results : Using different tetraspanins (CD9, CD63, CD81), we detected different EV subpopulations based on the presence of one or more of these markers, with absolute quantification and a sensitivity of down to 3 × 10 5 vesicles/µL, making PICO more sensitive or comparable to other available methods. EV detection was achieved from samples of different origins. The quantification values matched those determined by conventional methods (e.g., nanoparticle tracking analysis and nanoFCM), but without the need of a calibration standard. Incubating the EVs with lysis buffer allowed the detection and quantification of cytosolic markers (4EBP1) not detected in intact EVs. This enables the determination of the integrity of purified EVs, as well as the quantification of relevant biomarkers. Summary/Conclusion : Here, we present a novel and sensitive method that provides absolute quantification of EVs and their cargo using only 1 µL of sample as input. The multiplex nature of the assay (up to 4 antibodies) also allows simultaneous analysis of multiple markers, positioning PICO as an excellent tool for the study of EVs. Funding : The collabourative project EV‐surf (FKZ: 13GW0605B) is funded by the Federal Ministry of Education and Research (BMBF) within the funding measure “KMU‐innovativ.”

Wnt3A‐Loaded

Presenter: Yeo‐Jun Yoon Yonsei University College of Medicine, Republic of Korea Introduction : Salivary gland (SG) dysfunction, often induced by irradiation treatment for cancer therapy and Sjögren's Disease, can lead to fibrosis characterized by excess extracellular matrix accumulation. Enhancing stemness and promoting extracellular vesicle (EV) production from SG tissue stem cells may offer therapeutic strategies for restoring SG function. Methods : We developed a double‐layered microwell scaffold incorporating a biochemical niche to enhance stemness and EV production in SG tissue stem cells. The scaffold comprised a WNT3A protein‐loaded poly(D,L‐lactide‐co‐glycolide) (PLGA) electrospun nanofibre combined with a polycaprolactone (PCL) microwell array to ensure sustained WNT3A release and support three‐dimensional (3D) cell culture. Human SG‐derived epithelial stem cells (sgEpSCs) were cultured on various substrates: 2D plastic dishes, 3D PCL microwells, 3D bare PLGA + PCL microwells, and WNT3A‐loaded PLGA + PCL microwells. The regenerative effects of sgEpSC‐derived EVs were evaluated using irradiated murine SG models and a human SG organoid system. All animal experiments were approved by the institutional animal care committee, and human cells were obtained with informed consent under approved protocols. Results : The PCL microwells integrated with the WNT3A‐loaded PLGA nanofibre scaffold promoted the transformation of sgEpSCs into 3D spheroids, ensured sustained WNT3A release, and significantly increased EV production. EVs derived from sgEpSCs cultured in WNT3A‐releasing microwells (3DWNT‐EVs) were injected into the SG ducts of irradiated mice, resulting in reduced epithelial and progenitor cell death and preserved SG function. In vitro organoid assays demonstrated that 14‐3‐3 protein zeta/delta, highly expressed in 3DWNT‐EVs, significantly enhanced the proliferation of SG progenitor cells and increased the expression of phosphorylated phosphoinositide 3‐kinases/protein kinase B (p‐PI3K/AKT). Summary/Conclusion : The use of WNT3A‐releasing microwell scaffolds to grow 3D spheroids enhances EV production from sgEpSCs, which in turn can mitigate SG dysfunction post‐radiation exposure. These findings suggest that the engineered microwell scaffold supports stem cell maintenance and EV‐mediated regenerative processes, offering a promising approach for restoring SG function after radiation‐induced injury. Funding : This work was supported by the National Research Foundation (NRF) of Korea and funded by the Ministry of Science and ICT (2020M3A9I4039045); and the NRF grant funded by the Korea government (RS‐2024‐00432946).

Anti‐Tumoural

Luca Giacchi 1 , Elisa Pucci 1 , Argia Ucci 1 , Chiara Puri 1 , Letizia Clementi 1 , Maria Maggi 2 , Antonio Angelo D'Archivio 1 , Adriano Angelucci 1 , Nadia Rucci 1 1 University of L'Aquila, L'Aquila, Italy; 2 Hortus Novus Srl, Rome, Italy Introduction : Gold standard chemotherapy for osteosarcoma is the combined administration of high dose methotrexate (MTX), doxorubicin (DXR) and cisplatin (CPT). Currently, synthetics liposomes and nanoparticles are investigated as potential vehicle for drug administration, to reduce related side effects and increase specificity. Methods : We employed extracellular vesicles (EVs) isolated from the human foetal osteoblast (hFOB) cell line to deliver MTX, DXR, and CPT to MNNG/HOS human osteosarcoma cells. FOB‐EVs were loaded with MTX by incubation for 30 min with 20 mg/mL MTX in 0.1 M NaOH at 37°C (hFOB‐EVs + MTX). Results : MNNG/HOS treatment with hFOB‐EVs + MTX significantly reduced cell metabolic activity, number, and proliferation, while increasing apoptosis, compared to tumour cells treated with empty hFOB‐EVs or vehicle. However, passive incubation and electroporation failed to load DXR or CPT into hFOB EVs, so we adopted an endogenous loading strategy: hFOBcells were treated with 4.6 µg/mL DXR or 30.0 µg/mL CPT, and EVs were harvested from conditioned medium. hFOB EVs + DXR showed a similar cytotoxic effect on MNNG/HOS as exerted by hFOB‐EVs + MTX—significantly reducing viability, proliferation, and increasing apoptosis compared to tumour cells treated with empty hFOB‐EVs or vehicle—whereas hFOB‐EVs + CPT produced a marked trend toward reduced metabolic activity ( p  = 0.0633) while significantly reducing cell number, proliferation and increasing apoptosis, when compared to tumour cells treated with empty hFOB‐EVs or vehicle. Intriguingly, hFOB cells treated with drug‐loaded EVs maintained normal metabolic function, underscoring the safety of this delivery platform. Summary/Conclusion : These findings demonstrate that hFOB derived EVs can efficiently deliver MTX, DXR, and CPT to osteosarcoma cells with no observable toxicity on normal cells, offering a promising strategy to enhance chemotherapeutic specificity and minimize off target toxicity. Funding : This research was funded by an AIRC investigator grant to NR, grant number 24823.

Cell‐Specific

Zeinab Ghesmati 1 , Mohsen Rashid 1 , Shabnam Fayezi 2 , Effat Alizadeh 1 , Masoud Darabi 3, 4 1 Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. 2 Department of Gynecologic Endocrinology and Fertility Disorders, Women's Hospital, Heidelberg University, 69120, Heidelberg, Germany. 3 Department of Hematology and Oncology, Division of Experimental Oncology, University Hospital Schleswig‐Holstein, Lübeck, Germany. 4 University Cancer Center Schleswig‐Holstein (UCCSH), Lübeck, Germany Introduction : Adipose tissue functions as a secretory organ, influencing processes beyond energy storage. White and brown adipocytes, which play opposing roles in energy metabolism, exhibit unique paracrine and endocrine roles. Extracellular vesicles (EVs) from these adipocytes show therapeutic potential, yet their cell‐specific effects remain poorly understood. This study aimed to assess the impact of EVs from white and brown adipocyte‐like cells (WALCs and BALCs) on various human cell types. Methods : This study received local ethics committee approval, and all cell samples were collected under informed consent. Human adipose‐derived stem cells (hADSCs) were differentiated into WALCs and BALCs using induction media. Differentiation was verified by lipid droplet accumulation and quantification of adipocyte markers. EVs were isolated from conditioned media (CM) using differential ultracentrifugation and characterized by light scattering, electron microscopy, and marker immunoblotting. Four human cell types—representing stem, somatic, and cancer cells—were treated with purified EVs or crude CM. Cellular responses were evaluated using flow cytometry, qPCR, electrochemiluminescence, and lipidomic profiling via liquid chromatography‐tandem mass spectrometry. Results : In hADSCs, all EVs and CM promoted osteogenic differentiation and reduced stemness markers Oct4 and Sox‐2, with BALC‐derived EVs showing the greatest potential to enhance differentiation and WALC‐derived EVs most effectively suppressing stemness. When treated with BALC‐derived EVs, hADSCs displayed the highest number of differentially expressed lipids, primarily upregulated hits, while umbilical cord stem cells exhibited minimal responses to the treatment. In MCF‐7 cancer cells, CM from both adipocyte types had a greater pro‐apoptotic effect than EVs. Additionally, BALC‐derived EVs induced higher estradiol secretion in ovarian granulosa cells compared to WALC‐derived EVs. Pathway analysis indicated that BALC‐derived EVs promoted lipid storage and membrane remodelling in hADSCs, decreased lipid storage reliance in umbilical stem cells, reduced fatty acid oxidation in MCF‐7 cells, and enhanced energy metabolism in granulosa cells. Summary/Conclusion : EVs from WALCs and BALCs cells exert distinct, cell‐specific effects compared to soluble factors, underscoring their role in mediating cell‐cell communication. These findings provide insights into the potential applications of adipocyte‐derived EVs in non–cell‐based therapies. Funding : This study was supported by the Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 66664) and the University Medical Center Schleswig‐Holstein, Leubeck, Germany. The presentation was supported by the COST Action CA21113 SENESCENCE2030, awarded to the last author.

Characteristics

Csenge Szász 1 Ani Barbulova 2 , Kameswari Priyanka Devarakonda 2 , Immacolata Fiume 2 , Angela Mary Joseph 2 , Veronika Kralj‐Iglic 3 , Anna Romolo 3 , Lilla Turiák 4,5 , Mirjam Balbisi 4,5 , Ádám Vannay 1,6 Gabriella Pocsfalvi 2 1 Pediatric Center, MTA Center of Excellence, Semmelweis University, Hungary. 2 Institute of Biosciences and BioResources, National Research Council of Italy. 3 University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Slovenia. 4 MTA‐TTK Lendület (Momentum) Glycan Biomarker Research Group, Institute of Organic Chemistry, Hungary 5 HUN‐REN Research Centre for Natural Sciences, Hungary. 6 HUN‐REN—SU Peditarics and Nephrology Research Group, Hungary Introduction : Extracellular vesicles (EVs) from plant cell cultures can be advantageous over prokariotic and mammalian cell lines as therapeutic agent or drug delivery system. The plant cell wall is a firm barrier that restricts the exchange of metabolites between the cell and the extracellular space thereby also limiting EVs production. Therefore the aim of this study was to investigate the effect of cell wall removal on the yield and characteristics of EVs isolated from BY‐2 protoplast culture, a model species in plant cell biology. Methods : Protoplast isolation circumstances, including digestion time, enzyme concentration and media composition from BY‐2 cells were optimized, viable protoplasts were purified and cultured for three days. The state of the cultures was followed by cell counting and viability measurement using fluorescein diacetate. EVs were isolated from the culture supernatant using ultracentrifugation and resuspended in phosphate buffered saline. The protein concentration, protein profile, morphology, particle number and size distribution were determined by Qubit assay, SDS‐PAGE, cryo‐electron microscopy and Videodrop analysis, respectively. Results : In our experiment conducted in three biological replicates the average protoplast yield was 10.4% determined by cell counting. The protoplasts showed high viabilty, round morphology and loss of cell‐cell connections during the whole course of the experiment. 10 mL of protoplast culture yielded 5.25 µg of protein and 1.11×109 particles on average. On the electron microscopic images of the isolated EVs, an intermediate density plasma and the surrounding phospholipid bilayer is visible. The median diameter was 260.2, 272.9 and 340 nm in the three samples. Summary/Conclusion : We were able to obtain and culture viable protoplasts from BY‐2 cell suspension culture and successfully isolate and characterize EVs from their culture supernatant. The optimization of EVs isolation from plant resources might provide multiple opportunities both for basic and translational research fields. Funding : This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Staff Exchange project “FarmEVs” grant agreement N. 101131175 and the National Research, Development and Innovation Office, K‐142728; EKÖP‐2024‐53, EKÖP‐2024‐160, EKÖP‐2024‐162 New National Excellence Program of the Ministry for Culture and Innovation; Semmelweis University, TKP2021‐EGA‐24; Hungarian Academy of Sciences, János Bolyai Research Scholarship.

Cross‐Species

Presenter: Renan E. A. Piraine University of São Paulo, Brazil Introduction : Extracellular vesicles (EVs) play crucial roles in fungal communication and host immune modulation, representing potential therapeutic targets for fungal infections. This study investigates the role of EVs in intra‐ and interspecies interactions among pathogenic fungi from the Candida and Cryptococcus genera. Methods : We performed co‐incubation experiments with EVs from Candida albicans and Candida auris in planktonic cells and biofilms of C. albicans , as well as Cryptococcus neoformans and Cryptococcus gattii EVs in planktonic cells and biofilms of C. neoformans . Scanning electron microscopy confirmed the association of EVs with the cell surface of recipient cells, and further validation was achieved by labelling EVs and incubating them with fungal cells and human macrophages. The biological effects of EVs on intraspecies and interspecies interaction were assessed, including assays for biofilm adhesion, dispersion, and antifungal drug tolerance. Immunomodulatory effects on macrophages were analysed by assessing cell viability, gene expression, and cytokine production. Results : EVs were observed associating with recipient cell surfaces, suggesting subsequent internalization. Functional assays revealed that EV exposure led to increased expression of Cap59, Lac1, Ure1, and Erg11 genes, correlating with reduced antifungal susceptibility in both planktonic and biofilm forms. Additionally, EVs facilitate cross‐species communication, which enhances biofilm adhesion and dispersion, highlighting their role in fungal phenotypic modulation. Macrophages stimulated with fungal EVs exhibited receptor‐specific gene expression changes, notably the upregulation of galectin‐3, along with a pro‐inflammatory phenotype characterized by increased expression of iNOS and elevated cytokine levels (IL‐1β, IL‐6, and IL‐8). Summary/Conclusion : Collectively, these findings underscore the critical role of fungal EVs in interspecies communication, biofilm regulation, and immune modulation, offering valuable insights into fungal pathogenicity mechanisms. Funding : This project is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant number 2023/05800‐7.

Dual‐Function

Sin‐Yu Chen 1# , Hsin‐Tung Chen 1 , Tai‐Shan Cheng 1 , Guan‐Wan Liu 1 , Ly James Lee 1 , Chi‐Ling Chiang 1 *, Dau‐Ming Niu 2 *, Chi‐Ying F. Huang 1,2 * 1 Institute of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan; 2 Institute of Clinical Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan Abstract unavailable

Electrochemical

Presenter: Maria Carmen Blanco‐López University of Oviedo, Oviedo, Spain Introduction : Extracellular vesicles (EVs) are a source of diagnostic, prognostic, and prognostic biomarkers for several diseases. However, the methods for their isolation and subsequent characterization have not yet been standardized for clinical uses [1]. Moreover, sensitive quantification of EV and EV biomarkers is still a technological challenge. The actual techniques do not allow a full characterization due to the heterogeneity of the samples and complexity of the biological matrix. Additionally, most of these techniques require bulky and expensive instruments, and skilled operators. There is therefore an increasing need for simple methods and devices for quantification and characterization of EV biomarkers with good sensitivity and user‐friendly. Methods : We have developed a biosensor based on the coupling of a lateral Flow immunoassay (LFIA) for EV biomarkers. By using anti‐CD9 as a capture antibody and anti‐CD63 as detection, and using only 1 mL of isolated EVs, a limit of detection of 3.19 × 10 4 EV/µL by recording the fluorescent signal of Eu NPs. On the other hand, the LFIA was coupled to screen‐printed electrodes with two methods: one of them was based on silver enhancement over gold nanoparticles, and the other one by using gold‐starch nanocomposites doped with an electrochemical probe [2]. Results : The limits of detection achieved were 26 and 39 EV/µL. These biosensors were used directly in the plasma of colorectal cancer patients for quantification of EV biomarkers. Summary/Conclusion : Up to the authors knowledge, these are the lowest limits of detection for EV quantification and EV biomarkers characterization with these types of devices. Funding : This work was funded by the Ministerio de Ciencia e Innovación (PID2020‐119087RB‐I00). Clara Saweres‐Argüelles acknowledges her grant FPU22/00762 from the Ministry of Science, Innovation and Universities.

Exozirkel—The

Presenter: Giorgia Campione German Cancer Research Center (DKFZ), Heidelberg, Germany Introduction : In the field of oncological diagnostics, there are increasing efforts to reduce invasive tissue sampling and replace it with ‘liquid biopsy’ (LB) procedures. In the currently established LB procedures, nucleic acid‐based techniques are primarily used. Yet, the predictive power of these methods is limited. To fully exploit the potential of LB, protein‐based methods need to be established. Unlike free plasma proteins, EVs are specific to their (tumour) cells of origin and therefore are of central importance for protein‐based LB. However, EVs vary greatly intra‐ and interindividually, which currently limits their diagnostic utility. In cell culture models, diurnal dynamics have been described as one possible reason for this variability. The aim of this project is to investigate the extent to which the protein loading, plasma concentration, and characteristics of EVs in peripheral blood follow circadian rhythm in healthy individuals and patients with systemic tumour diseases. Methods : We recruited 15 patients from University Medical Centre Mannheim (UMM) and 12 healthy volunteers. EVs were isolated from peripheral blood samples taken every 3 h over 24 h using ‘top‐down’ Iodixanol DGs followed by SEC. The particle number, concentration and protein load were measured, and the EV‐derived proteome was subjected to mass spectrometric analysis. Concurrently, various parameters were determined from saliva samples to validate interindividual circadian fluctuations and control non‐circadian confounding factors. Our clinical study was approved by the ethics committee of UMM (2022‐46). Written informed consent was obtained. Statistical analysis is performed using ANOVA or multiple contrast testing. Results : In healthy volunteers, EV particle concentration and protein loading of EVs remained constant during the day, with a trend towards less interindividual variance in the morning. Circadian proteomic signatures were assessed in healthy individuals and cancer patients. Summary/Conclusion : The aim of the study is to determine whether the circadian rhythm of EVs indicates particularly suitable times of day for liquid biopsy, thereby improving the clinical application of this diagnostic method. By understanding which specific EV components fluctuate throughout the day, numerous scientifically interesting approaches arise, which are of significant interest for the entire field of EV research.

Internalization

Presenter: Shinya Nakai Osaka Metropolitan University, Osaka, Japan Introduction : Exosomes are extracellular vesicles (EVs, approximately 30‐200 nm in diameter) characterized by containing physiologically active molecules and contributing greatly to intercellular communications. In addition, EVs are known to have high advantages from pharmaceutical perspectives and are expected to be a next‐generation drug delivery tool. In this study, we are evaluating the transfer of cell‐penetrating peptides (CPPs) into multivesicular endosomes (MVEs), which are the production sites for EVs, during their cellular uptake, and the effects on EVs secretion. Methods : All peptides were synthesized by Fmoc‐solid phase methods. Cellular uptake of fluorescently‐labelled peptides was analysed using a confocal laser microscope. Isolation of secreted EVs was conducted by ultracentrifugation methods. In this research, CD63‐GFP‐stably expressing cells were used for assessments of intracellular locations of internalized fluorescently‐labelled peptides. Results : Using a confocal laser microscope, we observed cellular uptake of fluorescently‐labelled octaarginine (R8‐Alexa568), a typical CPP, into HeLa cells expressing the EVs marker protein CD63‐GFP. By pretreatment with the hydrophobic anion pyrenebutyrate (PyB), highly efficient cell membrane penetration of the R8‐Alexa568 and promotion of diffusion into cytosol were observed. In addition, colocalization of the CD63‐GFP and R8‐Alexa568 was observed over time, suggesting accumulation of the peptides to the MVEs. After cellular uptake of the R8‐Alexa568 into HeLa cells, EVs secreted from the cells were isolated using ultracentrifugation. As a result of evaluating the encapsulation of isolated EVs using the quenching effect of trypan blue (TB), the R8‐Alexa568 was judged to be encapsulated within EVs. Cryo‐TEM observation showed multivesicular formation in the isolated EVs, suggesting possible induction of membrane fusion in MVEs that affect encapsulation of delivered CPPs. Furthermore, the isolated EVs were taken up by other cells; it became clear that the R8‐Alexa568‐encapsulating EVs were also taken up into other cells after their secretion. We are still studying the effects of the number of arginine residues in the peptide sequences that affect cellular uptake pathways (e.g., endocytosis and cytosolic release) and membrane curvature formation on encapsulation in EVs. Summary/Conclusion : These results not only provide a basic technology for artificial inclusion in EVs using the CPPs but also provide basic knowledge regarding the effects of the CPPs on intercellular communication via EVs.

Intra‐Nephron

Janina Kern, Sindhu Thiagarajan, Nina Sopel, Janina Müller‐Deile Uniklinikum Erlangen, Erlangen, Germany Introduction : Insufficient cell‐cell communication of glomerular endothelial cells (GECs) and podocytes leads to a dysfunctional glomerular filtration barrier, and not much is known about the intra‐glomerular as well as the glomerular to tubular signalling. Exosomes are 30 to 160 nm small extracellular vesicles released by multivesicular bodies from various cell types. These transport vehicles encapsulate signalling molecules such as mRNAs, microRNAs or proteins. Exosomes secreted from cells facing the urinary space might be taken up by tubular cells. However, it is unclear if exosomes can serve as mediators of GEC to podocyte crosstalk in vivo, as it is unknown if they are able to pass the glomerular basement membrane (GBM). Methods : RNA‐seq analysis and proteomics were used to characterize exosome cargo of glomerular cells. A CD63 (surface protein of exosomes) plasmid tagged with mScarlet and pHluorin (labelling intracellular exosomes in red and secreted exosomes in green) was used to investigate exosome secretion in glomerular cell culture models. By injecting zebrafish eggs in the one‐cell stadium with a pHluorin CD63 reporter plasmid, including one with a podocin promoter for podocyte‐specific exosome labelling exosomes could be visualized in zebrafish larvae with a multiphoton microscope in high resolution. Moreover, in vitro labelled exosomes were injected into the zebrafish circulation and visualized with immunofluorescence and electron microscopy to examine their ability to pass the GBM. Results : In vitro podocytes and GECs secreted exosomes that could be live‐tracked with the help of a spinning disc microscope. Live tracking analyses in 2D and 3D glomerular co‐culture detected exosome‐transfer from one cell type to another. Incubation of GEC‐derived exosomes changed expression in podocytes, and likewise podocyte‐derived exosomes had effects on tubular cell expression. Injecting fluorescently labelled exosomes into the zebrafish circulation allowed to study the permeability of exosomes for the GBM. Using CD63 plasmids encoding podocyte‐specific labelled exosomes helped to investigate cell‐cell communication via exosomes originating from podocytes. Summary/Conclusion : Live tracking experiments of glomerular cell‐type‐derived exosomes will help to get more insights into inter‐glomerular and glomerular‐to‐tubular cell–cell communication. Funding: DFG, TRR‐374.

Lab‐On‐Chip

Presenter: Alessia Foscarini CNR NANOTEC—Institute of Nanotechnology, Bologna, Italy Introduction : Liquid biopsy and the use of selected biomarkers from biological fluids, greatly enhance an increasingly patient‐centred approach, avoiding invasive tests and tissue biopsies. Extracellular Vesicles (EVs) act as a snapshot of the cells from which they originate and as a repository of crucial information, facilitating the direct extracellular transfer of proteins, lipids, and miRNAs/mRNAs/DNAs. Despite EVs showing great potential as powerful biomarkers, their isolation and characterization remain challenging. Lab‐on‐Chip (LoC) technologies represent innovative tools to overcome the limits of standard methods, and we aim to implement these technologies for EV investigation. Methods : We design customizable LoC devices based on the experimental needs, starting from fluid dynamic simulations and using microfabrication techniques (micro‐milling and 3D printing). We develop two different LoCs exploiting the microfluidic approach and electrochemical detection for EV enrichment and characterization. Large and small EVs were isolated by ultracentrifugation and characterized by high‐resolution flow cytometry, western blot, and transmission electron microscopy. Results : We developed an in‐flow device using moulded‐plastic substrates assembled on a glass slide to create a microfluidic chamber for dynamic cell culture. Fluorescent neuroblastoma cell lines (SH‐SY5Y) were seeded into the device to allow a complete replacement of the medium in the dynamic condition within 24 h at a controlled flow rate. We observed increased EV production and decreased EV size in dynamic versus static conditions. Moreover, we realized a biosensing platform for EVs electrochemical characterization. Functionalized microelectrodes were used for differential pulse voltammetry measurements to build a calibration plot considering different EVs membrane proteins. With this method we were able to demonstrate (i) the possibility to detect very low concentrations of EVs and (ii) the retention of sample integrity. Summary/Conclusion : The key goals of the LoC technologies are to eliminate high‐impact procedures, reduce time and cost, and preserve EV morphology. Our systems allow us to study the release of EVs under dynamic cell culture conditions that mimic the physiological scenario at the cell surface, and to identify arrays of biomarkers associated with EV subclasses. The next step is to integrate microfluidic sorting and electrochemical characterization into a benchtop device that can be customized to meet clinical needs.

M2‐Macrophage

Melis Isik, Burak Derkus Department of Chemistry, Faculty of Science, Ankara University, 06560 Ankara, Turkiye Introduction : The crosstalk between cancer and immune cells adds a layer of complexity to our understanding of metastasis formation. This study explores the intricate dynamics between immune cells and colon cancer cells (HT‐29) during the post‐tumourigenic phase. The hypothesis is based on the premise that M2 macrophages, known to promote cancer metastasis, exert their effects through extracellular vesicles (EVs). To test this hypothesis, EVs were obtained from M0, M1, and M2 macrophages (M0‐EVs, M1‐EVs, and M2‐EVs), and their effects on colon cancer metastasis were studied in vitro. Methods : THP‐1 cells were polarized into M1 and M2 phenotypes, induced by GM‐CSF and M‐CSF, respectively. The success of polarization was examined for CD80 (M1) and CD206 (M2) markers using flow cytometry (FC) and immunoblotting. EVs were isolated from M0, M1, and M2 macrophages through serial ultracentrifugation. Briefly, a gradual centrifugation was applied for cell culture conditioned media (CCM, 400 mL), and CCM was ultracentrifuged (Himac CP100WX) for 90 min at 120,000 ×  g to obtain an EV pellet. The obtained EVs were characterized by immunoblotting for CD63 and CD81, nanoparticle tracking analysis, and transmission electron microscopy. To test our hypothesis, we induced HT‐29 cells by M0‐EVs, M1‐EVs, and M2‐EVs (10, 50, and 100 µg) to investigate their role in cancer metastasis. For this purpose, we performed transcriptome analysis in addition to microscopic and immunophenotypic evaluations. Results : The findings revealed a significant decrease in epithelial gene/protein expression (Ecad) and an increase in mesenchymal gene/protein expression (NCad) in cells exposed to M2‐EVs, indicating that M2‐EVs facilitate epithelial‐mesenchymal transition (EMT). Comprehensive transcriptomic analyses of M2‐EVs‐treated HT29 cells identified a subset of genes, including FAM83A, which is upregulated upon M2‐EVs induction, but not in the case of co‐culture with M2 macrophages, indicating that EVs play a direct role in EMT. Strikingly, targeted silencing of FAM83A via siRNA abrogated M2‐EVs‐mediated FAM83A overexpression and inhibited EMT activation, highlighting its potential as a therapeutic target. Summary/Conclusion : The findings in this work elucidate the immunological mechanisms underpinning cancer metastasis through EV‐mediated interactions. Identifying immune cell functions and key molecular targets may advance the development of immunotherapies aimed at inhibiting metastasis. Funding : TUBITAK, Grant No: 123S856.

Multiparametric

Ghazal Narimanfar 1 , Filippo Maltoni 1 , Dorian Forte 1 , Roberto Maria Pellegrino 2 , Simone Tiberi 1 , Martina Barone 3 , Filippo Branzanti 3 , Hillary Catellani 4 , Alessia Tieghi 4 , Carla Emiliani 2 , Francesca Palandri 3 , Lucia Catani 1,3 1 University of Bologna, Bologna, Italy; 2 University of Perugia, Perugia, Italy; 3 IRCCS Azienda Ospedaliero‐Universitaria di Bologna, Bologna, Italy, 4 Azienda Unitа Sanitaria Locale‐IRCCS di Reggio Emilia, Reggio Emilia, Italy Introduction : The inflammatory microenvironment supports the malignant clone in myeloproliferative neoplasms (MPNs) like polycythemia vera (PV) and essential thrombocythemia (ET), making it critical to explore therapeutic strategies. Extracellular vesicles (EVs), carrying proteins, lipids, and nucleic acids, play a role in intercellular signals and also in the regulation of immunity and inflammation. This study aims to provide an integrated and sensitive liquid biopsy platform of circulating EVs, offering new insights into MPN pathology and potential therapeutic targets. Methods : We combined lipidomic (liquid chromatography/mass spectrometry), transcriptomic, and surface protein profiling (flow cytometry) to analyse EVs isolated from plasma of PV ( n = 10) and ET ( n = 10) patients and sex/age‐matched healthy donors (HD = 10) by size‐exclusion chromatography. Results : Comparing the mRNA cargo of PV and HD EVs, we found the overexpression of haemostasis/coagulation and platelet activation (GPIBB, ITGA2B, GP1BA, CD226), inflammation (LCN2) and tumour‐associated (MYL9, MYBL2, CMTM5, TSC22D1, LCN2) transcripts. We also report how the transcriptome of circulating EVs captures PV‐specific features: NF‐E2, GFI1B, TIMP1, all overexpressed, play a role in erythrocytosis, development and maturation of erythrocytes and megakaryocytes, and bone marrow fibrosis, respectively. When we compared the mRNA cargo of ET and HD EVs, we found that most of the transcripts mirrored the PV counterparts, and no significant differences were observed between the transcriptomes of ET and PV EVs. Consistently, surface protein profiling revealed significant enrichment of immune/inflammatory (CD8, CD29), EV (CD9, CD63, CD81), and platelet‐associated markers (CD41a, CD42b, CD62P) in PV EVs versus those from HD. Elevated expression of CD9, CD29, and CD63 was also observed in ET‐EVs. Lipid class analysis revealed that PV EVs were significantly enriched in ether phospholipids (plasmenyl‐linked phosphatidylcholine (PC)‐P and ether‐linked PC‐O) in comparison with the HD counterparts. PC‐P typically signifies a protective or adaptive response to oxidative stress, inflammation, or metabolic demands. Conversely, no significant differences were observed between ET and HD EVs in lipid‐class cargo. Summary/Conclusion : Our work demonstrates that circulating EVs in MPN patients are enriched for tumour‐ and biology‐related molecules with the potential to contribute to the inflammatory microenvironment and proves the feasibility of comprehensive circulating EV profiling to study MPN.

Neuroprotective

Presenter: Dawson Hollingsworth UNMC, USA Introduction : Parkinson's disease (PD) is the second most common multi‐system neurodegenerative disorder, linked to immune dysfunction that impacts the clinical symptoms. Current PD treatments for motor and non‐motor symptoms are palliative, with a failure to affect the cause of nigrostriatal degeneration. In past decades, our labouratories have focused on innate and adaptive immune pathways that control neuroinflammation and provide neuroprotection in animal disease models and the clinic. Recently, bovine colostrum‐derived EVs (CEVs) have garnered interest as immune modulators, with high safety profiles and yields, and have been shown to traffic to the brain. Considering their favourable biodistribution pattern, safety, scalability, and anti‐inflammatory properties, we evaluated their potential as neuroprotective agents in the methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of PD. Methods : CEVs were isolated by differential ultracentrifugation from bovine colostrum, collected 24 h after birth. They were characterized for size, distribution, concentration, and morphology by biochemical and molecular tests and by positron emission tomography for tissue distribution. Parallel studies evaluated the expression of neuroinflammatory and neurodegeneration biomarkers after intravenous injection of the CEVs into the MPTP‐intoxicated mice. Results : CEVs had a size range of 50‐150 nm, were positive for exosome markers, and had a cup‐shaped double membrane morphology when imaged by transmission electron microscopy. RNA sequencing and transcriptomic analysis from the substantia nigra and striatum of MPTP‐intoxicated mice treated with CEVs demonstrated changes in biomarkers associated with reductions in immunocyte recruitment and activation, controlled microglial activation, and reduced pro‐inflammatory gene expression. This was in addition to diminished canonical inflammasome activation, and cytokine and chemokine responses in CEV‐treated MPTP mice. These changes corresponded with decreased expression of upstream regulatory molecules linked to inflammation. Concomitantly, the regulatory T‐cells increased in the blood and spleen of CEV‐administered MPTP mice. Anti‐inflammatory responses led to neuroprotection, as evidenced by a significant increase in the survival of tyrosine hydroxylase nigral dopaminergic neuronees following CEV treatment in the MPTP mice. Summary/Conclusion : CEVs control neuroinflammatory activities and demonstrate dopaminergic neuronal protection in a PD model. CEVs could be developed as part of a comprehensive therapeutic strategy, offering a unique disease‐modifying PD treatment. Funding : This research was supported by the University of Nebraska Foundation.

Non‐Canonical

Diego Baranda Martínez Abascal 1,2* , Thomas Glynn 1* , Julieta Sánchez 5,4,2,7 , Marc Moltó‐Abad 1,2 , Giovanni Lerussi 1 , Verónica, Villagrasa 1,2 , José Luis Corchero 2,5,4 , María Fidel‐Lledó 1 , Roser Ferrer 1,3 , Simó Schwartz 1,2,3 , Ibane Abasolo 1,2,3,6 , Joaquin Seras Franzoso 1,2,4 1 Vall d'Hebron Research Institute (VHIR), Spain; 2 Centro de Investigación Biomédica en Red, Bioingeniería; 3 Biomateriales y Nanomedicina (CIBER‐BBN), Spain; 4 Universitat Autònoma de Barcelona (UAB), Spain; 5 Institut de Biotecnologia i de Biomedicina (IBB), Spain; 6 Instituto de Química Avanzada de Cataluña (IQAC), Spain; 7 Instituto de Investigaciones Biológicas y Tecnológicas, Argentina *Diego Baranda Martínez Abascal and Thomas Glynn equally contributed to this work . Introduction : Interest in protein therapeutics is driven by their broader functional range and better biocompatibility than small drugs. Recombinant proteins are particularly suited for restoring missing biological activities via replacement therapies. However, they usually exhibit a short half‐life, may be difficult to produce and often display a poor ability to cross biological barriers. In this context, evolutionarily conserved delivery systems such as extracellular vesicles (EVs) have been shown to naturally surpass these barriers to deliver proteins and other biomolecules with unexpected efficiency. Here, we have recombinantly produced three structurally and functionally distinct proteins with potential for replacement therapies in lysosomal storage disorders (LSDs): alpha‐galactosidase, cathepsin K and NPC intracellular cholesterol transporter 1. We have loaded them in vivo into EVs and characterized the protein intermolecular interactions and the subsequent supramolecular protein architecture to unravel structural cues supporting EV's outstanding performance. Methods : EVs were isolated by a combination of TFF and SEC procedures from transiently transfected high‐density HEK293F cell suspension cultures. EV characterization included NTA, TEM and WB. Protein conformation was assessed by FTIR and TEM. The biological activity of the protein was determined in vitro via specific enzymatic and/or cell‐based assays. Results : The protein of interest was found in EVs in all cases after transient overexpression, and it was able to retain its biological activity. Looking at the protein nanoarchitecture within the EVs, significant amounts of intermolecular antiparallel beta sheets were detected. Additionally, TEM direct observation of proteinase K (PK) digested EVs revealed a fibrillar core of PK‐resistant protein. Such findings were complemented with a remarkably higher stability of the EV‐loaded protein in comparison to its soluble counterpart. Note that only EV‐loaded protein exhibited measurable amounts of enzymatic activity after 14 days of incubation at RT and a 5‐fold higher cumulative signal, even after EV membrane disruption by sonication. Summary/Conclusion : Such protein supramolecular organization, characteristic of amyloid structures, may endow the EV‐loaded protein with higher mechanical and enzymatic stability, providing also a sustained and slow‐release profile capable of outperforming conventional recombinant proteins. Funding : Instituto de Salud Carlos III and co‐funded by the European Union: DipEV (PI22/1301), NEX‐TRY (PI21/0936); European Commission: Mimic‐KEY ( GA964386 ).

Pharmacokinetic

Presenter: Hyun‐Suk Jung Yonsei University College of Medicine, Seoul, Republic of Korea Introduction : Exosomes are small vesicles released by all human cells, playing a crucial role in intercellular signalling and containing cellular information. Given their potential in disease diagnosis and therapy, there is increasing interest in utilizing exosomes for therapeutic purposes. However, concerns remain about their practical use, particularly because exosome concentrations in in vitro experiments are much higher than those found in vivo. This discrepancy has led to scepticism about whether the efficacy observed in vitro can be replicated in human bodies. In this study, we perform a pharmacokinetic analysis to examine the distribution of intravenously administered exosomes and assess their potential for therapeutic applications in realistic in vivo conditions. Methods : We calculated the distribution and dilution of intravenously administered exosomes across various organs. Based on these calculations, we estimated the intravenous dosage required for exosomes to exhibit efficacy in different organs, such as the liver, spleen, lungs, and kidneys. Results : Modelling calculations indicated that exosomes administered intravenously were predominantly distributed to the liver, followed by the spleen, lungs, and kidneys, which is consistent with observed trends in in vivo experiments with mice. To deliver the same exosome concentration to liver cancer cells as in vitro, approximately 10 15 to 10 17 exosomes would need to be administered. This is significantly higher than the 10 12 exosomes typically present in the bloodstream. Summary/Conclusion : Our study presents a pharmacokinetic model that helps understand the distribution of exosomes following intravenous administration. The results show that significantly higher exosome doses (10 15 to 10 17 ) are required in vivo to achieve the same efficacy observed in vitro. This highlights the need for exosomes with improved tropism and modified cargo to enhance therapeutic potential. Funding : This work was supported by the National Research Foundation of Korea (NRF) and the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea government (Ministry of Science and ICT) (RS‐2024‐00432946), the NRF grant funded by the Korea government (MSIT) (No. 2021R1A2C3011254), and the Technology Development Program funded by the Ministry of SMEs and Startup s(MSS, Korea) (RS‐2024‐00506850).

Pharmacological

Xingzhi Cheng 1 , Jonathan Stephens 1 , Wolfgang Kuebler 2 , Masao Takata 1 , Sanooj Soni 1 1 Imperial College London, London, UK; 2 Charité—Universitätsmedizin Berlin, Berlin, Germany Introduction : Leukocyte extracellular vesicles (EV) are vital mediators of intercellular communication within the alveolar space and have been implicated in the pathophysiology of acute lung injury (ALI). Previous work has demonstrated the importance of acid sphingomyelinase (ASM) in the formation of EVs and packageing of their molecular cargo. We hypothesise that inhibition of ASM will diminish pro‐inflammatory EV production, thereby ameliorating ALI. Methods : C57BL/6J mice received intratracheal instillation of lipopolysaccharide (LPS) to induce ALI. Imipramine, a functional inhibitor of ASM activity (FIASMA), was intratracheally administered into some mice 24 h prior to the LPS challenge. Bronchoalveolar lavage fluid (BALF) was collected for analyses of leukocyte EVs and albumin levels, and the lungs were harvested to measure leukocyte infiltration and surface ASM expression in single‐cell suspension by flow cytometry. EVs were defined and analysed by flow cytometry using their size and surface markers, with CD45+CD11c+ events as alveolar macrophage (AM) EVs and CD11b+Ly6G+ events as neutrophil (N) EVs. Results : LPS stimulation at 1 h caused increases in BALF AM‐EVs, accompanied by upregulation of ASM expression on AMs, and both were significantly reduced by pre‐treatment with imipramine (EVs: 1563 ± 318 vs. 1164 ± 201 EVs/µL, n = 8, p < 0.01. ASM: 13270 ± 3025 vs. 9935 ± 1502 MFI, n = 6, p < 0.05). LPS stimulation at 4 h caused substantive increases in N‐EV release as well as upregulated ASM expression on neutrophils, which were massively ablated with imipramine (EVs: 1639 ± 445 vs. 977 ± 379 EVs/µL, n = 7, p < 0.05. ASM: 1747 ± 431 vs. 1163 ± 417 MFI, n = 5, p < 0.05). Furthermore, imipramine attenuated the increase in BALF albumin levels post LPS stimulation (2.16 ± 0.374 vs. 1.67 ± 0.290‐fold change, n = 6, p < 0.05), suggesting ASM inhibition may have a protective effect against pulmonary permeability and oedema. Summary/Conclusion : Our study demonstrates that ASM is crucial in leukocyte EV formation in vivo, and inhibition of ASM activity reduces consequent EV release and offers protection against LPS‐induced alveolar oedema and inflammation. These data provide valuable insights into the therapeutic potential of repurposing FIASMAs for prevention and treatment of ALI. Funding : Friends of Hammersmith Hospital Charity.

Physicochemical

Presenter: Naomie Zhou University Medical Centre Utrecht, Utrecht, The Netherlands Introduction : Cells secrete extracellular vesicles (EVs) into their environment, where they participate in various physiological and pathological processes. For research purposes and therapeutic applications, EVs need to be separated from contaminants present in complex media or biofluids. However, in pursuit of effective EV isolation methods, balancing EV purity, yield, and integrity remains challenging. This study evaluates anion exchange chromatography (AEX) against routine EV enrichment methods—tangential flow filtration (TFF) and size exclusion chromatography (SEC). By comparing particle characteristics and uptake efficiency, we highlight how the isolation method impacts downstream applications. Methods : Conditioned medium from MDA‐MB‐231 cells expressing HiBiT‐tagged CD63 was concentrated using TFF and ultrafiltration. Following storage (TFF) or further purification (AEX and SEC), EV‐containing fractions were buffer‐exchanged and concentrated by ultrafiltration. Particle size and yield were assessed via nanoparticle tracking analysis, and protein quantity and profiles were analysed by micro BCA and western blot. After EV treatment, HiBiT‐tagged EV uptake was measured via luminescence in recipient HeLa cells after removing bound EVs. Results : EVs eluted from AEX in two distinct peaks, named AEX1 and AEX2. AEX1 contained the smallest particles (D10 = 76.4 to D90 = 163.8 nm, modal size at 89.6 nm), followed by SEC (84–160.7 nm, modal size = 104.7 nm), TFF (85.5–181.7 nm, modal size = 103.5 nm) and AEX2 (81.5–170 nm, modal size = 116.8 nm). Combined AEX1 and AEX2 particle yields, with most EVs present in AEX1, were similar to yields obtained by TFF. SEC caused significant particle loss, reducing yield by 46% compared to TFF. AEX1‐ and TFF‐EVs had higher protein contamination, whereas SEC provided the lowest protein‐to‐particle ratio. Interestingly, AEX1‐ and TFF‐EVs were internalized significantly more efficiently than SEC‐ and AEX2‐EVs. Summary/Conclusion : TFF delivers the highest yield but with notable protein contamination. SEC achieves the best protein‐to‐particle ratio, although with reduced yields. AEX offers a middle ground; AEX1‐EVs show increased uptake alongside higher protein contamination and yield, while AEX2‐EVs demonstrate an improved protein‐to‐particle ratio but reduced uptake and yield. Results indicate a positive correlation between the presence of protein in EV preparations and EV uptake. In future studies, we will explore how differences in EV purity and uptake efficiency impact their functional effects.

Physiologically

Presenter: Julia K. Monola University of Helsinki, Helsinki, Finland Introduction : Conventional 2D cell culture in 5% CO 2 with uncontrolled oxygen does not necessarily represent physiological conditions, leading to production of less in vivo‐like extracellular vesicles (EVs). Stem cell‐derived EVs can enhance the regenerative capacity of damaged tissues, but reproducible production of therapeutic in vivo‐like EVs remains challenging. This study aims to find an optimised protocol to produce therapeutic EVs from mesenchymal stromal cells (MSCs) for wound‐healing applications. Methods : Human mesenchymal adipose stem cells are cultured under normoxic (∼21% O 2 ) and physioxic (5% O 2 ) conditions in two 3D culture systems: ultra‐low attachment (ULA) conditions and NFC hydrogel (NFCh). The EVs were isolated by ultracentrifugation and size‐exclusion chromatography. The protein phenotype will be evaluated by western blotting and immunocytochemistry. The EV yield was determined by NTA and other EV characteristics will be evaluated by proteomics and qPCR. The effects of EVs with NFC hydrogel on wound healing were studied using an in vitro scratch wound model to model the rates of cell migration and proliferation. Results : Cells produced EVs for 7 days after which, the experiment was terminated. The metabolic activity of the cells was higher in ULA compared to NFCh and in physioxia compared to normoxia. The EV yield was greater in ULA plates than NFCh and in physioxia compared to normoxia. Cells cultured under all conditions exhibited typical mesenchymal stem cell markers (CD73, CD90, CD105) but differences in the amount of expression were observed that were favouring ULA over NFCh and 2D and physioxia over normoxia. The physioxic EVs accelerated relative wound closure compared to normoxic EVs and control (no EV treatment). Summary/Conclusion : Oxygen levels and culture conformation influences EV yield and phenotype. Additionally, EVs produced under lower oxygen levels seem to accelerate wound healing. Next, the impact of 3D culture and controlled release of EVs from NFC hydrogel on wound‐healing will be studied, along with a detailed analysis of EV characteristics and effects on skin cells during scratch wound healing.

Plant‐Derived

Roberta Gasparro 1 , Christian Sanchez‐ López 2 , Vincenza Tinnirello 1 , Giulia Duca 1 , Mari Cruz Manzaneque‐López 2 , Riccardo Alessandro 1 , Antonio Marcilla 2 , Stefania Raimondo 1 1 Department of Biomedicine, Neurosciences and Advanced Diagnostics (Bi.N.D), Section of Biology, University of Palermo, Palermo, Italy. 2 Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot, Valencia, Spain. Introduction : Plant‐derived nanovesicles (PDNVs) have recently garnered significant interest within the scientific community due to their functional properties. They carry lipids, proteins, nucleic acids, and metabolites, playing roles in cross‐kingdom communication and influencing mammalian cells. PDNVs, extensively studied by our research group, show promising potential with demonstrated anti‐inflammatory, antioxidant, and anticancer effects. Additionally, various plant's compounds, such as Citral and polysaccharides, are known to activate immune cells, potentially enhancing bacterial recognition. In this study, we aim to explore the ability of PDNVs to enhance the action of macrophages against bacteria. Methods : Lemon (LNVs), Tangerine (TNVs) and Pomegranate (PgNVs)‐derived nanovesicles were isolated from the juice through Tangential flow filtration and Size Exclusion Chromatography and characterized by Transmission Electron Microscopy and Nanoparticle Tracking Analysis. We established a protocol for evaluating the effect of human monocytes differentiated into macrophages (THP‐1 M0) against Enterococcus faecalis or Escherichia coli. The THP‐1 M0 cells were first pre‐treated for 24 h with LNVs/TNVs/PgNVs and then grown for 3 h in the presence of bacteria. THP‐1 M0 cells and bacteria were co‐plated in LB Agar and colony‐forming units (CFU) were counted. Results : We characterized LNVs, TNVs and PgNVs, confirming their integrity, size distribution, and positivity for HSP70 and TET8 markers. After evaluating their internalization in THP‐1 M0 cells, we demonstrated that these nanovesicles enhance macrophage activity, essential for pathogen elimination. We observed that the pre‐treatment with nanovesicles influences bacterial growth. In particular, THP‐1 M0 cells pre‐treated with LNVs/TNVs significantly reduced the bacteria's CFU compared to untreated cells. We validated the results related to Escherichia coli also on monocytes isolated from blood samples of healthy donors and differentiated into macrophages. Summary/Conclusion : These results represent an interesting starting point for understanding how LNVs, TNVs and PgNVs can modulate the activity of macrophages and improve their response against bacteria. Funding : This work supported by Eurostart and FFR 2024 (University of Palermo, Italy) to Stefania Raimondo, by Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Spain and Conselleria d'Educació, Cultura, Universitats i Ocupació, Generalitat Valenciana, Valencia, Spain. R.G. is the recipient of Post‐doc fellowship funded by Fondazione Umberto Veronesi, C.M.S‐L of Post‐doc fellowship funded by AEI/10.13039/501100011033.

Platelet‐Free

Jillian W. P. Bracht 1 , Mendel Engelaer 1,2 , Adam Ceroi 3 , Marco Guadagnoli 3 , Edwin van der Pol 1,2 , Rienk Nieuwland 1 1 Department of Clinical Chemistry, Amsterdam Vesicle Center, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands; 2 Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands; 3 Miltenyi Biotec B.V., Leiden, The Netherlands Introduction : Human blood plasma contains clinically relevant information that can be used to develop disease biomarkers. Despite intensive research, >99.9% of initially promising biomarkers never reach the clinic. A possible explanation is the inconsistent composition of plasma samples stored in biobanks. Although believed to be “cell‐free”, human plasma still contains 1E5‐1E8 erythrocyte ghosts (ery‐ghosts) and platelets per mL, even after two centrifugation steps. Moreover, the concentration of remaining cells varies, and depends on both the operator and centrifugation protocol (> 200 existing protocols). Although for some analytes the presence of remaining cells will not matter, for others, such as extracellular vesicle (EV) concentration and EV‐miRNA analyses, remaining cells disturb measurement and interpretation of results (Bracht et al. J Extracell Vesicles 2023). Since the concentration of remaining cells is too low for visual confirmation and because downstream effects on other analytes (DNA, proteins) are unknown, this is an unrecognized and possibly underestimated problem. Methods : We developed a procedure to specifically and simultaneously sort and concentrate ery‐ghosts and platelets from double centrifuged human plasma (MACSQuant Tyto, Miltenyi). Cell concentrations pre‐ and post‐sorting were measured by flow cytometry (Northern Lights, Cytek). The presence of ery‐ghosts and platelets was confirmed by super‐resolution microscopy (dSTORM, ONi), using specific capture‐ and fluorescent detection antibodies. We also developed a filtration method to remove remaining cells and cell ghosts from plasma. Results : Using a novel sorting procedure, the concentration of ery‐ghosts and platelets increased 78‐fold and 37‐fold post‐sorting, respectively. We confirmed that the sorted particles were ery‐ghosts and platelets using dSTORM imaging. Ery‐ghosts were both membrane dye and CD235a positive, whereas platelets showed characteristic pseudopods and were positively labelled with a membrane dye and CD63. Our filtration method removed > 98% of the remaining cells from plasma, whereas the detectable concentration of EVs by flow cytometry remained unaffected. Summary/Conclusion : Taken together, we have developed methodologies to (1) remove remaining cells from plasma and (2) isolate remaining cells, allowing us to characterize and quantify their effects directly on downstream analyses. By standardizing the quality and composition of plasma samples, a more uniform playing field is being prepared for enhanced biomarker development. Funding : Marie Sklodowska‐Curie (No. 101067982).

Post‐Exercise

Ludovic Giloteaux, Maureen R. Hanson Cornell University, Ithaca, New York, USA Introduction : Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating condition characterized by fatigue, cognitive dysfunction, and other symptoms. A hallmark of ME/CFS is post‐exertional malaise (PEM), where exertion leads to prolonged symptom worsening. This highlights the impaired response to physical stress in ME/CFS. Recent studies have focused on the cargo of extracellular vesicles (EVs) in ME/CFS. Our particular interest is mitochondrial DNA (mtDNA) within these EVs. mtDNA can act as damage‐associated molecular patterns (DAMPs), triggering immune signalling and cellular stress. Plasma EVs may help clear out damageing mtDNA, modulating inflammatory responses that worsen ME/CFS symptoms. Investigating EVs and their mtDNA content offers insights into PEM mechanisms and potential biomarkers or therapeutic targets in ME/CFS. Methods : Twenty‐eight ME/CFS patients and 26 healthy controls completed a cardiopulmonary exercise test (CPET). Blood plasma was collected at baseline, 15 min, and 24 h post‐exercise. Plasma‐derived EVs were isolated using size exclusion chromatography and characterized per MISEV2023 guidelines. DNA was extracted from EVs, and mtDNA was quantified using quantitative real‐time PCR. Group differences and exercise‐induced changes were analysed using linear mixed models, while linear regression assessed associations between mtDNA levels and physiological parameters. Results : Isolated EVs were positive for CD9 and CD81, with sizes between 100 and 200 nm and typical morphology, showing no group differences. NTA analysis indicated no significant differences in particle numbers between ME/CFS patients and controls at baseline or post‐exercise. Within‐group analysis showed significant EV concentration increases from baseline to 15 min post‐CPET, followed by a decrease during recovery (15 min to 24 h post‐CPET). EV‐mtDNA levels changed dynamically in both groups, rising after CPET and declining during recovery. Notably, EV‐mtDNA levels were significantly correlated with ME/CFS symptoms. Summary/Conclusion : This study highlights significant post‐exercise changes in EV abundance and mtDNA levels, with differences between ME/CFS patients and controls. These findings enhance the understanding of EVs and their cargo's role in PEM, suggesting the potential of EV‐mtDNA as a biomarker for symptom exacerbation and providing a foundation for future diagnostic and therapeutic approaches for ME/CFS. Funding : This project was funded by NIH NINDS/OD/NIDA/NHLBI/NHGRI through NINDS U54NS105541 and NIH NIAID U54AI178855 to the Cornell Center for Enervating Neuroimmune Disease.

Proinflammatory

Fadi Haddadin 1 , Rejina Roufegarinejad 2 , Luigi Licanti 3 , Luciano, Licanto 1 , Floyd Sarsoza 4 and Charisse Winston 4 1 University of California, San Diego, La Jolla, CA, USA; 2 University of San Diego, San Diego, CA USA; 3 San Diego State University, San Diego, CA, USA; 4 University of Southern California, Alzheimer's Disease Therapeutic Research, San Diego, CA, USA Introduction : Saliva is becoming more recognized as a promising source for discovering biomarkers related to Alzheimer's disease (AD) because it can be collected non‐invasively and is readily accessible to diverse populations. Exciting preliminary data in blood demonstrated that brain‐derived neuronal and glial extracellular vesicles (EVs) can accurately predict the conversion of mild cognitive impairment (MCI) to AD; however, these studies have yet to be fully explored in saliva. Here, we aim to assess the feasibility of extracting EVs based on cellular origin from human saliva to predict cognitive decline amongst asymptomatic individuals at risk for AD. Methods : EVs were extracted, precipitated, and enriched against neuronal (L1CAM, NRXN1) and glial sources (TMEM119, GLAST, PLP1) using magnetic immunocapture from patient samples enrolled in the UC San Diego Nathan Shock Healthy Aging Study and the New Ideas Study. Three methods for EV precipitation (ExoQuick (EQ)‐TC, EQ + OASIS, and Norgen) were employed per the manufacturer's recommendations. Particle size distributions were quantified using nanoparticle tracking analysis. EV concentrations and tetraspanin profiling were quantified using super‐resolution microscopy (ONI). Quantification of proinflammatory‐related EV cargos was characterized by MSD bioassay. Results : EQ‐TC and EQ + OASIS methods successfully isolated high concentrations of saliva EVs, while the Norgen kit failed to isolate high concentrations of EVs from human saliva. Saliva EVs isolated using EQ‐TC generated subpopulations of EVs that were predominantly CD9+, while saliva EVs isolated using EQ + OASIS were predominantly CD63+ and CD9+. Concentrations of IL‐8, TNF‐α, and IL‐12p70 were significantly reduced in NRXN+ saliva EVs while IL‐6 was significantly reduced in NRXN+ and GLAST+ EVs. Summary/Conclusion : Extracellular vesicles can be effectively isolated from human saliva, although the concentrations of tetraspanins in these EVs differ depending on the isolation method used. The levels of proinflammatory‐related cargos from EVs precipitated using EQ‐TC also varied by cellular origin, showing significantly lower concentrations in NRXN+ EVs. The positivity for tetraspanins and the specificity to cell types may influence the biomarker potential of these EVs, warranting further research. Metabolomic profiling and biomarker analyses will be conducted to assess changes in EV cargo loads concerning tetraspanin and cellular origin in asymptomatic individuals at risk for AD. Funding : U19AG074879 4R00AG070390.

Serum‐Derived

Presenter: Ayako Muraoka Nagoya University Hospital, Nagoya, Japan Introduction : Endometriosis can cause infertility, and evaluation methods for predicting clinical pregnancy outcomes are desired. Extracellular vesicles (EVs) exist in blood, and small non‐coding RNAs (ncRNAs) in EVs may reflect disease severity. In this study, we investigated small ncRNAs in serum EVs to identify specific biomarkers for predicting clinical pregnancy. Methods : Serum samples were collected from 48 patients who underwent assisted reproductive technology (ART). EVs were isolated by the serial centrifugation method, which is follows the MISEV2023 principal, and characterized using nanoparticle tracking assays, electron microscopy, and western blotting for EV markers. We performed small RNA sequencing and analysed microRNA (miRNA) profiles in the infertility patients with and without endometriosis to detect pregnancy‐predicting biomarkers. Results : Candidate miRNAs in serum EVs were selected by comparing patients without endometriosis who became pregnant ( n = 13) with those who did not ( n = 21). A total of 241 miRNAs were detected; however, no trends separated the two groups. Next, EVs from patients with endometriosis were analysed and divided into pregnant ( n = 4) and non‐pregnant ( n = 10) cases. Among the 224 candidate miRNAs, miRNA profiles of pregnant women with endometriosis were separated from those of non‐pregnant women by receiver‐operating characteristics curve analysis (area under the curve > 0.8). Finally, we used small RNA sequencing of the tissue to demonstrate that pregnancy‐predicting miRNAs in serum EVs were produced from endometriosis lesions. Summary/Conclusion : miRNAs in serum EVs of patients with endometriosis could provide novel noninvasive biomarkers to predict pregnancy and have potential clinical applicability in ART.

Size‐Specific

Prima Dewi Sinawang 1 , Fernando J. Garcia‐Marques 1 , Abel Bermudez 1 , Chenchen Zhu 1 , Demir Akin 1 , Emily Ding 1 , Lars Steinmetz 1 , James D. Brooks 1 , Manish Kohli 2 , Andrew J. Gentles 1 , Sharon J. Pitteri 1 , Utkan Demirci 1 * 1 Stanford University, Stanford, California, USA; 2 University of Utah, Salt Lake City, Utah, USA Introduction : Extracellular vesicles (EVs) play a crucial role in medical diagnostics. However, the size heterogeneity and the lack of convenient size fractionation methods pose challenges in identifying size‐specific vesicles with distinct markers. Common techniques, such as ultracentrifugation and size‐exclusion chromatography, are often time‐consuming and labour‐intensive, yet size fractionation of EVs is essential, as their dimensions influence intercellular signalling, disease progression, and cellular responses to therapies. Methods : To address these challenges, we developed a platform that simplifies the isolation, enrichment, and size fractionation of EVs from various biological samples. This system can fractionate EVs into size categories, including 100–200 nm, 50–100 nm, and 30–50 nm, all achieved with a simple button push. We isolated EVs from in vitro cancer culture models: benign (BPH‐1), non‐metastatic (22Rv1), and metastatic cell lines (LNCaP, DU145, and PC3), and performed proteomic and transcriptomic analyses to study their roles in prostate cancer progression. Markers identified in vitro were validated in relevant clinical samples (IRB‐approved and obtained informed consent) using Western blot and PCR. Size subpopulations were characterized using NTA and cryo‐EM. Results : We correlated the size and cargo of EVs with the metastatic potential and androgen receptor (AR) expression of the cancer cell lines, providing insights into disease stage. Our analysis revealed that different EV‐size subpopulations carry different cargos, highlighting the importance of size and metastatic characteristics. Unique proteins were identified for each cell line within each EV size subpopulation. Overlaps among the metastatic lines may indicate pro‐metastatic proteins, while few to no overlaps were found with the non‐metastatic lines. In metastatic cell lines, proteins in AR‐negative cell lines—compared to the AR‐positive one—were predominantly found in smaller EVs (30‐50 nm). Furthermore, different RNA types were distributed in different EV size subpopulations. Summary/Conclusion : Our innovative approach not only streamLines the isolation, enrichment, and size fractionation of EVs but also generates valuable insights into EV biology and their size heterogeneity. We believe these findings could contribute to the field of EV research and provide a new perspective on their utility in personalized medicine. * Information Disclosure : Due to a pending IP application, information disclosure is limited. Funding : Stanford Bio‐X Interdisciplinary Initiatives Program Seed Grant.

Standardization

Rikke W. Rasmussen 1 , Rikke B. Eschen 1 , Morten H. Nielsen 1 , Rikke Bæk 2 , Malene M. Jørgensen 2,3 , Aase Handberg 1,3 , Maiken Mellergaard 1,3 1 Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark; 2 Department Of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark; 3 Department Of Clinical Medicine, Aalborg University, Aalborg, Denmark Introduction : High‐resolution flow cytometry (hFCM) is highly efficient for characterization of phenotype on a single‐EV level. Detailed protocols are required from the collection of samples to data analysis and publication of reliable and translatable results. To document the credibility of data, relevant controls are required (MISEV 2023). However, hFCM analysis is complex and results are affected by the optical and physical configuration of the applied instrument as well as scatter characteristics of a given sample. To be able to reproduce and compare hFCM data between laboratories, calibration and conversion of signals such as arbitrary scatter signals to particle diameter are crucial. However, this is challenged by the variability in hFCM instrument properties and currently, no tools for size scatter calibration of medium angle light scatter (MALS) data are available. Finally, the risk of introducing bias in data analysis and gating strategy even within the same research group challenge the outcome. Methods : We have optimized the analysis of EVs detected directly in platelet poor plasma samples by our hFCM instrument (Apogee A60) designed to include a MALS detector to increase sensitivity. We have used Rosetta calibration of data to convert particle scatter signals to size in nm to enable us to specifically report the concentration within a certain size range of particles (MIFlowCyt‐EV, 2020,). Moreover, we tested the robustness of our gating strategy and subsequent reporting of our findings by comparison of data analysis from the same study performed independently by two different researchers. Results : We found that the limit of detection (LoD) is significantly lowered using MALS (∼110 nm) over LALS (∼250 nm) in hFCM and report how to perform size scatter calibration using Rosetta beads for data acquired by MALS analysis. Moreover, our data analysis test revealed coefficient of variations of 2.8%, 13%, and 10% for the gating and analysis of CD9+ EVs, CD81+ EVs, and CD9+CD81+ EVs, respectively. Summary/Conclusion : Our data demonstrate the advantage of using MALS for EV studies and a need for proper tools for reporting calibration of LoD. Moreover, we report a robust data analysis strategy especially for the CD9+‐positive EVs. Funding : This study was supported by the Novo Nordisk Foundation: NNF22OC0080036 and Research Cube AAUH 2022.

Ageing‐Induced

Efthymios Fousekis‐Papakonstantinou 1 , Arghavan Gholamalipoortehrani 1 , Maria Camila Melo‐Narváez 1,2 , Amine Chahin 1 , Anna‐Lena Rupp 1 , Isabell‐Valerie Burhorst 1 , Thomas Heimerl 4 , Gert Bange 4 , Katrin Laakmaan 1 , Bernd Schmeck 1,3,5 , Anna Lena Jung 1,3 , Mareike Lehmann 1,2,5 1 Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps University Marburg, Member of the German Center for Lung Research (DZL), Marburg, Germany; 2 Comprehensive Pneumology Center (CPC), Institute of Lung Health and Immunity (LHI), Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Germany; 3 Core Facility‐Extracellular Vesicles, Philipps University Marburg, Marburg Germany; 4 Center for Synthetic Microbiology (SYNMIKRO), Philipps‐Universität Marburg, Marburg, Germany; 5 Institute for Lung Health (ILH), Giessen, Member of the German Center for Lung Research (DZL) Introduction : Chronic lung diseases (CLDs) are among the leading causes of death worldwide, with incidence increasing with age. A major ageing hallmark is cellular senescence, a stress‐induced response reducing proliferative capacity. The Senescence‐Associated Secretory Phenotype (SASP) impacts CLD pathogenesis, with extracellular vesicles (EVs) playing a key role in SASP‐related cell communication. The lung's gas exchange unit includes multiple cell types, such as endothelial cells, fibroblasts, and alveolar epithelial cells, which show senescence markers in ageing‐associated chronic lung diseases. We hypothesize that ageing differentially alters EV protein cargo and secretion in those cells, thereby altering their SASP. Methods : We induced senescence in alveolar epithelial cells (A549), fibroblasts (MRC5), and endothelial cells (HULEC) by different inducers (H 2 O 2 and doxorubicin) before EV isolation and characterization. We compared the quantity, size, protein cargo, and morphology of EVs from untreated and treated cells using NanoFCM, transmission electron microscopy (TEM), and flow cytometry. Furthermore, we isolated EVs from mouse bronchoalveolar lavage (BAL) fluid and examined differences between young and old mice regarding EV quantity, quality, protein cargo, and size using NanoFCM, TEM, ExoView, and flow cytometry. We also investigated the difference in pro‐inflammatory cytokine levels between soluble and EV‐bound fractions by flow cytometry. Results : Treatment with H2O2 and doxorubicin induced senescence in a dose‐dependent manner. We detected senescence‐associated alterations in EV numbers and cargo. Senescent lung cells produced increased EV numbers compared to untreated cells, while EV size and shape remained unchanged. Moreover, we detected an age‐associated increase of EV surface proteins, including CD29 and CD81. We also observed an age‐associated size reduction on mouse BAL EVs. Finally, we detected an increase in proinflammatory cytokine concentration in BAL EVs compared to soluble BAL proteins. Summary/Conclusion : Our results showed senescence‐dependent differences in EV production and protein cargo. Senescent lung cells secreted higher levels of EVs that carried specific cargo. Furthermore, we detected age‐dependent changes in EV size and protein cargo, including cytokines. Our findings suggest a specific EV‐SASP secreted by senescent lung cells and warrant further functional studies exploring their contribution to age‐associated chronic lung diseases.

Cancer‐Derived

Presenter: Fereshteh Shojaeighahrizjani McGill University, Montreal, Quebec, Canada Introduction : EVs are a heterogeneous group of lipid‐bound cellular particles with the size of 50–150 nm in diameter and they act as a natural delivery system. Our aim is to evaluate the tumour tropism of glioma cell‐derived EVs towards tumour and to harness this tropism to deliver siRNA against epidermal growth factor receptor variant III (EGFRvIII) in glioma. Methods : Glioma cell line (U373P) was cultured and EVs were extracted from the conditioned media. The first EV preparation included filtration of conditioned media with syringe filters and concentration with 100 KDs filters followed by ultracentrifugation while the second preparation, the filtration steps were avoided. Then, EVs were labelled with fluorescent dye indocyanine green (ICG). After modelling a mouse model with U373vIII tumour, the in vivo biodistribution was evaluated through IVIS imaging. For the second part of our project, we evaluated different methods for loading therapeutic siRNA into EVs such as endogenous loading and exogenous loading (passive incubation, sonication, electroporation and transfection) with the aim of delivering this siRNA directly to the tumour. The efficiency of each method was evaluated by nano flowcytometry followed by uptake assays and immunoblot analysis. Results : ICG‐labelled U373P EVs showed a preferential accumulation at the tumour site compared to control groups while filtration could interfere with the tropism by removing the EV protein corona. Regarding the cargo loading part, endogenous loading, passive loading and sonication showed the least efficiency and electroporation showed false positive results as it caused aggregation of siRNA. Transfection of U373P Evs with siRNA by cell penetrating peptide showed better functionality in terms of knocking down the EGFRvIII expression in the cells. Summary/Conclusion : The tropism of cancer‐derived EVs needs further validation, and if the challenges regarding external loading are addressed, they could be considered a promising tool for the direct delivery of therapeutic cargoes to tumours. Funding : University of Milan and McGill University

Characterisation

I. Fichtner 1 , F. Tafuri 2 , A. Rizzuto 3 , A. Corsini 1 , C. Macchi 1 , I. Giusti 4 , V. Dolo 4 , G. Esposito 5 , C. Perrino 5 , S. Carugo 2,3 , M. Vicenzi 2,3 , M. Ruscica 1,3 1 Department of Pharmacology and Biomolecular Sciences, University of Milan, Milan, Italy; 2 Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy; 3 Department of Cardio‐Thoracic‐Vascular Area, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 4 University of L'Aquila, L'Aquila, Italy; 5 University of Naples—Federico II, Naples, Italy Introduction : Heart failure (HF) is a clinical syndrome involving multi‐organ dysfunction as vessels, muscles, kidneys, liver, and haematopoiesis. HF is classified as either reduced (HFrEF) or preserved (HFpEF) based on the ejection fraction (EF) percentage of the left ventricle. Extracellular vesicles (EVs) show potential as a liquid biopsy tool, as their circulation patterns and cargo can indicate the onset and severity of cardiac diseases. We analysed plasma‐derived EVs of 40 HF patients (13 HFpEF and 27 HFrEF) and 21 volunteers (CTR). Methods : Echocardiographic and clinical data were collected from both groups. Following the MISEV 2023 guidelines, EVs were isolated from plasma using size‐exclusion chromatography and ultracentrifugation, then characterised via NTA, TEM, WB, and FACS analyses. Protein content of EVs was determined by targeted proteomic analysis and by ELISA. Results : Diagnosis of HF relied on echocardiographic (e.g., %EF, E/é) and biochemical parameters (e.g., NT‐proBNP). EV‐specific protein (CD63, CD9, CD81, Alix and Beta1 integrin) expression and integrity were confirmed by FACS, WB and TEM. Median EV concentration was lower in HF vs CTR (EV/mL/cell count: 2.8 × 10 9  vs. 3.9 × 10 9 ), while the size was larger (mm: 204 vs. 187). Among the different subpopulations analysed, HF showed significantly reduced EVs from cardiomyocytes (CD172a+), endothelial cells (CD62E+) and endothelial progenitor cells (CD309+). Conversely, EVs from lymphocytes T and B (CD8+, CD4+ and CD19+) and lymphatic vessels (CD310+) were all significantly increased. Interestingly, platelet‐derived EVs (CD41a+) decreased along with the total plasma platelet count, while EVs from activated platelet (CD154+/CD41a+) increased in HF patients. Proteomic analysis of EVs from HF showed increased levels of surfactant proteins (SFTPD) and markers of fibrosis (Galectin‐3 and Chitinase‐3‐like‐1 protein). These findings were confirmed through ELISA. When EV subtype analysis was stratified by clinical presentation (HFrEF vs HFpEF), the main differences between patients and CTR were driven by HFrEF. Summary/Conclusion : EVs isolated from patients with HF displayed a unique fingerprint aligned with the haemodynamic characteristics of the condition. Additionally, the characterization of EVs allows us to distinguish HFpEF from HFrEF, showing different activity of systemic mechanisms (e.g., endothelial and inflammation). Funding : (1) PRIN ‐ 2022ZPS49L, (2) ANTHEM: “AdvaNced Technologies for Human‐Centred Medicine PNC0000003 – CUP B53C22006670001– Spoke”.

Characterization

Lucrezia Luisotti 1 , Rebecca Piccarducci 1 , Lorenzo Germelli 1 , Alessandra Falleni 2 , Benedetta Masciulli 3 , Ranieri Bizzarri 3,4,5 , Martina De Felice 6 , Francesca Cella Zanacchi 4,6 , Chiara Giacomelli 1,4 , Laura Marchetti 1,4*, and Claudia Martini 1,4 1 Department of Pharmacy, University of Pisa, Pisa, Italy; 2 Department of Experimental and Clinical Medicine, University of Pisa, Pisa, Italy; 3 Department of Surgical, Medical and Molecular Pathology, and Critical Care Medicine, University of Pisa, Pisa, Italy; 4 Center for Instrument Sharing, University of Pisa, 56126, Pisa, Italy; 5 NEST, Istituto Nanoscienze‐CNR and Scuola Normale Superiore, Pisa, Italy; 6 Department of Physics, University of Pisa, Pisa, Italy Introduction : Exosomes are cell‐derived small extracellular vesicles (EVs) essential for cell‐to‐cell communication; indeed, exosome bioengineering is an appealing approach to tune the exosome cargo as well as modify the exosome surface to target desired recipient cells. Consequently, exosome journey monitoring is essential. Although fluorescent lipophilic dyes are the gold standards for exosome labelling, they lack selectivity, due to their unconditional affinity for lipids. An alternative exosome fluorescent labelling strategy is herein evaluated, by taking advantage of green fluorescent protein farnesylation (GFP‐f), a post‐translational modification that allows the direct anchor of GFP to the exosome membrane. The current study analyses the performance of GFP‐f, in terms of selectivity and efficiency, in tracking engineered exosomes during uptake studies. Methods : Naïve or engineered HEK293T‐derived exosomes were isolated by size exclusion chromatography and physically and biochemically characterised. Then, they were employed for uptake studies. Results : Firstly, the advantage of directly anchoring the GFP to the exosome membrane for labelling exosomes was demonstrated. Then, GFP‐f‐bearing exosomes were further compared to VybrantTM DiD lipophilic dye labelled ones in uptake studies, by capturing intracellular fluorescence in a time‐ and concentration‐dependent manner. The internalization assay revealed a particular ability of GFP‐f to monitor the uptake of tagged exosomes into recipient cells, with a significant peak of intensity reached 12 h after administration by GFP‐f, while it was negligible in VybrantTM DiD labelled exosomes. Finally, the GFP‐f tagging capability was tested in the presence of a surface‐modification of exosomes, that is, LAMP2B‐Exenatide, a GLP1 analogue moiety tailored for targeted delivery into pancreatic b‐cells. Results showed that GFP‐f performance is influenced by surface engineering compared to naïve GFP‐f exosomes, but the fluorescence is importantly preserved. Therefore, the ability of LAMP2B‐Exenatide to selectively target human pancreatic b‐cells is currently under investigation, by monitoring the internalization of targeted exosomes into different recipient cells through GFP‐f labelling. Summary/Conclusion : Overall, these data provide a direct insight into the advantages and limitations of GFP‐f as a tagging protein for selectively and accurately tracking exosome routes from isolation to uptake in recipient cells, also in the context of EV bioengineering applications. Funding : This study was funded by the European Union‐NextGenerationEU: Mission 4‐Component 2‐CUP B93D21010860004.

Clinical‐Grade

Johanna Gamauf 1 , Alessia Brancolini 1 , Matthias Wieser 1 , Matthias Postl 1 , Giulia Corso 1 , Madhusudhan Reddy Bobbili 2,3,4 , Johannes Grillari 2,3,4 , Regina Grillari 1,4 , Marieke Roefs 1 1 Evercyte GmbH, Austria; 2 BOKU University, Austria; 3 Ludwig Boltzmann Institute for Traumatology, Austria; 4 Austrian Cluster for Tissue Regeneration, Austria Introduction : Extracellular vesicles (EVs) derived from immortalized MSCs hold great therapeutical potential for a range of regenerative and age‐related diseases. To develop MSC‐EVs further towards a scalable clinical application, prolonged EV storage with maintained therapeutic potential is required. Therefore, more information about MSC‐EV physical and biological stability over time is needed. Here, we compared EVs produced by telomerized MSCs that were derived from different tissues, produced in 2D or 3D culture vessels, and isolated in different storage buffers, for physical and functional characteristics up to 1 year after isolation. Methods : MSCs were isolated from bone‐marrow (BM‐MSC), Wharton's‐jelly (WJ‐MSC) and adipose tissue (ASC), and immortalized by non‐viral introduction of the catalytic subunit of human TERT (hTERT). EVs were isolated from serum‐free 2D or 3D hollow‐fibre cell cultures, enriched by 300 kDa tangential flow filtration (TFF), diafiltrated and stored in PBS, 20 mM HEPES or Ringer's lactate. Aliquoted EVs were stored at ‐80°C until further use. Particle and protein concentrations were measured directly after isolation and after thawing at different time‐points using nanoparticle tracking analysis and BCA. In vitro anti‐fibrotic activity was assessed by adding EVs to TGF‐β stimulated hTERT‐immortalized fibroblasts and evaluating the subsequent reduction in α‐SMA expression. Anti‐inflammatory activity was determined by adding EVs to LPS‐stimulated RAW264.7 cells and measuring the resulting decrease in NO levels. Results : BM‐MSC, WJ‐MSC and ASC‐derived EV particle concentrations were not greatly influenced by one freeze thaw cycle and prolonged storage at −80°C. Storage over time did not influence total protein content. MSC‐EVs maintained anti‐fibrotic and anti‐inflammatory activity for up to 1 year after isolation, irrespective of the used storage buffer and 2D or 3D MSC culture conditions. Summary/Conclusion : EVs isolated from different telomerized human‐derived MSCs maintained in vitro biological activity after long‐term storage at −80°C, demonstrating biological stability over time. Total particle and protein concentration did not markedly reduce over time, probably due to the high protein content in TFF‐enriched EV preparations. This observed stability supports the upscaling of EV production and long‐term storage, reducing the variability associated with different cell batches and isolation parameters, and advancing the development of MSC‐EV therapeutics. Funding : EU‐Horizon‐2020 #101072766; #721975. Evercyte GmbH, TAmiRNA GmbH.

Evca—Advancing

Presenter: Rúben Silva University of Lisbon, Lisbon, Portugal Introduction : EVCA stands for Extracellular Vesicles in Clinical Applications and is an acronym of the project titled ‘Centre for Excellence in Diagnostic and Advanced Therapeutics based on Extracellular Vesicles.’ EVCA focuses on methods and standard operating procedures for EVs and advanced therapeutics and diagnostics based on EVs. By partnering with internationally leading institutions—Institut Curie, France, and CIC bioGUNE, Spain—EVCA will leverage the existing multidisciplinary knowledge, experience, research and training best practices, widen collabourative networks, and step up the excellence of the researchers and research management at NOVA Medical School in order for it to become an internationally‐recognized Centre of EVs in clinical applications, research and innovation. Methods : Since the start of the project, excellent dissemination, outreach, training, and mentoring activities have been implemented, supported by many initiatives, including Think Tank sessions, winter retreat, summer school, training workshops, and staff exchanges. The essential information about the project is disseminated through a well‐structured website (http://evca‐twinning.eu), carefully curated mailing lists and active social media channels, including LinkedIn and X. Results : In the first reporting period of the EVCA, 4 high‐quality papers were published on (i) gut‐diet EV interaction in obesity, (ii) the role of EVs in skin intercellular communication, pigmentation and skin cancer, (iii) EV‐based bladder cancer diagnosis, and (iv) urinary EV‐based biomarkers for prostate cancer. Additionally, EVCA members were selected for 16 oral presentations and 13 poster presentations. Moreover, the previously mentioned organized events had more than 100 participants, and the project also supported the attendance of 50+ researchers in other initiatives and conferences, including MOVE, ISEV, PNEV23 and SINAL24 among others. Finally, EVCA was present in outreach events such as the European Researcher Night and reached 900+ followers across the social media platforms. Summary/Conclusion : During this time progress was made towards the goals of the project. One of the main impacts was the consolidation of the collabourations between partners to make them long‐lasting—common participation in different initiatives and common scientific publications. Remarkably, the EVCA project had a profound impact on the institutional scientific landscape of NMS, fostering over 200% growth in the number of research groups studying EVs.

First‐In‐Man

Presenter: Lishan Lin Sun Yat‐Sen University, Guangzhou, People's Republic of China Introduction : ER2001 is a genetic circuit (plasmid) encoding both a neuronee‐targeting rabies virus glycoprotein (RVG) tag and an HTT siRNA. This circuit is able to reprogram liver cells to transcribe and self‐assemble HTT siRNA into RVG‐tagged exosomes after intravenous administration. The RVG‐guided HTT siRNA is further delivered through the exosome‐circulating system to the cortex and striatum. The study is to evaluate the safety and tolerability of ER2001 in patients with early manifest HD, including the pharmacokinetics (PK) of ER2001 in plasma and the exposure of ER2001 in cerebrospinal fluid (CSF). Methods : This open label, single centre pilot study evaluates a single dose of ER2001 followed by multiple doses in eligible patients with early manifest HD. The primary endpoints were incidence of adverse events and abnormal clinical Laboratory results. Participants received a total of 8 intravenous injections of ER2001, and the treatment period of 14 weeks was followed by an observation phase of 84 days after the last injection. Results : A total of 10 HD patients, including 5 males and 5 females, aged between 27 and 51 years, were enrolled in this study. Up to now, there were a total of 27 adverse effects, and none of the AEs was related to ER2001. A significant reduction in plasma mHTT from baseline was detected in HD patients. The PK, PD, and clinical outcomes will be updated once available. Summary/Conclusion : Intravenous injection of ER2001 is safe and feasible in patients with early manifest HD, thus warranting further evaluation in randomized clinical trials.

Gabarapl2‐Alix

Naveen Soni, Megha Chaudhary, Bhawana Bissa Department of Biochemistry, Central University of Rajasthan, Ajmer, India Introduction : Autophagy, in addition to its primary function of nutrient recycling, has been identified as a growth regulator for both healthy and tumour cells. On the other hand, exosomes are small extracellular vesicles secreted by most cells that carry genetic information such as DNA, RNA, and proteins from host cells. Multivesicular bodies (MVBs) facilitate the crosstalk between both pathways through the formation of a hybrid amphisome and facilitate tumour cell homeostasis. Methods : We used ultracentrifugation to isolate exosomes from control and chloroquine‐treated MDA‐MB‐231 cells. Exosomes were characterized by DLS, NTA, TEM and western blotting. We analysed the change in autophagy and exosome pathway genes after inhibition of autophagy and exosome pathways with CQ and GW4869, respectively. We also performed knockdown of GABARAP, GABARAPL1 and GABARAPL2 and analysed the change in exosome gene expression. Additionally, we also performed confocal microscopy to evaluate the localization of Alix and CD63 inside the MDA‐MB‐231 cells after inhibition of autophagy. Results : Inhibition of exosome biogenesis led to an increase in breast cancer cell proliferation. However, we observed a significant reduction in cancer cell proliferation when both pathways are inhibited simultaneously. To assess the reciprocal regulation of two pathways, we blocked the autophagy pathway and observed an increase in the secretion of exosomes from MDA‐MB‐231 cells, along with decreased expression of Alix and CD63. On the contrary, inhibition of exosome biogenesis led to an increase in the expression of ATG5 and ATG16L1, which caused a significant decrease in the expression of GABARAPL2. Interestingly, the knockdown of GABARAPL2 rescues the decrease in Alix expression upon autophagy inhibition, thus highlighting the essential role of GABARAPL2 in Alix secretion. Our study highlights for the first time the synergistic effects of autophagy and exosome pathway inhibition in restricting cancer cell growth as well as the involvement of GABARAPL2 in the regulation of exosome secretion via modulating the Alix expression. Summary/Conclusion : We conclude that combined inhibition of autophagy and exosome pathways promotes cancer cell death. Furthermore, inhibition of exosome secretion enhances tumour cell proliferation. Moreover, autophagy protein GABARAPL2 interacts with Alix and regulates its secretion. Funding : SERB‐SRG (F.30‐528/2020(BSR)), SERB‐POWER Grant (SPG/2021/002833), ICMR extra‐mural research grant (52/27/2020‐BIO/BMS).

Gp130‐Enriched

Presenter: Feng Chen Tsinghua University, Beijing, People's Republic of China; Southeast University, Nanjing, People's Republic of China Introduction : Sepsis is a fatal disease with high morbidity and mortality, of which acute lung injury is the earliest and most serious complication. Excessive inflammatory response is an important mechanism in the development of sepsis‐induced acute lung injury (S‐ALI). Interleukin‐6 (IL‐6) is a pleiotropic cytokine whose abnormal activation of signal transduction plays an important role in the inflammatory cascade of sepsis, thus promoting SALI. Although extracellular vesicles derived from mesenchymal stem cells (MSC‐sEV) have anti‐inflammatory and repair promoting effects, they also have problems of low efficacy. How to develop potent EVs‐based therapeutic systems remains to be studied. Methods : We first obtained MSC‐sEV and HEK293F‐sEV by differential ultracentrifugation combined with size‐exclusion chromatography, and then characterized their properties using transmission electron microscopy, nanoparticle tracking analysis, nano‐flow cytometry and western blot. First, we used MSC‐sEV to intervene in the mice with SALI and screened the effective active components of MSC‐sEV and their mechanisms of action by proteomic analysis. Then, we transfected HEK293F cells using plasmids to overexpress the effective active component. Further, we obtained engineered HEK293F‐sEV for the treatment of SALI mice to evaluate its real effect. Results : We successfully isolated MSC‐sEV and HEK293F‐sEV and confirmed that their characteristics were consistent with MISEV. First, MSC‐sEV was significantly enriched in the lungs of mice with SALI and was effective in ameliorating inflammatory injury and overall survival. Second, the proteomics results of MSC‐sEV showed that its membrane was enriched in gp130, and sEV carrying gp130 could competitively bind IL‐6 when injected into mice via tail vein, thus blocking the cascade amplification of inflammation and alleviating the inflammatory injury in the lungs. To facilitate future clinical translational applications, the engineered HEK293F‐sEVs express high levels of gp130 and also show significant therapeutic effects. Summary/Conclusion : MSC‐sEV carrying gp130 competitively binds IL‐6 and thus attenuates SALI, while gp130‐enriched engineered HEK293F‐sEV likewise exerts a significant protective effect, which facilitates future clinical translational applications.

Hypoxia‐Driven

Presenter: Rawan Almasri Trinity College, Dublin, Ireland Introduction : Neratinib benefits many patients with HER2+ breast cancer, but innate or acquired resistance to such drugs undermines their benefit. Our group has previously discovered that expression of hypoxia‐inducible carbonic anhydrase 9 (CAIX), which is associated with poor outcome in breast cancer, is increased in neratinib‐resistant compared to neratinib‐sensitive HER2+ cells. Separately, in prostate cancer, our group also made the original discovery that extracellular vesicles (EVs) can transmit drug‐resistance to formerly drug‐sensitive cells. Thus, we aimed to evaluate (i) if EVs carry CAIX reflecting their cells of original and (ii) if blocking CAIX can block EVs release. Methods : Neratinib‐sensitive (HCC1954, SKBR3, EFM192A) and neratinib‐resistant (HCC1954‐NR, SKBR3‐NR, EFM192A‐NR) cells, established in our Laboratory, were cultured in medium containing EV‐depleted FBS under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. EVs were collected from their conditioned media using a differential ultracentrifugation in a 70Ti fixed‐angle rotor at 120,000 ×  g at 4°C. EVs were fundamentally characterised using immunoblotting, NTA, and TEM. Cellular and EVs CAIX was quantified by ELISA. Any effect of the CAIX inhibition by S4 on EVs (CD81+ and CD63+) release was evaluated via Amnis image stream flow cytometry. Results : Hypoxic compared to normoxic conditions significantly (1.5–4.6‐fold; p = 0.01–0.0001) increased the expression of CAIX in all cell line variants, as expected. Induction of CAIX was not substantially different whether the cells were neratinib‐resistant or neratinib‐sensitive. Interestingly, CAIX was detected as cargo of the corresponding EVs, significantly increased ( p = 0.03–0.004) under hypoxia versus normoxia. Under hypoxia, S4 treatment led to a significant reduction of EVs (CD81+ and CD63+) release ( p = 0.01–0.001). Treatment with S4 also reduced CAIX expression in both cell lysates and the cargo of their corresponding EVs ( p = 0.01–0.001). Summary/Conclusion : The CAIX cargo of EVs may have potential as a biomarker reflecting the hypoxic environment of a tumour. S4 blocking of CAIX and its reduction of CD81+ and CD63+ EVs release presents a possible way of reducing EVs release in cancer and thus their unwanted influences on recipient cells. Funding : This research was supported by the Research Council Advanced Laureate Award EVIC to LOD [IRCLA/2019/49].

Immunomodulation

Pamina Contreras‐Kallens 1 , Hannah Kimingi 1 , Yeyu Shen 1 , Hannah Aris 1, Dessi Malinova 2, Meadhbh Brennan 1 1 University of Galway, Ireland, 2 Queen's University, Belfast Introduction : Autoimmune diseases (ADs) are characterized by B cell function dysregulation. Novel therapies such as antibody‐mediated B cell depletion have significant side effects. Mesenchymal stem cells (MSCs) have been proposed as an alternative therapy for ADs, as these cells bear potent immunomodulatory capacities, particularly when exposed to inflammatory conditions known as ‘licensing’. MSCs‐derived extracellular vesicles (EVs) have been proposed as a safer alternative to MSCs. The aim of this study was to evaluate the effect of EVs derived from licensed and unlicensed MSCs on B cell function and activation. Methods : Bone marrow murine MSCs (BM‐MSCs) were cultured on tissue culture plastic in EV‐depleted media with IFN‐γ and TNF‐α to ‘license’ them, or in standard conditions. MSCs‐EVs were isolated from the conditioned media by size exclusion chromatography. EVs samples’ particle and protein concentrations were measured by NTA and BCA, respectively. For assessing the effect of EVs on B cells, primary B cells were isolated from an allogenic strain mouse and activated in vitro. The effect of licensed and unlicensed EVs on the expression of activation surface markers and proliferation was measured by spectral flow cytometry. Results : The presence of pro‐inflammatory cytokines in the media increased the expression of both constitutive markers, such as CD73, and inducible markers, like MHCII, on the BM‐MSCs. These cells were also capable of secreting more EVs per cell, and these EVs contained a higher content of protein per particle compared to unlicensed EVs. BM‐MSCs‐derived EVs could modulate the expression activation markers CD69, CD24 and PD‐L1 on allogeneic murine activated B cells. The EVs capacity of modulating B cell proliferation was also studied. Summary/Conclusion : Our findings demonstrate that BM‐MSC‐derived EVs can modulate B cell activation and function in vitro. Moreover, our study highlights the influence of MSCs' culture conditions on EV effects, suggesting potential therapeutic versatility in diseases characterized by immune dysregulation, such as ADs. Further elucidation of the underlying mechanisms is crucial to fully harness the clinical potential of MSCs‐derived EVs as immune therapies. Funding : European Research Council (ERC) Starting Grant #852152 and Higher Education Authority (HEA) Ireland funding through the North‐South Research Program (ExoBcell project).

Immunomodulatory

Kimingi W. Hannah, Buckley Fiona, Contereras Kallens Pamina, Shen Yeyu, Aris M. Hannah, Treacy Oliver, Brennan Á. Meadhbh University of Galway, Ireland Introduction : Dendritic cells (DCs) are professional antigen‐presenting cells (APCs) that link the innate and adaptive immune systems. Overactive DCs are implicated in inflammation‐related bone loss by activating T‐cells as reported in rheumatoid arthritis (RA) and chronic periodontitis. Therefore, there is a need for therapies that can modulate DCs towards tissue‐repair effectors. It has been shown that mesenchymal stromal cells (MSCs), and more recently their secreted extracellular vesicles (EVs), can influence the immune system. This study investigates the influence of MSC‐EVs' potential to modulate dendritic cell differentiation and activation. Methods : Murine MSCs were cultured on tissue culture plastic in EV‐depleted media for 48 h. EVs released by the MSCs were isolated by using differential centrifugation, ultrafiltration, and size exclusion chromatography (SEC). EVs were characterized by particle size and concentration using nanoparticle tracking analysis (NTA), by protein content using bicinchoninic acid (BCA), for morphology, as well as surface marker expression. MSC‐EVs were treated on monocyte‐derived dendritic cells isolated from C57BL/6 mice. EVs were treated at increasing dosages (0, 4e+009, 8e+009, and 2.25e+010 EV particles per mL) on days 0, 3, and 6 for differentiation assay and one day after activation for activation assay. The uptake of the EVs by DCs was monitored by tracking palmitoylated‐tandem dimer Tomato (PalmtdTomato) EVs using confocal microscopy. DC surface markers analysed using flow cytometry, and cytokine secretion, were employed to assess the effect of MSC‐EVs on differentiation, activation, and intracellular cytokine release by dendritic cells. Results : The size of the isolated MSC‐EVs ranged between 30 and 380 nm with the majority of EVs having the size of 130 ± 40 nm. MSC‐derived EVs caused a significant dose‐dependent reduction in the expression of MHC‐II, CD11c, CD40, and CD86 surface markers and increased expression of CD11b surface marker and PD‐L1 immune checkpoint marker. Summary/Conclusion : MSC‐EVs inhibited the differentiation and activation of monocyte‐derived dendritic cells. To understand the underlying mechanisms behind these findings we are currently investigating the role of EV‐treated dendritic cells on the activation and proliferation of T‐cells. Further, we will investigate the transcriptome, miRNA, and protein cargo profiles of the murine MSC‐EVs. Funding : Science Foundation Ireland (SFI).

Immunoregulatory

Patrizia Ghidotti 1 , Claudia Proto 2 , Marta Brambilla 2 , Francesca Pontis 1 , Mattia Boeri 1 , Benedetta Bussolati 3 , Cristina Grange 3 , Daniela Boselli 4 , Annalisa Radeghieri 5 , Miriam Romano 5 , Rossella Crescitelli 6 , Gabriella Sozzi 1 , Elena Jachetti 7 , Orazio Fortunato 1 1 Epigenomics and Biomarkers of Solid Tumors Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2 Thoracic Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 3 Department of Medical Sciences, University of Torino, Turin, Italy; 4 FRACTAL Facility, San Raffaele Hospital, Milan, Italy; 5 Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; 6 Sahlgrenska Center for Cancer Research, University of Gothenburg, Gothenburg, Sweden; 7 Molecular Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Introduction : Tumour PD‐L1 is the only predictive biomarker used in clinical practice for the use of immune checkpoint inhibitors (ICIs) in NSCLC. Extracellular vesicles (EVs) are membrane‐limited particles described as biomarkers for cancer progression and as modulators of anticancer immune response. In this study, we aimed to evaluate EVs of advanced NSCLC patients with low PD‐L1 expression to find biomarkers for combination therapy (chemotherapy plus ICIs) and to investigate their role in the anti‐tumour immune response. Methods : Plasma EVs were isolated using ultracentrifuge and sucrose density gradient from responder (R), stable disease (SD) and non‐responder (NR) advanced NSCLC patients ( n = 40) with PDL1 < 50%, treated with combination therapy. EV characterization was performed following MISEV guidelines. Tetraspanin expression was assessed via super resolution microscopy and nano‐scale flow cytometry. miRNA cargo was evaluated by using the miRCURY LNA miRNA Panel and digital PCR. Proteomic analysis on EVs was done by Tymora Analytical. EV subpopulations were sorted through FACS or immune‐capture beads. Results : Super‐resolution microscopy revealed a significant increase in CD9+ EVs in NR samples. Instead, in nano‐scale flow cytometry analysis, R‐EVs resulted enriched for CD81+EpCAM+ and CD63+EpCAM+ events. We found 6 miRNAs differentially represented between the three groups of patients. Among them, miR‐142‐3p was the most expressed in R patients both at baseline and after the start of the treatment. Via sucrose density gradient, we confirmed that this miRNA was mainly detected in fractions containing small‐EVs. Moreover, miR‐142‐3p levels were modulated during T cells activation. The transfection of T cells with a miR‐142‐3p mimic slightly increased IFN‐γ production in both CD4 and CD8 T cells. Proteomic analysis found 13 proteins up‐regulated in R‐EVs and another 13 proteins in NR‐EVs at baseline. Instead, 113 proteins changed after combination therapy. We successfully isolated EV subpopulations based on CD9 and CD45 expression. Summary/Conclusion : Our preliminary results showed that R‐, SD‐ and NR‐EVs differed in terms of tetraspanins, miRNAs and protein content, suggesting a potential use as biomarkers for therapy response. Further studies are needed to elucidate the role of EV subpopulations in immune response modulation in lung cancer. Funding : The study was supported by grants from the Italian Ministry of Health (GR‐2019‐12369047).

Nanobody‐Based

Suzan Glänzer 1 , Jenny Ge 1 , Mana Nedjabat 1 , Natasja Dits 2 , Joke Veldhoven‐Zweistra 2 , Ardalan Mansouri 2 , Aline Carvalho Tahyra 2 , Alba Martin Barreiro 2 , Guido W. Jenster 2 , Martin E. van Royen 1* 1 Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands; 2 Department of Urology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands Martin E. van Royen is the presenting author . Introduction : High‐throughput detection of disease biomarkers on individual EVs in clinical samples is challenged by the low abundance of disease‐derived EVs in clinical samples and the low number of biomarkers on the surface of individual EVs. To address these challenges, we aim to develop an EV‐capture variant of the EVQuant assay where we use biomarker‐specific immune capture to enrich for biomarker positive EVs and use high fluorescence nanobody‐bacteriophage conjugates for the detection of low‐abundance EV surface markers on individual EVs using high‐throughput confocal microscopy. Methods : Using biotin conjugated CD9/CD63 or PSMA antibody coating of 96‐well imaging‐compatible well plates, we aim to capture specific subpopulations of EVs from conditioned culture medium and clinical samples. Sensitive detection of scare EV surface markers was performed using custom nanobody‐M13 bacteriophage conjugates that were fluorescently labelled using Atto488 NHS‐ and Alexa647 NHS‐ester dies. The large size of the M13 bacteriophage allows sensitive detection of individual nanobodies using the Opera Phenix high‐throughput confocal screening microscope platform (Revvity). Nanobody‐bacteriophage binding of EVs was further evaluated using dSTORM super resolution microscopy. Results : After protocol optimization and evaluation of experimental parameters, we were able to quantitatively detect CD9 on individual DU145 and LNCaP derived EVs. In contrast, PSMA was only quantitatively detected on PSMA+LNCaP derived EVs, but not on PSMA‐DU145 EVs. The EV origin of the CD9 and PSMA signals was confirmed in colocalization analysis with fluorescently labelled EVs and the efficiency of immune detection was evaluated in multi‐colour colocalization analysis using Alexa 488. dSTORM analysis showed surprisingly mobile nanobody‐bacteriophage conjugates bound to individual EVs. Summary/Conclusion : We have successfully developed a microscopy based high‐throughput analysis of individual EVs using biomarker dependent immune capture and sensitive nanobody‐bacteriophage detection of EV surface markers. We will further develop this assay towards more disease‐specific EV biomarkers in more complex clinical samples also by introducing innovative labelling strategies. Funding : This work was supported by the Health–Holland TKI‐LSH (Match) grant EMCLSH22011, the Dutch Cancer Society (“IMMPROVE,” EMCR2015‐8022) and a Daniel den Hoed Foundation grant on Cancer Nanobodies.

Nanoerythrosomes

Kulzhan Berikkhanova, Alexandr Gulyayev, Ernur Zakirov, Nurzhan Bikhanov, Askhat Zhilkaidarov Nazarbayev University, Kazakhstan Introduction : Red blood cell‐derived nanocarriers, known as nanoerythrosomes, are remarkable nanovesicles that enable controlled drug release and targeted delivery. Administering antibiotic‐loaded nanoerythrosomes to an inflammatory site achieves higher and more prolonged drug concentrations compared to conventional free‐drug administration. This strategy aligns with physiological nature of the inflammatory process, as purulent tissues have increased blood flow and numerous macrophages. The antibiotic‐loaded nanoerythrosomes are selectively captured by macrophages. In purulent‐inflammatory areas, extensive phagocytosis leads to the release of a high concentration of the encapsulated drug. Nanoerythrosomes are believed to enhance targeted antibiotic delivery to sites of microbial inflammation, providing sustained drug release into the affected tissue. To model this release process, an in vitro study was conducted to observe the release of ceftazidime from these carriers. Methods : Nanoerythrosomes loaded with 1 g of Ceftazidime were obtained by the hypoosmotic hemolysis method in our original modification. This study received approval from institutional ethics committee. Venous blood samples were collected from volunteers after obtaining their signed informed consent. To assess the degree of ceftazidime release from nanoerythrosomes, method of equilibrium dialysis with semipermeable membrane was used. Samples were collected after 15 min, 30 min, 1 h, 3 h, 17 h, 24 h. The concentration of ceftazidime in the collected samples was determined spectrophotometrically. Results : The maximum release of ceftazidime from nanoerythrosomes was observed at 15 min (1.72 µg/mL ± 0.15), then until 17 h there was a period of stable release, in average of 1.5 µg/mL in each sample. From 17 h to 24 h concentration of antibiotic released from nanoerythrosomes decreased from 0.17 µg/mL ± 0.07 to zero. Summary/Conclusion : The release of ceftazidime from nanoerythrosomes occurs gradually over 24 h, with peak concentrations sustained for up to 17 h. The results suggest that nanoerythrosomes are effective carriers for targeted ceftazidime delivery to inflammatory sites, potentially improving treatment of purulent diseases. These data will be considered in pharmacokinetic preclinical in vivo study of ceftazidime deposited in nanoerythrosomes. Funding : This research is funded by Science Committee of Ministry of Science and Higher Education of Kazakhstan, Grant №AP19676272 and by Nazarbayev University under Collabourative Research Program Grant № 211123CRP1614, A.G.

Pharmacokinetics

Zach Troyer, Miranda Soumakis, Erin Shirk, Olesia Gololobova, Sarah Marquez, Martina Fabiano, Natalie Castell, Isabella Baumann, Suzanne Queen, Kenneth W. Witwer Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Introduction : It is increasingly recognized that extracellular vesicles (EVs) fusion do not fuse promiscuously with cellular membranes. Fusion proteins of several exogenous viruses have been used to enhance EV‐cell fusion; however, immunogenicity may limit clinical applications. Endogenous retroviruses (ERVs) are found in various primate genomes, and their fusogenic envelope proteins may be less immunogenic due to expression in early development. Here, we engineered the human endogenous retrovirus protein (HERV) Syncytin‐1 onto EVs and assessed pharmacokinetics, biodistribution, and immunogenicity after intravenous injection into to pig‐tailed macaques ( Macaca nemestrina ). Methods : Expi293F cells were used to produce EVs loaded with PalmGRET, an EGFP‐Nanoluciferase (NLuc) fusion and displaying Syncytin‐1. EVs were separated by ultrafiltration and size exclusion chromatography and characterized per MISEV by electron microscopy, Western blot, and nanoflow cytometry. EVs were administered to macaques intravenously every two weeks for four doses total. Pharmacokinetics and biodistribution in the primate model were assessed by NLuc assay with blood plasma and organ/tissue homogenates. Immunogenicity was assessed by detecting cytokines and EV‐specific IgG in plasma. Results : Syncytin‐1 EVs displayed a significantly different distribution of association with blood cell subtypes compared with those of unmodified EVs from a previously published study. In particular, T cell and monocyte associations were diminished. In addition to negligible cytokine responses at early administrations, we now also report on adaptive immune responses and tissue distribution of these EVs. Summary/Conclusion : Syncytin‐1+ EVs display different cell association characteristics compared with unmodified EVs, providing proof‐of‐principle that not all exogenous EVs are processed identically in the primate bloodstream and beyond.

Plasma‐Derived

Presenter: Kamal Baswal All India Institute of Medical Sciences, India Introduction : Non‐small cell lung cancer (NSCLC) remains a major health challenge worldwide, accounting for a significant portion of cancer‐related deaths. Early detection and diagnosis are crucial for improving patient outcomes; however, reliable biomarkers are still limited. Exosomes—small extracellular vesicles secreted into bodily fluids—have emerged as promising candidates for biomarker discovery due to their role in cell communication, cargo protection and disease specificity. This study aims to identify exosomal miRNA signatures in NSCLC patients, providing insights into novel biomarkers for NSCLC diagnosis. Methods : Exosome isolation was performed on blood samples using ultracentrifugation. The isolated exosomes were validated through the expression of characteristic markers CD63, ALIX, and CD9, and morphological confirmation was achieved using transmission electron microscopy (TEM). RNA extraction from exosomes was conducted and next‐generation sequencing (NGS) was subsequently performed to profile miRNAs across all samples. Differential expression analysis identified specific miRNAs of interest, with miR‐16‐5p used as a housekeeping gene due to its stable expression across samples. Validation of differentially expressed miRNAs, was carried out using quantitative PCR (qPCR). In addition, expression levels in plasma‐derived exosomes were compared to those in plasma alone to evaluate miRNA exclusivity to the exosomal fraction. Results : The sequencing analysis revealed hundreds of miRNAs across the patient and control samples, with a subset displaying differential expression between the NSCLC and control groups. Notable miRNAs, including hsa‐miR‐7b‐5p, hsa‐miR‐486‐5p, hsa‐miR‐122‐5p, and others, were significantly upregulated or downregulated in the NSCLC samples. qPCR validation confirmed these expression trends, underscoring their potential as diagnostic markers. Additionally, comparison of plasma versus plasma‐derived exosome miRNA profiles revealed specific miRNAs, that were exclusively present in the exosomal fraction, further suggesting a role for these miRNAs in NSCLC pathophysiology. Summary/Conclusion : we identified several differentially expressed miRNAs, including hsa‐miR‐7b‐5p, hsa‐miR‐486‐5p, and hsa‐miR‐122‐5p, that show promise for diagnostic applications. The exclusive presence of certain miRNAs within the exosomal fraction, as compared to plasma alone, further highlights the diagnostic specificity of exosomal miRNAs. These findings provide a foundation for developing minimally invasive, blood‐based diagnostic tools for NSCLC, which could significantly improve early detection and patient outcomes. Funding : INDIAN COUNCIL OF MEDICAL RESEARCH, NEW DELHI

Polyelectrolytes

Elżbieta Karnas 1 , Mateusz Zając 1 , Katarzyna Kmiotek‐Caller 1 , Kamil Kamiński 1 , Shin‐Ichi Yusa 2 , Sylwia Kędracka‐Krok 1 , Patrycja Dudek 1 , Krzysztof Szczubiałka 1 , Maria Nowakowska 1 , Ewa K. Zuba‐Surma 1 1 Jagiellonian University, Kraków, Poland; 2 University of Hyogo, Himeji, Japan Maria Nowakowska and Ewa K. Zuba‐Surma are co‐senior authors Introduction : Extracellular vesicles (EVs) are currently recognized as potential new‐generation cell‐free therapeutic factors in the field of tissue regeneration. However, clinical application of EVs requires the development of standardized procedures of their long‐term storage without the loss of structural integrity and biological activity following cryopreservation. Thus, the aim of this study was to develop a method of EV cryoprotection based on their coating with bilayer of biocompatible polyelectrolytes. Methods : EVs secreted by human umbilical cord‐derived mesenchymal stem/stromal cells (AT‐MSCs) were isolated using ultracentrifugation method and examined according to their size distribution and presence of selected surface markers. Subsequently, EVs were coated with ultrathin bilayer of designed polyelectrolytes, including (1) cationic poly(ethylene glycol‐block‐(3‐(methacryloylamino)propyl)trimethylammonium chloride) block copolymer (PEG46‐b‐PMAPTAC52; P1) and (2) anionic of poly(2‐acrylamido‐2‐methylpropanesulfonic acid) (PAMPS18, P2), using layer‐by‐layer technique. Based on the nanoparticle tracking analysis (NTA), high‐resolution flow cytometry, as well as mass spectrometry, we analysed vesicle integrity following single or multiple freezing‐thawing cycles, as well as long‐term storage. Additionally, we evaluated the effect of cryopreservation on the functional activity of EVs in vitro, using human osteoblasts as exemplary target cells. Results : Obtained data indicate, that coating with P1 and P2 polymers stabilizes structural integrity of EVs following deep freezing and thawing, preserving their biological activity in vitro. Additionally, proteomic analysis has confirmed an effect of particle stabilization, as well as an enrichment in EV‐ and MSCs‐related proteins in samples cryopreserved in the presence of tested polymers, indicating protective effect on EV protein cargo. Summary/Conclusion : Taking together, our study indicates that the use of P1/P2 polyelectrolyte bilayer may be a novel, effective way of long‐term storage of EV preparations for the purpose of basic research and their clinical applications. Funding : This study was funded by NCBR grant STRATEGMED III (STRATEGMED3/303570/7/NCBR/2017) and NCN grant MAESTRO 11 (2019/34/A/NZ3/00134) to E.Z.S.

Pre‐Analytical

Yohan Kim 1 , Chisom Uzendu 2 , Mohamed Ahmed 1 , Jack Andrews 3 , Ree Erickson 1 , Edwin Van Der Pol 4 , Fabrice Lucien 1,5 1 Department of Urology, Mayo Clinic, Rochester, Minnesota, USA; 2 Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA; 3 Department of Urology, Mayo Clinic, Scottsdale, Arizona, USA; 4 Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands; 5 Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA Introduction : Nanoscale flow cytometry (nFCM) is a suitable platform for high‐throughput, single extracellular vesicle (EV) enumeration and characterization from biofluids with minimal handling and processing. However, the small size of EVs and low number of surface antigens make detection above the background challenging. There is a need to develop strategies to augment the signal‐to‐noise ratio for the sensitive detection of rare EV populations. Therefore, we conducted a systematic evaluation of pre‐analytical labelling conditions and their impact on the quantification of different EV subsets from clinical samples. Methods : The Apogee A60‐Micro Plus nFCM was calibrated and standardized using Exometry and Bangs Labouratories fluorescence beads according to the MIFlowCyt‐EV guidelines. We tested the impact of the following conditions on the limit of detection (LoD) and fluorescence quantification of EVs from patient platelet‐poor plasma samples and recombinant PSMA+ EV spiked samples: direct and indirect EV labelling, pre‐ and post‐labelling size exclusion chromatography (SEC). Lastly, we assessed the impact of multiplexing on the quantification of EVs from blood samples collected from localized prostate cancer patients ( n = 134) and healthy donors ( n = 7). Results : Direct labelling of PSMA+EVs with a fluorescent primary antibody increased the percentage of PSMA+ EVs detected compared to indirect labelling with a fluorescent secondary antibody (45% vs. 35%, p = 0.0005) due to a reduction in LoD. Both pre‐ and post‐labelling SEC successfully removed ApoB+ lipoproteins (6‐fold decrease, p = 0.008). Although both pre‐ and post‐labelling SEC reduce LoD (2.5‐fold, p = 0.0156), only post‐labelling SEC leads to higher PSMA+ EV concentrations measured (2.0‐fold, p = 0.03). In the multiplex assay, using PSMA and STEAP1 antibodies, we observed increased concentrations of both single‐positive and double‐positive PSMA+ and STEAP1+ in samples from localized prostate cancer patient samples compared to healthy individuals ( p < 0.0001). Summary/Conclusion : SEC is beneficial to improve the signal‐to‐noise ratio for the sensitive detection and quantification of plasma‐derived EVs. Careful selection of fluorophore and optimized fluorophore antibody conjugation improves fluorescence‐based quantification of EVs. This work serves as a framework for reproducible EV analysis by flow cytometry, offering new opportunities for the development of non‐invasive diagnostic tests.

Size‐Exclusion

Hedayatullah Hayat, Dianne Cooper, Lucy V. Norling, Mauro Perretti Queen Mary University of London, London, UK Introduction : We compared centrifugation‐based with size exclusion chromatography (SEC) protocols, taking advantage of NanoFCM and other EV analytical approaches. Methods : Heparinized blood ( n = 3 donors; Queen Mary Ethics Research Committee approval 0391) was centrifuged at 200 × g 20 min to prepare plasma. Then, two protocols were followed. (A) Plasma (500 µL) underwent three centrifugation steps: (i) 4400 × g for 15 min at 4°C; (ii) 13,000 × g for 2.5 min at 4°C; (iii) 13,000 × g for 2.5 min at 4°C. Supernatants stored at −80°C for further use. (B) Chromatography columns (BioRad Econo‐Pac, cat#7321010) were loaded with 14 mL Sepharose CL‐2B (cat#10217754) followed by 10 mL EV‐free, double filtered PBS (buffer) overnight at 4°C. After three washes, plasma (500–250–100 µL) was loaded. Elution was done with 10 mL buffer + 0.03% Tween and fifteen 0.5 mL eluted fractions were collected. In all cases, EVs were stained with CellMaskGreen (CMG) and analysed with NanoFCM, calibrated using dual‐laser quality control beads and Silica Nanospheres Cocktail #1 (65–155 nm). Quality control beads, sizing beads and fluorescently labelled EVs were all acquired with a sampling pressure of 1.0 kPa, and events were recorded for 1 min. The proportion of total events and CMG+ events was calculated. NanoSight NS300 (Malvern Instruments Ltd., Malvern, UK) analyses were also conducted. In some cases, the Oxford Nanoimager (ONI) dSTORM microscope (Nanoimager S, Oxford Nanoimaging) and Western blotting were run. Proteins were quantified with the Bradford Assay (Sigma). Results : Centrifuged plasma EVs gave values of 30% to 40% CMG+ events. SEC fractionation of plasma yielded high EV numbers in fractions 5 to 10. Protein concentration was elevated from fraction 10 to 15. NanoFCM analyses indicated 70% ± 15% CMG+ EVs out of total events, with 500 µL plasma starting volume being optimal. NanoSight analyses calculated a mode of 157 ± 12 nm for SEC samples, against 120 ± 12 nm for centrifuged plasma samples ( N = 3 donors). Cell‐marker staining is ongoing with ONI microscopy and Western blotting analyses. Summary/Conclusion : Reliable characterization is of paramount importance for diagnostic and prognostic use of EV numbers and types. We perfected a protocol for EV separation for plasma based on SEC and subsequent quantification of EVs, to be applied for clinical investigations, including an RCT for myocardial infarction ( NCT06465303 ). Funding : Barts Charity Grant MGU0509.

Spleen‐Derived

Presenter: Jihoon Han Korea University, Seoul, Republic of Korea Introduction : Cancer immunotherapy, particularly immune checkpoint blockade (ICB), has transformed cancer treatment but often struggles in tumours lacking pre‐existing immune responses. Our novel hierarchical hydrogel platform encapsulating spleen‐derived extracellular vesicles (EVs) addresses this by fostering tertiary lymphoid structure (TLS) formation, thereby enhancing ICB efficacy. Combined with ICB therapy, this approach shows potential to significantly boost anti‐tumour immunity. Methods : We followed MISEV2023 guidelines for EV separation and characterization. All animal experiments were approved by the animal department at KIST. Results : We engineered EV‐encapsulating hydrogels using an EV‐binding CP05 peptide sequence and verified its ability to capture spleen‐derived EVs via binding assay. We then implanted spleen EV‐loaded hydrogels into mice and observed that after 10 days, TLS‐like cell aggregates were formed inside the hydrogel. Finally, we assessed the antitumour effect of this TLS hydrogel on a murine CT26 colon cancer model along with anti‐PD‐1 antibody therapy and demonstrated that the combination group showed dramatic tumour regression with enhanced immune cell infiltration. Summary/Conclusion : Our study highlights the immune‐enhancing effects of spleen EV‐loaded hydrogels in promoting tertiary lymphoid structure (TLS) formation within tumour environments. By using spleen‐derived EVs, we successfully triggered high immune cell infiltration, aggregation, vessel formation, and germinal centre‐like structures. This immune‐rich environment significantly boosts the efficacy of immune checkpoint blockade (ICB) therapy, converting the tumour microenvironment (TME) into an “immune hot” phenotype. This approach addresses limitations in current immunotherapies, offering broad applicability across diverse cancer types.

Stress‐Induced

Elie Beit‐Yannai 1 , Valeria Feinshtein 1 , Boris Knyazer 1 , Sofia Schreiber‐Avissar 1 , Padmanabhan Pattabiraman 2 , Christina Ferrara 2 , Isana Veksler‐Lublinsky 1 1 Ben‐Gurion University of the Negev, Be'er Sheva, Israel; 2 Ophthalmology Department, Indiana University, Indianapolis, Indiana, USA Introduction : Primary open‐angle glaucoma (POAG) is a leading cause of irreversible blindness, characterized by progressive optic neuropathy often associated with elevated intraocular pressure (IOP). The non‐pigmented ciliary epithelial (NPCE) cells produce aqueous humour, while the trabecular meshwork (TM) regulates its outflow, together maintaining IOP homeostasis. Oxidative stress has been implicated in POAG pathogenesis, particularly in TM dysfunction. However, the molecular mechanisms of communication between NPCE and TM cells under stress conditions remain poorly understood. Extracellular vesicles (EVs) are emerging mediators of intercellular communication, yet their lipid composition and functional significance in ocular physiology are understudied. We investigated how oxidative stress modifies NPCE‐derived EV lipidome and microRNA content and characterized their effects on TM cells. Methods : We employed a three‐phase experimental approach: (1) human NPCE cells were exposed to oxidative stress using AAPH under acute (15 mM, 90 min) or chronic (1.5 mM, 24 h) conditions. (2) EVs were isolated from control and stressed NPCE cells, characterized by nanoparticle tracking analysis and CD9 Western blotting, then analysed for lipid composition using Bligh‐Dyer extraction with MRM‐based untargeted lipidomics and microRNA content via microarray. (3) Human TM cells were treated with these characterized EVs, and subsequent transcriptional changes were assessed through RNA sequencing. Data analysis utilized MetaboAnalyst 6.0 for lipidomics and standard RNA‐seq pipelines. Results : Oxidative stress significantly modified NPCE‐derived EVs, altering both their lipid and microRNA cargo. Lipidomic analysis revealed distinct changes in bioactive lipid species, while microarray identified 54 differentially expressed microRNAs in stress‐derived EVs. When TM cells were treated with these stress‐modified EVs, RNA sequencing revealed 146 differentially expressed genes (88 upregulated, 58 downregulated). Gene ontology analysis showed enrichment in pathways governing extracellular matrix organization and cell proliferation, processes crucial in glaucoma pathogenesis. Summary/Conclusion : This study provides new insights into stress‐induced EV lipid modifications in the eye and their functional impact on recipient cells. Our findings indicate that EV lipids actively participate in intercellular communication, influencing recipient cell behaviour. This work advances our understanding of EV‐mediated cell‐cell communication in ocular health and disease, suggesting potential therapeutic strategies targeting EV lipid composition in glaucoma. Funding : Israel Science Foundation 1545/20.

Thermoresponsive

Beyza Nur Küçük 1,2 , Eylul Gulsen Yilmaz 1,2 , Yusuf Aslan 1,2 , Özgecan Erdem 1 , Bengi Özgün Öztürk 3 , Fatih Inci 1,2 1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey; 3 Hacettepe University, Department of Chemistry, Ankara, Turkey Introduction : Extracellular vesicles (EVs) are key in cell communication, carrying biomolecules that reflect their origin cell. Pure isolation of EVs is essential but challenging with traditional methods, which risks non‐vesicular impurities and EV damage. This study introduces a smart thermoresponsive polymer on a metasurface sensor decorated with anti‐CD63 antibodies, enabling the sensitive capture and release of EVs from MCF‐7 breast cancer cells under surface plasmon resonance (SPR). By raising the local temperature above the polymer's lower critical temperature (LCST), EVs are isolated without contamination. The sensor's nanostructure offers real‐time, label‐free EV detection. Methods : Poly(N‐isopropylacrylamide) (PNIPAM) was polymerized for its thermoresponsive and biocompatible properties. EVs from MCF‐7 cells were isolated by centrifugation, filtered, and introduced into a microfluidic device. Characterized using NTA and SEM. EVs were captured on PNIPAM‐modified metasurface at 25°C, then released at 37°C, with SPR signal tracking capture and release events. Results : SEM results confirmed the microfluidic device's effectiveness with 50–150 nm particles that have expected spherical morphology. NTA analysis revealed exosomes sized at 137.7 ± 3.6 nm with a concentration of 1.01 × 10 1 ⁰ ± 5.23 × 10⁸ particles/mL. Following, 1 × 10 9 particles/mL were introduced to the microfluidic chip under SPR at 25°C and washed. Temperature increased to 37°C for release, and cooled to room temperature.A 1.07 ± 0.4 nm signal shift occurred after capture, slightly reduced by washing. After heating, a 0.71 ± 0.4 nm downward shift was observed, indicating approximately 34% EV release. Western blot analysis of released EVs was confirmed with Anti‐CD63 antibody. Summary/Conclusion : Pure EV solution was collected in real‐time via SPR with 34% efficiency and confirmed with Western Blot and SEM. This study is paving the way for detailed EV studies, overcoming limitations of traditional isolation methods. Funding : All authors acknowledge the support of Turkish Academy of Sciences (TÜBA) and The Scientific and Technological Research Council of Türkiye (TÜBİTAK) 2232 International Fellowship for Outstanding Researchers (Project No: 118C254).

Time‐Dependent

Zeynep Tavukcuoglu 1 , Zhilin Qiu 1 , Alessandra V. de Sousa Faria 1 , Saara Laitinen 2 , Mari Palviainen 1 , Pia R‐M Siljander 1,2 1 University of Helsinki, Finland, 2 Finnish Red Cross Blood Service, Finland Introduction : A dynamic crosstalk between platelets and cancer cells promotes haematogenous metastasis. Depending on the activating receptors, platelets generate different extracellular vesicles (PEVs) that may educate cancer cells in the circulation or within the tumour microenvironment. We engaged multiple receptors to generate a spectrum of PEV types and investigated how they altered cell transcriptomes in a melanoma model at different time‐points. Methods : Human melanoma cell lines (MV3 and A2058) were studied in 3D spheroid cultures. Human platelets were activated with collagen related peptide (CRP), fucoidan from Fucus vesiculosus (FFV), thrombin and collagen co‐stimulus (TC) and Ca2+ ionophore (Ca2+). PEVs were isolated by size‐exclusion chromatography and characterized according to the MISEV guidelines. Spheroids were treated twice with PEVs for 5 days. On Day 5, the treatment lasted either 6 or 24 h before RNA extraction and sequencing. Results : The number of differentially expressed genes (DEGs) was higher at the 6 h time‐point in A2058 spheroids and at the 24 h time‐point in MV3 spheroids, and time point was a main factor for clustering. The most significant differences in DEGs were induced by CRP and FFV PEVs, whereas TC and particularly Ca2+ PEVs, altered far fewer DEGs. In A2058 spheroids, both CRP and FFV PEVs enriched mitochondrial metabolism‐related pathways at 6 h. The PI3K‐Akt and MAPK signalling pathways were commonly activated by both CRP and FFV PEVs; however, they displayed distinct temporal dynamics that were also cell‐type dependent. Finally, CRP and FFV PEVs boosted different immunomodulatory pathways (TGF‐β and type‐I interferon, respectively), which also had cell‐type and time‐dependent patterns. Summary/Conclusion : The impact of PEVs on the melanoma transcriptome depends on multiple factors: time, cell type and PEV induction. Understanding the shared and unique signalling pathways could help the development of antiplatelet strategies in cancer therapy.

Tissue‐Derived

Presenter: Xinrui Wu Renji Hospital Affiliated to Shanghai Jiaotong University, Shanghai, People's Republic of China Introduction : Tissue‐derived extracellular vesicles (Ti‐EVs) play a significant role in tumour development, yet their potential as diagnostic markers for various pathological types of renal cell carcinoma (RCC) and their specific roles in tumour progression remain unclear. Methods : In this study, we analysed tissue samples from 25 RCC patients and their matched Ti‐EVs to identify diagnostic panels for different pathological types of RCCs (DPRCCs) using machine learning. To validate our findings, we extracted preoperative and postoperative urinary extracellular vesicles (uEVs) and supernatants from 178 samples. Based on single‐cell RNA sequencing (scRNA‐seq) data from 14 samples, we further explored the cellular origins of these diagnostic panel EVs, validated by extracting Ti‐EVs from primary tumour‐associated macrophages (pTAMs) of 6 low‐grade ccRCC (low‐ccRCC) and 6 high‐grade ccRCC (high‐ccRCC) samples. Results : We identified NEAT1 and MMP15 in Ti‐EVs as markers associated with papillary RCC (pRCC); DSG2 and AC026888.1 with chromophobe RCC (chRCC); SERPINA1, VEGFA, EGLN3, CPE, and C6orf223 with low‐ccRCC; APOC1, TGFBI, and LINC00887 with high‐ccRCC; and NDUFA4L2 as a diagnostic marker for both low‐ and high‐ccRCC. We achieved an Area Under the Curve (AUC) of 0.922 for low‐ccRCC detection and 0.874 for high‐ccRCC detection in uEVs, demonstrating superior predictive accuracy for low‐ and high‐ccRCC. Additionally, we observed that Ti‐EVs secreted by endothelial cells in pRCC were rich in MMP15, and those from malignant cells (MCs) in chRCC contained DSG2. In low‐ccRCC, pTAM‐derived Ti‐EVs expressed high levels of SERPINA1 and VEGFA, while high‐ccRCC was characterized by APOC1 and TGFBI expression. NDUFA4L2 expression was notably elevated in ccRCC MCs, with higher levels in high‐ccRCC, underscoring its potential role in ccRCC progression. Summary/Conclusion : In summary, we provide a comprehensive exploration of the entire EV secretion axis from Ti‐EVs to uEVs, presenting new insights for noninvasive RCC diagnostics and unveiling a potential mechanism of ccRCC invasion.

Transglutaminase

Maria Pia Savoca 1 , Timothy S. Johnson 2,3 , Elisabetta AM Verderio 1,4 1 Nottingham Trent University, United Kingdom, 2 University of Sheffield, United Kingdom, 3 Mestag Therapeutics Limited, United Kingdom, 4 University of Bologna, Italy. Introduction : Increased externalization of the crosslinking enzyme transglutaminase 2 (TG2) is linked with fibrosis progression in CKD and occurs via extracellular vesicles (EVs) (Furini, JASN 2018). We have hypothesised a novel role for EVs‐TG2 in tuning EVs‐dynamics in the extracellular space. Methods : Clinical urine samples were obtained by informed consent from the Sheffield and Patras University biorepositories. TG2 was characterised in urinary EVs (uEVs) by enzyme‐activity assay, immune electron microscopy and single uEVs quantifications (Exoview R200). Mouse embryonic fibroblasts MEFTG2WT and MEFTG2KO were from a transgenic model. Results : Small uEVs isolated from samples of CKD patients, stratified as “progressive” according to eGFR‐loss (> 4 mL/min/year, n = 18), displayed a significantly increased TG2 level on the uEV surface and higher transamidase activity ( p < 0.05) compared to uEVs from “stable” patients (< 1.2 mL/min/year, n = 16). Transglutaminase activity was detected in both intact uEVs and total lysates, confirming that TG2 is present on the uEV surface. To understand the role of TG2 in vesicular uptake by recipient cells, we employed PKH67‐labelled small EVs isolated from MEFTG2WT and MEFTG2KO conditioned medium. EVs‐TG2WT uptake by MEF was significantly higher than the uptake of EVs‐TG2KO ( p < 0.0001) and EVs‐TG2WT entered the cell cytosol. On the contrary, EVs‐TG2KO led to a slight punctuate fluorescence in the recipient cells. Moreover, EVs‐TG2WT led to the visualisation of TG2 cross‐linking in situ on the surface and inside MEFTG2KO, confirming EVs‐transfer of TG2 biological activity in recipient cells. In the fibronectin extracellular matrix (ECM), EVs‐TG2WT better adhered to the ECM than EVs‐TG2KO ( p = 0.024), suggesting that EVs‐TG2 could affect EV‐migration and adhesion. To identify cell‐surface targets of cross‐linking by EVs‐bound TG2, MEFTG2KO were incubated with either EVs‐TG2WT or negative control EVs‐TG2KO in the presence of the biotinylated TG2 amine‐substrate cavaderine, and the specific biotinylated substrates were revealed by mass spectrometry. Desmoplakin, vinculin, fibronectin emerged as specifically modified by EVs‐TG2. Summary/Conclusion : We report for the first time that protein transamidation mediated by EVs‐TG2 contributes to the collabourative role of EVs and ECM/cell surface in the extracellular space. Funding : NTU Vice Chancellor studentship to EAMV and MPS.

Tumour‐Derived

Chan Ho Kim 1 , Kyung Hee Han 1 , So Hee Kim 1 , Jae Hyung Park 1,2,3* 1 School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu‐ro, Jangan‐gu, Suwon, 16419, Republic of Korea 2 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06351, Republic of Korea 3 Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, 2066 Seobu‐ro, Jangan‐gu, Suwon, 16419, Republic of Korea Introduction : Tumour‐derived extracellular vesicles (T‐EVs) have been implicated in promoting tumour growth and metastasis and modulating the tumour microenvironment by influencing surrounding stromal cells. T‐EVs have been shown to suppress immune cell function and recruit immunosuppressive cell populations, including regulatory T cells and tumour‐associated macrophages, thereby facilitating immune evasion. Paradoxically, accumulating evidence suggests that T‐EVs carry tumour‐associated antigens, which can elicit anti‐tumour immune responses, underscoring their potential utility as components in cancer vaccine strategies. Methods : In this study, we aimed to develop a T‐EV‐based cancer vaccine by enhancing immune‐stimulatory molecules, including damage‐associated molecular patterns (DAMPs), while reducing immunosuppressive proteins. To achieve this, B16F10, a murine melanoma cell line, was treated with tunicamycin, known as an endoplasmic reticulum (ER) stress inducer, and the resulting T‐EVs (tT‐EVs) were subsequently isolated from the supernatants. Results : Western blot analysis revealed that representative DAMPs, such as HMGB1 and HSP70, were upregulated in tT‐EVs, whereas immunosuppressive proteins, including CD47 and S100A9, were reduced. Treatment of DC2.4, a murine dendritic cell line, with tT‐EVs resulted in enhanced maturation, evidenced by increased expression of CD80, CD86, and CCR7 compared to DCs treated with bare T‐EVs (bT‐EVs). In a therapeutic efficacy study using a B16F10 tumour‐bearing mouse model, tT‐EVs significantly suppressed tumour growth and triggered an anti‐tumour immune response, as shown by flow cytometry analysis revealing an increased population of cytotoxic T cells within the tumour. These outcomes were corroborated in both post‐surgery and bilateral tumour models, underscoring the ability of tT‐EVs to mediate systemic anti‐tumour immunity. Summary/Conclusion : One major advantage of cancer vaccines over other treatments is their ability to robustly induce a systemic anti‐tumour immune response, which can prevent tumour recurrence and inhibit metastatic growth following surgery. The tT‐EVs developed in this study showed their feasibility as a cancer vaccine, which showed enhanced DAMPs and reduced immunosuppressants. Also, as tT‐EVs can be isolated from resected tumours as a personalized cancer vaccine, tT‐EVs demonstrated the potential to treat tumours regardless of their heterogeneity.

Alcohol‐Exposed

Veronika Brezani, Radhika Joshi, Marti Ortega‐Ribera, Mrigya Babuta, Viliam Brezani, Adam Zivny, Gyongyi Szabo Beth Israel Deaconess Medical Center & Harvard Medical School, USA Introduction : Alcohol misuse induces inflammation both in the liver and brain. We showed that alcohol increases circulating exosomes containing miR‐122, a hepatocyte‐enriched miRNA, in humans and mice. Hepatic inflammation has been shown to induce encephalopathy and neuroinflammation. Therefore, we hypothesized that exosomes may mediate liver‐brain communication through miR‐122 transfer, particularly in the context of alcohol‐induced neuroinflammation in Alzheimer's disease (AD). Methods : Exosomes were isolated from mouse serum or supernatants of ethanol‐exposed hepatocytes, size and numbers were characterized by nanoparticle tracking analysis, and exosome markers were determined by western blot. BV‐2 mouse microglia were treated with hepatocyte exosomes before lipopolysaccharide (LPS) stimulation, and cytokine levels were assessed at 24 h. WT and APP/PS1 mice received daily alcohol binges (3.5 g/kg) for 30 days. Results : Alcohol increased exosome biogenesis and release from mouse hepatocytes. Furthermore, we found that the mature form of miRNA‐122 was enriched in the exosomes derived from ethanol‐treated hepatocytes compared to control exosomes. Co‐culture of PKH26‐stained hepatocyte exosomes with BV‐2 microglia confirmed microglial uptake of hepatocyte exosomes. Furthermore, exosomes from alcohol‐treated hepatocytes sensitized microglia to LPS stimulation to produce significantly increased MCP‐1, CXCL2, CCL3, and TNF‐α compared to control exosomes. Transfection of microglia with an miR‐122 mimic resulted in pro‐inflammatory sensitization and significantly higher protein levels of MCP‐1 and CCL3 at baseline, which were further elevated by LPS treatment. This supports our hypothesis that the transfer of miR‐122 from ethanol‐treated hepatocytes to microglia increases the pro‐inflammatory responses in microglia. In APP/PS1 mice, alcohol administration increased the number of circulating exosomes and miR‐122 levels in the brain compared to WT mice. Alcohol‐treated APP/PS1 mice showed increased levels of ALT, indicating liver damage and increased inflammatory markers. Moreover, we showed worsened amyloid beta pathology in AD brains after alcohol exposure. Summary/Conclusion : Alcohol increases circulating EVs and miRNA‐122 expression in the brain of APP/PS1 mice. Alcohol‐induced hepatocyte‐derived exosomes sensitize microglia to pro‐inflammatory responses via miR‐122 transfer. Our results suggest an exosome‐mediated crosstalk between hepatocytes and microglia in alcohol use and AD. The impact of the hepatocyte‐microglia crosstalk in AD and alcohol use deserves future investigations. Funding : This project is supported by NIH grants 5R01AG072899 and 5R01AA020744.

Apoe4/4‐Induced

Yang You 1 , Son N. Nguyen 2 , Sean D. Mann Jr 1 , Prakruthi Vadakattu 1 , Takahisa Kanekiyo 1 , Maria Paz. Gonzalez‐Perez 2 , Seiko Ikezu 1 , Scott A. Shaffer 2 , Wayne W. Poon 3 , Tsuneya Ikezu 1* 1 Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA. 2 Mass Spectrometry Facility, University of Massachusetts Chan Medical School, Shrewsbury, MA, USA. 3 Department of Neurobiology and Behaviour, Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, California, USA. *Co‐responding author. Introduction : The APOE4 allele is the strongest genetic risk factor for late‐onset Alzheimer's disease (AD). Recent studies demonstrated APOE as a key driver to induce disease‐associated microglia and may in turn affect extracellular vesicle (EV) biogenesis, leading to neuronal vulnerability and neurodegenerative risk. However, the contribution of microglial APOE4 to AD pathogenesis and EV‐mediated disease progression remains poorly understood. Here we conduct a comprehensive proteomics analysis using APOE isogenic human induced pluripotent stem cells (iPSCs)‐derived microglia (iMG) and microglial EVs (iMDEV) to explore the impact of APOE4 on regulating the microglial response and the protein composition of EVs in AD pathology. Methods : Three pairs of APOE3/3 and APOE4/4 isogenic iPSC lines were generated by CRISPR/Cas9 and differentiated into iMG. Fibrillar Aβ was employed to induce the disease associated microglia phenotype between the APOE3/3 and APOE4/4 backgrounds. iMDEVs were isolated from homeostatic and Aβ‐induced iMG by size exclusion chromatography and characterized by nano‐flow cytometry. Data‐independent‐acquisition mass spectrometry (DIA‐MS) was performed to analyse the proteomic profiles of APOE isogenic iMG and iMDEVs under homeostatic and Aβ‐treated conditions. The iMG/iPSC‐neuronee and iPSC‐organoid co‐culture model was utilized to investigate the contributions of APOE isogenic microglia and iMDEVs to AD pathogenesis. Results : APOE isogenic iPSC lines were successfully differentiated into mature microglia. APOE4 altered the microglial phagocytic function compared to APOE3. Additionally, the production of EVs was significantly higher in APOE4 iMG than APOE3 iMG. Using DIA‐MS, we totally identified over 6000 iMG proteins and 600 iMDEV proteins in homeostatic and fibrillar Aβ‐treated iMGs. Proteomics analysis revealed increased levels of MHC class I proteins in APOE4 iMG compared to APOE3 iMG under homeostatic conditions. Upon fibrillar Aβ treatment, APOE3 iMG exhibited elevated expression of interferon‐γ signalling and MHC class II proteins, while APOE4 iMG showed induction of cGAS‐STING and apoptosis‐related pathways. Notably, fibrillar Aβ remarkably increased the expression of proteasomal proteins in APOE4 iMDEVs compared to APOE3 iMDEVs. The microglial co‐culture model further demonstrated that Aβ‐treated APOE4 iMG and iMDEVs resulted in neurotoxicity and impaired neuronal activity. Summary/Conclusion : APOE4 impairs the microglial response, triggers cGAS‐STING signalling and modifies the protein cargos of iMDEVs in an AD iMG and organoid model.

Facs‐Proteomics

Maria Concetta Cufaro, Alice Di Sebastiano, Ilaria Cicalini, Marianna Gabriella Rispoli, Giulia Catitti, Domenico De Bellis, Serena Pilato, Antonella Fontana, Valentina Tomassini, Serena Veschi, Alessandro Cama, Paola Lanuti, Luca Federici, Damiana Pieragostino, Piero Del Boccio University ‘G.d'Annunzio’ of Chieti‐Pescara, Italy Introduction : The isolation and proteomics characterization of Extracellular Vesicles (EVs) from biological fluids remains an open challenge due to their vast heterogeneity and their suitability for proteomics purposes, once purified. Circulating EVs can enable an extreme reduction in the high dynamic range of detectable protein concentration in whole biofluids, paving the way for the biomarker discovery in the so‐called “dark proteome”. We have recently optimized an innovative “FACS‐Proteomics” workflow for the isolation and subsequent proteomics characterization of EVs directly from untouched biological fluids taking advantage of a lipophilic cationic dye (LCD) able to probe intact EVs. Here we report an update of the workflow suitable for carrying out proteomics on specific EV populations purified by sub‐typing them with an appropriate panel of antibodies. Methods : FACS strategy was used to purify Leukocyte‐derived EVs (Leuko‐EVs, LCD+/CD45+/Phalloidin‐ events) from blood and tears of healthy volunteers and multiple sclerosis patients. After purification, Atomic Force Microscopy and Nanoparticle Tracking Analysis were used to investigate Leuko‐EVs size distribution, morphology and number. Moreover, EVs were analysed for the expression of CD63 and CD81 by flow cytometry. Label‐free proteomics approach was performed by nanoLC‐Orbitrap‐Fusion‐Tribrid‐Mass Spectrometer; raw data were processed by MaxQuant and loaded on Ingenuity Pathway Analysis for functional investigation. Results : proteomics study was carried out on LeuKo‐EVs sorted and characterized from blood and tears (1.4x105 and 2x106 events, respectively) displaying a high level of purity (>90%). More than 90% of proteins identified in both biofluids (78 in tears and 155 in blood) were mainly referred to the leukocyte phenotype. This strategy was applied for proteomics evaluation of Leuko‐EVs purified from tears of multiple scleorosis patients, demonstrating that they were packaged with a well‐defined encoded protein cargo, reflecting the neuroinflammatory condition of the disease (TGFB1 and NFE2L2, Upstream Regulators activated) together with an increase of vascular networks (AGNPT2 and VEGF, Upstream Regulators activated) which mirrors pro‐angiogenic processes predicted as downstream effects. Summary/Conclusion : Our data show that the combined FACS‐Proteomics strategy for EV single‐phenotype characterization could open groundbreaking avenues for biomarker discovery, exalting the clinical value of tears EVs and helping in a better understanding of the EV‐mediated processes in vivo.

Fully‐Automated

Anna‐Lena Felser 1 , Tanja Jasmin Kutzner 2 , Moritz Meyer 3 , Kai Janning 1 , Yasemin van Heuvel 3 , Tim Milfeit 2 , Marco Carvalho Oliveira 3 , Bernd Giebel 2 , Patrick Bongartz 3 , Laura Herbst 1 , Bastian Nießing 1 , Robert H. Schmitt 1,4 1 Fraunhofer Institute for Production Technology IPT, Aachen, Germany; 2 University of Duisburg‐Essen, Essen, Germany; 3 BioThrust GmbH, Aachen, Germany; 4 RWTH Aachen University, Aachen, Germany Introduction : Despite significant advancements in regenerative medicine, osteoarthritis (OA) remains a prevalent illness. Mesenchymal stromal cells (MSCs) are proposed as a therapeutic treatment for OA. The clinical efficacy of MSCs has been inconsistent; while several successful phase I and II trials have been conducted, phase III trials have raised doubts about their effectiveness, and no therapy has been approved for patients outside of these trials. These conflicting findings are partly due to significant biological variability and the lack of standardization in the MSC manufacturing process. Since MSCs exert their therapeutic functions mainly through their secretome, particularly via extracellular vesicles (EVs), MSC‐EVs have gained attention as a new drug product for various diseases, including OA. A major challenge in this field is to define robust and scalable production strategies. To this end, automation is a key technology to increase throughput and improve iMSC manufacturing efficiency in scalable EV production. Methods : Within the EU project AutoCRAT, we have developed a fully automated Laboratory platform for cultivating induced pluripotent stem cells (iPSCs), differentiating them into iMSCs, and bioreactor‐based expansion of iMSCs to generate iMSC‐EVs. Results : We incorporated bioreactors designed to accommodate three distinct vessels for maximum scalability. Liquid handling is performed by a dedicated pumping station. Samples can be taken automatically and analysed for cell count and viability at‐line, while temperature, pH, dissolved oxygen, glucose, and lactate are monitored online. An FPLC with an automated fraction collector is integrated into the EV module for downstream processing of the EV‐containing supernatant. We constructed a housing for the fraction collector with openings on both sides to allow for sterile purification of the EV‐containing supernatant. In our ongoing project, we confirmed that the platform enables the manufacturing of bona fide MSC‐EV products. Summary/Conclusion : The platform establishes the groundwork for the automated production of MSC‐EV products, including scalability and enhanced manufacturing efficiency. Coupled with the potential for automated, repeated quality control evaluations, measures can be implemented to regulate the process for optimal production of highly abundant, high‐quality EVs. Funding : This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 874671.

Functionalization

Presenter: Yanhang Hong Shenzhen Institute of Advanced Technology, China (People's Republic) Introduction : Extracellular vesicles (EVs) exhibit diverse functional components and sources, leading to challenges in developing universal engineering approaches. Traditional methods for modifying and loading isolated EVs are often complex and inefficient, limiting the development of EV‐based delivery systems. Here, we have developed a novel microfluidic platform for EV functionalization. This platform enables the engineering modification of EVs isolated from various sources and supports the surface modification with a variety of functional components, including proteins and peptides. Methods : EVs derived from bovine milk and HEK293T cells were isolated via ultracentrifugation. DSPE‐PEG2000‐MAL was embedded into the membrane of EVs using a microfluidic device. The device incorporates with Tesla valve‐shape structures, which facilitate rapid conjugation of thiol‐containing peptides and proteins to the maleimide groups on the EV surface. The characterization of EVs were performed using NanoFCM, fluorescence spectroscopy, TEM and WB. To investigate the functionality of the modified EVs, a glioblastoma‐targeting peptide was introduced into HEK293T‐derived EVs, their targeting efficiency and blood‐brain‐barrier (BBB) penetration were evaluated using flow cytometry and confocal microscopy. Results : Results demonstrated that the embedding efficiency of DSPE‐PEG2000‐MAL in EVs derived from HEK293T cells and milk was 97.93% and 98.47%, respectively, using the ExoNP chips, compared to 13.97% and 46.10% with co‐incubation. Within the same reaction time, the microfluidic chip facilitated the binding of 677 peptides per EV, compared to only 50 with co‐incubation. The binding efficiency of FITC‐BSA‐SH was also increased by 3.1‐fold. Furthermore, EVs modified with peptides exhibited significantly improved internalization in tumour cells and enhanced penetration capability across the BBB. Summary/Conclusion : In conclusion, a novel platform for EVs functionalization, based on thiol‐ene reaction and microfluidic manipulation, offers a universal approach for modifying EVs from various sources. This platform efficiently introduces multiple biofunctional structures onto the EV surface, paving the way for large‐scale EV production and clinical applications.

Gcc2‐Containing

Presenter: Byeong Hyeon Choi Korea University, Republic of Korea Introduction : Early diagnosis and accurate prognosis are crucial for initiating effective therapeutic interventions and improving lung adenocarcinoma survival rates. Emerging evidence suggests that tumour‐derived small extracellular vesicle (sEV) cargo could serve as a cancer‐specific biomarker. A previous study identified GRIP and coiled‐coil domain‐containing 2 (GCC2)‐enriched sEV (sEV‐GCC2) as a promising diagnostic biomarker for lung adenocarcinoma. However, the diagnostic, predictive, and tumourigenic roles of sEV‐GCC2 remain unclear. This retrospective multicentre study aimed to evaluate and validate the diagnostic and prognostic values and tumourigenicity of sEV‐GCC2 in early‐stage lung adenocarcinoma. Methods : We evaluated plasma samples from surgically treated patients with lung adenocarcinoma and controls, which were retrospectively obtained from five institutions. sEVs were isolated using size‐exclusion chromatography, and sEV‐GCC2 was quantified using enzyme‐linked immunosorbent assays. Immunohistochemical staining (IHC) was performed to validate the expression of GCC2 in tumour sites. Kaplan–Meier and Cox regression analyses were performed for survival analyses. GCC2 knockdown cell lines were used to assess the implications of GCC2 in sEVs in vitro and in vivo for lung cancer progression and lymph node metastasis. Results : Four hundred seventy plasma samples (150 controls, 320 patients) were analysed. The mean follow‐up duration was 34.7 ± 24.0 months. Patients had significantly higher sEV‐GCC2 levels than controls, with an area under the curve (AUC) of 0.856 (95% confidence interval [CI], 0.820–0.886; p < 0.0001). For patients with TisN0–T1miN0 disease vs. controls, the AUC was 0.802 (95% CI, 0.734–0.859). Multivariate analysis showed significant associations between sEV‐GCC2 concentration and pathological TNM stages and tumour location in the left lower lobe. IHC revealed significantly higher GCC2 expression in lung adenocarcinoma tissues than in controls ( p < 0.001). Higher sEV‐GCC2 concentrations were linked to greater recurrence rates and lower recurrence‐free survival, even in stages 0–IA1. Additionally, sEV‐GCC2 increased cancer cell proliferation, tumour growth, and lymph node metastasis, but not GCC2‐deficient sEV. Summary/Conclusion : We verified sEV‐GCC2 as a diagnostic and prognostic biomarker of early‐stage lung adenocarcinoma. sEV‐GCC2 could be clinically significant for better pathological evaluation and a key molecule in lung adenocarcinoma progression. Funding : This study was supported by the Korea Health Industry Development Institute: RS‐2024‐00436472. Ministry of Science and ICT: RS‐2024‐00459242. Korea Medical Device Development Fund: 1711138151, KMDF_PR_20200901_0094_02.

Glycosaminoglycan

Mirjam Balbisi 1,2 , Tamás Langó 1 , Virág Horváth 1 , Domonkos Pál 1,2 , Gitta Schlosser 3 , Zoltán Varga 1 , Kinga Ilyés 1 , Nikolett Nagy 1 , Otília Tóth 1 , Tamás Visnovitz 2 , Beáta G. Vértessy 1 , Lilla Turiák 1 1 HUN‐REN Research Centre for Natural Sciences, Hungary, 2 Semmelweis University, Hungary, 3 Eötvös Loránd University, Hungary Introduction : Proteoglycans play an important regulatory role in the extracellular matrix, the balance of which is disrupted in many cancers. Proteoglycans contain glycosaminoglycan (GAG) chains that consist of repeating disaccharide units, which can be sulfated at various positions. Here, we present the chondroitin sulfate (CS) GAG disaccharide analysis of small extracellular vesicles (sEVs) derived from A549 lung adenocarcinoma and BEAS‐2B non‐tumourigenic epithelial cell lines. Methods : A549 cells were cultured in fetal bovine serum‐free F12 medium, while BEAS‐2B cells were cultured in BEGM medium for 72 h. Next, the supernatant was collected and after centrifugation steps, small EVs were isolated by mini‐size exclusion chromatography on in‐house prepared columns. Freeze‐thaw cycles were followed by an overnight digestion step by chondroitinase ABC enzyme. The resulting disaccharides were purified by a cotton‐graphite solid phase extraction method and analysed by HPLC‐MS(MS). 6‐6 parallel samples were measured for both cell lines from separate EV isolations. Results : The most abundant CS disaccharides: the non‐sulfated D0a0, the monosulfated D0a4 and D0a6 (distinguished by tandem MS measurements), and the disulfated D0a10 disaccharides were successfully identified in each sample. Cancer EVs contained on average three times more CS disaccharides than non‐tumour EVs. In terms of relative amounts, the proportion of D0a4 increased and the proportion of D0a6 decreased in A549 EVs. Based on both absolute and relative amounts of the disaccharides, the two groups were well separated in principal component analysis and hierarchical clustering. Summary/Conclusion : In the current study, we performed CS GAG disaccharide analysis of sEVs derived from lung cancer and non‐cancer cell lines. To the best of our knowledge, this is the first study to characterize the glycosaminoglycan profile of EVs, which might be a new area of biological interest. Funding : Support of the Lendület (Momentum) Program of the Hungarian Academy of Sciences and Semmelweis 250+ Excellence PhD Scholarship is acknowledged.

High‐Resolution

Presenter: Aiden M. Jurcenko Johns Hopkins University, Baltimore, Maryland, USA Introduction : Super‐resolution microscopy (SRM) examines subcellular structures at high resolution and can be used to probe the surface and internal features of enveloped particles, including the human immunodeficiency virus (HIV) and extracellular vesicles (EVs). Potentially, SRM can be used to distinguish fully formed, infectious virions of viruses like HIV from non‐viral EVs that may carry a subset of viral proteins or other components, and to monitor the topology of viral markers in and on EPs. Methods : We initially searched the literature to investigate how SRM techniques, such as dSTORM and STED, have been used to study HIV components (i.e., envelope glycoproteins or group‐specific antigen (Gag) proteins) in virion structure and assembly. We then applied dSTORM imaging to examine extracellular particles released from PM1 cells, either uninfected or infected with HIV‐1 (BaL and MN strains) and concentrated by differential ultracentrifugation. Particles were captured by antibodies against CD81 and detected with fluorescent antibodies against CD63 and gp120 in combination with anti‐HIV smFISH probes (Bal or MN). Results : HIV‐infected cells release particles ranging from host EVs without detectable gp120 or viral RNA to fully infectious virions. Similarly, both virions and host EVs can display tetraspanins. Additionally, some particles had an unexpected topology of viral RNA: surface vs internal. Summary/Conclusion : Our results suggest that a combination of multiple labelling techniques, particularly smFISH for RNA and fluorescent antibodies to viral proteins, may be needed to distinguish HIV virions from virus‐like particles/host EVs. Our results also emphasize that tetraspanin capture cannot easily be used to separate host EVs from viruses. Additional SRM studies are needed to examine other features that are common to or distinguish virions and host EVs.

High‐Throughput

Juhwan Park Kookmin University, Seoul, Republic of Korea Introduction : Understanding the heterogeneity of extracellular vesicles (EVs) has a promising potential for disease diagnosis, but current methods for single EV analysis are limited to either low throughput or less multiplexity. Methods : EVs labelled with multiple antibodies conjugated with RCA templates were encapsulated into agarose droplets with RCA mixture. Cleavable chemistry was used to overcome steric hindrance of RCA products (hundreds of nm) over a single EV (∼100 nm). The RCA template is conjugated to antibodies through a disulphide bond that can be cleaved by a reducing agent originally included in the RCA mixture. Once EVs are encapsulated into agarose droplets with RCA mix, cleavage of RCA templates from antibodies and the RCA reaction occur simultaneously. After the RCA reaction followed by agarose gelation, agarose beads were transferred to the aqueous phase, and RCA products within agarose beads were labelled with fluorescent detection probes. Results : To validate the single EV assay, EVs from Mel‐624‐B7H1 cells were labelled with anti‐CD63 antibodies conjugated with an RCA templates and were encapsulated into 0.3% agarose droplets with RCA mixture. By titrating EVs concentration, the number of beads with RCA products decreases. EVs distribution over agarose beads reaches a digital regime by noting that less than 10% of agarose beads showed multiple RCA products whereas more than 90% of agarose beads did not have any RCA products. By analysing >100,000 agarose beads using a flow cytometer, the limit of detection was assessed to be 7 EVs/µL. The effect of the reducing agent was evaluated that enables single EV quantification at the single‐molecule level. To characterize the multiplex single EV assay, EVs were labelled with tetraspanin antibodies with different RCA templates. The positive ratio of each tetraspanin was measured by flow cytometry, which agrees with the EXOVIEW result. Furthermore, the co‐localization of tetraspanin was studied in a single EV level (> 10,000 EVs). We further measured PD‐L1 expression in single EV levels that play a key role in the immune checkpoint of cancer. Summary/Conclusion : We developed a novel method to quantitatively analyse single EVs with multiplexing capabilities, high throughput, and ultra‐sensitivity, both to single molecules on an individual EV and to rare EVs suspended in complex clinical specimens, using an agarose droplet‐based RCA technique.

Laminin‐Binding

Presenter: Mai Quyen Nguyen Paracelsus Medical University, Salzburg, Austria Introduction : Peripheral nerve injuries (PNIs) lead to sensory and motor impairments, with current treatments often causing suboptimal recovery. Schwann cells (SCs) support axonal regeneration via their laminin‐rich basement membrane. Extracellular vesicles (EVs) facilitate intercellular communication and show promise for regenerative medicine. However, precise EV targeting remains a challenge. This study investigates genetically modified laminin‐binding EVs for targeted nerve repair in a rat PNI model with direct and delayed reconstruction. Methods : Stable ASC lines expressing CD81‐LEL fusion proteins were generated using a lentiviral system. EVs were isolated, characterized (size, fluorescence, binding affinity), and tested for SC uptake and pro‐regenerative gene expression. In vivo, male Lewis rats underwent median nerve transection, followed by immediate or delayed (8 weeks) reconstruction using an autologous graft (ANT) or muscle‐vein conduit (MVC). Six groups ( n = 8 each) received either modified EVs, wild‐type EVs, or Vehicle Control (VC). Functional recovery was assessed weekly for 12 weeks using CatWalk gait analysis and Grip Strength tests. Electrophysiology was conducted before euthanasia, and muscle, median nerve, DRGs, and spinal cord were collected for molecular and histological analysis. Results : EV characterization confirmed a 100–150 nm size range and 75% GFP positivity. Fluorescence imaging showed enhanced laminin binding, with EV uptake by SCs. qRT‐PCR revealed increased p75, BDNF, and c‐Fos expression, suggesting a pro‐regenerative effect of modified EVs. Functional tests demonstrated better motor recovery in the ANT + modEVs group compared to controls. While wild‐type EVs also promoted good recovery, vehicle controls exhibited the poorest outcomes. Electrophysiology confirmed enhanced reinnervation in the modified EV group, correlating with improved nerve conduction velocity and muscle response. Post‐mortem analysis revealed that DRG and spinal cord molecular changes mirrored functional test results, with higher neuroregenerative marker expression in the ANT + modEVs group. Additionally, autologous nerve repair significantly outperformed MVC‐based repair, suggesting that the EV therapy was most effective in a structurally optimal regeneration environment. Summary/Conclusion : Laminin‐binding EVs enhance nerve regeneration by promoting SC repair, functional recovery, and reinnervation. Future research will focus on assessing EV homing after intravenous administration in a PNI model to optimize targeted delivery and therapeutic efficacy.

Limch1‐Enriched

Seiko Matsuo 1 , Akira Yokoi 1 , Takafumi Ushida 1 , Kosuke Yoshida 1 , Hironori Suzuki 2 , Masami Kitagawa 1 , Eri Asano‐Inami 1 , Rika Miki 3 , Sho Tano 1 , Kenji Imai 1 , Yusuke Yamamoto 2 , Hiroaki Kajiyama 1 , Tomomi Kotani 1 1 Nagoya University, Japan, 2 National Cancer Center Research Institute, Japan, 3 Nozaki Tokushukai Hospital Attached Research Institute, Japan. Introduction : Preeclampsia (PE) is a major complication of pregnancy, characterized by hypertension and various forms of end‐organ dysfunction. However, its exact pathogenesis remains incompletely understood. This study aimed to investigate a novel pathogenic mechanism related to increased vascular permeability in PE, using LC‐MS/MS analyses and detailed functional assays on serum small extracellular vesicles (sEVs) from women with PE. Methods : We isolated sEVs from the serum of 16 women, including those with early‐onset PE and healthy pregnant controls, using serial ultracentrifugation. The isolated sEVs were then subjected to LC‐MS/MS analysis. To generate sEVs enriched with the target protein, we developed a stable overexpression cell line for the target protein using the JAR choriocarcinoma cell line. The effects of those sEVs were subsequently evaluated on human umbilical vein endothelial cells (HUVECs) and in mice. Additionally, placental immunohistochemistry for the target protein and spatial transcriptomic analysis were conducted. The study protocol was approved by the Institutional Ethics Board of our university. Results : LC‐MS/MS analysis of serum sEVs identified LIMCH1 as an early‐onset PE‐associated EV protein. In the GEO dataset, LIMCH1 expression was significantly upregulated in PE placentas ( p < 0.01). Immunohistochemistry revealed predominant expression of LIMCH1 in syncytiotrophoblast, with a significantly higher H‐score in PE placentas compared to normal placentas ( p < 0.05). Spatial transcriptomic analysis further showed that LIMCH1 expression correlated with PLAP, a marker of placenta‐derived sEVs. Although administration of LIMCH1‐overexpressing JAR‐derived sEVs (LIMCH1‐enriched EVs) had no effect on HUVEC proliferation, the FITC‐dextran permeability assay demonstrated a significant increase in endothelial permeability with the addition of LIMCH1‐enriched EVs ( p < 0.01). Transcriptomic analysis revealed that LIMCH1‐enriched EVs impaired endothelial cell‐cell junction assembly by downregulating TJP1 (ZO‐1) expression. Furthermore, LIMCH1‐enriched EVs increased pulmonary vascular permeability in vivo. While LIMCH1‐enriched EVs promoted lung vascular permeability, they did not affect vascular permeability in the brain. Intravenous injection of sEVs into mice revealed greater uptake in the lungs than in the brain. Summary/Conclusion : We demonstrated EV‐mediated vascular endothelial permeability, a central feature of PE pathology, and provided insights into the underlying mechanism of PE‐related pulmonary edema, which has not yet been fully elucidated.

Next‐Generation

Celia Atalaya 1 , María Mantecón‐Oria 1 , Nazely Diban 1 , Ana Villar 2 , Ane Urtiaga 1 1 University of Cantabria, Santander, Spain; 2 Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), Santander, Spain Introduction : The clinical application of extracellular vesicles (EVs) is limited by the scalability of traditional 2D cell culture methods. Hollow fibre bioreactors (HFBR), as 3D culture systems, support high cell density and increased EV yield, addressing this scalability challenge. However, current commercial HFBR systems lack transparency, limiting cell culture monitoring. This study introduces an advanced HFBR designed for high EV yields and improved visualization of EV‐producing cells. The system incorporates translucent fibres with optimal porosity for EV enrichment. EVs were generated from genetically modified HaCaT cells with enhanced adhesion to fibres composed of 100‐200 nm pore‐polymer. Methods : We generated various polymer hollow fibres (HF) to build a hollow fibre bioreactor (HFBR), each offering different translucency levels and tailored material properties. These HFs were produced using a lab‐scale wet‐dry HF spinning system, and parameters were optimized to extrude fibres from synthetic polymers, including polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), polyethersulfone (PES), and an alginate hydrogel [1]. Porosity was carefully controlled between 100 and 200 nm. HaCaT cells were modified via viral infection with a CAR‐T‐like plasmid encoding a fibroblast activating protein (FAP) antibody variant, enhancing cell membrane adhesion for attachment to HFBR cartridges. Cell colonization on the fibres’ surface was tracked using epifluorescence visualization of the fluorophore‐tagged CAR‐T‐like FAP construct. Extracellular vesicles (EVs) were characterized for size and concentration by nanoparticle tracking analysis (NTA), morphology by transmission electron microscopy (TEM), and fluorescence by confocal microscopy. Results : We observed differences in terms of areas colonized by HaCaT CAR‐T cells and EVs yield among conventional 2D methods, commercial HFBR and transparency custom Laboratory‐engineered microfluidic HFBR. Summary/Conclusion : These findings provided strong evidence for the high potential of Laboratory‐engineered HFBRs and adherent engineered HaCaT cells as an efficient and monitorable system for EV production. This system showed potential to become a high‐efficiency platform for EV production with characteristics similar to biological production. [1] N. Diban, M. J. Rivero, A. Urtiaga, M. Mantecón‐Oria, 10 June, 2024, Spanish patent application P202400038 Fibras huecas poliméricas porosas de alta transmitancia. Funding : MICIU/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR [grant PDC2022‐133704‐I00]. SUBVTC‐2023‐0006 T. Gobierno de Cantabria. C: Fomento de la transferencia de conocimiento en la Comunidad Autónoma de Cantabria

N‐Glycosylation

Mirjam Balbisi 1,2 , Tamás Langó 1 , Virág Horváth 1 , Zoltán Varga 1 , Kinga Ilyés 1 , Gábor Kecskeméti 3 , Zoltán Szabó 3 , Nikolett Nagy 1 , Otília Tóth 1 , Beáta G. Vértessy 1 , Lilla Turiák 1 1 HUN‐REN Research Centre for Natural Sciences, Hungary, 2 Semmelweis University, Hungary, 3 University of Szeged, Hungary Introduction : Post‐translational modifications greatly influence protein function. However, their characterization in extracellular vesicles (EVs) has been limited. N‐glycosylation, essential in processes like cell‐cell interactions, is frequently altered in cancer. This modification occurs at asparagine residues in proteins at consensus sequences. The attached N‐glycans are classified as complex, hybrid, or oligomannose. Our aim was to develop a methodology to analyse the site‐specific N‐glycosylation of glycoproteins of small EVs isolated from A549 lung adenocarcinoma and BEAS‐2B non‐tumourigenic cell lines. Methods : A549 and BEAS‐2B cells were cultured in non‐completed F12 and BEGM medium for 72 h, respectively. Small EVs were isolated by mini‐size exclusion chromatography in 6 6 replicates per cell line. The size distribution of EVs was characterized by microfluidic resistive pulse sensing and shapes by transmission electron microscopy. Small EVs were subjected to freeze‐thaw cycles and digested with trypsin. The peptides obtained were enriched for N‐linked glycopeptides by acetone precipitation and analysed by nanoUHPLC‐MS(MS). Results : Altogether close to 1000 N‐glycopeptides were quantified by GlycReSoft software across the 12 samples. 72 N‐glycopeptides were overrepresented, while 161 were underrepresented in the A549 cell line‐derived sEVs compared to the BEAS‐2B cell line‐derived EVs. Overall, changes in fucosylation, sialylation, and galactosylation were observed. Among the significantly altered N‐glycopeptides, we also identified changes corresponding to vesicular marker proteins. Summary/Conclusion : We have demonstrated that site‐specific N‐glycosylation analysis of glycoproteins present in small EVs is feasible following isolation from cell culture media. Our results lay the basis for future studies to explore the roles of glycosylation of EV proteins in understanding mechanisms behind disease progression, such as lung cancer. Funding : Support of the Lendület (Momentum) Program of the Hungarian Academy of Sciences is acknowledged.

Pcbp2‐Dependent

Presenter: Hou‐Fu Xia Wuhan University, China (People's Republic) Introduction : The epidermal growth factor receptor (EGFR)‐driven angiogenesis is a crucial process observed across various solid malignancies, facilitating tumour progression and therapy resistance, particularly by modulating extracellular vesicles (EVs)‐mediated intercellular communication. Previous studies, including our own, have highlighted EGFR's influence on miRNA production and the pro‐angiogenic effects of EV‐miRNAs. However, the precise mechanism by which EGFR controls miRNA content within EVs remains unclear. Methods : Firstly, cell co‐culture and murine tumour models were used to investigate the effects of EGFR overexpression on the pro‐angiogenic ability and representative cargo composition of oral squamous cell carcinoma‐derived EVs (OSCC‐EVs). We then adopted small‐RNA sequencing to compare the differences in miRNAs carried by OSCC‐EVs with or without EGFR overexpression and conducted functional enrichment analysis and motif analysis on these differential miRNAs. Next, we used miRNA pull‐down experiments to identify candidate molecules involved in the sorting of these characteristic miRNAs. Finally, we validated the role of the candidate sorting protein in promoting the secretion of these characteristic miRNAs to regulate the EGFR‐driven tumour angiogenesis based on tissue specimens, cell lines, murine tumour models and reported data. Results : EGFR overexpression significantly enhances the pro‐angiogenic effects of OSCC‐EVs, accompanied by a marked increase in the content of nucleic acid cargo in OSCC‐EVs. Small‐RNA sequencing identified a group of miRNAs that were significantly enriched in OSCC‐EVs due to EGFR overexpression, which primarily functioned in angiogenesis and shared a characteristic ‘GGGU’ motif. EGFR overexpression significantly enhanced the binding of PCBP2 with ‘GGGU’ motif‐containing miRNAs, thereby increasing their secretion through EVs to play a tumour angiogenesis promoting role. Mechanistically, EGFR overexpression upregulates PCBP2 through transcriptional regulation in OSCC cells. What's more, PCBP2‐depletion impaired the EGFR‐driven tumour angiogenesis by inhibiting the secretion of pro‐angiogenic miRNAs via sEVs. Summary/Conclusion : EGFR overexpression upregulates PCBP2 through transcriptional regulation, followed by enhanced loading of angiogenesis‐associated miRNAs into sEVs by PCBP2 directly recognizing sEV motifs. Funding : This work was supported by the National Natural Science Foundation of China (82101036, 82341023).

Single‐Molecule

John Atanga 1 , Pablo Sánchez‐Martín 2 , Siobhan King 3 , Tobias Gross 2 , Irina Nazarenko 1 1 Faculty of Medicine, Institute for Infection Prevention and Control, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany; 2 Actome GmbH, Freiburg, Germany; 3 ONI UK, Linacre House, Oxford, UK Introduction : Extracellular vesicles (EVs) hold great potential in liquid biopsy applications, especially in disease diagnostics. However, translation of EV‐based findings from the bench to the bed faces significant drawbacks due to a lack of methods that allow for precise EVs quantification and the value of surface biomarkers on EVs. Here, we present a novel method using protein interaction coupling (PICO), enabling precise EV quantification and surface biomarker evaluation on intact EVs at the single‐vesicle level. The PICO assay utilizes oligonucleotide‐labelled antibodies, which are incubated with a sample to form a ternary complex comprising at least two antibodies and a target (e.g., a protein or EV), referred to as a ‘couplex’ in the so‐called ‘binding reaction,’ which will be the reference point for all later quantitative calculations. The assay is entirely homogeneous, requiring no washing or purification steps, as none of the reactants are bound to a solid phase or bead. In order to detect the complexes and antibodies, the oligonucleotide of the antibodies must be amplified and detected using a workflow that is analogous to a digital polymerase chain reaction. Methods : The present study utilised small extracellular vesicles (sEV) isolated from cell‐conditioned media supernatant employing a combination of tangential flow filtration (TFF) and size exclusion chromatography. Results : PICO enables absolute quantification, as validated through single‐molecule specific quantification of EV‐membrane proteins (CD9, CD63, CD81). Additionally, PICO allows for EV subpopulation analysis. This was evaluated using a 3‐plex antibody panel allowing for colocalization analysis as demonstrated by simultaneously quantifying the proportion of sEV harbouring cancer‐specific markers (HER2/HER3, HER2/CD9, HER2/CD81, HER2/CD63 and HER2/CD9/CD63) on tumour‐derived sEV isolated from the C4.2B cell line. We further illustrate PICO clinical applicability by quantifying HER2 and HER3 on single vesicles isolated from blood plasma of prostate cancer patients. Summary/Conclusion : PICO holds tremendous promise for robust, standardized, and precise EV surface biomarker quantification, providing valuable insights into EV‐protein biology and liquid biopsy applications. Funding : Federal Ministry of Education, Germany.

Single‐Particle

Presenter: Rachel Mizenko Northwestern University, Evanston, Illinois, USA Introduction : Fusion of extracellular vesicles (EVs) with liposomes can be used to alter the properties of EVs to enhance their drug delivery properties. However, fusion efficiency is often characterized in bulk, clouding distribution of fusion across heterogeneous EV populations. Here we sought to use orthogonal single‐particle techniques including nanoparticle‐tracking analysis (NTA), resistive‐pulse sensing (RPS), nanoscale flow cytometry, interferometric fluorescence imaging, and laser trapping Raman spectroscopy (LTRS), each with different limitations or biases, to examine the effects of mechanical fusion techniques on physical characteristics and the distribution of fusion across single particles. Methods : EVs were isolated via UC from HEK293Ts, and liposomes were produced via extrusion. An equivalent number, by NTA, were combined and subjected to various fusion methods (incubation, extrusion, sonication, freeze‐thaw) prior to analysis. Results : Here we found that concentration and, to a lesser degree, size were altered differentially across different mechanical fusion techniques. In addition, fusion efficiency varied greatly across analysis techniques, with nanoscale flow cytometry and interferometric fluorescence imaging showing concordant results of freeze‐thaw and sonication having relatively high fusion efficiency. The number of remaining liposomes and EVs varied greatly across mechanical fusion techniques, with freeze‐thaw producing the ‘purest’ engineered population. LTRS identified discordant fusion efficiencies compared with other analysis techniques, which may have been due to its relatively high limit of detection. Summary/Conclusion : Overall, this data shows that not only can single‐particle techniques be used to inform optimization of fusion technique for increasing fusion efficiencies, but also that the ingrained limitations of various equipment can change perceived fusion efficiencies. Funding : This work was funded by the NIH (RPC: R01 CA241666, RRM: F31 NS120590) and used equipment supported by NCI P30CA093373 and R00‐ GM080249 . This work was adapted from RRM's published dissertation, “EV heterogeneity: an essential feature for intercellular communication in health and disease and an underutilized resource in the clinic,” University of California Davis (currently under embargo).

Tumour‐Secreted

Presenter: Hilal Nur Sensoy Vrije Universiteit Amsterdam, Amsterdam, The Netherlands Introduction : Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSCs), though their origin and function remain unexplored. We demonstrate that cancer‐secreted extracellular vesicles (EVs) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function. Methods : The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV‐induced alterations of the MSC transcriptome were analysed and compared with scRNA‐seq data of osteosarcoma and multiple myeloma patient biopsies. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC‐induced resistance in vivo. Results : We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGF‐β signalling inhibition. Importantly, EV‐induced iMSCs faithfully recapitulate the inflammatory single‐cell RNA signature of stromal cells enriched in multiple myeloma and osteosarcoma patient biopsies. Mechanistically, cancer EVs act through two distinct mechanisms. EV‐associated TGFb induces IL6 production, while the EV‐RNA cargo enhances TLR3‐mediated chemokine production. We reveal that simultaneous blockade of downstream EV‐activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC‐induced resistance. Summary/Conclusion : Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as physiological triggers of iMSC development and highlight a promising combination strategy to improve therapy response in bone cancer patients.

Ultra‐Sensitive

Presenter: Arianna Bonizzi University of Milan, Milan, Italy Introduction : Extracellular vesicles (EVs) have gained significant interest as promising liquid biomarkers for breast cancer (BC). However, many aspects of their biological roles remain unexplored due to technical limitations in current EVs isolation and characterization protocols. A critical challenge is identifying markers enriched in cancer‐released EVs to distinguish them from the general EVs population. Cancer‐specific markers are present on EVs at minimal concentrations, underscoring the need for sensitive, user‐friendly methods to unlock their full clinical potential. Methods : Here we leverage single molecule array (SiMoA) technology, enabling ultra‐sensitive biomarker detection, to detect HER2‐positive EVs in BC patients. Two distinct SiMoA immunobeads were developed: magnetic beads functionalized with either an anti‐CD63 antibody or a membrane‐sensing peptide (MSP) with specific affinity for EVs curvature. These approaches were evaluated and compared as strategies for capturing EVs in biological samples, using CD9 and HER2 detector antibodies to differentiate between general and tumour‐specific EVs populations, respectively. Results : The two assays were tested on EVs isolated from patient‐derived organoids (PDOs) lines: one from a healthy sample and the other from a tumour sample. Both assays exhibited a clear dose‐dependent response to the loaded EVs. Importantly, the MSP/HER2 assay demonstrated superior performance compared to the CD63/HER2 assay, effectively distinguishing EVs from the two PDOs lines and revealing significantly higher HER2 expression in tumour‐derived EVs compared to those from the healthy sample. The two detection strategies were also evaluated directly on plasma samples collected from metastatic breast cancer (MBC) patients and healthy controls (HC). The results indicate that the signals obtained with MSP‐functionalized beads were higher than those obtained with CD63‐functionalized beads across HER2 assays. In both assays, general EVs levels appeared lower in MBC patients compared to HC, and HER2‐expressing EVs showed an enrichment trend in MBC patients. Summary/Conclusion : This study demonstrates the feasibility of SiMoA technology for detecting cancer‐released EVs, with its high sensitivity, and highlights how the use of magnetic beads conjugated with MSP can further improve the specificity of the assay. Coupling SiMoA technology with MSP thus offers a rapid and effective method for the detection of cancer‐released EVs in BC patients. Funding : PRIN, EV‐PRINT 2022CS9H53

Vesicle‐Cloaked

Presenter: Danmeng Shuai George Washington University, USA Introduction : Vesicle‐cloaked virus clusters (also known as viral vesicles) are emerging environmental pathogens. Compared to free viruses, viral vesicles are more infectious, environmentally persistent, and resistant to disinfection. Norovirus is the leading cause of worldwide gastroenteritis, and thus the presence and environmental transmission of norovirus vesicles raise significant public health concerns. In this study, we explore the inactivation of rotavirus and norovirus vesicles under ultraviolet light B (UVB) and peroxide treatment, which represents typical wastewater disinfection and natural attenuation processes for controlling the spread of waterborne pathogens. Methods : Norovirus vesicles were isolated from murine norovirus 1 stock via immunomagnetic selection. TIM4 protein has a strong and selective binding towards phosphyltidylserine in the vesicle membrane but it does not bind free noroviruses. Norovirus vesicles were lysed by a non‐detergent lysis buffer to release free viruses. Both norovirus vesicles and free noroviruses were subjected to UVB exposure and peroxide treatment, and viral infectivity loss was quantified via the integrated cell culture‐reverse transcription‐quantitative polymerase chain reaction. The damage to viral biomolecules and lifecycle was determined. Results : Norovirus vesicles were more resistant to UVB disinfection when compared to free noroviruses, especially under a low viral load. This could be due to en bloc transmission of viruses in vesicles, increased multiplicity of infection, and complementary and cooperative effects between viruses for successful infection. Neutral peroxide, i.e., peracetic acid, penetrated through the vesicle lipid membrane and inactivated noroviruses inside the vesicles. In contrast, negatively charged peroxides, i.e., peracetate and peroxymonosulfate, did not inactivate viruses inside vesicles. Both UVB and peroxide damaged vesicle/viral proteins but not viral genomes, and they did not compromise vesicle integrity. UVB oxidized a tyrosine residual in the receptor binding domain of the murine norovirus viral protein 1, resulting in reduced virus binding and infectivity. Peroxides oxidized sulfur‐containing amino acid residuals and impaired vesicle/viral internalization into host cells and corresponding infectivity. Summary/Conclusion : Our study discovered that norovirus vesicles were more resistant to UVB and peroxide disinfection in wastewater treatment and natural attenuation in surface water than free viruses. It advocates for future advancement of water disinfection strategies to protect public health. Funding : NSF and USDA‐NIFA

Bacteria‐Derived

Presenter: Marco Catania University of Siena / University of Catania, Italy Introduction : Bacterial Extracellular Vesicles (bEVs) are membranous structures released by both gram‐negative and gram‐positive bacteria. bEVs serve as an additional means of communication with host cells and other bacteria. They play a critical role in host‐pathogen interactions, transferring resistance and virulence factors and directly inducing host immune responses. This is relevant given the global health challenge posed by infections from antibiotic‐resistant bacteria, with resistant Enterococcus strains greatly contributing to mortality rates. Gaining insight into these novel mechanisms of interaction is thus essential for developing new therapeutic strategies and enhancing infection management. Methods : bEVs were isolated from three Enterococcus strains: E. faecalis vancomycin‐susceptible (VSE), E. faecalis vancomycin‐resistant (VRE), and E. faecium from clinical samples. Strains were inoculated in DMEM, grown overnight at 37°C and harvested upon reaching the early exponential phase, at approximately 18 h. Cultures were centrifuged at 8000 × g for 10 min, filtered (0.22 µm), and ultracentrifuged at 100,000 × g for 75 min. The pellets were washed in PBS and ultracentrifuged again at the same speed. Next, bEVs were analysed for size and concentration via Dynamic Light Scattering and Nanoparticle Tracking Analysis. DNA from bEVs was then isolated, quantified, and sequenced using Illumina MiSeq. Results : The analysis of size and concentration indicated significant quantitative differences in bEVs among the strains. Clinical samples and VSE produced more bEVs than VRE, and these vesicles were significantly larger than VRE‐bEVs. Notably, vesicles from clinical sample contained the highest DNA levels, while VRE‐bEVs, despite their lower abundance, held more DNA than VSE‐bEVs. Interestingly, DNA sequencing results revealed a concordance between the resistome and virulome of clinical strain and VRE cells with their respective vesicles. Summary/Conclusion : The differences among Enterococci in terms of size and concentration suggest that bEVs production is influenced by their strain of origin. Differences in DNA quantification and the concordance between cells and vesicles resistome and virulome suggest a possible involvement of bEVs in infection and the existence of specific cargo loading mechanisms. The potential role of bEVs in host‐pathogen interactions will be further investigated by functional assays in co‐culture models integrated with omics analysis.

Exercise‐Induced

Reine Khoury 1,2 , Dariusz Żurawek 2 , Georgia Kruck 1,2 , Sierra Codeluppi 2 , Minh Nguyen 2 , Corina Nagy 2,3 1 Integrated Program in Neuroscience, McGill University, Montreal, Québec, Canada; 2 McGill Group for Suicide Studies, Douglas Mental Health University Institute, Verdun, Québec, Canada; 3 Department of Psychiatry, McGill University, Montreal, Québec, Canada Introduction : Exercise is a crucial non‐pharmacological intervention for addressing mental health conditions like major depressive disorder (MDD), with known benefits for brain function, including enhanced plasticity and neuroprotection. Recent research highlights the brain‐muscle and brain‐bone axes, suggesting that exercise influences not only local adaptations but also systemic effects, including interneuronal communication. While the molecular mechanisms remain unclear, small extracellular vesicles (sEVs), released during exercise, are emerging as potential mediators of these effects. Given the role of miRNAs in regulating various diseases, including depression, this study explores the role of exercise‐induced sEVs and their miRNA content in mitigating MDD symptoms. Methods : The first objective is to characterize changes in sEV size, concentration, and molecular content (miRNA and proteins) in different tissues following an aerobic exercise regimen in mice. We conducted a 2‐week study with young‐adult mice undergoing treadmill running to examine the release of sEVs from plasma, bone marrow, and skeletal muscle. sEVs were collected and analysed for size, quantity, and molecular cargo, particularly focusing on miRNAs. The second objective is to assess the antidepressant effects of exercise‐induced sEVs by examining their impact on stress‐susceptible mice after treadmill exercise and chronic stress exposure. Results : The study revealed that exercise significantly increased the release of skeletal muscle‐derived sEVs in both male and female mice, with a transient spike that declined hours post‐exercise. These vesicles were consistent in size across groups. miRNA analysis showed that miR‐133b‐3p and miR‐199a‐5p were upregulated post‐exercise, while miR‐21a‐5p and miR‐200a‐3p were downregulated, among others. Notably, miR‐133b‐3p's upregulation points to its involvement in muscle adaptation, synaptic plasticity, neuronal communication, and cardiovascular function, emphasizing the broader physiological and neuroprotective benefits of exercise. Summary/Conclusion : These findings suggest that exercise‐induced sEVs from peripheral tissues like skeletal muscle may have systemic effects, including influencing neuronal communication. The enrichment of synaptic‐related pathways in miR‐133b‐3p targets points to the role of muscle‐derived EVs in muscle‐nervous system cross‐talk, supporting evidence of exercise's therapeutic potential via tissue‐derived EVs.

First‐In‐Human

Presenter: Matthias Krause University Hospital Salzburg, Austria Introduction : Spina bifida is a congenital neural tube defect that has a high risk of secondary neurological deterioration due to tethering of the spinal cord. We present the case of a child with spina bifida who underwent spinal cord release surgery accompanied by human umbilical cord‐derived mesenchymal stromal cell‐derived extracellular vesicle (UC‐MSC‐EV) therapy for the first time in a human. Here, we discuss the surgical procedure and post‐operative outcome and highlight the potential benefits of extracellular vesicle therapy in the management of spina bifida. The administration of extracellular vesicles containing potentially therapeutically active agents has emerged as a potential new treatment modality for neurological disorders. By direct intrathecal application during surgery, UC‐MSC‐EVs can deliver therapeutic payloads to target cells and the extracellular environment, offering a novel approach to neuroprotection and tissue repair. Methods : Case presentation—a 2‐year‐old female child with a diagnosis of spina bifida presented with progressive syringomyelia as a sign of secondary tethered cord syndrome with an intramedullary dermoid inclusion tumour after postnatal spina bifida repair. After thorough pre‐operative assessment and multidisciplinary consultation, it was decided to proceed with spinal cord release surgery combined with extracellular vesicle application. During the surgical procedure, the tethered spinal cord was released, and the dermoid as well as lipoma tissue were completely resected using intraoperative neurophysiologic monitoring to minimize trauma to neural structures. Concurrently, UC‐MSC‐EVs were administered directly onto the released and neurulated placode and the spinal cord. Results : The postoperative MRI demonstrated a good de‐tethering effect, no postoperative medullary oedema, and diminished syringomyelia. No adverse events were reported. The neurological deficit remained stable at the pre‐existing level. Summary/Conclusion : Intraoperative application of UC‐MSC‐EVs might be a feasible option to ameliorate intrathecal scarring and neurodegeneration following spina bifida surgery. Whether EVs will result in significant effects for the long‐term neurological outcome needs to be studied thoroughly in randomized controlled clinical trials. EV therapy might open new horizons to prenatal and postnatal spina bifida therapy if UC‐MSC‐EVs can efficiently diminish spinal cord tethering and neurodegeneration for the many severely handicapped children and thus reduce the burden of disease for the patients and their families.

High‐Dimensional

Daniel Bachurski 1 , Rahil Gholamipoorfard 1 , Yong Jia Bu 2 , Patrick Hölker 1 , Lisa Wessendorf 1 , Hendrik Jestrabek 1 , David Stahl 1 , Philipp Gödel 1 , Felix Gaedke 1 , Elias Ranjbari 3 , Selen Seyhan 4 , Ulrike Resch 5 , Luisa Marie Schmidt 1,6 , Tobias Tertel 7 , France Rose 1 , Cláudio Pinheiro 8 , Maribel L. Corona 9 , Alexander F. vom Stein 1 , Phuong‐Hien Nguyen 1 , Katrin Reiners 10 , An Hendrix 8 , Guillaume van Niel 9 , Marcus Krüger 1 , Katarzyna Bozek 1 , Lydia Meder 11 , Per Malmberg 3 , Paula Cramer 1 , Barbara Eichhorst 1 , Roland Ullrich 1 , Astrid Schauss 1 , Christian Pallasch 1 , Paul J. Bröckelmann 1,12 , Ron D. Jachimowicz 1,12 , Christian Preusser 13 , Elke Pogge von Strandmann 13 , Bernd Giebel 7 , Mark Nitz 2 , Michael Hallek 1 , Nima Abedpour 1 1 Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; 2 University of Toronto, Toronto, Canada; 3 University of Gothenburg, Gothenburg, Sweden; 4 Dokuz Eylül University, İzmir, Türkiye; 5 University of Vienna, Vienna, Austria; 6 University of Copenhagen, Copenhagen, Denmark; 7 University of Duisburg‐Essen, Essen, Germany; 8 Univesity of Ghent, Ghent, Belgium; 9 Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, van Niel's Team, Paris, France; 10 University of Bonn, Bonn, Germany; 11 University of Erlangen‐Nürnberg, Erlangen, Germany; 12 Max Planck Institute for Biology of Ageing, Cologne, Germany; 13 Philipps University Marburg, Marburg, Germany Daniel Bachurski, Rahil Gholamipoorfard and Yong Jia Bu are co‐first authors . Mark Nitz, Michael Hallek and Nima Abedpour are co‐senior authors . Daniel Bachurski is corresponding author . Introduction : EVs play a critical role in intercellular communication within the tumour immune microenvironment (TIME), emerging as crucial in immunomodulation. However, due to methodological limitations—such as reliance on bulk EV methods and the lack of routine implementation of single‐cell (sc) techniques—the bioimmune distribution and effects of EVs on individual recipient cells remain insufficiently understood. Methods : To address this gap and by considering the MISEV guidelines, we developed TeLEV. Our metabolic mass‐tag labelling approach using monoisotopic L‐tellurienylalanine (TePhe) to label cellular secretomes enables high‐dimensional sc analysis of EV recipient cells by (imaging) mass cytometry and nanoSIMS. Using TeLEV, we analysed the uptake and effects of the EV proteome and soluble protein fractions at the sc level in 14 million immune cells, examining EV uptake dynamics over 48 h, dose‐response relationships, and differential SEC fraction uptake. By this, we provided an immune atlas of EV uptake in 50 immune cell populations and subpopulations, comparing EVs from six primary, treatment‐naïve chronic lymphocytic leukaemia (CLL) cells purified from peripheral blood with those from nine cell lines. The findings were validated by analysing 6 million single cells from 33 CLL patient samples. Results : We discovered that primary CLL‐TeLEVs were predominantly taken up by classical, transitional, and non‐classical monocytes and myeloid dendritic cells in a CD11c‐ and BTK‐dependent manner, leading to immune alterations in T cell effector memory and NK cells. Healthy donor myeloid cells showed a specific CD25, IL‐3R, and IL‐7R expression signature only when exposed to primary CLL and Ramos cell line TeLEVs, demonstrating the specific functional role of malignant B cell‐derived EVs compared to different EV sources. Notably, CLL‐TeLEVs induced BCL‐2 expression in immune recipient cells, promoting myeloid cell selection of the CLL TIME. Finally, we employed integrative sc multi‐omics—CITE‐seq and CyTOF—to confirm the EV‐dependent selection mechanism in CLL. Summary/Conclusion : In conclusion, we developed TeLEV to enable comprehensive sc immune uptake studies of EVs from primary samples and cell lines. Applying TeLEV, we uncovered a novel CLL‐EV‐driven immune selection mechanism, underscoring the role of EVs as global mediators of immune cell communication.

Melanoma‐Derived

Raphaela Rebeca Silveira Assunção, Milena Perez Mak, Roger Chammas, Luciana Nogueira de Sousa Andrade Universidade de São Paulo, São Paulo, Brazil Introduction : Melanoma skin cancer is the 17th most incident tumour worldwide and there is still a high rate of non‐response to treatment. Extracellular vesicles (EVs) stand out as a potential biomarker for cancer prognosis; nonetheless, it has been shown that melanoma‐derived EVs influence immune cell response. In this study we aimed to evaluate the impact of EVs from human melanoma cells on proliferation, death and gene expression of monocytes, important players in cancer development. Methods : EVs from BRAF‐mutated primary and metastatic melanoma cell lines A375 and SKMel‐28, respectively, were isolated by differential ultracentrifugation. Plasma EVs from melanoma patients were isolated by ultracentrifugation and size‐exclusion chromatography columns after informed consent was obtained (ethical committee approval number 51680021.1.0000.0068). EV's mean concentration and size were determined by the nanoparticle tracking system (NTA) and electronic microscopy, and CD9, CD63 or CD81 were detected by western blotting. THP‐1 human monocytes were treated with EVs for 24 and 48 h, and cell proliferation was determined by counting, cell death by flow cytometry after propidium iodide staining and gene expression by RT‐qPCR using specific primers for VEGF‐A, IL‐1b, HLA‐DRA, CCL2 and MCL‐1. Statistical analyses were performed by one way ANOVA followed by Tukey. Results : EVs (10 10 EVs/mL) from A375 primary melanoma cells inhibited monocyte proliferation and downregulated VEGF‐A and MCL‐1 and upregulated CCL2, HLA‐DRA and IL‐1b genes. Conversely, EVs from SKMel28 metastatic melanoma cells upregulated all mentioned genes without alteration on monocyte proliferation. Concerning circulating EVs from melanoma patients, we noticed two different EVs populations characterized as large and small EVs based on NTA analysis, and their effect on monocyte phenotype is being investigated. Summary/Conclusion : EVs from melanoma cells modify monocyte phenotype towards a pro‐inflammatory and anti‐tumoural phenotype through the induction of the transcriptional levels of certain inflammatory genes. However, EVs from metastatic disease were also able to induce an increase in VEGF‐A levels, which could favour melanoma progression. Future functional assays will be conducted to better clarify the impact of these EVs on monocyte activity and tumour progression using EVs from metastatic and non‐metastatic melanoma patients. Funding : This study was supported by the FAPESP (grant number 2023/16355‐4).

Multi‐Parametric

Elena Scurti 1 , Johanna Puutio 1 , Martina Hanzlikova 1 , Iida Kähärä 2 , Elina Vuorimaa‐Laukkanen 2 , Saara Laitinen 3 , Pia Siljander 1 , Tapani Viitala 1,4 1 University of Helsinki, Helsinki, Finland, 2 Tampere University, Tampere, Finland; 3 Finnish Red Cross Blood Service, Helsinki, Finland; 4 Åbo Akademi University, Turku, Finland Introduction : The multi‐parametric surface plasmon resonance (MP‐SPR) technique is a versatile optical real‐time label‐free detection technique that can be adapted into several different sensing platforms for characterizing the size and amount of extracellular vesicles (EVs), as well as for monitoring the interaction kinetics of EVs with cells and induction kinetics of EVs. Here we use platelet derived EVs (PLT EVs) to demonstrate these unique analysis platforms. Methods : For the determination of particle size and concentration of differently prepared PLT EVs the MP‐SPR sensors were functionalized with biotinylated CD 41 for specific capture of PLT EVs. A two‐wavelength MP‐SPR analysis approach was used to determine the particle size and concentration of the captured EVs. Real‐time cell interactions of fluorescently labelled and non‐labelled PLT EVs were performed by seeding HeLa cells on fibronectin coated MP‐SPR sensors. Monitoring the induction of EVs from PLTs was achieved by immobilizing PLTs on MP‐SPR sensors and injecting an ionophore to stimulate PLT EV formation while simultaneously monitoring the MP‐SPR signal response in real time. Results : Ionophore induced PLT EVs were efficiently captured by CD 41, while PLT EVs prepared by ultracentrifuging the supernatant of PLT preparations contained impurities that did not allow to efficiently capture EVs for further MP‐SPR analysis. The size of the captured ionophore induced PLT EVs determined through the MP‐SPR analysis were 76 ± 16 nm and the concentration ranged between 4 and 40 µg/mL depending on the particle amount used in the measurements. Cell interactions of non‐labelled PLT EVs with HeLa cells showed concentration dependent MP‐SPR responses, and labelling of the EVs did not significantly affect their interaction with HeLa cells. Stimulation of PLTs immobilized on MP‐SPR sensors with an ionophore induced clear MP‐SPR signal changes that suggest that MP‐SPR could be used to monitor EV formation kinetics in real‐time without using labels. Summary/Conclusion : We envision that the MP‐SPR‐based analysis platforms presented here have a great potential to provide unprecedented information about EVs that is not accessible by any of the currently widely used methodologies in EV research. Funding : Business Finland, Academy of Finland, The Finnish Research Impact Foundation and Blood Service Research Fund.

Neurone‐Targeted

Jisook Moon 1 , Jae Hyun Park 1 , Chul‐Woo Lim 1 , Jinwoo Hawng 1 , Soo‐Hyeon Nam 1 , Tae Hee Han 1 1 Department of Biotechnology, CHA University, South Korea Jisook Moon and Jae Hyun Park contributed equally to this study. Jisook Moon is the corresponding author . Introduction : Early diagnosis and effective treatment of neurodegenerative diseases remain challenging due to the difficulty of capturing brain‐specific molecules in the blood. Amyloid precursor‐like protein 1 (APLP1), a membrane protein highly expressed in the brain, has emerged as a promising biomarker for isolating brain‐derived extracellular vesicles (EVs). However, differentiating APLP1 from homologous proteins like APLP2 and APP770 and developing targeted delivery systems have posed significant obstacles. Methods : This study addresses these challenges by designing APLP1‐specific peptide sequences that bind to its extracellular domain, enabling selective brain targeting. We developed a nanoparticle‐EV complex by integrating these peptides, creating a robust platform for precise delivery to neural tissues and brain regions. By analysing structural differences within the APLP family, we optimized two peptide sequences that specifically recognize APLP1. These sequences were conjugated onto EVs to enhance targeting efficacy, while the integration of nanoparticles allows for advanced applications such as controlled delivery and imaging. Results : The resulting Nanoparticle‐EV complex demonstrated high selectivity and efficiency in binding to APLP1‐expressing areas, validating its use in brain‐targeted delivery. Importantly, APLP1+ EVs isolated from human plasma were enriched with neuronal markers and neurodegenerative disease‐related proteins, including those implicated in Alzheimer's and Parkinson's diseases. Small RNA profiles further confirmed brain‐specific signatures, with putative miRNA targets highly expressed in brain tissues. Summary/Conclusion : This innovative approach, which combines APLP1‐targeted peptides with nanoparticles and EVs, establishes a foundational platform for the early diagnosis and treatment of neurodegenerative diseases. It provides an efficient and precise system for brain‐targeted delivery, offering significant advancements in neurotherapeutics and diagnostics. Funding : This work was supported by the Korea government (MSIT) (RS‐2024‐00462182) and a grant (C390000) from the Korea Basic Science Institute and the Korean Fund for Regenerative Medicine funded by the Ministry of Science and ICT and the Ministry of Health and Welfare (RS‐2022‐00070658, 22C0617L1‐12).

Phonups—Plotting

Bence Mélykúti, Gonzalo Bustos‐Quevedo, Tony Prinz, Irina Nazarenko Institute for Infection Prevention and Control, Medical Center—University of Freiburg, Freiburg, Germany Introduction : The processing of size and zeta potential measurements of extracellular vesicles is plagued by the lack of an output standard shared by the measuring instruments. We envision software which can process measurement data in a high‐throughput fashion. It should compute basic statistics and should plot histograms in a standard but also adjustable way. It should be extensible with the data formats of additional measuring instruments. Methods : We release the free/libre and open‐source software (FLOSS) PHoNUPS which can read multiple measurement data formats, which computes simple statistics of the input data and plots their histograms. The program PHoNUPS is in the programming language R. Results : PHoNUPS is currently able to read data from the nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) instruments of a certain German manufacturer. PHoNUPS displays the statistics that the instruments do: total concentration, and mean, median, mode/peak of the particle size distribution. It processes multiple measurements in one run, potentially from different instruments. It plots their histograms both in one chart and in separate subcharts in a joint plot. The plots can be shown on screen, or they can be saved in a range of vector and raster graphics formats, such as .svg and .png. The vector graphics output enables further graphical editing in batch for the user, even for disparate input instruments. Summary/Conclusion : PHoNUPS is made with extensibility in mind, and we call on the community to contribute the capability to parse further input formats. Funding : This development was funded by the German Federal Ministry of Education and Research.

Platelet‐Derived

Presenter: Mandy Chan STEMCELL Technologies, Vancouver, Canada Introduction : Platelet‐derived extracellular vesicles (PLT‐EVs) are the most abundant EV subtype in blood and are often further elevated during illnesses. The increased level of PLT‐EVs is a double‐edged sword as PLT‐EVs may contain valuable cargoes for immunological functions (e.g., coagulation or inflammation), but their high abundance can mask the signals from other EV subtypes and hinders biomarker discovery. In this study, we developed new immunomagnetic isolation methods (EasySep) to either positively select or deplete CD61+ PLT‐EVs. Methods : Plasma was obtained from blood collected in acid‐citrate‐dextrose tubes. Bulk plasma EVs were obtained using differential ultracentrifugation (dUC‐EVs). Starting with either untreated plasma or dUC‐EVs, PLT‐EVs were labelled with anti‐CD61 antibody complexes and magnetic particles for positive selection or depletion. The tube containing the labelled sample was then placed in an EasySep magnet. Magnetically labelled PLT‐EVs were retained on the tube wall, while the fraction containing PLT‐EV depleted plasma or dUC‐EVs was poured off into a new tube. To assess depletion efficiency, pan‐EVs were isolated from PLT‐EV depleted or untreated plasma using immunomagnetic isolation targeting CD9, CD63, and CD81 or size exclusion chromatography (SEC). PLT‐EV recovery and depletion were assessed by western blotting. Results : CD61+ EVs that were isolated by positive selection had the expected PLT‐EV phenotype: CD61+ (platelet marker), CD63+ and CD9+ (common EV markers), and CD81‐ (non‐PLT EV marker). The depletion method efficiently removed CD61+ EVs from plasma (94.7% ± 3.5%, N = 17) and dUC‐EVs (95.7% ± 4.9%, N = 10). All PLT‐EV‐depleted and control samples had similar non‐PLT EV recoveries. In preliminary experiments, the signal‐to‐noise ratio (non‐PLT‐EV:PLT‐EV) increased from 0.2 to 4.4 after CD61 depletion when 1.3 × 10 10 depleted or control EVs were analysed. Pilot data demonstrated the feasibility of performing both depletion and positive selection from the same plasma sample. Summary/Conclusion : Immunomagnetic isolation or depletion of PLT‐EVs can easily fit into common EV isolation workflows. PLT‐EV positive selection allows researchers to specifically analyse PLT‐EVs in diagnostic applications, while PLT‐EV depletion improves signal‐to‐noise ratio for biomarker discovery and functional characterization of EVs.

Tanycyte‐Derived

Presenter: Rafik Dali University of Lausanne, Switzerland Introduction : Energy balance requires precise communication between the periphery and the central nervous system. In the hypothalamus, tanycytes—specialized ependymoglial cells—line the walls and floor of the third ventricle and extend processes into the brain parenchyma. Thanks to their strategic location, tanycytes integrate peripheral metabolic signals and modulate neuronal function accordingly. However, the mechanisms by which tanycytes communicate with neural cells remain largely unknown. Methods : Our study investigates Annexin A1 (ANXA1) as a tanycyte‐derived signalling molecule involved in neural modulation in response to energy imbalance. We analysed ANXA1 expression and localization along the third ventricle in vivo and in vitro, revealing its colocalization with the CD9 marker in the presence of glucose. Proteomic analysis of isolated tanycyte‐derived extracellular vesicles confirmed the presence of ANXA1 among classical vesicular proteins. Using publicly available single‐cell and single‐nucleus RNA sequencing data, we identified microglia and neuronees as potential targets of tanycyte‐derived ANXA1. Results : Our findings show that ANXA1 is primarily expressed by dorsal tanycytes and classical ependymal cells, and its expression and localization are regulated by energy imbalance. In vitro, ANXA1 colocalizes with CD9 in the presence of glucose, suggesting its secretion via extracellular vesicles during positive energy balance. Functional experiments demonstrate that tanycyte‐derived ANXA1 modulates microglial number and morphology, as well as the activation of SF1‐expressing neuronees. These neural modifications ultimately influence brown adipose tissue thermogenesis in response to feeding. Summary/Conclusion : Our study highlights a tripartite communication between tanycytes, microglia, and neuronees to regulate energy balance. These findings represent a first step toward understanding the complex hypothalamic networks involved in metabolic control.

Adipocyte‐Derived

Presenter: Xilal Y. Rima The Ohio State University, Columbus, Ohio, USA Introduction : The prevalence of obesity is continuously rising, with half the global population predicted to be overweight or obese by 2035. Insulin resistance (IR), atherosclerosis, and metabolic‐dysfunction‐associated steatohepatitis (MASH) have paralleled the rising trend in obesity. The adipocyte, the dominant cell in adipose tissue (AT), performs innate and adaptive immune functions by releasing adipokines and contributing to antigen presentation. However, the mode by which the adipocyte affects distant organs remains unclear. Extracellular vesicles (EVs) are key mediators of interorgan communication, which harbour microRNAs (miRNAs). 65% of circulating EV‐encapsulated miRNAs are derived from the adipocyte. Therefore, we hypothesize that adipocyte‐derived EVs (AdEVs) affect obesity‐associated comorbidities. Methods : AdEVs were collected from the visceral AT (VAT) of lean and obese mice and lean and obese bariatric patients who voluntarily participated under informed consent. AdEVs were characterized using transmission electron microscopy, tunable resistive pulse sensing, western blot, and single‐EV phenotyping. Mice modelling the metabolic syndrome, middle‐aged Ldlr − / − mice given a western high‐fat diet, were retro‐orbitally injected with AdEVs from lean and obese mice. Insulin tolerance tests, en‐face aortae analyses, and histology were conducted to measure IR, atherosclerotic extent, and MASH. In vitro experiments were performed to elucidate human disease. Lastly, miRNA sequencing was executed to uncover candidates for disease progression. Results : Mice administered AdEVs from obese mice had increased IR ( p < 0.05), accelerated atherosclerosis by ∼50% ( p < 0.0001), and higher MASH activity scores ( p < 0.01) than mice delivered AdEVs from lean mice and saline injections, which were comparable. Human AdEVs were internalized by macrophages and trafficked intracellularly into endosomal/lysosomal compartments. AdEVs from obese patients induced greater pro‐inflammatory responses in recipient macrophages ( p < 0.01) compared to AdEVs from lean patients and saline injections. AdEVs from lean and obese patients differentially packaged miRNAs that may have direct effects on the obesity‐associated comorbidities accelerated by AdEVs from obese AT. Summary/Conclusion : Our results offer insight into the role of adipocyte interorgan signalling in the pathophysiology of IR, atherosclerosis, and MASH, which are major complications of obesity. Furthermore, we have identified therapeutic targets that may alleviate these diseases and benefit an increasing patient population.

Anti‐Inflammatory

Slavomira Gulova 1 , Alexander Otahal 2 , Karina Kramer 2 , Markus Rothammer 2 , Zsombor Lacza 3 , Denisa Harvanova 1 , Stefan Nehrer 2 , Andrea De Luna 2 1 Associated Tissue Bank, Faculty of Medicine, P. J. Safarik University and L. Pasteur University Hospital in Kosice, Kosice, Slovakia; 2 Center for Regenerative Medicine, Department for Health Sciences, Medicine and Research, University for Continuing Education Krems, Krems, Austria; 3 Department of Sport Physiology, University of Physical Education, Inst. Clinical Experimental Research, Semmelweis University, Budapest, Hungary Introduction : Osteoarthritis (OA) is the most common form of degenerative joint disease in adults. Effective OA treatment should confer regenerative and immunomodulatory properties. The therapeutic effect of mesenchymal stem cells (MSCs) is mediated through secretome rich in soluble factors and extracellular vesicles (EVs). The aim of this study was to determine how Hoffa's fat pad MSC (HFP‐MSC)‐derived EVs could be applied as a cell‐free therapy for OA. Methods : Primary HFP‐MSCs were isolated from Hoffa tissue of 3 patients undergoing total knee replacement after informed consent and ethical approval. HFP‐MSCs were cultured in vertical wheel bioreactors on microcarriers in xeno‐free media supplemented with human blood products (citrate‐anticoagulated platelet‐rich plasma or hyperacute serum). EVs were isolated from serum‐free supernatant via ultrafiltration (100 kDa cut‐off) and characterized via nanoparticle tracking analysis (NTA) in scatter and fluorescence mode. Western blot was used to assay the presence of EV marker proteins. To assess the therapeutic potential of HFP‐MSC‐EVs, patient‐derived chondrocytes or synovial fibroblasts were co‐cultured with M1 macrophages in an inflammation model. Treatment effects of HFP‐MSC EVs were determined via gene expression analysis using RT‐qPCR. Cytokine release was assayed via ELISA. Uptake of HFP‐MSC‐EVs was analysed via fluorescent labelling and confocal microscopy. Results : CD73 and CD9 were colocalised on HFP‐MSC‐EVs, indicating their MSC origin. Labelled HFP‐MSC‐EVs were internalised by both cell types. RT‐qPCR analysis showed that HFP‐MSC‐EVs decreased the expression of inflammatory genes (interleukin‐6, interleukin‐8, cyclooxygenase‐2 (COX‐2), chemokine (C‐C motif) ligand 2 (CCL‐2) and 5 (CCL‐5)) in synovial fibroblasts and catabolic enzyme genes (matrix‐metalloproteinases 3 (MMP‐3) and 13 (MMP‐13)) in chondrocytes. HFP‐MSC‐EVs modulated the release of IL‐6, TNF‐α, IL‐4 and IL‐10 by chondrocytes and synovial fibroblasts. Summary/Conclusion : HFP‐MSCs cultured in vertical wheel bioreactors on microcarriers in xeno‐free conditions yield EVs capable of modulating inflammatory and catabolic gene expression in cells central to OA progression. Owing to their potential disease‐modifying effects, HFP‐MSC‐EVs offer a promising cell‐free therapy approach for OA. Funding : BMBWF (No. SK11/2024), VUaVP35 UPJS (No. vvgs‐2023‐2730), APVV (No. SK‐AT‐23‐0005).

Cancer‐Associated

Presenter: Kazuki Oshima Keio University, Japan Introduction : Recent advances in RNA sequencing technology revealed the presence of circulating RNA fragments that are not derived from the human genome (Max et al. PNAS 115: E5334, 2018). Since bacterial RNAs are encapsulated in EVs released from bacteria (bacterial EV; bEV), we investigated whether bacterial RNAs can be identified in human blood and their levels can be associated with the host disease condition, such as cancer. Methods : Serum samples obtained from cancer patients ( n = 1406) and non‐cancer individuals ( n = 383) were prospectively collected and subjected to small RNA‐seq analysis. The obtained data were mapped to the genomes of bacteria reported to be associated with cancer. We collected bEVs released from the identified bacteria by ultracentrifugation of bacterial culture supernatants followed by sucrose density gradient centrifugation. Collected bEVs were then administered into the tail vein of STAM mice, a MASH‐based hepatocellular carcinoma (HCC) model. Functional analysis of the identified bacterial RNAs was performed using an immortalized human hepatic stellate cell line (LX‐2). Results : We identified transfer RNA‐derived small RNAs (tsRNAs) derived from bacteria, the levels of which were associated with the presence of HCC with AUROC > 0.75 compared to non‐cancer individuals. We focused on the tsRNAs from Klebsiella pneumoniae ( K. pneumoniae ), which were detectable in bEVs purified from K. pneumoniae culture supernatants (Kp‐bEVs). Tail vein administration of Kp‐bEVs to STAM mice resulted in increased tumour growth in the liver and increased expression of the inflammatory cytokines, such as IL‐1β and IL‐6. The expressions of these cytokines were increased by transfection of K. pneumoniae tsRNAs into LX‐2. Summary/Conclusion : Several bacterial tsRNAs were found in the human blood and associated with HCC. The bEVs released from K. pneumoniae accelerated liver carcinogenesis, which can be explained by the increased expression of the inflammatory cytokines in hepatic stellate cells. Thus, measuring bacterial tsRNAs could be useful for predicting the risk of HCC.

Context‐Dependent

Grace Richmond, Rose Nguyen, Alanna Sedgwick, Jeff Schorey, Crislyn D'Souza‐Schorey University of Notre Dame, USA Introduction : Extracellular vesicles (EVs) have emerged as an important conduit for intercellular communication within the tumour ecosystem. Different types of EVs have been identified based on cellular origin and biogenesis mechanisms and may be classified as ‘large EVs’ (> 200 nm) or ‘small EVs’ (< 200 nm). Angiogenesis, a key cellular process, is co‐opted during tumour growth and development and is markedly impacted by EV‐based paracrine signalling. Here we show that large EVs (lEVs) derived from highly invasive tumour cell lines promote pro‐angiogenic behaviours in endothelial cells through mechanisms that are distinct from those documented for small EVs (sEVs) or soluble growth factors. Methods : Tumour EV subtypes were assessed for their effects on endothelial cells using tube‐forming assays. To this end, large and small EVs shed from tumour cell conditioned media were isolated and characterized for size and morphology per MISEV guidelines. Endothelial cytokine expression and release were determined by cytokine arrays and validated by qRT‐PCR and ELISA. EV angiogenic cargo was further studied via immunofluorescence microscopy, protein biochemistry and western blotting. Results : Our findings reveal that lEVs from highly invasive tumour cell lines promote angiogenesis via unique mechanisms that are distinct from those reported for other EV subtypes. In response to lEV uptake, endothelial cells exhibit increased induction of pro‐angiogenic phenotypes, including increased tubulation and branching, altered cytokine expression and increased autocrine signalling by pro‐angiogenic factors. Our study also highlights differences in pro‐angiogenic cargos in tumour EV subtypes. Summary/Conclusion : We suggest that the effects of EVs on angiogenesis are likely tumour‐type and EV subtype dependent. Of particular bearing to the findings described is that lEV production appears to be usurped as tumour cells transition to metastatic and more aggressive ‘amoeboid’ phenotypes. Funding : This work was supported by NIH 1R01GM148708, NIH 1R03CA273598‐01, and the Boler Foundation.

Fourier‐Transform

Martyna Durak‐Kozica 1,2 , Andrzej Wróbel 1,2 , Ewa Ł Stepień 1,2 , 1 Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland; 2 Center for Theranostics, Jagiellonian University, Kraków, Poland Introduction : A 2016 World Health Organization report on diabetes states that over 400 million people worldwide have diabetes mellitus (DM). Recent studies indicate that extracellular vesicles (EVs) act as new bio‐mediators in T2D. Research suggests that hyperglycaemia triggers increased secretion of EVs, with elevated levels of endothelial EVs observed in individuals with pre‐diabetes compared to those with normal glucose tolerance. In vitro studies further show that high glucose levels enhance EV release compared to normal glucose concentrations. However, there is limited experimental data on how high glucose impacts vesicular transport in insulin‐secreting pancreatic β‐cells. Methods : 1.1B4 cells were cultured in RPMI1640 medium with supplements. Cells were grown in 2 glucose concentrations: normoglycemic (NG, 5 mM) and hyperglycaemic (HG, 25 mM). After 3 passages, cells were starved for 24 h in serum‐free medium, and EVs were isolated as previously described. EVs were visualized using CryoTEM and TEM microscopy. EV markers were analysed using spectral flow cytometry. ATR‐FTIR spectra were collected using a Nicolet 6700 spectrometer equipped with a diamond ATR accessory. Results : CryoTEM, TEM, and spectral flow cytometry confirmed the presence of EVs in the isolated samples. PCA of infrared spectra showed no impact of hyperglycaemia on cells compared to normoglycaemia, with no significant differences in key spectral regions (900–3150, 900–1800, 2800–3105, 1484–1720). PCA effectively differentiated cells from EVs across all ranges. Based on literature data, we showed that the 1452/1656 region and its area can detect DM in EVs samples, whereas the 1540/1343 region enables detection in cells (HG vs. NG). Summary/Conclusion : This study explored hyperglycaemia's effect on EV secretion in insulin‐secreting pancreatic β‐cells, crucial for understanding metabolic stress in DM. Our results align with studies suggesting glucose levels influence EV release, supporting EVs as potential biomarkers for T2D. PCA proved effective in distinguishing EVs from cells under different glycaemic conditions, highlighting spectral analysis's potential for detecting metabolic changes. Findings support using spectral markers for early T2D detection, as EV‐based biomarkers hold promise for non‐invasive diabetes diagnostics. Funding : This study was financed from Polish NCN grants: 2019/33/B/NZ3/01004 and 2022/45/B/NZ7/01430 for E.S.

Hydrogel‐Assisted

Yu‐Chen Fa 1 , Bor‐Luen Chiang 2,3,4 , Li‐Chen Wu 5 , Ja‐an Annie Ho 1,6,7,8 1 BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan; 2 College of Life Science, National Taiwan University, Taipei, Taiwan; 3 Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan; 4 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; 5 Department of Applied Chemistry, National Chi Nan University, Nantou, Taiwan, 6 Department of Chemistry, National Taiwan University, Taipei, Taiwan; 7 Center for Emerging Materials and Advance Devices, National Taiwan University, Taipei, Taiwan; 8 Center for Biotechnology, National Taiwan University, Taipei, Taiwan Bor‐Luen Chiang, Li‐Chen Wu, and Ja‐an Annie Ho are co‐senior authors . Introduction : Psoriasis is a chronic inflammatory skin condition characterized by red, scaly lesions due to an overactive immune response that accelerates keratinocyte proliferation. While topical corticosteroids are commonly used, prolonged use leads to significant side effects, affecting 70%–90% of patients globally. This study aims to develop a novel transdermal drug delivery system utilizing extracellular vesicles‐containing hydrogel (hydrogel@EVs) to reduce inflammation in psoriasis. hydrogel@EVs are designed to suppress keratinocyte activity, potentially providing a safer alternative treatment. Methods : We developed a biocompatible hydrogel loaded with EVs and evaluated it for moisture retention, stability, and transdermal delivery efficiency. In an Imiquimod (IMQ)‐induced psoriasis mouse model, we compared the therapeutic effects of hydrogel@EVs with the hydrogel alone. Results : The hydrogel@EVs exhibited significant anti‐inflammatory effects in an inflamed keratinocyte model, effectively targeting both the epidermal and dermal layers and showing excellent moisture retention. In the IMQ‐induced psoriasis mouse model, all treatments reduced symptoms, but hydrogel@EVs achieved the greatest improvements in the psoriasis area severity index (PASI), histopathological assessments, and immunohistochemical analysis, with results nearing those of healthy controls. Summary/Conclusion : The hydrogel@EVs offers a promising topical treatment for psoriasis, combining moisture retention, EV protection, and strong anti‐inflammatory effects. This approach could serve as a safer, long‐term alternative to corticosteroids, meeting the need for more effective psoriasis therapies.

Mhc‐Ii‐Positive

Adam M Bourke, James R Edgar Department of Pathology, University of Cambridge Introduction : Major Histocompatibility Complex class II (MHC‐II) molecules traffic through the endosomal system to the multivesicular body (MVB), where peptide loading and editing occurs. Here, MHC‐II molecules are specifically enriched on intraluminal vesicles (ILVs). To ensure MHC‐II presentation at the cell surface, ILV retrofusion has been proposed—allowing a route back from ILVs to the plasma membrane. Recently, it has been shown that small extracellular vesicles can be retained and tethered to producer cells, a property dependent on the expression of the antiviral protein, Tetherin. My research investigates the prominence of this phenotype in Lymphoblastoid Cell Lines (LCLs) and the role that tethered, endosomal derived exosomes play in MHC‐II presentation. Methods : Tetherin knockout 721.221 LCLs were generated using CRISPR/Cas9, where the presence of tethered small EVs was scrutinised using conventional and cryo‐immunogold transmission electron microscopy. Immunofluorescence was used to further compare MHC‐II distribution, particularly in relation to endosomal markers. Small EVs were isolated from wild type and knockout cells using differential centrifugation, and protein composition and abundance determined by western blot. Results : We found small extracellular vesicles tethered to the cell membranes of tetherin‐expressing 721.221s which contain CD63 and MHC class II molecules. Immunofluorescence showed MHC‐II and CD63 colocalising in puncta at the cell surface, a phenotype lost in tetherin KO 721.221s. Western blotting confirmed that tetherin KO 721.221s secrete increased levels of MHC‐II, CD63, and syntenin positive EVs. Summary/Conclusion : Our findings demonstrate, on the surface of LCLs, tethered sEVs enriched in MHC‐II specifically. Tetherin knockout cells exhibit decreased tethering, and increased release of sEVs into the culture media. The finding of MHC‐II positive tethered sEVs on the 721.221 cell surface highlights that ILV retrofusion may not occur in LCLs, and highlights a new route for MHC‐II presentation at the cell surface. Funding : This work is funded by a PhD studentship from the Department of Pathology, University of Cambridge. JRE's lab is funded by a Sir Henry Dale Fellowship funded by the Wellcome Trust and Royal Society (216370/Z/19/Z). I have received research grants, such as through the British Society of Cell Biology and Pembroke College, Cambridge, to aid my learning of cryoimmuno‐gold electron microscopy.

Probiotic‐Derived

Hannah J. O'Toole 1 , Trina A. Knotts 1,2 , Neona M. Lowe 1 , Dustin Hadley 1 , Luis Valdiviez 1 , Jean Debédat 1,2 , Oliver Fiehn 1 , Sean H. Adams 1,2 , Randy P. Carney 1 1 University of California, Davis, California, USA; 2 UC Davis Health, Davis, California, USA Introduction : Probiotic bacteria‐derived extracellular vesicles (bEVs) are a promising therapeutic and preventative modality for metabolic and gut health. bEVs containing natural microbial metabolites (xenometabolites), such as anti‐inflammatory indole derivatives and peroxisome proliferator activated receptor (PPAR) activating cyclopropane fatty acids (CpFAs), are examples of probiotic‐derived molecules with potential gastrointestinal (GI) health benefits. Here, we engineered bEVs (e‐bEVs) to augment the natural cargo of xenometabolites (i.e., CpFAs) to assess their bioactivities in vitro and in vivo. Methods : Probiotic Lactobacillus reuteri ATCC PTA 6475 was cultured under low pH (3–4 pH) conditions to optimize bEV yield. bEVs were isolated via differential ultracentrifugation from bacterial cell culture media. bEV size, concentration, and morphological confirmation were performed via nanoparticle tracking analysis and cryogenic electron microscopy. Protein concentration was analysed via BCA assay. CpFA loading into isolated bEVs was performed via a novel microfluidic mixing approach. Size‐exclusion chromatography was used to remove free CpFA, then gas chromatography time of flight mass spectrometry (GC‐TOF(MS)) was performed to quantify native indole and CpFA metabolite cargo and determine the absolute concentration of target CpFA loading into e‐bEVs. Cell media and native bEVs were analysed as negative controls. Results : As evidenced by MISEV2023 guidelines, L. reuteri bEVs were successfully enriched. As expected, MRS media did not contain detectable particles. bEVs isolated from low pH conditions had ∼10‐fold higher number of particles from an equivalent number of cells when compared to cultures grown under standard conditions. GC‐TOF(MS) metabolomics analysis of native L. reuteri bEVs confirmed the presence of indole‐3‐lactate and indole‐3‐butyric acid. GC‐TOF(MS) analysis of CpFA‐loaded e‐bEVs had > 40‐fold enrichment in dihydrosterculic acid CpFA in comparison to native bEVs, confirming our ability to engineer bEVs to contain xenometabolites of interest. Notably, our high‐throughput microfluidic CpFA loading scheme did not compromise the native indole cargo, nor majorly alter EV morphology or concentration. Summary/Conclusion : This work demonstrates that native probiotic bEVs are carriers of xenometabolite cargo, which can be further increased by exogenous loading of target molecules. These e‐bEVs and our microfluidic loading workflow will have a broad impact in developing more robust therapeutics and probiotic‐derived supplements to improve GI health.

Three‐Dimensional

Presenter: Maulee Sheth University of Cincinnati, Cincinnati, Ohio, USA Introduction : Biophysical cues such as extracellular matrix (ECM) stiffness affect tumour progression in vivo. Physical properties of ECM not only affect intracellular signalling but also impact the tumour microenvironment through secreted extracellular vesicles (EVs). Small EVs (sEVs)‐mediated intercellular communication and horizontal transfer of biomolecular cargo to recipient cells facilitate interplay and rewiring between different components of the TME, promoting tumourigenesis and metastasis. Here, we engineered a 3D spheroid culture system with varying mechanical properties to study the role of EVs in matrix stiffness‐triggered oral squamous cell carcinoma (OSCC) invasiveness. Methods : EVs were extracted from day 14 culture media of Cal27 spheroids cultured on soft (EVsoft) and stiff (EVstiff) matrices (3 and 12 mg/mL Matrigel, respectively) using a lab‐on‐a‐chip dielectrophoretic device and characterized using nanoparticle tracking analysis (NTA), western blot (CD63, HSP70, and TSG101), imaging flow cytometry, and super‐resolution microscopy. Genomic analysis included directional total RNA and microRNA (miRNA) sequencing. Pathway enrichment was performed for target genes of differentially expressed EV miRNAs (DEMs) found to be differentially expressed in parental spheroids (DEGs). Results : NTA and western blot indicated a stiffness‐induced increase in the secretion of EVs. miRNA‐seq revealed upregulation of frequently deregulated EV miRNAs such as miR‐1246, ‐122‐5p, and ‐574‐5p, which corroborate with increased tumourigenic behaviour, including modulation of epithelial to mesenchymal transition, metastatic potential, and chemoresistance in OSCC in EVstiff. Long non‐coding RNAs (Set and Anp32b) involved with cancer promotion were also upregulated in EVstiff, while certain mitochondria‐encoded genes, dysregulation of which contributes to progression, were upregulated in EVsoft. Overlay of target genes of EV‐DEMs and spheroid‐DEGs showed 110 and 120 genes corresponding to up‐ and down‐regulated DEMs, respectively. Pathways including MAPK cascade, cell migration and motility, and cell signalling by electrical coupling were enriched in the downregulated DEMs‐upregulated DEGs overlay for EVstiff. Summary/Conclusion : We report that 3D ECM stiffness alters EV production and composition. Our findings provided the first evidence that parental biomechanical Piezo1 is expressed in spheroid‐derived EVs. Our data suggests that matrix stiffness modulates carcinogenic signatures in EVs in head and neck cancer. References : (1) Sheth et al. 2024, APL Bioengineering; (2) Sharma et al. 2023, Scientific Reports. Funding : This study was supported by the NSF CAREER ECCS (No. 2046037).

Astrocyte‐Enriched

Hind Haj Ahmad 1 , Santra Brenna 1 , Ali Biabani 2 , Jelena Navolic 3 , Yvonne Pechmann 4 , Frank Heisler 4 , Julia Neumann 3 , Matthias Kneussel 4 , Tim Magnus 1 , Mathias Gelderblom 1 , Berta Puig 1 1 Neurology Department, Experimental Research in Stroke and Inflammation, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 2 Section for Mass Spectrometry and Proteomics, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 3 Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 4 Institute for Molecular Genetics, Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg‐Eppendorf, Hamburg, Germany Introduction : Following an ischaemic stroke, brain cells directly supplied by the affected vessel (the core of the stroke) die within min. However, the neuronees surrounding this area (the penumbra) remain metabolically active yet enter a state of electric silence. Due to the energetic demands of synaptic activity, this temporary suppression of synaptic function may act as an adaptive response to sustain cell viability. Depending on the signals these neuronees receive in the hours and days that follow the ischaemic insult, they may either survive or undergo cell death. Current therapies are focused on rapid reperfusion, but a lot of effort is put into understanding how to rescue these neuronees to improve neurological recovery. We have previously shown that 24 h after ischaemia/reperfusion, the relative proportion of astrocyte‐enriched EVs (AEVs) within the pool of brain‐derived EVs (bdEVs) was increased in the affected hemisphere in a mouse stroke model (transient middle cerebral artery occlusion, tMCAO), and in the present study we aimed to analyse the content of AEVs and investigate their functional implications. Methods : We isolated both small and medium/large AEVs using antibody‐based magnetic sorting and performed mass spectrometry analysis on samples from both stroke and sham‐operated mice at 24 h and 7 days after stroke. Additionally, we performed functional studies using calcium imaging by incubating AEVs with primary neuronal cultures subjected to oxygen‐glucose‐deprivation (OGD) as an in vitro model of stroke. Results : Our results show that one day after stroke, proteins related to synaptic formation, integrity, plasticity and activity were upregulated. At 7 days after stroke, while some proteins related to synaptic processes remained upregulated, proteins related to metabolic pathways showed increased expression. Functional studies point to a role of AEVs in synaptic preservation and neuronal survival after ischaemic insult. Summary/Conclusion : In this study, we have developed a protocol suitable for directly studying AEVs from the brain and providing new insights into the role of AEVs in neuroprotection, laying the groundwork for identifying potential therapeutic targets. Funding : Werner Otto Stiftung, Deutsche Forschungsgemeinschaft (DFG), and Hermann und Lilly Schilling Stiftung für Medizinsiche Forschung.

Escrt‐0‐Mediated

Xiao‐Le Wang 1,2 , Hou‐Fu Xia 1,2 , Wei Zhang 1,2 , Gang Chen 1,2 1 State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China 2 Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan, 430079, China Introduction : To elucidate the intricate mechanisms governing the secretion of programmed death‐ligand 1 (PD‐L1) in small extracellular vesicles (sEVs) and to further explore its role in establishing a tumour‐promoting microenvironment and inducing systemic immunosuppression. Methods : Flow cytometry was employed to analyse the expression levels of sEV PD‐L1 in tumour tissues and peripheral blood samples from patients with oral squamous cell carcinoma (OSCC). A tissue microarray was utilized to detect the expression levels of proteins associated with sEV secretion in OSCC tissues. The secretion of sEV PD‐L1 by tumour cells under conditions of ESCRT‐0 activation and silencing was assessed using flow cytometry and nanoparticle tracking analysis (NTA). Co‐immunoprecipitation (Co‐IP) was conducted to identify molecules implicated in the sorting of sEV PD‐L1. Western blot and immunofluorescence assays were applied to investigate the mechanisms regulating the secretion of sEV PD‐L1 by these molecules. Ultimately, animal models were established to evaluate the therapeutic efficacy of targeting ESCRT‐0 members or tumour‐derived sEV PD‐L1 in a tumour‐bearing mouse model. Results : STAM2, a component of the ESCRT‐0 complex, plays a pivotal role in the trafficking of PD‐L1 to sEVs. Through GST‐pulldown assays, we demonstrate that STAM2, rather than STAM1 or HRS, directly interacts with PD‐L1, forming a STAM2‐PD‐L1 complex on the plasma membrane. This complex further recruits another ESCRT‐0 protein, HRS, on endosomes, thereby facilitating the routing of PD‐L1 to sEVs. In vitro studies reveal that knockout of STAM2 significantly suppresses the expression of PD‐L1 in OSCC cell‐derived sEVs, and these sEVs markedly inhibit the proliferation of CD8+ T cells, underscoring the central role of STAM2 in regulating sEV PD‐L1 secretion. Moreover, silencing STAM2 enhances the therapeutic efficacy of PD‐L1 blockade in tumour models, as inhibiting STAM2 simultaneously suppresses membrane PD‐L1 recognition and endosomal HRS recruitment. Summary/Conclusion : ESCRT‐0 members sequentially participate in the secretion of sEV PD‐L1, highlighting their crucial role in modulating this process. This finding offers valuable insights into diseases characterized by dysregulated PD‐L1 secretion, particularly in the context of cancer.

Macrophage‐Derived

Presenter: Bingbing Ru Southern Medical University, China (People's Republic) Introduction : Sepsis, a lethal organ dysfunction caused by dysregulated immunity, claims millions of lives annually. Macrophages (Mφ) drive sepsis pathogenesis, and their extracellular vesicles (EVs) may propagate systemic immune dysregulation. We identified macrophage‐derived EVs carrying bacterial components (chimeric EVs, CEVs) in sepsis patients, hypothesizing CEVs mediate remote monocyte activation and serve as biomarkers. Methods : To elucidate the role of macrophage‐derived extracellular vesicles (Mφ‐EVs) in immune regulation during sepsis, we isolated Mφ‐EVs from serum EVs of sepsis patients and healthy controls using magnetic bead‐based specific isolation and performed proteomic analysis. Bacterial components in Mφ‐EVs were characterized via immunogold labelling, EV‐specific ELISA, and nano‐flow cytometry. CEVs were extracted from E. coli‐phagocytosing THP‐1 cells using magnetic beads and intraperitoneally injected into sepsis mouse models. Systemic immune effects of CEVs were assessed by flow cytometry analysis of peripheral blood and peritoneal lavage cells (monocytes, macrophages, neutrophils) and ELISA quantification of serum TNF‐α and IL‐6. CellMask‐labelled CEVs and M1 Mφ‐EVs were intravenously injected into mice, and their cellular uptake was analysed via flow cytometry. Primary murine monocytes and sepsis mice were used to evaluate CEV‐induced monocyte recruitment and inflammatory differentiation. Transcriptomic and enrichment analyses of CEV‐treated THP‐1 cells were conducted to identify regulatory mechanisms. To assess whether CEV levels reflect macrophage functional states, macrophages from sepsis mice at different disease stages were isolated for functional evaluation. Results : Sepsis patient Mφ‐EVs showed elevated immune proteins involved in pathogen recognition, inflammation, chemotaxis, complement activation, and antimicrobial defense. Bacterial proteins, including LPS colocalized with the EV marker CD63, were uniquely detected in sepsis patient Mφ‐EVs. In murine models, pre‐injection of CEVs significantly increased circulating monocyte percentages, reduced bone marrow and peritoneal monocytes, and elevated CD80, CD86, CCR2 pro‐inflammatory markers and IL‐6, TNF‐α. CEVs activated TLR/NF‐κB pathways in monocytes, driving inflammatory differentiation. Circulating CEV levels positively correlated with macrophage phagocytosis, inflammatory polarization, and cytokine secretion (r = 0.75–0.89, p < 0.001). Summary/Conclusion : CEVs drive sepsis‐associated immune dysregulation by shuttling bacterial components to monocytes, triggering pro‐inflammatory differentiation via NF‐κB/IRF3 pathways. The novel CEV liquid biopsy technique offers a robust tool for real‐time immune monitoring and immunotherapy evaluation in sepsis.

Microalgae‐Derived

Presenter: Sabrina Picciotto National Research Council of Italy, Italy Introduction : Extracellular vesicles (EVs) represent an innovative advance in nanotechnology for targeted therapeutic delivery, efficiently transporting endogenous or exogenous bioactive compounds across tissue barriers. EVs derived from microalgae, known as (nano)Algosomes (Alg), offer a natural, sustainable platform for developing pharmaceutical formulations. Our previous studies have revealed that Alg possesses impressive antioxidant and anti‐inflammatory capabilities without causing allergic‐like responses. Gene expression analysis demonstrated no genetic alterations following Alg treatment, with no DNA damage observed. Additionally, our biodistribution research in mice showcased a distinctive organotropism, particularly observed in the bone tissue. These findings emphasize Alg's promising potential for safe and effective therapeutic applications. Methods : Alg are obtained by isolating from a suspension of Tetraselmis chui microalgal cells through tangential flow filtration. Our innovative platform has been developed and validated for the medium‐scale production, characterization, and bioengineering of Alg. This cutting‐edge system was specifically designed to address the limitations associated with current sources of EVs and existing EV‐based nano‐delivery systems. Results : Our research investigates the properties of Alg in alignment with MISEV2023 guidelines, focusing on their bioactivity, biocompatibility, and biodistribution, particularly highlighting their tropism for bone. Based on these findings, we intend to capitalize on the inherent organ‐targeting capabilities of Alg for the development of innovative pharmaceutical applications for osteosarcoma treatment. Alg demonstrated a strong capacity for loading, significantly improving the cellular uptake of small molecules such as doxorubicin. Functional assays performed on osteosarcoma cells, including variants resistant to doxorubicin, revealed that the drug's effectiveness was markedly greater when delivered via Alg compared to conventional free drug administration. Summary/Conclusion : Alg present a natural and highly effective delivery system for both naturally occurring compounds from microalgae and exogenous bioactive compounds or synthetic drugs, particularly targeted for bone health enhancement.

Multi‐Instrumental

Nadim Tawil 1 *, Laura Montermini 1 , Mahsa Jalali 2 , Molly L. Shen 2 , David Juncker 2 , Sara Mahshid 2 , Thupten Tsering 1 , Julia Burnier 1 , Hélène Pagé‐Veillette 1 , Ekaterina Iourchenko 1 , Dongsic Choi, Brian Meehan 1 , Anthoula Lazaris 1 , Peter Metrakos 1 , Janusz Rak 1,2 * 1 Research Institute of the McGill University Health Centre, Montreal, Québec, Canada; 2 McGill University, Montreal, Québec, Canada Introduction : Current and emerging nanotechnologies offer diverse and often incomplete perspectives on the complexity of extracellular vesicle and particle (EVP) landscapes. In order to obtain a more comprehensive picture, we employed complementary technologies available at the Center for Applied Nanomedicine (CAN) at the Research Institute of the McGill University Health Centre (RI‐MUHC) in Montreal, Canada. Here we describe the capacities and results that can be generated through the use of orthogonal particle counters and single EVP analysis platforms using fluorescent probes and label free detection techniques as tools that enable dissection of unsuspected EVP subsets released by a range of donor cells. Methods : CAN features a robust, high‐resolution EV research pipeline that includes nanoparticle sizing and counting, nanoflow cytometry, imaging cytometry, super‐resolution microscopy, and chip‐based immunofluorescence for molecular EVP profiling. These technologies facilitate detailed EVP characterization including surface marker detection, cargo analysis, and localization of EVP‐associated moieties. Results : Leverageing CAN's capabilities, a series of pioneering studies has emerged. Using well characterised cellular sources of EVPs we have conducted instrumental and molecular analysis of particle subpopulations in cellular conditioned media and biofluids. Using the integrated CAN pipeline enabled identification of unsuspected subsets of EVPs carrying signalling membrane receptors, tetraspanins and functional markers. Chip technologies revealed shifts in EVP subpopulations following disruption of biogenic mechanisms dependent on syntenin‐1, neutral sphingomyelinase, and other effectors. Extending CAN capacity to support novel platforms enabled fingerprinting of cancer EVPs at single particle resolution using molecular Surface‐Enhanced Raman Spectroscopy (MoSERS) and machine learning‐based detection of hallmarks associated with progression of glioblastoma (GBM). Multi‐instrumental approaches afforded by CAN have also propped the development and assessment of novel EV isolation microfluidic devices as well as various fusion‐protein systems that take advantage of membrane‐localizing peptides as tools for prolonged and non‐disruptive EV tracking in both in vivo and in vitro settings. Summary/Conclusion : Integrated technology platforms, such as CAN, enable objective and orthogonal analysis of complex EVP populations and benchmarking novel nanotechnologies aimed for biomedical applications. Our recent experience with CAN infrastructure illustrates how essential the multi‐instrumental approach is for EV‐focused discovery and facilitating cross‐disciplinary collabourations in nanomedicine and nanotechnology.

Neutrophil‐Derived

Mátka Nagy 1 , Kristóf Molnár 1 , Vanda Vikman 1 , Katalin Kohán 1 , Viktória Szeifert 1 , Delaram Khamari 1 , Ágnes Kittel 2 , Erzsébet Ligeti 1 , Roland Csépányi‐Kömi 1 , Ákos M. Lőrincz 1,3 1 Semmelweis University, Hungary, 2 HUN‐REN, Hungary, 3 Szent György Hospital, Hungary Introduction : Neutrophils (PMNs) are the predominant leukocyte population in human blood, they facilitate intercellular communication through the release of extracellular vesicles (EVs). Our Laboratory focuses on EVs generated in three biologically characteristic states of neutrophils: spontaneously released spEVs from resting neutrophils, apoEVs produced by neutrophil undergoing apoptosis and ozEVs formed during complex biological activation with opsonized zymosan. The aim of our current work is to investigate the PMN EVs effect on other immune cells viability. Methods : Neutrophils, lymphocytes and monocytes were isolated from human blood, macrophages were differentiated from monocytes using M‐CSF. PMNs were incubated with or without activating agent for 20 min or left in a pro‐apoptotic environment for 24 h. After cellular elimination, PMN EVs were isolated by two‐step centrifugation and gravitational filtration. Right after isolation, leukocytes were treated with EVs for different incubation periods. Cell count and viability was assessed by measuring annexin V and propidium iodide positivity using FC, LDH release was measured from cell culture supernatants. Caspase‐3 activity of EV treated cells was also assessed. During the macrophage differentiation, cell culture supernatants was measured by FC and LDH assay, fluorescence microscopy images were taken of the fully differentiated macrophages. Results : All three PMN EVs improved the viabilityof lymphocytes, while they had no effect on neutrophils. Monocytes cell viability was slightly enhanced by apoEVs and spEVs. In the case of the monocyte‐derived macrophages, spEVs and apoEVs significantly increased the differentiation success. Based on the supernatant measurements, spEVs and apoEVs improved the viability of the cells from the beginning of the differentation, resulted in three times more cells in the spEV and apoEV‐treated samples compared to the untreated samples. However, there was no difference after EV treatment in the viability of the differentiated macrophages. Summary/Conclusion : Depending on the prevailing circumstances, neutrophils produce extracellular vesicles that can selectively enhance the viability of partner immune cells. This selectivity underscores the complex ways in which neutrophils dynamically modulate inflammation through EV production. By exploiting this property, neutrophil EVs can be utilized for therapeutic applications. Funding : EFOP‐3.6.3‐VEKOP‐16‐2017‐00009; NKFIH FK137770 ; TKP2021‐EGA‐24; János Bolyai Research Scholarship of the Hungarian Academy of Sciences

Tgf‐Β‐Activated

Chao Li 1 , Agustin Enciso‐Martinez 1,2,3 , Lizhe Zhu 1 , Sarah A. Rotman 4 , Peter A. van Veelen 4 , Roman I. Koning 5 , Hailiang Mei 6 , Peter ten Dijke 1 1 Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands; 2 Amsterdam UMC, University of Amsterdam, Biomedical Engineering & Physics; Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands; 3 Amsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry; Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Meibergdreef 9, Amsterdam, The Netherlands; 4 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands; 5 Electron Microscopy Facility, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands; 6 Department of Biomedical Data Sciences, Sequencing Analysis Support Core, Leiden University Medical Center, Leiden, The Netherlands Introduction : Cancer‐associated fibroblasts (CAFs) are a major component of tumour stroma in various cancer types. The cytokine transforming growth factor‐β (TGF‐β) is frequently present at high levels in the tumour microenvironment and is a potent inducer of CAF activation. Activated CAFs produce and remodel the extracellular matrix and release soluble factors and extracellular vesicles (EVs) to communicate with other cell types in the tumour microenvironment. The content of EVs, including proteins and nucleic acids, has mainly been studied for its association with EV function. However, the EV surface can function as a versatile platform for presenting surface proteins and extracellular matrix components, which directly mediate intercellular communication. The EV surface is in direct contact with extracellular space, enabling EVs to act as sponges that adsorb additional proteins and matrix components after release. In this study, we highlight the surface of CAF‐derived EVs and reveal the functions and mechanisms of EV surface‐associated proteins in promoting immune evasion and cancer development. Methods : EVs were isolated from quiescent and TGF‐β‐activated CAFs using size exclusion chromatography for quantitative proteomic analysis and functional studies. The presence of EV surface‐associated protein was validated by super resolution microscopy and western blot. The biological functions of EVs were tested on spheroid models with cancer cells, CAFs and CD8+ T cells, and monocultures. The interactions between EV surface proteins and cell receptors were investigated using a proximity ligation assay. Results : TGF‐β can induce CAF activation and upregulate the quantity of EV proteins. Quantitative proteomic analysis revealed that the top upregulated proteins are matrix proteins, including TSG6. The upregulated matrix proteins and extracellular matrix components localized on the EV surface and interacted with receptors on EVs; for example, hyaluronan, which colocalized with CD44+ EVs. The EV surface‐associated TSG6 and hyaluronan interacted with cell receptors CD44 and ALK5 and induced receptor clustering, thereby triggering signalling cascades before the internalization of EVs. This process can sustain CAF activation and induce immunosuppression in CD8+ T cells. Summary/Conclusion : EV surface proteins and matrix components from TGF‐β‐activated CAFs interact with receptors and induce receptor clustering on target cells, thereby further inducing CAF activation and immunosuppression in CD8+ T cells.

Eps‐Urine‐Derived

Chiara Ansermino 1,2 , Denise Drago 1 , Radmila Pavlovic 1 , Jordi Capellades Tomàs 3 , Óscar Yanes Torrado 3 , Vito Cucchiara 4 , Francesco Montorsi 4 , Alberto Briganti 4 , Annapaola Andolfo 1 1 Proteomics and Metabolomics Facility (ProMeFa), Center for OMICS Sciences (COSR), IRCCS San Raffaele Scientific Institute, 20132 Milan; 2 Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy; 3 Department of Electronic Engineering, Universitat Rovira i Virgili, Institut d'Investigació Sanitària Pere Virgili, 43007 Tarragona, Spain; 4 Department of Urology and Division of Experimental Oncology, Urological Research Institute (URI), IRCCS San Raffaele Scientific Institute, Milan 20132, Italy Abstract unavailable

Hoffa‐Msc‐Derived

Alexander Otahal 1 , Karina Kramer 1 , Slavomira Gulová 1,2 , Markus Neubauer 1,3 , Zsombor Lacza 4,5 , Stefan Nehrer 1,3 , Andrea De Luna 1 1 Center for Regenerative Medicine, University for Continuing Education Krems, Krems, Austria; 2 Associated Tissue Bank, Faculty of Medicine, Pavol Jozef Safarik University and Luis Pasteur University Hospital, Kosice, Slovakia; 3 Universitätsklinikum Krems, Krems, Austria; 4 University of Physical Education, Department of Sport Physiology, Budapest, Hungary; 5 Semmelweis University, Inst. Clinical Experimental Research, Budapest, Hungary Introduction : Adipose mesenchymal stromal cells (MSCs) are increasingly studied for regenerative therapies due to their ability to modulate inflammation and support tissue homeostasis. Recent findings suggest MSC‐derived extracellular vesicles (EVs) play a central role in therapeutic effects. The infrapatellar Hoffa fat pad is a promising MSC source for EVs. Conventional EV production in 2D cultures can limit EV yield and functional relevance due to its artificial conditions. Here, we developed a 3D dynamic culture approach using vertical wheel bioreactors, supplemented with different human blood products such as citrate‐anticoagulated platelet‐rich plasma or hyperacute serum to generate EVs in a cost‐efficient manner. Methods : Hoffa MSCs were isolated from surgical waste from joint replacement surgeries, characterized by flow cytometry, and assessed for adipogenic, osteogenic, and chondrogenic differentiation. Cells were cultured on animal‐free microcarriers in 3D bioreactors and standard 2D flasks, both with blood products to simulate clinical treatment settings. EVs were isolated by ultrafiltration and characterized using nanoparticle tracking analysis in scatter and fluorescence mode and western blotting. EV miRNA cargo was analysed through RNA sequencing. Biological potency was assessed by evaluating gene expression changes in osteoarthritic chondrocytes via RT‐qPCR. Results : Hoffa MSCs displayed expected stemness markers before and after EV production as well as tri‐lineage differentiation potential. Approximately 10,000 particles per cell were obtained in 3D culture versus 1000 in 2D. EV marker proteins were confirmed via western blot, and EV preparations were not contaminated with lipoproteins. miRNA profiles were consistent across treatments, with miR‐100‐5p most abundantly present in EVs. EVs from 3D culture significantly inhibited inflammatory and hypertrophic genes, while preserving extracellular matrix gene expression, exemplified by iNOS, type 10 and type 2 collagen, in contrast to EVs from 2D culture. Summary/Conclusion : The 3D culture of Hoffa MSCs in bioreactors, combined with ultrafiltration, supports efficient EV production for potency assays and preclinical research. Dynamic culture conditions appear to enhance EV release and could prevent EV reuptake, while yielding high amounts of EVs. This method facilitates scalable EV production having substantially higher anti‐inflammatory and tissue homeostatic potency than conventional EV production approaches. Funding : The study was funded by Gesellschaft für Forschungsförderung NÖ (GFF), grant number LSC18‐014.

Hypoxia‐Upregulated

Presenter: Yousheng Wu The First Affiliated Hospital of Jinan University, Guangzhou, People's Republic of China Introduction : Cellular hypoxia results in an elevated release of small extracellular vesicles (sEV), which can be more readily absorbed by surrounding cells. This process may lead to the dissemination of harmful substances and initiate the ischaemic cascade, a response associated with ischaemic stroke. Nonetheless, sEV derived from various cell types have different effects, and there is presently an absence of specific markers to identify sEV originating from hypoxic neuronees. Methods : Isolating membrane proteins of SH‐sy5y‐derived sEV for proteomic analysis. Identifying sEV membrane proteins through Western blotting, immunoelectron microscopy, and ELISA. Extracting sEV from ischaemic brain tissue to verify the presence of membrane proteins. In vitro labelling of sEV is performed using PKH67 and CFSE to evaluate the efficiency of sEV uptake. Administering sEV from ischaemic brain tissue via stereotactic injection into mice prior to middle cerebral artery occlusion (MCAO), with or without monoclonal antibodies against membrane proteins, and evaluating behavioural and neurological outcomes in mice three days post‐surgery. Results : Biotin successfully isolated the sEV membrane protein and identified the hypoxia‐upregulated membrane protein Ataxin2 (ATXN2). ATXN2 was found to be expressed in the sEV membrane protein fractions of hypoxic neuronees and ischaemic brain tissue, and it was present on the surface of the sEV membrane. Fluorescently labelled hypoxic sEVs were taken up by cells more readily than normoxic sEVs; however, the uptake of hypoxic sEVs was significantly reduced following the knockdown of ATXN2, and the use of an ATXN2 monoclonal antibody greatly diminished the uptake of hypoxic sEVs. Furthermore, the stereotactic injection of hypoxic neuronee‐derived sEVs into mice subjected to MCAO facilitated the recovery of neurological function. The outcome for MCAO mice receiving sEVs derived from infarcted brain tissue was more favourable than that of MCAO mice treated with a combination of infarcted brain tissue sEVs and the ATXN2 monoclonal antibody at the three‐day mark. Summary/Conclusion : Hypoxic conditions lead to an elevated expression of the ATXN2 membrane protein in neuronal sEV and improve the uptake of these vesicles.

Macrophage‐Mediated

Chieh Shen 1 and Chu‐An Wang 2 1 Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2 Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan Introduction : Chronic inflammation is a key risk factor in pancreatic cancer development, with inflammatory responses playing a significant role in cancer progression. Extracellular vesicles (EVs) are essential mediators of cell communication. This study examines the role of immune cells in regulating EV biogenesis in pancreatic cancer cells and the consequent promotion of cancer malignancy. Methods : We conducted single‐cell RNA sequencing on human pancreatic ductal adenocarcinoma (PDAC) tumours. We performed co‐culture experiments to investigate molecular changes in pancreatic cancer cells interacting with macrophages. EVs were isolated by size exclusion chromatography; particle numbers were determined by nanoparticle tracking analysis. Immunofluorescence staining and light‐sheet microscopy were utilized to observe the membrane network and track intracellular particles. Results : Single‐cell RNA sequencing of pancreatic tumours revealed increased macrophage populations from early to advanced cancer stages. Macrophages maintained a high level of ERK status and promoted metastatic ability in pancreatic cancer cells through the secretion of TIMP metallopeptidase inhibitor 1 (TIMP‐1). Concurrently, the expression of CD63, the cell‐surface receptor for TIMP‐1, was increased in pancreatic cancer cells during interactions with macrophages. Knockdown of CD63 not only resulted in the loss of responsiveness to macrophage induction but also significantly affected cell adhesion and the vesicle network in pancreatic cancer cells. CD63 knockdown in pancreatic cancer cells led to notable membrane aggregation, suggesting defects in the vesicle biogenesis. Nanoparticle tracking analysis demonstrated that EV secretion in pancreatic cancer cells increased following treatment with macrophage‐conditioned medium (MCM), while CD63 knockdown resulted in decreased EV secretion and altered composition of particle size compared to control cells. Co‐culturing PKH67‐labelled PANC‐1 cells with pancreatic stellate cells (PSCs) indicated that EV uptake may be mediated by CD63 expression. Pancreatic cancer cell‐derived EVs were able to promote collagen expression in PSCs. Last, analysis of TIMP‐1/CD63 expression levels in clinical samples revealed an association with poorer outcomes in pancreatic tumours. Summary/Conclusion : Our study revealed that the interactions between macrophages and pancreatic cancer cells lead to the upregulation of CD63 and promote EV secretion, facilitating the transition from the early to advanced stages of pancreatic cancer.

Membrane‐Associated

N.A. Basalova 1, O.A. Grigorieva 1 , U.D. Dyachkova 1 , M.A. Vigovsky 1 , D.V. Bagrov 2 , O.S. Sokolova 2 , E.S. Novoseletskaya 1,3 , A.Yu. Efimenko 1 1 Centre for Regenerative Medicine, Medical Research and Educational Institute, Lomonosov MSU, Moscow, Russia. 2 Faculty of Biology, Lomonosov MSU, Moscow, Russia. 3 Shenzhen MSU‐BIT University, Shenzhen, China. Introduction : Mesenchymal stem/stromal cells (MSCs) are important regulators of tissue homeostasis and regeneration mostly due to the secretion of bioactive factors, including molecules within extracellular vesicles (EV). Recently, a new type of EVs has been discovered—membrane‐associated vesicles (MAV). However, their certain characteristic and role in the regulatory effects of MSCs, as well as reliable methods of isolation, have not been previously studied. Methods : For the isolation of MAV from MSCs (ASC52telo, ATCC), we proposed methods based on disruption of MAV from cell membranes—the use of TritonX100; trypsin; hyaluronidase and proteinase K. Free‐secreted EV, isolated from MSC conditioned medium by ultrafiltration(300 kDa), were used as control. The isolated fractions were characterized according to MISEV2023 criteria by size (SEM, NTA), morphology (SEM) and markers (WB‐CD81, CD9, CD73, HSP70, Alix—positive marker, H2Ax—negative marker for cell contamination verification). Since one of the important homeostatic functions of MSCs is ability to suppress fibrosis, the functional activity of the isolated fractions in equal concentration was tested on the TGFβ‐induced fibroblast‐to‐myofibroblast differentiation model (WB; ICC–αSMA). Results : We have shown that the optimal protocol for isolating MSC‐MAV includes hyaluronidase treatment, which allows obtaining the highest yield of particles homogeneous in size and save cells in a native state. Isolated particles have a morphology and average size similar to EV (MAV 133±69 nm, EV 139±64 nm). However, the peak concentration of MAV is significantly lower ‐ 110±5 nm vs 135±10 nm for EV. Alix is not expressed on MAV, unlike EV, which contain all studied positive markers. In model of fibroblast‐to‐myofibroblast differentiation MAV suppresses the expression of αSMA 30 times less than EV. Summary/Conclusion : Among the tested approaches for the separation of MAV from MSCs we suggest the use of hyaluronidase. The obtained MAV have the characteristic morphology of EVs, but their size is smaller than EV and they do not carry Alix. MAV are able to suppress the differentiation of fibroblasts into myofibroblasts, but less effectively than EV from the same MSCs, which may indicate MAV distinguished cargo and function. Funding : Support by Interdisciplinary Scientific and Educational School of Moscow State University “Molecular Technologies of Living Systems and Synthetic Biology”,#24‐Ш04‐14.

Mesenchymal‐Derived

Mimma Maggio 1 , Carolina Martins 1 , Rawan Almasri 1 , Stéphane Petrousek 1,2 , Mathieu Y. Brunet 1 , Tara Ní Néill 1,2 , Conor T. Buckley 1,2,3 , Lorraine O'Driscoll 1 , David A. Hoey 1,2,3 1 Trinity College Dublin, Ireland; 2 Advanced Materials and Bioengineering Centre (AMBER), Ireland; 3 Royal College of Surgeons in Ireland, Ireland Introduction : Osteoporosis is a skeletal disease arising from an imbalance in bone remodelling leading to increased fracture risk. As key regulators, osteocytes mediate osteoclastogenesis via secreted paracrine factors in a mechanical loading‐dependent manner. Recently, extracellular vesicles (EVs) have emerged as major physiological mediators of cell‐cell communication in bone. However, their involvement in osteoclastogenesis remains unestablished. Hence, this study aimed to investigate the role of mesenchymal‐derived bone cells and secreted EVs in osteoclastogenesis upon the lineage commitment stage and mechanical environment of parental cells. Methods : Human marrow stromal/stem cells (MSC), human osteoblasts (OB) and MLO‐Y4 osteocyte‐like cells (OCY) were mechanically stimulated by oscillatory fluid shear (OFS). EVs from static (EV‐S) and mechanically activated (EV‐F) bone cells were obtained via conditioned media (CM) differential ultracentrifugation (70Ti fixed‐angle rotor, 110,000 ×  g , 75 min, 4°C) and characterized via NTA, TEM and flow cytometry. The osteoclast differentiation of human CD14+ monocytes was assessed upon CM and EV treatments. EV‐microRNA sequencing was performed, and selected miRNA was transfected into monocytes. Results : CM from MSC cultured in static (CM‐S) and exposed to OFS (CM‐F) significantly inhibited osteoclastogenesis. Inhibition was observed to a greater extent upon treatment with CM‐S. Static‐cultured OB secretome didn't affect osteoclast differentiation; however, OB CM‐F significantly inhibited osteoclastogenesis compared to CM‐S. Osteocyte CM‐S and CM‐F both inhibited osteoclastogenesis, with stronger inhibition following mechanical stimulation. Following EVs collection, MSC‐EVs inhibited osteoclastogenesis independent of the mechanical environment. Similarly, OB‐EVs elicited osteoclast inhibition, with stronger inhibition upon treatment with EV‐F. OCY‐EVs strongly inhibited osteoclastogenesis, evidenced to a greater extent following mechanical stimulation of the parent cell, on par with what was observed in the whole secretome. Finally, miR‐150‐5p was identified within OCY‐EVs, and its transfection suggested a dose‐dependent decrease in osteoclastogenesis. Summary/Conclusion : These results showcased osteocytes as regulators of osteoclastogenesis via a novel EV‐mediated mechanism. MSC and OB secretomes inhibited osteoclast differentiation to differing degrees dependent on their mechanical environment, found to be EV‐mediated. These findings offer an important stepping stone towards the understanding of EV‐mediated osteoclastogenesis, paving the way for the development of cell‐free osteoporosis therapies. Funding : SFI FFP (19/FFP/6533) (DH), EU Horizon 2020 IND, EVPRO [814495] and IRC Advanced Laureate Award EVIC (LOD) [IRCLA/2019/49]

Pomegranate‐Derived

Christian M. Sánchez‐López 1,2 , Miquel Martínez‐Navarrete 3 , M. Isabel Torres‐Cuevas 4 , Antonio J. Guillot 3 , Ana Melero 3 , Salvador Pérez‐Garrido 4 , Antonio Marcilla 1,2 1 Àrea de Parasitologia, Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot (Valencia), Spain. 2 Joint Research Unit on Endocrinology, Nutrition and Clinical Dietetics, Health Research IIS La Fe‐Universitat de València, Valencia, Spain. 3 Àrea de Farmàcia i Tecnologia Farmacèutica, Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Universitat de València, Burjassot (Valencia), Spain. 4 Departament de Fisiologia, Universitat de València, Burjassot (Valencia), Spain Introduction : Pomegranate is an edible fruit that has garnered increasing scientific interest due to its potential health benefits. Our research group first described that pomegranate juice (PgJ) contains nanovesicles (NVs) with antioxidant, anti‐inflammatory, and wound‐healing properties in vitro, highlighting their therapeutic properties (1). We now further evaluate the therapeutic potential of PgNVs in two mouse models of inflammatory diseases like acute pancreatitis and atopic dermatitis. Methods : PgNVs were isolated using size exclusion chromatography (SEC) and characterized through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Acute dermatitis (AD) and chronic dermatitis (ChD) were induced in CD1‐Swiss female mice via oxazolone (OXA) topic administrations, with PgNVs administered subcutaneously on the same days as OXA. Acute pancreatitis (AP) was induced in C57BL/6J mice by administering three intraperitoneal L‐arginine injections (3 g/kg body weight) at 1 h intervals; a single subcutaneous dose of PgNVs was given prior to the first L‐arginine injection. Results : In AD mice, PgNVs administration significantly lowered IL‐4 and IL‐1β levels, although it did not lead to marked changes in skin condition. In contrast, ChD mice treated with PgNVs exhibited notable reductions in skin histological damage, including reductions in epidermal thickening, collagen deposition, and inflammatory cell infiltration, along with increased IL‐10 and decreased IL‐1β levels. In the AP model, PgNVs markedly reduced oedema and inflammatory infiltrates, while limiting the rise in plasma amylase activity observed after AP induction. The improvements in AP were strongly associated with the regulation of IL‐6 expression and PgNV's antioxidant activity through NQO1 upregulation. Summary/Conclusion : Our findings suggest that PgNVs have significant therapeutic potential for inflammatory diseases, effectively modulating cytokine profiles and reducing markers of inflammation in ChD and AP models. These results support PgNVs as a promising natural therapeutic with anti‐inflammatory and antioxidant properties, meriting further investigation for clinical applications. (1) C.M. Sánchez‐López et al. Food Funct 13 (2022) 12870–12882. Funding : This work was supported by Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Spain (Grants PID2020‐114530GA‐I00 and PID2023‐146116NB‐I00 and ERDF/EU), and Conselleria d'Educació, Cultura, Universitats i Ocupació, Generalitat Valenciana, Valencia, Spain (Grant CIPROM/2023/54). C.M.S.‐L. is the recipient of a postdoctoral fellowship funded by AEI /10.13039/501100011033.

‘Cassette‐Like’

Presenter: Miho Nakabo Osaka Metropolitan University, Japan Introduction : Extracellular vesicles (EVs, exosomes) are secreted by almost all cells constituting a living organism. Due to their pharmacological advantages, EVs have been highly expected to be next‐generation drug carriers in DDS [1,2]. Meanwhile, there are urgent problems to be improved, especially in the low efficiency of cellular uptake of EVs [1,2]. As a bacterial vesicular marker, the curvature‐sensing peptide, nFAAV5 was developed with inspiration from the BAR protein [3]. nFAAV5, which has a helix structure, binds to vesicles less than 100 nm in diameter [3]. Because of the diameter ranges of EVs (approximately 30∼200 nm), nFAAV5 was considered to bind to the surface of EV membranes. We also synthesized a linked peptide of the nFAAV5 and octaarginine (R8) that is one of the representative cell‐penetrating peptides for macropinocytosis induction and increased cellular uptake (nFAAV5‐R8) [4]. Methods : All peptides were synthesized by Fmoc‐solid phase methods. Isolated EVs secreted from CD63‐GFP‐expressing cells were used for visualization of their cellular uptake after the peptide modification on the EVs. Results : Internalization of EVs by cells was evaluated using a confocal laser microscope and a flow cytometer, and we confirmed increased cellular uptake of nFAAV5‐R8‐modified EVs. Meanwhile, in the case of the non‐crosslinked nFAAV5 and R8, cellular uptake of EVs was not significantly altered. nFAAV5‐R8‐modified EVs were observed using a cryo‐TEM system, and the vesicular morphology was maintained. Furthermore, the application of this technology to boron neutron capture therapy (BNCT) [5] demonstrated effective uptake of boron (10B) compounds and enhanced cancer cell killing activity by thermal neutron irradiation, suggesting its high potential for drug delivery in future therapy. Summary/Conclusion : The developed technique for ‘cassette’‐like modification of functional peptides on EV membrane will be widely applicable in EV‐based molecular delivery system and controlled cellular communications. [1] I. Nakase, Processes, 9, 224 (2021), [2] I. Nakase, T. Takatani‐Nakase, Drug Metab., Pharmacokinet., 42, 110435 (2022), [3] K. Kawano, et al., Chem. Pharm. Bull., 69, 1075 (2021), [4] S. Futaki, I. Nakase, Acc. Chem. Res. 50, 2449 (2017), [5] S. Hirase, et al., Mol. Pharmaceutics, 19, 4, 1135 (2022)

5′‐Trf‐Gly‐Gcc

Presenter: Yuya Monoe Osaka University, Japan Introduction : Tumour‐infiltrating T cells have been reported to be important in the acquisition of platinum drug resistance in ovarian cancer, but the underlying mechanisms are unknown. Previously, we have found that colon cancer EVs harbor large amounts of 5′tRF‐GlyGCC, a known T cell inhibitor (ISEV2023). Therefore, we aimed to determine the expression of tRFs in ovarian cancer EVs and its effect on T cells and platinum drug resistance. Methods : Clinical specimens: The ovarian cancer specimens were obtained from patients undergone primary resection at the Osaka Medical and Pharmaceutical University, Japan. Written informed consent was obtained from each patient, and the study was approved by the ethics review board of the Osaka Medical and Pharmaceutical University. Tumour tissue‐derived EVs: Tissue‐immersed medium (normal ovaries, platinum‐resistant and sensitive ovarian cancer tissues) were centrifuged at 2000 ×  g , for 30 min, and the collected supernatants were subjected to the ultracentrifuge method (100,000 ×  g × 2) for recovery of tissue‐exudative EVs (Te‐EVs). The size and concentration of EVs were determined using qNano. RNA‐seq of Te‐EVs: RNA were isolated from Te‐EVs by using miRNeasy kit. RNA libraries were constructed using the NEBNext Small RNA Library Prep Set and sequenced on a HiSeq 2500 platform. Results : Compared to normal ovarian Te‐EVs and platinum‐sensitive ovarian cancer Te‐EVs, platinum‐resistant Te‐EVs were significantly taken up by CD4 T cells and their activity was suppressed. RNAseq analysis showed that ovarian cancer Te‐EVs harbored more 5′tRF‐GlyGCC than that of normal ovarian Te‐EVs. Overexpression of 5′tRF‐GlyGCC in normal ovarian Te‐EVs suppressed T cell activity. Furthermore, ovarian cancer EVs contribute to the acquisition of platinum drug resistance in a 5′tRF‐GlyGCC‐dependent manner in a mouse ovarian cancer model. Summary/Conclusion : 5′tRF‐GlyGCC in ovarian cancer EVs suppress T cells and contribute to the acquisition of platinum drug resistance.

Electrokinetic‐Based

Premanshu K. Singh 1 , Ali F. Usmani 1 , Patricia Sarchet 2 , Federica Calore 2 , Debmalya Halder 1 , Raphael E. Pollock 2 , Shaurya Prakash 1,2 1 Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, Ohio, USA; 2 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA Introduction : Liposarcoma (LPS), with 1500 US cases/year, has a 10‐year survival rate of only 10% due to late detection and high recurrence. Tissue biopsies and radiologic scans are costly and/or invasive, while liquid biopsy offers a minimally invasive alternative. We identified EV‐bound MDM2 DNA in serum as a biomarker for LPS recurrence, though limited sample volumes restrict analysis. Urine, offering larger volumes, could support broader analysis but requires improved isolation methods due to its variability and low EV concentrations, while conventional ultracentrifugation (UC) is labour‐intensive with low EV yield and purity. We developed a multi‐layered micro‐nanofluidic device with a sample injection channel (top) and a separation channel (bottom; each 3cm × 500µm × 60µm) integrated with a 200 nm nanocapillary membrane (NCAM) for selective capture of small EVs (< 200 nm) through immunocapture. We hypothesize that applying an electric field across the NCAM enhances the migration of negatively charged EVs into separation channel, improving capture efficiency. Methods : Cells from the LPS cell line Lipo246 were cultured in serum‐free media for 48 h to produce conditioned media (LCCM), centrifuged at 2000 × g for 20 min to remove debris, and stored for EV isolation. Voltage biases (0–100 V) were tested on LCCM with anti‐CD81 for EV capture in the separation channel. EV isolation from pooled human urine was performed to assess device performance with clinical samples alongside UC for comparative EV isolation. Isolated EVs were characterized using Nanotracking Analysis, DNA quantification (Nanodrop), and MDM2 DNA analysis (qPCR). All experiments were conducted in triplicates. Results : EV isolation from LCCM was optimal at 50 V, yielding 438 ± 59 ng/µL from a 450 µL sample, a 2.6× increase over 0 V. For urine samples (450 µL), 50 V resulted in a 1.5x increase in EVs ( p = 0.04, n = 2) and enhanced EV‐DNA yield to ∼98 ng, compared to ∼71 ng at 0 V. MDM2 DNA was detected in EVs from LCCM and urine. The device at 50 V isolated 98 ± 16 ng EV‐DNA from 450 µL of urine, offering 7.9× more EV‐DNA/mL than UC, which yielded 553 ± 111 ng from 20 mL. Summary/Conclusion : We report an electrokinetic‐based microfluidic device capable of isolating 7.9x higher EV‐DNA/mL of urine than UC with a 75% reduction in time. Funding : This work was supported by the US DoD through the PRCRP under Grant‐ CA210874 .

Bioinformatics‐Driven

Chin‐Ya Sophie Chiang 1,2 , Uta Erdbruegger 3 , Samantha Upson 3 , Fabian Braun 4,5,6 , Benedetta Bussolati 7 , Cristina Grange 7 , Helen G. Healy 1,2,8 , Andrew J. Kassianos 1,2,8 , Monica Suet Ying Ng 1,2,8 1 Kidney Health Service, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia; 2 Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Queensland, Australia; 3 Division of Nephrology, University of Virginia Health System, Charlottesville, Virginia, USA; 4 3rd Department of Medicine, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 5 Hamburg Center for Kidney Health, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 6 Martin Zeitz Center for Rare Diseases, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany; 7 Department of Medical Sciences, University of Turin, Turin, Italy; 8 Faculty of Medicine, University of Queensland, Herston, Queensland, Australia Introduction : The majority of kidney‐specific biomarkers identified in urine extracellular vesicles (uEVs) are derived from bulk multi‐omic (protein/RNA) analyses. However, only a limited number of these kidney‐specific markers will be present on the surface of uEVs and amenable to single uEV immunostaining. We present a bioinformatics‐driven approach to identify kidney‐specific surface markers on uEVs, followed by confirmation using single uEV flow cytometry. Methods : A bioinformatics pipeline was established to generate a list of kidney‐specific proteins predicted to be on the uEV surface. uEV protein data was extracted from Vesiclepedia (http://www.microvesicles.org; version 5.1) and crosslinked with subcellular location data from the UniProt database. Functional enrichment analyses using the STRING database (https://string‐db.org) were used to identify kidney‐specific uEV proteins localised to plasma and/or intracellular membranes. The resultant list was crosschecked against single‐cell RNA sequencing data from the Kidney Precision Medicine Project (KPMP, doi: 10.48698/mgd0‐gz70) to identify the potential nephron source of proteins. Protein expression in the kidney was confirmed using confocal microscopy. Subsequent marker validation was performed in uEVs isolated by sequential centrifugation followed by size exclusion chromatography and examined as per MISEV2023 guidelines using (i) single uEV flow cytometry and (ii) cryogenic electron microscopy and EV marker positivity. Results : 5062 uEV proteins were extracted from 95 experiments: 1463 proteins localised to membranes, 1642 proteins localised to the cytoplasm, 859 proteins localised to the nucleus, 714 proteins localised to intracellular organelles and 435 secreted proteins (some proteins identified in multiple compartments). 190 (∼13%) of the membrane proteins were identified as kidney‐specific. Confocal microscopy of kidney tissue confirmed nephron segment location of putative markers. Validation studies identified PODXL (podocytes), LRP2 (proximal tubule), SLC12A1 (loop of Henle) and AQP2 (collecting duct) positive uEVs at mean concentrations of 3.5 × 10 8 EVs/mL urine, 9.0 × 10 8 EVs/mL urine, 5.3 × 10 8 EVs/mL urine and 6.1 × 10 8 EVs/mL urine, respectively. Summary/Conclusion : Existing bulk uEV proteomics and single‐cell RNA sequencing datasets can be leveraged to identify surface markers for selecting kidney uEVs. This bioinformatics pipeline provides novel diagnostic targets for improved characterization of discrete glomerular versus tubular uEVs for the clinical and scientific evaluation of kidney diseases. Funding : Queensland Advancing Clinical Research Fellowship, Metro North Health Clinician Research Fellowship.

Extracellular‐Vesicle

Omnia M. Elsharkasy 1 , Charlotte V. Hegeman 2 , Ivana Lansweers 2 , Olaf L. Cotugno 1 , Ingmar Y. de Groot 1 , Zoë E.M.N.J. de Wit 2 , Antonio Garcia‐Guerra 3,4 , Niels J. A. Moorman 2 , Willemijn S. de Voogt 1 , Jerney J. Gitz‐François 1 , Xiuming Liang 5,6,7 , Samir El Andaloussi 5,6,7 , Raymond M. Schiffelers 1 , Sander A.A. Kooijmans 8,9 , Enrico Mastrobattista 2 , Pieter Vader 1 , Olivier G. de Jong 1,2 1 University Medical Center Utrecht, Utrecht, The Netherlands; 2 Utrecht University, Utrecht, The Netherlands; 3 University of Oxford, Oxford, Oxfordshire, UK; 4 Institute of Developmental and Regenerative Medicine, Oxford, Oxfordshire, UK; 5 Karolinska Institutet, Stockholm, Sweden; 6 Karolinska University Hospital Huddinge, Huddinge, Sweden; 7 Karolinska ATMP Center, Stockholm, Sweden; 8 Wilhelmina's Children's Hospital, Utrecht, The Netherlands; 9 Regenerative Medicine Center Utrecht, Utrecht, The Netherlands Olivier G. de Jong is the presenting author . Introduction : CRISPR/Cas9 is a prokaryotic endonuclease capable of targeting genomic sequences with high specificity and efficiency, generating double‐strand DNA breaks that may result in frameshift mutations. Additional functionalization of Cas9 with enzymatic groups, such as adenine base editors (ABE), which replace adenine with guanosine in specific targeted genomic sequences, allows for more specific genomic repairs. One major hurdle for therapeutic applications of such Cas9‐based moieties is their intracellular delivery. Extracellular vesicles (EVs) hold the potential to overcome this hurdle due to their biocompatibility and intrinsic capability of efficient intercellular transfer of RNA and proteins. Here, we present a modular strategy for EV‐mediated loading and delivery of Cas9 and Cas9‐ABE. Methods : To facilitate loading of either spCas9 or Cas9‐ABE RNP complexes, sgRNAs with high‐affinity MS2 coat protein‐interacting aptamers were generated and expressed alongside spCas9 or ABE8e and EV‐enriched proteins fused to the MS2 coat protein (MCP). The MCP, lacking the Fg loop to prevent capsid formation, was cloned in tandem on the N‐terminus of CD9 and CD63, linked by a UV‐sensitive photocleavable protein (PhoCl). To study spCas9 and ABE8e delivery, we used previously published fluorescent Stoplight reporter systems (De Jong, Nat Commun. 2020; Öktem, Pharmaceutics 2023) and analysed endogenous genomic targets by T7E1 assays. Results : EV loading of Cas9, as well as UV‐mediated cleavage of the PhoCl fusion proteins, was confirmed by western blot analysis and flow cytometry. Using EVs with MS2‐PhoCl‐CD63/CD9 fusion proteins, we observed efficient spCas9 and ABE8e delivery, where CD9 showed notably higher (> 2‐fold) delivery than CD63. Interestingly, ABE8e activity was hampered by the incorporation of MS2 aptamers in the second stemLoop of the sgRNA, but only in the presence of MCPs, suggesting interference by steric hindrance of the deaminase. This was resolved by solely incorporating an MS2 aptamer in the sgRNA tetraloop, resulting in high levels of EV‐mediated ABE8e delivery (> 60% reporter activation). Summary/Conclusion : Here, we describe a novel modular platform for EV‐mediated loading and delivery of Cas9 RNPs. Our results demonstrate that EVs are capable of functional spCas9 and ABE8e RNP delivery. Moreover, these data indicate that additional modifications for regulated cargo release strongly increase Cas9‐RNP delivery.

Fetoplacental‐Derived

Juliana S Powell, William J Shufesky, Adriana T Larregina, Yoel Sadovsky, Adrian E Morelli University of Pittsburgh, USA Introduction : Pregnancy constitutes an ‘immunological paradox,’ where despite the competent maternal immune system, the semi‐allogeneic foetus evades rejection. Mechanisms at the maternal‐foetal interface and the mother's secondary lymphoid tissues (SLTs) that dodge the immunologic attack on the foetus, remain unknown. Passage of small extracellular vesicles (EVs) is a mechanism of cell‐to‐cell communication that transfers antigens (Ags), regulatory mediators, and RNAs. In transplantation, graft‐derived EVs carried through blood or lymph promote anti‐donor T‐ and B‐cell responses in the maternal SLTs. Thus, akin to transplantation, fetoplacental‐derived EVs transported via blood could represent a mechanism by which fetoplacental Ags and immune‐regulatory mediators could be delivered to maternal SLT immune cells for T and B cell recognition. Methods : We tested whether fetoplacental EVs could transport paternal Ags to maternal SLTs. Results : In female BALB/c mice impregnated with mOVA C57Bl/6 (B6) males, the surrogate paternal Ag OVA accumulated in maternal SLTs. In a mouse model in which EVs released by the fetoplacental unit are labelled with the green fluorochrome mNeonGreen linked to the small EV‐associated marker CD81, mNeonGreen‐CD81 was detected by fluorescence microscopy in APCs in the maternal spleen. Immunoprecipitation followed by western blot of EVs from primary cultures of trophoblasts from BALB/c females impregnated with mOVA B6 males revealed OVA on EVs. Administration of these trophoblast‐derived EVs to virgin females induced defective activation and proliferation of OVA‐specific CD4 (OT‐II) and CD8 (OT‐I) TCR transgenic T cells in the spleen. In Rab27a‐/‐ mice in which vesicle release is deficient, OVA‐specific OT‐I proliferation is reduced. In B6 females impregnated with Act‐mOVA males, paternal Ag, OVA, is retained in maternal tissues until at least post‐partum (PP) day 30. In humanized mice, i.v. injection of CM‐DiI (red)‐labelled EVs from primary human trophoblasts were captured by human antigen presenting cells (APCs) in the spleen. Summary/Conclusion : Therefore, systemically released fetoplacental EVs carry paternal/trophoblast Ags that reach the maternal APCs in the mother's SLTs, where the Ags are presented in a manner that promotes deficient activation of T cells as occurs during a normal pregnancy. Funding: National Institutes of Health, USA. 5R01AI168142‐03, 5R01AI148690‐05, 5R01AI174273‐03.

Hydrogel‐Encapsulated

Presenter: Qi Chen Shanghai Jiao Tong University, China (People's Republic) Introduction : Due to the low immune cell infiltration and the suppressive tumour immune microenvironment (TIME), the efficacy of immunotherapy for prostate cancer is currently quite limited. This study aims to design a cancer vaccine using supramolecular peptide hydrogel encapsulated with ex vivo tumour tissue‐derived exosomes (TEXs) and adjuvants to inhibit the growth and recurrence of prostate cancer, as well as improve the effect of immune checkpoint inhibitors. Methods : Here, we design an amphiphilic self‐assembling peptide, FK12, capable of forming hydrogels in situ after subcutaneous injection. TEXs are extracted through the lysis and ultracentrifugation of tumour tissue. The positively charged self‐assembler FK12F can adsorb negatively charged TEXs, CDA (a STING agonist), and GM‐CSF through electrostatic interactions to create the Gel‐Vaccine system. We characterize the controlled release of the vaccine components through the gel by in vitro and in vivo experiments. We then assess the therapeutic effects and related molecular mechanisms of gel vaccines in different mouse models of prostate cancer. Results : Significant tumour regression was observed in mice vaccinated with the gel vaccine across multiple mouse models of prostate cancer. This effect was even more pronounced in mice treated with the combination of aPD‐1. We detected a higher number of DC aggregates at the site of gel vaccination, with an increased presence of mature and migratory DCs. Furthermore, we observed an elevated count of mature DCs, along with an increased presence of CD4+ and CD8+ T lymphocytes in the lymph nodes. Additionally, a greater number of CD8+ T cells with cytotoxic capabilities and fewer regulatory T cell infiltrates were detected within the tumour tissue. Summary/Conclusion : TEXs are enriched with tumour antigens, making them potential stimulators of anti‐tumour immune responses. After subcutaneous inoculation, the exosome‐gel scaffold can be triggered in situ to form an artificial secondary lymphoid organ, continuously recruiting antigen‐presenting cells through cytokines and achieving highly efficient antigen processing induced by adjuvants. This orchestrated process induces immune infiltration in the TIME. When combined with aPD‐1 immunotherapy, it triggers a potent and long‐lasting tumour‐specific immune response, effectively inhibiting prostate cancer growth and recurrence.

Myofibroblast‐Derived

Presenter: Rahul Sanwlani University of Surrey, Guildford, UK Introduction : Hypertrophic cardiomyopathy (HCM) is the most prevalent genetic cardiomyopathy. Previously, we reported paracrine signalling‐mediated regulation of hiPSC‐derived cardiomyocyte electrophysiology by myofibroblasts. The role of extracellular vesicles (EVs) has been increasingly highlighted as mediators of pathophysiology. Methods : HCM patient biopsies were processed for cardiac fibroblast (CF) isolation from tissue explants. To understand the role of CFs and their EVs in regulating cardiomyocyte function, in this study, we used the ALK5 inhibitor SD‐208 to characterise its effects on HCM CFs. Utilising this model of revering CF activation, we studied the effect of HCM CF small EVs in regulating cardiomyocyte function and electrophysiology. Results : SD‐208 treatment did not significantly alter the viability and metabolic potential of HCM CFs. However, molecular characterisation suggested a significant reduction in mRNA levels of activation markers ACTA2, POSTN, FAP, COL1A1, IL‐6 and IL‐11 ( p < 0.0001) following treatment. A significant reduction in α‐SMA positive cells ( p = 0.0043) in flow cytometry and reduction in total collagen secretion ( p = 0.00062) occurred following treatment. CF EVs collected using size exclusion chromatography were observed to be in the small EV range and were enriched with EV marker proteins. Interestingly, SD‐208 treatment led to a 6.5‐fold reduction in EV particles secreted by HCM CFs ( p < 0.0001). Similarly, SD‐208 treatment had no significant impact on viability and metabolic potential of HEK‐293 cells but led to a dose‐dependent reduction in EV yield (< 0.01). HCM CF EVs were observed to regulate contractility and calcium signalling in hIPSC derived cardiomyocytes. Following treatment, the cardiomyocytes demonstrated the HCM phenotype. Summary/Conclusion : Overall, these findings demonstrate antifibrotic activity of SD‐208. As SD‐208 treatment reverted myofibroblasts to fibroblasts, this allowed for investigations to understand the role of CF EVs in regulating cardiomyocyte function. These findings have also highlighted the potential role of SD‐208 as a regulator of EV biogenesis and secretion.

Polydopamine‐Modified

Presenter: Bodeng Wu Southern Medical University, China (People's Republic) Introduction : Plant‐derived nanovesicles are gaining interest for their role in interspecies communication, yet challenges persist in distinguishing them from their source plants and understanding their regulatory mechanisms and clinical applications. Our previous studies identified turmeric‐derived nanovesicles (TDNVs) as pharmacological complexes that modulate the Nrf2/HO‐1 pathway in fibroblasts and the TLR4/MyD88 pathway in macrophages, enhancing the diabetic wound microenvironment. To address the frequent occurrence of multidrug‐resistant infections in diabetic wounds, this study developed an engineered TDNVs@PDA hydrogel system as a potential therapeutic strategy. Methods : TDNVs were modified with polydopamine to create near‐infrared (NIR) responsive vesicles TDNVs@PDA (TP). Characterization of TP involved transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering for assessing morphology, size, and zeta potential. A PLGA‐PEG‐PLGA hydrogel was used as the matrix to load TP, forming TP@Gel. The photothermal effect, uptake efficiency, and antibacterial activity of TP@Gel against multidrug‐resistant Pseudomonas aeruginosa (MDRPA) were evaluated in vitro. The therapeutic efficacy of TP@Gel in diabetic wound infections was investigated, and transcriptomic analysis assessed changes in the tissue microenvironment. Results : TP was synthesized with a uniform cup‐shaped structure, displaying a particle size of 100–200 nm. The TP@Gel system demonstrated effective TP release and significant uptake in MDRPA, enhancing antibacterial efficacy under NIR irradiation. In vitro studies showed that TP@Gel reduced reactive oxygen species levels in fibroblasts, enhanced Nrf2/HO‐1 signalling, and promoted macrophage polarization to the M2 phenotype, resulting in decreased pro‐inflammatory cytokines TNF‐α and IL‐1β. Gelation of TP@Gel at physiological temperatures effectively cleared wound infections and facilitated healing. Transcriptomic analysis revealed modulation of inflammation, antioxidant responses, and extracellular matrix remodelling pathways compared to controls. Summary/Conclusion : TP@Gel serves as a multifunctional therapeutic approach that addresses bacterial infections while enhancing antioxidant defence and reducing inflammation in diabetic wounds. This study underscores the potential of TDNVs in regenerative medicine and their applicability as promising solutions for diabetic wound healing in clinical settings. Funding : National Science Fund for Distinguished Young Scholars of China (82025024). National Key R&D Program of China (2021YFA1300604).

Hyperglycaemia‐Induced

Anastasiia Artuyants 1,2 , Zi Yin Enid He 1 , Bianca Nijmeijer 1 , Sophia Bebelman 1 , Martin Middleditch 3 , Deanna Shea 3 , Cherie Blenkiron 1,2 1 Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand; 2 Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand; 3 School of Biological Sciences, The University of Auckland, Auckland, New Zealand Introduction : Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries, with rising incidence partly driven by obesity and metabolic disorders like type 2 diabetes. Hyperglycaemia, a defining feature of diabetes, contributes to a pro‐tumourigenic environment through enhanced cellular growth, inflammation, and oxidative stress, which can accelerate cancer progression. However, the impact of hyperglycaemia on extracellular vesicles (EVs)—crucial mediators of cell‐to‐cell communication in the tumour microenvironment, remains underexplored. In this study, we investigated how high glucose conditions affect EV production, protein cargo, and functionality in EC cell lines, potentially revealing mechanisms through which hyperglycaemia may influence EC progression. Methods : Three EC cell lines—MFE280, MFE296, and Ishikawa, representing different EC grades, were cultured in 3D bioreactor systems under normal and high‐glucose conditions for 10 to 23 weeks. EVs were isolated twice weekly via differential centrifugation and size exclusion chromatography. Cell tolerance to long‐term high glucose exposure was monitored through particle counts, glucose and pH testing. Selected EV samples were analysed using proteomics and functional assays. Results : 3D bioreactor models enabled continuous culture and large‐scale EV production. High glucose conditions did not significantly change EV particle count or size, though a pH reduction in the conditioned media suggested increased lactate production. Proteomic analysis revealed significant alterations in EV protein composition under high glucose conditions, indicating potential shifts in pathways related to extracellular matrix remodelling, immune response, energy metabolism, stress signalling, and oxidative stress. These changes suggest that hyperglycaemia may promote modifications in the tumour microenvironment by altering cell signalling, supporting metabolic adaptation, and enhancing survival mechanisms within cancer cells. The effects on EC line proliferation, activity, colony formation and adhesion are being analysed. Summary/Conclusion : These findings indicate that hyperglycaemia induces specific changes in EV protein cargo that may alter the tumour microenvironment, potentially facilitating EC progression. This research provides insights into the role of EVs in hyperglycaemia‐related tumour biology. Further work could clarify the role of EV‐mediated signalling in hyperglycaemic conditions and its implications for EC progression.

Micromixer‐Facilitated

Özgecan Erdem 1 , Kadriye Ölmez 1,2 , Beyza Nur Küçük 1,2 , Eylul Gulsen Yilmaz 1,2 , Fatih Inci 1,2 1 Bilkent University, UNAM‐National Nanotechnology Research Center, Ankara, Turkey; 2 Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey Introduction : Molecularly imprinted polymers (MIPs) mimic antibodies or enzymes, incorporating binding sites for target analytes. Conventional MIP production is slow, typically relying on closed, batch systems. Microfluidics offers a modern approach to nanoparticle synthesis, enabling precise mixing and increased productivity. Extracellular vesicles (EVs), critical biomarkers for kidney disease diagnosis, provide more specific information than traditional markers like serum creatinine, which may lack sensitivity. This study introduces an innovative convergence‐divergence micromixer for synthesizing EV‐MIPs with high precision. Additionally, we developed a nanoplasmonic sensor using EV‐MIPs for real‐time detection of kidney‐related EVs, offering excellent specificity and binding affinity. Methods : EVs were first isolated from HEK 293 cells using on‐chip ultrafiltration and then characterized via Nanoparticle Tracking Analysis (NTA) and Scanning Electron Microscopy (SEM) before being applied to EV‐MIP synthesis. Soft lithography‐fabricated microfluidic channels, shaped as ellipses (600 × 500 µm) merging into rectangular (100 × 300 µm) and smaller ellipsoidal (300 × 200 µm) channels, facilitated the flow. Mixing efficiency was optimized through COMSOL Multiphysics simulations at varied flow rates, resulting in successful nanoparticle synthesis. EV‐MIPs underwent analysis with NTA, Dynamic Light Scattering (DLS), SEM, and Fourier‐transform infrared spectroscopy (FTIR). A plasmonic metasurface was also modified with EV‐MIPs for EV detection. Results : The size of the isolated EVs were measured around 180 nm with a concentration of 7 × 10⁹ particles/mL using NTA. SEM images confirmed their spherical shape and dimensions. COMSOL simulations at flow rates of 100, 120, and 150 µL/min identified 150 µL/min as optimal for producing nanoparticles with narrower size distribution. EV‐MIPs were measured around 84 nm, with a concentration 1 × 10⁹ particles/mL. EV detection by the microfluidic plasmonic sensor was indicated by wavelength shifts signalling EV‐MIP binding on the surface. Binding of EVs shifted the baseline signal to 1.34 nm, confirming EV attachment to EV‐MIPs on the sensor. Summary/Conclusion : This study demonstrates the efficient use of a micromixer for continuous‐flow EV‐MIP synthesis and detection of kidney disease‐related EVs using a nanoplasmonic sensor. This approach shows promise for real‐time diagnostics in renal health applications. Funding : Supported by TÜBİTAK (Project No: 118C254), Turkish Academy of Sciences (GEBİP), and Science Academy's (BAGEP) awards.

Glycopolymer‐Engineered

Presenter: So Hee Kim Sungkyunkwan University, Republic of Korea Introduction : Acute kidney injury (AKI), commonly induced by nephrotoxic agents, leads to high morbidity due to limited repair treatments. Interventions like dialysis or transplantation relieve symptoms but do not restore kidney function. Effective recovery requires protecting renal tubular epithelial cells (RTECs) and regulating M1 macrophages. Stem cell‐derived extracellular vesicles (SC‐EVs), rich in bioactive molecules, offer a cell‐free regenerative approach. However, since most injected EVs accumulate in the liver, surface modifications are needed. This study developed glycopolymer‐engineered EVs(PH‐EVs) with PEGylated hyaluronic acid(PEG‐HA) to target CD44 and TLR4 on RTECs and macrophages, achieving precise kidney targeting and promoting renal protection. Methods : To synthesize PEG‐HA, PEG was conjugated to HA (5:100 ratio), followed by DBCO‐PEG‐HA synthesis for SC surface engineering via click chemistry. PH‐EVs were isolated from PEG‐HA‐modified SCs using TFF. The physicochemical properties of PH‐EVs were confirmed by TEM and ELISA. In vivo bio‐distribution of PH‐EVs were compared to unmodified‐EVs. The therapeutic efficacy of PH‐EVs was verified in both cellular level and cisplatin(CP)‐induced AKI mice model. Results : Surface modification of EVs was confirmed by TEM, with HA polymer quantification via ELISA. Cisplatin (CP) was used to model drug‐induced AKI in vitro and in vivo. PH‐EVs showed enhanced uptake compared to unmodified‐EVs, only in CP‐treated HK‐2 cells. PH‐EVs protected RTECs from apoptosis and inhibited macrophage polarization into a pro‐inflammatory state in vitro. In CP‐induced AKI mice, PH‐EVs selectively accumulated in injured kidneys, co‐localizing with CD44, thereby significantly reducing AKI markers, including creatinine, BUN, and NGAL levels. Also, immunohistochemistry showed PH‐EV‐mediated renal protection by reducing apoptotic cells and inducing proliferation of cells. Summary/Conclusion : Our PH‐EVs demonstrate strong potential as a regenerative AKI treatment by selectively targeting injured kidneys via AKI‐related receptors (CD44 and TLR4), thereby alleviating AKI symptoms. This precisely targeted approach highlights the therapeutic promise of extracellular vesicle engineering, offering a cell‐free strategy to overcome limitations in current AKI therapies and enhance kidney repair.