ISEV2023 Abstract Book

A

Mohammed T. Hussain 1 ; Shani Austin‐Williams 2 ; Dianne Cooper 3 ; Lucy Norling 2 1 Biochemical Pharmacology, William Harvey Research Institute, Queen Mary University of London, United Kingdom; 2 William Harvey Research Institute, Queen Mary University London, London, United Kingdom; 3 William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, United Kingdom, United Kingdom Introduction : Osteoarthritis (OA) is the most prevalent age‐related degenerative disorder and severely reduces the quality of life of those affected. Whilst management strategies exist, no cures are currently available. Virtually all joint resident cells generate extracellular vesicles (EVs), which even seed articular cartilage; a structure whose health is central to the initiation and propagation of OA. There are reported changes in the cargo of joint‐derived EVs during OA. However, whether EVs play a homeostatic role that is compromised in OA is unknown. Here we investigate how chondrocyte EVs may have a homeostatic role in maintaining chondrogenic gene expression. Methods : EVs from C28/I2 chondrocytes in monolayers (2D) or pellet cultures (3D) were isolated using differential centrifugation with a final step at 20,000g for 30 minutes at 4°C. EVs were characterised by nano‐flow cytometry, transmission electron microscopy and western blotting. Culture media supplemented with insulin‐selenium‐transferrin (ITS) promoted anabolic gene expression whereas EV‐free foetal bovine serum (FBS) decreased it. Secondary EV release and anabolic gene expression was evaluated following treatment of chondrocytes with 2 × 106 EVs from 2D or 3D cultures for 24 hours. Additionally, the uptake of fluorescently labelled EVs was assessed via confocal microscopy following 4 hours of treatment of EVs from 2D or 3D cultures in either FBS or ITS conditions. Results : Culturing chondrocytes in 3D significantly increased EV generation compared to cells cultured in 2D. EV accumulation in 3D cultures was similar irrespective of culturing in FBS or ITS supplemented media. However, EV release decreased in 2D cultures with FBS compared to those in ITS. Treatment of 2D chondrocytes cultured in FBS with 2D EVs had no effect on anabolic genes; however, treatment with EVs from 3D cultures significantly improved chondrogenic gene expression and promoted secondary EV release. EV uptake was greatest under ITS conditions and irrespective of culture conditions; uptake of EVs was greater from a 3D source compared to 2D. Summary/Conclusion : These results indicate chondrocyte EVs have an autocrine homeostatic role that maintains chondrocyte phenotype. How this role is perturbed under OA conditions remains the subject of future work. Funding : Funding provided by the Centre of Osteoarthritis Pathogenesis versus Arthritis/Centre for Innovation and Therapeutic Innovation (CiTI) Doctoral Training Programme (grant number 21621). Keywords : osteoarthritis, chondrocytes, cartilage

L

Agnieszka Razim 1 ; Deepshika Parthasarathy 2 ; Eliška Krčmářová 3 ; Magdalena Wysmołek 4 ; Paweł Migdał 5 ; Catherine Daniel 6 ; Ursula Wiedermann 4 ; Irma Schabussova 7 1 Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria, Wien, Austria; 2 Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria, Vienna, Austria; 3 Institute of Immunology and Microbiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republik, Austria; 4 Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria, Austria; 5 Hirszfeld Institute of Immunology and Experimental Therapy PAS, Wroclaw, Poland, Poland; 6 Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 ‐ UMR 9017 – Center for Infection and Immunity of Lille, F‐59000 Lille, France, France; 7 Medical University of Vienna, Vienna, Austria Introduction : Allergy is still a pressing issue and the only causative and disease‐modifying treatment for allergies is immunotherapy, which is burdened with several drawbacks e.g. risk of anaphylaxis induction. The beneficial properties of probiotics in fighting allergies have been recognized, however, the use of living organisms is not without its flaws. Derivatives of probiotics, such as extracellular vesicles (EVs), due to their modulatory and adjuvant activity, can be an excellent replacement of whole bacteria, but there is a gap in the knowledge on Gram+ probiotic EVs. Methods : L. plantarum NCIMB8826 was grown anaerobically in MRS medium (37°C) until it reached the OD of 0.5, 1.0, 1.5 or 2. L. plantarum EVs (LpEVs) were isolated by centrifugation (4 000 x g, 20 min), filtration (0.22 μm filter), concentration by Amicon Stirred Cell (300 kDa filter) and ultracentrifugation (3 h, 150 000 x g). LpEVs were purified with IZON qEV original column and characterized by Zetasizer (size and particle counts), TEM, SDS‐PAGE (with MS protein identification) and Bioanalyzer (RNA, DNA and protein content). In vitro analysis of immunostimulatory properties and stability studies are ongoing. Results : Our data show that the profile of produced LpEVs is OD‐dependent. Longer cultures of bacteria result in a heterogenic profile of isolated LpEVs. We are able to isolate up to 2 × 1012 of EVs from a 5 l culture of bacteria. Produced EVs are less than 100 nm in size and they contain diverse proteins and small RNA. Interestingly, we did not detect DNA. Summary/Conclusion : L. plantarum species were already shown to have an immunomodulatory effect. We have shown that this Gram+ bacteria produces EVs and we characterized them. This is the first step in designing a platform for antigen‐specific, intranasal, anti‐allergy therapy based on probiotic EVs. Funding : This research was funded by HORIZON‐MSCA‐2021‐PF (project no. 101066450), Danube Allergy Research Cluster and Foundation for Polish Science (FNP). Keywords : allergy, probiotics, extracellular vesicles

An

Jingyuan Zheng 1 ; Brian V. Hong 1 ; Joanne K. Agus 2 ; Xinyu Tang 1 ; Nola Klebaner 1 ; Fei Guo 1 ; Wyatt Vreeland 3 ; Olesia Gololobova 4 ; Kenneth W. Witwer 5 ; Carlito Lebrilla 2 ; Izumi Maezawa 1 ; Lee‐Way Jin 1 ; Angela M. Zivkovic 2 1 University of California, Davis, USA; 2 University of California, Davis, Davis, USA; 3 National Institute of Standards & Technology, Gaithersburg, USA; 4 Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, USA; 5 Johns Hopkins University, Baltimore, USA Introduction : The smallest of the biologically important nanoparticles, high‐density lipoproteins (HDL, 5–12nm diameter) are too small for most particle sizing methods. Negative‐stain transmission electron microscopy (NS‐TEM) allows direct visualization and precise size quantification of individual particles down to this size range. Methods : HDL were isolated from 26 plasma samples, negatively stained (2% uranyl formate), and micrographs were obtained using a JEOL 1230 model EM at 40k magnification, 120 kV with a bottom‐mounted Ceta camera (4k × 4k pixels). Particle size was analyzed with ImageJ using an in‐house automation script with different circularity, roundness, and aspect ratio cutoff combinations and compared to manual selection. Particle sample sizes from 1 to 5,000 were selected randomly 100 times from a population of 43,494 particles in one representative sample, and the sample size required for deviation from population mean by 1%, 5%, and 10% within 95% of the time was simulated. The coefficient of variation (CV) across 15 images per sample was calculated and compared to batch dynamic light scattering (DLS). Results : The optimal parameters of circularity < 0.5, roundness 1.5 yielded 78% particle recovery and 87% correct rate compared to manual particle selection, in a fraction of the time. A sample size of at least 3,000, 120, and 50 particles resulted in a mean particle size within 1%, 5%, and 10% deviation from the mean population particle size 95% of the time. Inter‐run CVs of 0.34% vs. 2.8% for size standards, and 3.503% vs. 10.43% for biological samples were obtained for the NS‐TEM method vs. batch DLS, respectively. Summary/Conclusion : Our optimized method using NS‐TEM followed by automated image analysis is reproducible and amenable to particle sizing of HDL in large numbers of clinical samples due to its accuracy and high‐throughput. Funding : This project was funded by NIH Grants R01AG062240, R01GM147545, UH3CA241694. Keywords : nanoparticles, high‐density lipoprotein, electron microscopy

Do

Vera Tscherrig 1 ; Valérie Haesler 2 ; Sophie Cottagnoud 2 ; Patricia Renz 1 ; Daniel Surbek 2 ; Andreina Schoeberlein 2 ; Marianne Jörger‐Messerli 2 1 1) Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, Switzerland and Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland, 2) Graduate School for Cellular and Biomedical Sciences (GCB), University of Bern, Bern, Switzerland, Bern, Switzerland; 2 1) Department of Obstetrics and Feto‐maternal Medicine, University Women's Hospital, Inselspital, Bern University Hospital, Bern, Switzerland and Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland, Bern, Switzerland Introduction : Preterm birth is the leading cause of childhood morbidity and mortality. Preterm birth often results in neurological complications such as premature white matter injury (WMI). WMI leads to lifelong disabilities. There is no cure for premature WMI. Mesenchymal stroma cell‐derived small extracellular vesicles (MSC‐sEV) show promising results as a therapeutic agent for neurological injuries. MSC‐sEV carry microRNAs (miRNAs), predicted to target mRNAs encoding for proteins belonging to signaling pathways of premature WMI. We hypothesize that miRNAs, released by MSC‐sEV upon uptake in their target cells, have a key function in the observed beneficial effects from MSC‐sEV. Methods : sEV were purified from Wharton's jelly MSC by ultracentrifugation followed by size exclusion chromatography and characterized according to morphology, protein content, size, and zeta potential. The miRNA content was measured by qPCR. A luciferase assay and a DROSHA knock‐down of the MSC were established to evaluate the regulatory activity of sEV miRNA. The regulatory potential of the sEV was validated in cell differentiation and cell death in vitro assays. Results : The samples were positive for sEV markers CD81, CD63, and CD9, and contained miRNAs being involved in WMI, such as hsa‐miR‐22‐3p, hsa‐miR‐21‐5p, hsa‐miR‐27b‐3p, and the hsa‐let‐7 family. sEV significantly reduced the luciferase signal in a luciferase assay with a vector containing a 3'UTR of TP53 and TAOK1, genes involved in WMI, indicating an inhibitory effect of sEV miRNA. sEV enhanced differentiation in the oligodendrocyte lineage. After oxygen‐glucose deprivation, sEV reduced apoptotic markers in neuroblastoma cells. sEV from WJ MSC with DROSHA knock‐down had less miRNA cargo and a lower effect on the in vitro assays than sEV from untreated cells. Summary/Conclusion : Our results show the functionality of the miRNA cargo in the therapeutic effect of MSC‐sEV in preclinical WMI. To confirm the in vitro results, in vivo experiments in a model of premature WMI are ongoing. Keywords : white matter injury, mesenchymal stromal cells, small extracellular vesicles, therapeutic potential, microRNA

Ev

Shruti Jain 1 ; Laura Lehtinen 2 ; Kim Pettersson 3 ; Karin Sundfeldt 4 ; Janne Leivo 3 ; Kamlesh Gidwani 5 1 Department of Life Technologies and InFLAMES Flagship Research Center, University of Turku, Turku, Finland., Turku, Finland; 2 University of Turku, Turku, Finland, Paimio, Finland; 3 Department of Life Technologies and inFLAMES Flagship Research Center, University of Turku, Turku, Finland., Turku, Finland; 4 Sahlgrenska Center for Cancer Research, University of Gothenburg, Gothenburg, Sweden; 5 University of Turku, Turku, Finland, Turku, Finland Introduction : Epithelial ovarian cancer (EOC) is usually diagnosed at later stages with 5 year survival rate less than 30%. There is a need for better diagnostic markers. Glycosylation changes is a universal phenomenon of cancer cells and could be targeted for potential biomarker discovery. Glycans are strongly associated to integrins (ITG) and tetraspanins (e.g. CD9, CD63, CD81) stability and function, and their interaction partners are dysregulated in tumorigenic processes. CD63 is a marker for exosomes and glycans are major constituents of extracellular vesicles (EV). EVs carry an array of cargo which includes integrins. Methods : EVs were purified from OC002 cell line using Exo‐spin SEC columns (CGS, UK). Purified EV glycovariants were screened with the use of integrin and tetraspanin specific antibodies. The most promising ITG and CD63 glycovariants (STn, WGA, UEA) were detected from 10 EOC and 5 benign ovarian cyst fluid samples on microtiter wells using antibodies or lectins conjugated on europium nanoparticles. Total ITG and CD63 immunoassays were also performed. Finally, the best performing glycovariants were clinically evaluated on the whole cohort of 77 ovarian cyst fluid samples. This study was approved by the ethics committee in Gothenburg and informed consent was obtained from all individual participants included in the study. Results : STn glycovariant of both ITGα3 and CD63 was detected in EVs purified from EOC cell line and performed better than corresponding protein epitope‐based immunoassays, ITGα3‐IA and CD63‐IA respectively in clinical samples. Combined ITGα3 based assays (ITGα3‐IA + ITGα3‐STn) detected 49 out of 55 malignant and borderline cases without detecting any of the 22 benign or healthy cysts. Summary/Conclusion : Our findings indicate the potential diagnostic application of STn glycovariants of ITGα3 and CD63 along with total ITGα3‐IA, which could help reduce the unnecessary surgeries. The results encourage studying further the potential use of these novel EV based assays to detect EOC at earlier clinical stages. Funding : N/A. Keywords : integrin, CD63, glycosylation, cancer, diagnostic

In

Lavinia Flaskamp 1 ; Christine Ried 2 ; Jan Kranich 3 ; Thomas Brocker 2 1 Institute for Immunology, Biomedical Center (BMC), Faculty of Medicine, LMU Munich, Munich, Germany, Munich, Germany; 2 Institute for Immunology, Biomedical Center (BMC), Faculty of Medicine, LMU Munich, Munich, Germany, Planegg‐Martinsried, Germany; 3 Institute for Immunology, Biomedical Center (BMC), Faculty of Medicine, LMU Munich, Munich, Germany, USA Introduction : Phosphatidylserine (PS) is a phospholipid normally retained on the inner leaflet of cellular membranes. However, it has been shown to be exposed on the surface of apoptotic cells, activated platelets and importantly also on extracellular vesicles (EV). EV detection based on PS, using for example fluorescent Annexin V, has been widely established in vitro, but Ca2+‐dependence prohibits its application in vivo. In contrast, Milk fat globule‐EGF factor 8 (MFG‐E8) represents a Ca2+‐independent PS‐binding protein. Previously, we have used fluorescent MFG‐E8, or its C1‐domain, to detect EV‐associated cells in vivo by imaging flow cytometry (IFC). The aim of the present study was to extend the application of C1‐reagents to bind and detect circulating PS+ EVs in serum in vivo. Methods : For in vivo labelling of PS+ EVs, C57BL/6 mice were intravenously injected with 50 μg of fluorescent labelled mC1‐multimers. Serum EVs were isolated by size exclusion chromatography (SEC) using qEV35 columns (Izon) and EV containing fractions were confirmed by nanoparticle tracking analysis, bicinchoninic acid assay and western blotting. Following isolation, EVs were analyzed by IFC (ImageStreamTM) and dSTORM microscopy (ONI). Results : EVs were bound by the PS‐specific mC1‐multimer in vivo and the label remained intact following their isolation from serum, as assessed by IFC. This analysis revealed that the majority of endogenous circulating serum EVs were PS+ (>90%). Furthermore, co‐incubation of differentially labelled EVs did not show dye transfer between EVs after isolation via SEC. Summary/Conclusion : We could demonstrate a novel method for detection of PS+ EVs in vivo, which enables the study of endogenous EVs without the need for introduction of reporter genes. Moreover, we showed that mC1‐multimer labelled EVs can be used for distinct downstream analyses, including quantitative measurements and characterization of surface molecules. Funding : This project was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research foundation) – Project‐ID 210592381 ‐ SFB 1054 B03 to TB and SFB 1954 Z02 to JK. Keywords : extracellular vesicles, phosphatiylserine, dSTORM, imaging flow cytometry, MFG‐E8, lactadherin

No

Rossella Crescitelli 1 ; Daniele D´Arrigo 2 ; Cecilia Lässer 3 ; Kyong‐Su Park 4 ; Roger Olofsson Bagge 5 ; Jan Lötvall 4 1 1Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Gothenburg, Sweden; 2 Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 3Regenerative Medicine Technologies Laboratory, Ente Ospedaliero Cantonale, Bellinzona, Switzerland, Sweden; 3 Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Gothenburg, Sweden; 4 Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Gothenburg, USA; 5 Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Department of Surgery, Sahlgrenska University Hospital, Region Västra Götaland, Gothenburg, Sweden, Gothenburg, USA Introduction : Solid tissues are emerging as a relevant source of extracellular vesicles (EVs). EVs have been isolated from both fresh and from frozen tissues, even though a thoroughly analysis of the potential effects introduced by the freezing process on EVs has not been performed yet. The aim of this study is therefore to evaluate the effects of tissue freezing process on EV purity and composition. Methods : Human melanoma metastatic tissue (n = 6) were collected and divided in two parts. The first part was immediately used to isolate EVs by enzymatic treatment followed by ultracentrifugation and density cushion. The second part was frozen in dry ice and stored at ‐80°C for 2 weeks. After thawing, the EVs were isolated in exactly the same way as from fresh tissue. Tissue‐derived EVs from both fresh and frozen tissues were characterized by transmission electron microcopy (TEM) and nanoparticle tracking analysis (NTA), and the ratio particle/protein was calculated to evaluate sample purity. Moreover, quantitative mass spectrometry was used to compare the EV protein composition from the two conditions in depth. Results : From both fresh and frozen melanoma tissue, TEM showed the presence of round elements with typical EV size (80‐500 nm), and no significative difference in particle concentration, size and purity was observed. The proteomic analysis showed strong similarity between the two groups and few proteins were differently expressed. Moreover, no significant difference was observed when typical marker of EVs (CD9, CD63, CD81, ADAM10 and Mitofilin) were analyzed. Summary/Conclusion : Our results demonstrated that the purity and the protein composition of EVs isolated from frozen tissue was not affected by the freezing process, and they were comparable to those of the EVs obtained from the same fresh sample. This opens the possibility to isolate and analyze EVs from tissues stored in biobanks, further strengthening research studying EVs as potential biomarkers for disease. Keywords : extracellular vesicle from tissue, human melanoma, fresh tissue, frozen tissue

Csf

Laura R. Cechinel 1 ; Madeleine Goldberg 1 ; Brennan Harmon 1 ; Rachael Batabyal 2 ; Robert J. Freishtat 1 ; Ionara Rodrigues Siqueira 3 1 Children's National Hospital, Washington, USA; 2 Children's National Hospital, Columbia, USA; 3 Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Introduction : Interactions between the peripheral organs, such as adipose tissue, and brain have been widely described. In this context, a growing body of evidence indicates that adipose tissue dysfunction, such as in aging process, can be involved with obesity‐induced susceptibility to tissue injury even in distant sites, as well as the role of adipocytes‐derived extracellular vesicles and particles (EVPs) as a potential mechanism for the spread of bioactive molecules has been raised. We hypothesized that CSF EVPs derived from adipocytes can be involved to the cerebral aging process via their microRNA cargo. Our aim was to study the impact of aging process on microRNA profiles of CSF EVPs derived from adipocytes (FABP4+), performing an in silico prediction of their downstream signaling effects. Methods : Fatty acid‐binding protein 4 (FABP4) was used as a marker for adipocyte‐derived EVPs that were isolated from CSF of young adult and aged Wistar rats (3 and 21 months old) using the commercially available kit. microRNA isolation and microarray expression analysis were performed. Canonical pathways, Disease & Functions, and Upstream Regulator analyses were performed using IPA‐Qiagen. Results : The analysis revealed that 78 miRNAs were differentially expressed between groups (p < 0.05; fold change ≥ |1.1|), of which 30 miRNAs were up‐regulated and 48 were down‐regulated in adipocyte‐derived EVPs obtained in CSF from aged animals compared to young adults. Interestingly, the “Neuroinflammation Signaling Pathway” was listed as a significant canonical pathway (z‐score = 2,425). MiRNAs from adipocyte‐derived EVPs obtained in CSF reduced by aging, such as miR‐1‐3p, miR‐24‐3p, miR‐3065‐5p, miR‐16‐5p, miR‐17‐5p, can target key molecules of neuroinflammation. For instance, the downregulation of miR‐17‐5p that targets C‐X‐C motif chemokine ligand 8 (CXCL8) and tumor necrosis factor (TNF) can be related at least in part to higher levels of these cytokines in brain areas during aging process. Summary/Conclusion : miRNA signature in CSF adipocyte‐derived EVPs may be involved with susceptibility to neuroinflammation conditions and consequently with deleterious effects of obesity in aging process. Funding : This study was financed in part by the CNPq (Dr. I.R. Siqueira ‐ 307980/2018‐9) and CAPES (Dr. Laura Reck Cechinel ‐ Finance Code 001, # 88881.189257/2018‐01).

Ebc

Afsareen Bano 1 ; Rashmi Bhardwaj 2 1 Maharshi Dayanand University Rohtak, Rohtak, India; 2 Maharshi Dayanand University, India, Rohtak, India Introduction : Being the leading cause of worldwide mortalities, the burden of early diagnosis of lung cancer is the utmost requirement of the present research. Due to the invasive nature of Blood and Biopsy samples, noninvasive sample sources such as EBC can be screened for early predictive lung cancer biomarkers. EBC, originating from the lower part of the respiratory tract can be a promising source of biomarkers that reflects lung cancer pathogenesis. Akin to blood and tissue, EBC consists of a colossal array of molecules such as lipids, proteins, nucleic acids, cell metabolites, and tiny intercellular messengers called exosomes. These nanoparticles can be screened as lung cancer biomarkers based on the discrepancy in their size, composition, concentrations, and their existence. Methods : EBC samples were collected from three study groups: non‐smokers (NSE), smokers (SME), and lung cancer (LCE) followed by exosome isolation. DLS, NTA, and TEM were performed to evaluate exosomal properties such as size, concentration, and morphology. Exosomal protein was quantified by using the Bradford assay. Results : We successfully isolated EBC exosomes using a commercially available kit with slight modifications per the requirement. DLS (NSE 61.08 ± 24.12 nm, SME 94.45 ± 26.45 nm, LCE 126.4 ± 20.55 nm) and NTA (NSE 91.6 ± 10.46 nm, SME 129.2 ± 10.91nm, LCE138.2 ± 2.86 nm) results confirm the size range of EBC exosomes (30‐150 nm). We found significant differences (*P 0.05) among NSE and LCE exosome size as per NTA results. TEM images validate the EBC exosome morphology. Isolated EBC exosomes are quite quantifiable (NSE 2.6E+09 ± 2.64E+08 particles/ml, SME 4.15E+09 ± 1.26E+09 particles/ml, LCE 7.22E+09 ± 2.79E+09 particles/ml) and exosomal protein (NSE758 ± 252.7 mg/mL, SME1903 ± 633.2 mg/mL, LCE1090 ± 133.9 mg/mL) can be used for downstream processing. Summary/Conclusion : Being novel and non‐invasive in nature EBC exosomes can be used to screen biomarkers for LC diagnosis, prognosis as well as therapeutics. Funding : Indian Council of Medical Research, India. Keywords : exhaled breath condensate, exosomes, lung cancer, protein, novel, biomarker source

Evs

Bianca C. Pachane 1 ; Blanca Rodriguez 2 ; Zhaohao Liao 3 ; Olesia Gololobova 4 ; Erin Shirk 5 ; Suzanne Queen 5 ; Bess Carlson 2 ; Kenneth W. Witwer 6 1 Department of Comparative and Molecular Pathobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Physiological Sciences, Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil, Baltimore, USA; 2 Department of Comparative and Molecular Pathobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, USA; 3 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, USA; 4 Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, USA; 5 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, USA; 6 Johns Hopkins University, Baltimore, USA Introduction : Exogenous EVs from different sources may interact with different cell types when introduced into a complex organism for therapeutic purposes. However, the biodistribution of EVs in larger animals is still obscure. Following up on a recent study reporting the association of EVs with peripheral blood mononuclear cell (PBMC) subtypes in vivo (Driedonks et al, JExBio 2022), we used an ex vivo blood system to interrogate EV cell tropism and interaction mechanisms. Methods : Expi293F cells were transfected with pLenti‐palmGRET, and EVs were separated from culture medium by tangential flow filtration and size‐exclusion chromatography, then characterized by Western blotting, flow NanoAnalyzer, single particle interferometric reflectance imaging sensing and transmission electron microscopy. Whole blood samples from Macaca nemestrina were incubated with palmGRET EVs for 10–30 min at 37°C. PBMCs and CD20+ B cells were separated for confocal imaging analysis. EV‐spiked PBMCs were also phenotyped by flow cytometry, tested for cell viability, and treated with trypsin for 15–30 min. Results : palmGRET EVs bound to different PBMC subtypes with different affinities, with the greatest association with CD20+ B cells, CD14+ monocytes, and CD14dim monocytes. palmGRET signal decreased with exposure to trypsin, suggesting that at least some EVs remain at the cell surface or in trypsin‐accessible compartments. Cell viability and EV integrity were not altered by trypsin treatment. Confocal imaging shows that EVs are internalized by CD20+ B cells within the first hour of exposure, with the diffusion of palmGRET fluorescence in unlabeled cells. Summary/Conclusion : palmGRET EVs spiked into ex vivo macaque blood show preferential tropism to CD20+ B cells, CD14+ monocytes, and CD14dim monocytes. Funding : São Paulo Research Foundation [2022/04146‐9], Michael J. Fox Foundation for Parkinson's Research [00900821]; National Institute of Allergy and Infectious Diseases [ AI144997 ]; NIH Office of the Director [U42OD013117]; National Institute on Drug Abuse [ DA047807 ]; National Cancer Institute [ CA241694 ]; National Institute of Mental Health [ MH118164 ]. Keywords : B cells, monocyte, EV uptake

Hcc

Na Zhang 18292861052, China (People's Republic) Introduction : Hepatocellular carcinoma (HCC) is the second leading cause of cancer‐related death and the fifth most common cancer in the world. Recently, exosomes have been focused on tumor diagnostic biomarkers research and transformation. However, the glycosylation modification of exosomes has not been reported in HCC. Methods : Using western blot and RT‐PCR, we measured the expression of proteins and mRNAs of HCC cell models. We detect the core fucosylation of HCC derived exosomes using Lectin‐ELISA. Cell proliferation and apoptosis were detected by flow cytometry. Results : Our previous study found that the level of core fucosylation in HCC patients changed significantly with the progression of the disease, and the level of core fucosylation significantly increased in serum exosomes. Moreover, exosomes derived from serum of HCC patients with high level of core fucosylation can enhance the proliferation and migration of HCC cells, through activing PI3K/AKT signal pathway. The purpose of our study is to detect and evaluate the clinical application of exosomal core‐fucosylation level in the early diagnosis of HCC, using the HCC cell model and mice model, as well as the clinical study, to explore the related mechanisms and provide scientific basis for the clinical diagnosis of HCC. Summary/Conclusion : In our study, we demonstrate exosomal core fucosylation promotes malignant behavior of hepatocellular carcinoma cells through activation of PI3K/AKT signaling pathway.

How

Mehdi Kabani 1 ; Luc Bousset 1 ; Hong Boon Ong 2 ; Han Wei Hou 3 ; Marc Dhenain 1 1 Laboratoire des Maladies Neurodégénératives, UMR9199, CNRS, CEA MIRCen, Université Paris‐Saclay, Fontenay aux Roses, France, Fontenay aux Roses, France; 2 School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, Singapore, Singapore; 3 School of Mechanical and Aerospace Engineering, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore, Singapore 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 via a prion‐like mechanism of self‐replication and spreading of diffusible Tau species (‘seeds’). Extracellular vesicles (EVs) recently emerged as major vehicles for Tau dissemination (Leroux et al., Mol. Ther. 2021; Ruan et al., Brain 2021). Yet, the extent to which free or EV‐associated Tau species contribute to the spatiotemporal spreading of Tau misfolding is unclear. To address these issues, we are using different methodological approaches to isolate and characterize free and vesicle‐associated Tau seeds from post‐mortem AD brain tissues. Methods : Post‐mortem human brain tissue samples from AD (Braak VI) and non‐demented age‐matched control subjects were obtained from the NeuroCEB brain bank, in accordance with French bioethics laws. After dissociating the tissues with collagenase, EVs and other extracellular particles were isolated either by size‐exclusion chromatography, ultracentrifugation and iodixanol density gradients (Hurwitz et al., J Neurosci Methods 2018), or using an inertial‐based microfluidic device (Tay et al., Lab Chip 2021). Extracellular fractions were analyzed by Western blot, nanoparticle tracking analysis, negative staining and immunogold transmission electron microscopy (TEM) and cryo‐electron microscopy. Results : Our preliminary observations suggest that free Tau aggregates frequently co‐isolate with brain‐derived EVs, which could lead to misinterpretations as to the roles the latter play in AD. We will present the results of our ongoing methodological comparisons at the meeting and discuss their implications. Summary/Conclusion : We envision that characterizing the diversity of extracellular Tau seeds and their interactions with EVs, with components of the extracellular matrix and with neuronal and glial cells will allow for a deeper understanding of the mechanisms involved in AD progression. Keywords : EV, tau, alzheimer's disease, prion‐like propagation, amyloids, brain tissue

Il2

Kyungmoo Yea 1 ; Moon‐Chang Baek 2 ; Noh Soojeong 3 ; Suyeon Ryu 3 ; Dokyung Jung 2 ; Sanghee Shin 3 ; Inseong Jung 3 ; Sung‐Min Kang 4 ; Christine Seulki Kim 3 ; Sung‐Jin Choi 3 ; Hanchae Cho 4 ; Melanie Schwämmle 5 ; Youngtae Jeong 3 ; Felicitas Bucher 5 ; Shin Yup Lee 4 ; Sin‐Hyeog Im 6 1 Department of New Biology, DGIST, Daegu, Republic of Korea; 2 School of Medicine, Kyungpook National University, Daegu, Republic of Korea; 3 DGIST, Republic of Korea; 4 School of Medicine, Kyungpook National University, Republic of Korea; 5 University of Freiburg, Freiburg, Germany; 6 POSTECH, Republic of Korea Introduction : Interleukin‐2 (IL2) induces anticancer effects by activating immune cells expressing the IL2 receptor complex (IL2R). Interestingly, IL2R is also expressed in cancer cells, such as melanoma cells; however, previous studies are focused on the influence of IL2 on immune cells, not on cancer cells with regard to anticancer immunity. Here, we suggest that IL2 modulates the immunosuppressive tumor microenvironment (TME) by inhibiting sEV release and cancer‐derived PD‐L1, suggesting a novel effect of IL2 on cancer cells under immune surveillance, in addition to its systemic immunomodulatory efficacy. Methods : We investigated whether IL2 affects cancer cells to reduce small extracellular vesicle (sEV) release and suppress cellular programmed death ligand 1 (cPD‐L1) and exosomal PD‐L1 (ePD‐L1) expression and in melanoma cells through MAPK/ERK signaling. These anticancer immune responses were observed even in animal models wherein IL2 induces cancer‐specific activity by transplantation with IL2‐tethered melanoma cells. Notably, the reduction of PD‐L1 and Rab27a expression by IL2 was observed in both IL2R‐positive lung cancer cell lines and lung cancer cells from human patients. Results : We show that IL2, identified from a cytokine screening assay, significantly inhibits sEV secretion along with downregulation of cPD‐L1 and ePD‐L1 levels in melanoma cells and increases their sensitivity to CD8+ T cell‐mediated cytotoxicity. Mechanistically, IL2 induces these effects in melanoma cells by activating IL2R‐MAPK/ERK signaling. To analyze the direct effects of IL2 on melanoma cells in vivo, a unique mouse model is developed by introducing melanoma cells engineered to express IL2 on their surface via a flexible peptide linker for self‐stimulation. In this model, melanoma cell‐specific IL2 stimulation strongly inhibited tumor growth and downregulated both the expression of cPD‐L1 in tumor tissues and the plasma ePD‐L1 levels. IL2R‐expressing lung cancer cell lines and clinical samples were employed to confirm the same effects on human samples. Summary/Conclusion : This study revealed that IL2 enhances immune surveillance by directly reducing both EV secretion and PD‐L1 expressions in melanoma cells via the IL2R‐MAPK/ERK signaling. This study presents the potential of IL2 as a novel immunoOncology strategy in IL2R‐positive cancers. Funding : This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) of Korea funded by the Korean government (MSIT) (2020M3A9I4039539 and 2019M3A9H1103607), the NRF grant funded by MSIT (2021R1A5A2021614), the DGIST Program of the Ministry of Science and ICT (21‐DGRIP‐01), the 2020 Joint Research Project of Institutes of Science and Technology, and National Cancer Center, Korea (NCC‐203205). Keywords : interleukin‐2, PD‐L1, sEV, exosomal PD‐L1, cancer immunotherapy

New

Tamás Beke‐Somfai 1 ; Imola Szigyártó 2 ; Zoltan Varga 3 ; Judith Mihály 2 ; Priyanka Singh 4 ; Maria Ricci 4 ; Anikó Gaál 2 ; Mayra Quemé‐Pena 2 ; Diána Kitka 2 ; Livia Fülöp 5 ; László Drahos 2 1 Research Centre for Natural Sciences, Budapest, Budapest, Hungary; 2 Research Centre for Natural Sciences, Budapest, Hungary; 3 Biological Nanochemistry Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Budapest, Hungary, Budapest, Hungary; 4 Research Centre for Natural Sciences, Hungary; 5 Research Centre for Natural Sciences, Szeged, Hungary Introduction : Host defense membrane active peptides (HDPs) play important roles as part of the innate immune system from antimicrobial activities, through biofilm inhibition, immunomodulation, tissue repair to wound healing. Some of the HDPs are overexpressed at infection sites where they could disrupt the lipid bilayer of EVs that are either mammalian or bacterial origin, where the former interaction could help molecule distribution in wound healing while the latter could hinder spread of important components of the cell‐cell communication in bacterial biofilms. In our initial investigations (Singh et al. 2020), we have observed that some peptides have only minor disrupting effect, but they could directly interact with the surface of the vesicles, removing the surface adsorbed protein corona from them. This phenomenon could potentially be exploited to manipulate protein corona of EVs and other nanoparitcles enabling separation of proteins from different origins. To explore both the potential in vivo role of this phenomenon, and also to progress towards better surface engineering components for EV protein corona modulation, here a set of HDPs were selected and studied with a model system, red blood cell‐derived vesicles (REVs) to gain an overview on their interactions and to reach a broader understanding on how they could be used to manipulate EV content. Methods : The interaction of HDPs with REVs were investigated using flow‐linear dichroism, circular and infrared spectrocopy, microscale resistive pulse sensing, microscale thermophoresis, transmission electron microscopy and proteomics analysis. The experimental parameters were submitted to EV‐TRACK knowledgebase ( EV220311 ). Results : Based on these results, HDPs can be categorized based on their action mechanism, some of them removes the surface proteins of vesicles at lower concentrations, or can disrupt the vesicles forming lamellar bilayer structures, or can penetrate without disrupting the membrane. Importantly, the proteomic results enabled us to identify fifteen proteins as external protein corona members. Summary/Conclusion : The removal of protein corona enables the separation of proteins from different origin: Those adsorbed on the surface, from those located inside the EVs. It is hoped that these results will aid the use of HDPs and related membrane active peptides in surface engineering of EVs and other biological nanoparticles. Funding : This work was funded by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021‐EGA‐31, the KKP_22 Project n.o. 144180 and the 2020‐1‐1‐2‐PIACI‐KFI_2020‐00021 funding schemes. Support from Eötvös Loránd Research Network, grant n.o. SA‐87/2021, is also acknowledged. Keywords : protein corona, membrane active peptides, proteomics, membrane biophysics

Rna

Yuya Monoe 1 ; Kentaro Jingushi 1 ; Yoshiaki Takano 2 ; Kohei Taniguchi 3 ; Kazumasa Komura 4 ; Kazutake Tsujikawa 1 1 Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan; 2 Department of Gastro Surgery, Osaka Medical and Pharmaceutical University, Takatsuki, Japan; 3 Department of Translational Research, Osaka Medical and Pharmaceutical University, Tkatsuki, USA; 4 Department of Translational Research, Osaka Medical and Pharmaceutical University, Takatsuki, USA Introduction : We have previously shown that 5'tRF‐GlyGCC encapsulated in colon cancer EVs acts in a tumor‐promoting manner by promoting the production of inflammatory cytokines via TLR8 in macrophages. However, it is not clear how 5'tRF‐GlyGCC regulate TLR8 reactivity in macrophages. Since RNA modifications have been reported to modulate TLR8 reactivity, we focused on RNA modifications on 5'tRF‐GlyGCC to determine the mechanism of action of tumor EVs on macrophages. Methods : Clinical specimens: The colon 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. Isolation of tumor tissue‐derived EVs: Tissue‐immersed medium was centrifuged at 2,000 g, for 30 min, and the collected supernatants were subjected to the ultracentrifuge method (100,000 g x 2) for recovery of tissue‐exudative EVs (Te‐EVs). The size and concentration of EVs were determined using qNano. Isolation of 5’tRF‐GlyGCC from Te‐EVs: EV‐RNA were isolated from Te‐EVs by using miRNeasy kit. 5’tRF‐GlyGCC were collected from EV‐RNA by Dynabeads magnetic beads conjugated with anti‐sense oligo targeting 5’tRF‐GlyGCC. RNA modification analysis of EV‐RNAs: Quantitative analysis of RNA modification levels was performed by UHPLC‐UniSpray‐MS/MS using 10 ng of EV‐RNA sample. Results : UHPLC‐MS/MS analysis identified tumor characteristic RNA modification landscape in tumor Te‐EVs. Among the modifiers reported to contribute to TLR8 reactivity, m6A levels were decreased in tumor Te‐EVs compared to normal Te‐EVs. The 5'tRF‐GlyGCC in tumor Te‐EVs also showed a significant decrease in m6A content as well. The reduction of m6A levels in tumor Te‐EVs by the recombinant protein ALKBH5, an m6A demethylase, promoted inflammatory cytokine production in macrophages. Summary/Conclusion : The reduction of m6A modification level on 5'tRF‐GlyGCC in tumor Te‐EVs, was found to act in a tumor‐promoting manner by modulating the reactivity of TLR8 in macrophages. To our knowledge, this is the first report showing that dysregulation of RNA modifications in EV‐RNA functions as a tumor‐promoting factor in colorectal cancer.

Ros

Hongbo Chen 1 ; lu xingyu 2 ; Fang Cheng 3 1 School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat‐sen University, Shenzhen 518107, China, Shenzhen, USA; 2 School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat‐sen University, Shenzhen 518107, China, China (People's Republic); 3 Sun Yat‐sen University, Shenzhen, China (People's Republic) Introduction : Heart transplantation is the best treatment for end‐stage heart failure. Due to cold ischemia‐reperfusion injury and transplant rejection during transplantation, which reduces the utilization rate of donor hearts, there is an urgent need for new mechanisms and therapeutic targets to develop effective therapies to treat I/R injury and inhibit rejection in heart transplantation rejection. In this study, we screened plant exosomes with antioxidant damage and anti‐inflammatory properties, fused them with IPSC‐derived vesicles containing phagocytotropic targets for drug delivery, and adopted click‐chemistry to promote delivery of drug delivery systems, providing a new strategy for the treatment of heart transplant rejection Methods : 1. Characterization of materials by electron microscopy /particle size/ potential 2. Synthesis of ROS responsive Tetraacetated N‐azide acetyl‐D‐mannosamine(ros‐n3) 3. Evaluation of ros‐n3 targeted modified grafts by imaging in vivo in small animals 4. In vitro, the anti‐inflammatory and anti‐myocardial oxidative damage effect of FV@RAPA was detected by flow /QPCR/ELISA 5. Construction of mouse heart transplantation model, and caudal vein injection and therapy 6. MASSON/HE/ flow assays the in vivo efficacy of DBCO‐FV@RAPA Results : 1. Tomentose Pummelo Peel derived exosomes have anti‐myocardial oxidative damage and anti‐inflammatory effects. 2. Targeting macrophages a multifunctional stem cells (IPSCs) vesicles with calreticulin that promote the phagocytosis and efferocytosis of macrophages. 3. IPSCs vesicles with calreticulin that promote phagocytosis, fused with Tomentose Pummelo Peel derived exosomes, and loaded with rapamycin (FV@RAPA). In vitro, the FV@RAPA not only maintain the bioactivity of TEVs, RAPA and IPSC membranes, but also can promote uptake of apoptotic cardiomyocytes by macrophages under the action of calreticulin, thus promoting the efferocytosis of macrophages. 4. In a mouse xenograft heart model, ros‐n3 labeling at the transplanted heart site promoted more targeted delivery of DBCO‐FV@RAPA, thereby alleviating ischemia‐reperfusion injury to cardiomyocytes and inhibiting early macrophage immune activation. Summary/Conclusion : In this study, we first constructed ROS‐responsive tetraacetyl N‐acetylazide‐D‐mannoamine, which can mark the transplanted heart site and promote more targeted delivery of DBCO‐FV@RAPA, thereby alleviating ischemia‐reperfusion injury and inhibiting early macrophage immune activation to achieve therapeutic effect. Funding : This research was supported by National Key R&D Program of China (No. 2022YFA1104900, China. Keywords : early rejection of heart transplantation, CALR, fusion vesicles, ROS response biological orthonormal chemistry

Tau

Zhengrong Zhang 1 ; Yang You 1 ; Kaiwen Yu 2 ; Clara Scholes 3 ; Seiko Ikezu 1 ; Michael DeTure 3 ; Dennis Dickson 3 ; Junming peng 2 ; Tsuneya Ikezu 1 1 Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL 32224, USA, Jacksonville, USA; 2 Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States, USA; 3 Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL 32224, USA, USA Introduction : Extracellular vesicles (EV) play critical roles in transferring pathological proteins related to Alzheimer's disease (AD). Recent studies demonstrate tau‐containing EVs are transmissible and induce tau propagation in the brain, however, the underlying mechanism regarding EV‐mediated tau pathology is still largely unknown. Methods : We performed the immuno‐affinity purification of tau in EVs derived from 14 AD and 14 normal controls (CTRL) brains and mass spectrometry to characterize EV associated tau interactome. We applied the Monolith to validate the interaction of potential EV‐tau interactors with purified recombinant tau. We tested super‐resolution microscopy (ONI) to further visualize their colocalizations in a single‐EV level. We also designed siRNAs to silence EV‐tau interactors in human neuronal SH‐SY5Y cells overexpressing human P301L tau and assessed tau containing EVs secreted from the cells by Nanoanalyzer (NanoFCM). EVs from cells after silencing tau interactors are evaluated with live‐cell imaging in human iPSC‐derived neurons to determine tau uptake and seeding. Results : A total of 764 proteins were identified as tau interactome in brain‐EV isolated from CTRL and AD patients. The proteins are enriched in exocytic vesicle, transmembrane transport and cytoskeleton pathways as determined by bioinformatics. The comparisons of tau interacting proteins demonstrated that 65 proteins (e.g.,CYCS, KRAS, SIR2, SYT1), significantly downregulated in AD brain EVs, are enriched in pathways associated with vesicle‐mediated transport in synapse; whereas 5 proteins (e.g., ATP1B3, ANXA5, ANK3), significantly upregulated in AD brain EVs, are involved in transmembrane localization. Correlation analysis showed the top differentially expressed EV‐tau interacting proteins including CYCS, SYT1, ATP1B3 and ANXA5 were significantly correlated with Braak tangle stage. SYT1 level showed a significant prediction of AD (AUC = 0.86) as determined by ROC curve. The purified recombinant proteins of CYCS (Kd 0.34 μM), SYT1 (Kd 1.19 μM), ATP1B3 (Kd 0.833μM ) and ANXA5 (Kd 1.88μM) showed binding affinities with purified recombinant tau in vitro as determined by Monolith. Furthermore, Nanoimager validated the reduced colocalization of SYT1, and increased colocalization of ATP1B3 or ANXA5 with tau in AD‐derived EVs at a single‐particle level. Finally, we silenced ANXA5 or ATP1B3 in SH‐SY5Y cell lines and found reduction of tau+ EV populations. Summary/Conclusion : We performed a novel EV‐associated tau interactome and found the changes in tau interactors in AD‐derived EVs: SYT1 and CYCS were reduced to affect synaptic vesicle transportation; Localization of specific membrane proteins ATP1B3 and ANXA5 preferably interacted with tau. Finally, silencing of ANXA5 or ATP1B3, the enriched tau interactors in AD EV, diminished tau loading in EVs. Targeting of these molecules may suppress tau dissemination, highlighting their therapeutic potential in AD.

The

Simon Swift 1 ; Joni R. White 1 ; Jiwon Hong 1 ; Priscila Dauros Singorenko 2 ; Frederique Vanholsbeeck 3 ; Anthony Phillips 1 1 University of Auckland, Auckland, New Zealand; 2 University of Auckland, New Zealand; 3 Frederique Vanholsbeeck, Auckland, New Zealand Introduction : Human extracellular vesicles (hEVs) were hypothesised to be possible vehicles for a communication axis from eukaryotic cells to bacteria in the gut. Methods : Small EVs (sEVs) were isolated from a human colorectal adenocarcinoma cell line, HT29, grown in a CELLine bioreactor for 230 days in Advanced RPMI with EV‐depleted FBS. sEVs isolated using differential centrifugation (2,000 × g, 10,000 × g, 100,000 × g), were purified with size exclusion chromatography. The sEVs were characterised by nanoparticle tracking analysis, protein analysis, transmission electron microscopy, and Western blots. Pathogenic enteroinvasive Escherichia coli (EIEC) and probiotic E. coli Nissle were treated with three doses of HT29‐EVs: ∼2 × 108, ∼2 × 109, and ∼2 × 1010 EVs/mL, and grown in physiological iron‐restricted media (RPMI‐1640) or iron‐replete media (RPMI + 10 μM FeCl3 (RPMIF)). Results : In RPMI for each increasing HT29‐EV dose, E. coli Nissle growth was increased by ∼20%, ∼55%, and ∼130%; EIEC growth was increased by ∼80%, ∼40%, and ∼580% (plate counts at 8 h). In RPMIF, hEVs did not affect growth of E. coli Nissle or EIEC. Bacteria were treated with CMTPX‐Red labelled HT29‐EVs in RPMI (no phenol red). Bacteria were counterstained with SYTO9 (green). Fluorescence‐activated cell‐sorting separated bacteria‐only, EV‐only, and bacteria+EV populations. Confocal microscopy showed co‐location of live, green‐stained E. coli Nissle and red fluorescent EVs, indicating a biological interaction. Transcriptomic analysis of E. coli Nissle and EIEC total RNA was performed after treatment with unlabelled HT29‐EVs (∼2 × 109 EVs/mL) in RPMI and RPMIF. RNA‐Seq (Illumina Hi‐Seq) showed that genes involved in molybdenum uptake were upregulated, while genes involved in molybdenum cofactor synthesis were downregulated. Summary/Conclusion : This work provides new insight into the complex host‐microbe relationship in the context of health and disease and indicates that hEVs may play an important role. Funding : This work was supported in part by a grant from Manatū Hauora Health Research Council of New Zealand (HRC) (18/735). Jiwon Hong is funded by the Hugo Charitable Trust. Joni White is funded by a University of Auckland Doctoral Scholarship. Keywords : human extracellular vesicles, bacteria, interkingdom communication

Use

Salomé Araujo‐ Abad 1 ; Enrique Rodríguez‐Cañas 1 ; María Fuentes‐Baile 2 ; Pilar García‐Morales 1 ; Ricardo Mallavia 1 ; Miguel Saceda 3 ; Camino de Juan Romero 4 1 Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández, Avda, Universidad s/n, Ed. Torregaitán, Elche, 03202 Alicante, Spain., Spain; 2 Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Camí de l'Almazara 11, Elche, 03203 Alicante, Spain., Spain; 3 Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Camí de l'Almazara 11, Elche, 03203 Alicante, Spain, Spain; 4 Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Camí de l'Almazara 11, Elche, 03203 Alicante, Spain, Elche, Spain Introduction : Pancreatic Ductal Adenocarcinoma (PDAC), is the most common aggressive cancer of the pancreas. The standard care of PDAC include tumor resection and chemotherapy, but the lack of early diagnostic and the limited response to the treatment worsens the patient's condition. In order to improve the efficiency of chemotherapy, we look for more efficient ways of drug delivery. Methods : We isolated and fully characterized small Extracellular Vesicles (EVs) from the RWP‐1 cell line. We loaded them with two chemotherapeutic drugs (Temozolomide and EPZ015666) by direct incubation method and, the amount of drug loaded was measured by high‐performance liquid chromatography (HPLC). Finally, we tested their antiproliferative effect on different cancer cell lines. Results : Our study indicates that the direct incubation method was the most efficient loading protocol and that a minimum total amount of drug triggers an effect on tumor cells. Moreover, we have seen that RWP‐1 small EVsTMZ, were more efficient than RWP‐1 small EVsEPZ015666. Summary/Conclusion : RWP‐1 derived small EVs represent a promising drug delivery tool that can be further investigated in preclinical studies and its combination with PRMT5 inhibitor can be potentially developed in clinical trials for the treatment of PDAC. Funding : This research was funded by the Miguel Servet Program to C.d.J.R. from Instituto de Salud Carlos III (CP19/00095) and GVA (CIAICO/2021/135). M.S was funded by FISABIO intramural grants UGP‐20‐135 and UGP‐20‐254. R.M was recipient of the following grants PID‐2021‐123253‐OB‐C21.S.A.A was recipient of “Carolina foundation predoctoral fellowship” 2020. The FESEM equipment used in this work was funded by Generalitat Valenciana (Spain) and co‐financed with ERDF funds (OP ERDF of Comunitat Valenciana, Spain) GVA‐IDIFEDER_2018/020, “Una forma de hacer Europa”. Keywords : pancreatic ductal adenocarcinoma, small EVs, nanocarriers, chemotherapy, FESEM.

Aged

Laura Hueser 1 ; Yash Chhabra 1 ; Olesia Gololobova 2 ; Vania Wang 3 ; Mitchell Fane 1 ; Murilo Ramos Rocha 3 ; Kenneth W. Witwer 1 ; Ashani Weeraratna 1 1 Johns Hopkins University, Baltimore, USA; 2 Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, USA; 3 Johns Hopkins University, USA Introduction : Age is an important prognostic factor in cutaneous melanoma, commonly arising in the elderly. Melanoma progression is the concerted outcome of changes occurring in tumor cells and within the tumor microenvironment (TME). Aging causes remodeling of the TME, making it conducive to melanoma progression. Due to the important role of extracellular vesicles (EVs) in tumor progression in different cancer types we investigated how the aged TME can alter EV attributes, cargo, and function. Methods : Conditioned media of age‐stratified dermal fibroblasts (dFs), the predominant cell type in the melanoma TME, was concentrated and EVs were isolated via Izon size separation and characterized according to MISEV2018 guidelines. Next, mass spectrometry was performed to investigate how systemic aging of dFs changes the cargo of their secreted EVs. We then evaluated the influence of these EVs on melanoma progression in vitro and in two mouse models using the EV biogenesis inhibitor PDCC and EV injection. Results : We found that the tetraspanin CD9 was reduced both in aged dFs and EVs released compared to young dFs. Modulating the CD9 expression in dFs was sufficient to alter its levels in EVs. CD9 is a crucial member in cargo sorting of EVs, and mass spectrometry analysis of EVs released by CD9 knock down vs. control cells revealed a significant increase in ANGPTL2 (angiopoietin‐like protein 2), a promoter of angiogenesis. Our in vitro analysis of primary endothelial cells confirmed increased sprouting in HUVEC under CD9 KD conditions. Inhibiting EV biogenesis in aged mice reduced the amount mice with metastasis by 27 % compared to vehicle‐treated mice. Moreover, injecting aged dF derived EVs in the primary melanoma in vivo resulted in 17% more mice with metastasis compared to the injection of young EVs. Summary/Conclusion : Our data shows modulation of EV cargo owing to intrinsic aging that promotes tumor metastasis via angiogenesis and identifies novel therapeutic avenues to alleviate age‐associated melanoma progression. Keywords : cancer, aging, tumor microenvironment

Bulk

Paola Loreto Palacio 1 ; Jacelyn Greenwald 2 ; Jingjing Zhang 3 ; Yongseok Kim 2 ; Vicki Wysocki 2 ; Eduardo Reátegui 3 ; Setty M. Magaña 4 1 Translational Neuroimmunology, Center for Clinical and Translational Research, Nationwide Children's Hospital, Columbus, USA; 2 Department of Chemistry and Biochemistry, The Ohio State University, USA; 3 Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, USA; 4 Translational Neuroimmunology, Center for Clinical and Translational Research, Nationwide Children's Hospital, USA Introduction : Multiple sclerosis (MS), a chronic demyelinating disorder of the central nervous system (CNS), and its cause remains unknown. The most common form of MS, relapsing‐remitting MS (RRMS), is characterized by discrete relapses followed by neurological recovery. Most RRMS convert to the secondary progressive form of MS (SPMS), in which accumulation of neurological dysfunction occurs in the absence of relapses. No reliable biomarkers exist to distinguish RRMS from SPMS or inform mechanisms of MS progression over time. EVs have emerged as potential prognostic biomarkers in various neurological disorders and may also provide mechanistic insights into neurodegenerative pathomechanisms. Methods : Archival serum samples from RRMS and SPMS (n = 5 per group) were processed via tangential flow filtration (TFF) for EV isolation. Microfluidic Resistive Pulse Sensing (MRPS) measured particle size and concentration. EVs were characterized via immunoblotting, Exoview, and Total Internal Reflection Fluorescence (TIRF) microscopy for candidate CNS and EV markers. EV morphology was assessed by Transmission Electron Microscopy (TEM). Quantitative label‐free LC‐MS/MS proteomics analysis was performed on serum EVs. Results : TEM of serum EVs demonstrated a cup‐shaped morphology. Serum EVs were positive for known EV markers (i.e., CD63, CD81). Bulk analysis of EVs revealed the presence of GFAP+ particles. Single EV analysis via TIRF microscopy showed EVs positive CNS markers such as Tau, beta‐amyloid, GFAP, and alpha‐synuclein. Proteomics analysis showed proteins related to metabolic processes, protein binding, and cytoskeleton proteins. Cross‐validation of Exoview tetraspanin profiles is ongoing. Summary/Conclusion : In this study, we present an experimental workflow that outlines the enrichment and orthogonal characterization of serum EVs from MS patients for prognostic biomarker discovery. Our preliminary results demonstrate the presence of CNS markers in serum EVs of MS patients, assessed by bulk and single EV analysis—identifying a potential liquid biopsy for neurodegenerative diseases of the CNS.

Cd81

Madhusudhan Bobbili 1 ; Johannes Grillari 2 ; Gordana Wozniak‐Knopp 3 ; Florian Rüker 3 ; Nuria Gimeno Magan 4 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria; 2 Ludwig Boltzmann Institute for Traumatology, Vienna, Austria, Vienna, USA; 3 Christian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria,, Vienna, USA; 4 Ludwig Boltzmann Institute for Traumatology, Vienna, Austria, USA Introduction : Despite the recent successes with lipid nanoparticles (LNPs) for drug delivery, LNPs can display toxicity and can be limiting. Thus ineffective drug delivery problem needs to be addressed urgently. For this reason, extracellular vesicles (EVs) as a drug delivery vehicles has become an intense topic of research. EVs have an innate therapeutic potential with the promise of them functioning as target‐directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the recipient cell that are vital for health of the host organism. To enhance the ability of their targeted delivery, we employed an intrinsically overrepresented protein, CD81, to serve for recognition of the desired target antigen. Methods : Yeast libraries displaying mutant variants of the large extracellular loop (LEL) of CD81 have been selected for binders to EGFR and HER2 as an example target. Their specific interaction with EGFR and HER2 was confirmed in a mammalian display system by multiple rounds of screenings. Derived sequences were introduced in to full‐length CD81 tagged either with eGFP or luciferase and stably expressed in EVs producer cell line HEK293 to target EGFR and HER2 tumors. EVs were isolated from producer cell lines by tangential flow filtration (TFF). Results : TFF isolated EGFR and HER2 targeting EVs were characterized for size and number by nanoparticle tracking analysis (NTA) and for surface marker profile by bead‐based flow cytometry. Additionally, the purity of the EV preps were confirmed by immunoblotting. To assess the novel functionality of antigen‐binding CD81 LEL variants, internalization of such EVs into EGFR and HER2 overexpressing cells was compared with the wild‐type CD81 EV internalization. Additionally, EGFR and HER2 targeting EVs loaded with doxorubicin can induce apoptosis in recipient cells more effectively compared to wild‐type CD81 EVs. Currently, we are accessing the doxorubicin loaded EVs targeting EGFR and HER2 tumors in xenograft mouse models. Summary/Conclusion : To our knowledge, this is the first example of harnessing an EV membrane protein as a mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various therapeutic settings. The advantage of the method presented here is that it can rapidly deliver binders to any antigen of choice, which can simply be ‘clicked’ into the full‐length CD81, recombinantly expressed on the EV surface, enabling specific EV‐mediated delivery to a large variety of cells and tissues. Keywords : CD81, extracellular vesicles, targeted drug delivery, EGFR, HER2

Cell

Seokhwan Yun 1 ; Hyun jae Kim 2 ; Seung‐Jin Kim 2 ; Keunsun Ahn 3 1 Sphebio Co.Ltd, Seoul, Republic of Korea; 2 Sphebio, USA; 3 Sphebio, Seoul, USA Introduction : The construction of a spheroid to mimic the human physiological condition is highly desired for drug discovery and clinical applications because conventional monolayer cell culture is inaccurate compared to human physiological condition. However, current spheroid production technologies are limited in ability to control uniformity of diameter and cell number. Methods : This study presents a method for producing homogenous spheroid using precisely controlled dispensing system incorporating a thermo‐sensitive bioink and human adipose derived stem cell. The spheroid can be successfully generated in the core shell structure at a speed of approximately 3,000 spheroids per hour and with a uniform diameter and accurate cell number. Exosomes from cell culture media were isolated by the TFF method. Results : The spheroids allow dynamic culture without any aggregation and secrete a large amount of biologics such as exosome. The amount of exosomes derived from spheroids increased more than 90 times compared to the amount of exosomes derived from 2D culture cells (ADSC, BM‐MSC, NTSC, Fibroblast). The amount of exosomes was measured using NTA(Nanoparticle tracking analysis). In addition, spheroid‐derived exosomes showed enhanced skin improvement effect in vitro and in vivo. ADSC‐spheroid exosomes increased the proliferation and migration of fibroblast and keratinocyte. Also, these exosomes enhanced collagen1 secretion of fibroblast. In big data analysis (NGs and Proteomics) indicated that these exosomes have proliferative, migratory, and anti‐inflammatory effects. Summary/Conclusion : In summary, these results suggest that spheroid derived exosomes were improved its function and efficacy because of its in vivo similarity. Further, we plan to conduct multi‐cell spheroid research to improve its in vivo similarity. Funding : This research was funded by TIPS(Tech Incubator program for startup) by Korean Goverment (Grant number S3197974, 20105847). Keywords : spheroid, dynamic culture, exosome

Does

Angelo Musicò 1 ; Rossella Zenatelli 2 ; Miriam Romano 3 ; Andrea Zendrini 4 ; Silvia Alacqua 2 ; Selene Tassoni 2 ; Lucia Paolini 5 ; Chiara Urbinati 2 ; Roberto Frigerio 6 ; Marina Cretich 7 ; Marco Rusnati 2 ; Paolo Bergese 3 ; Giuseppe Pomarico 8 ; Annalisa Radeghieri 3 1 Department of Molecular and Translational Medicine, University of Brescia, Italy and CSGI,Research Center for Colloids and Nanoscience, Florence, Italy, Italy; 2 Department of Molecular and Translational Medicine, University of Brescia, Italy, Italy; 3 Department of Molecular and Translational Medicine, University of Brescia, Italy and CSGI,Research Center for Colloids and Nanoscience, Florence, Italy, Brescia, Italy; 4 CSGI, Research Center for Colloids and Nanoscience, Florence, Italy, Brescia, Italy; 5 Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Italy and CSGI,Research Center for Colloids and Nanoscience, Florence, Italy, Brescia, Italy; 6 Istituto di Scienze e Tecnologie Chimiche, Consiglio Nazionale delle Ricerche, Milano, Italy; 7 Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Milano, Italy; 8 Department of Molecular and Translational Medicine, University of Brescia, Italy, Brescia, Italy Introduction : One strategy to improve EV targeting properties for drug delivery includes EV surface engineering with tissue‐specific peptides or protein ligands. This procedure requires the incubation of EVs with the ligand, followed by washing cycles. On the other hand, it has recently been reported EVs immersed in a biofluid containing biomolecules can adsorb proteins on their surface. This phenomenon leads to the formation of a so‐called Biomolecular Corona (BC). While BC role and composition in natural biofluids have started to be investigated, its formation and eventual role in EV surface engineering by biomolecules has not been yet studied. We will introduce the problem and present a first investigation of this kind on Red Blood Cell (RBC)‐derived EVs engineered with the monoclonal antibody Cetuximab (CTX), the target ligand of EGFR. Methods : MISEV 2018 compliant EVs, separated from healthy donors' RBCs, were reacted with CTX under conditions leading to the formation of covalent bond (chemisorption, through click chemistry) or weaker non‐covalent interactions (physisorption). Both EV sets were evaluated for physicochemical properties, molecular recognition performances, and in vitro cellular uptake. Results : Both EV sets showed a comparable amount of bound CTX. Surprisingly, the EV set functionalized by CTX physisorption showed the same affinity for EGFR as the EV set functionalized by chemisorption at Surface Plasmon Resonance and microarray assay. Nevertheless, only the chemisorbed EV set showed improved uptake ability by EGFR‐positive cells in physiological conditions compared to physisorbed EV set and native EVs. Summary/Conclusion : CTX physisorbs and chemisorbs with the same efficiency onto EV surface. CTX physisorbed on EVs bears the same binding affinity of covalently bound CTX towards EGFR however it is not able improve EV uptake in vitro. These findings add new perspectives and approaches to the study of EV biomolecular coronas. Funding : 2019‐08 to 2022‐08 | GRANT_NUMBER: 2017E3A2NR_004, Development of a biotechnological nanoparticle platform for the delivery of antitumor therapies using Patient Derived‐Organoid library of Breast Cancer, Ministero dell'Istruzione, dell'Università e della Ricerca (Roma, IT). Keywords : extracellular vesicles, EV corona, cetuximab, EV surface engineering

Dual

Wan Ki W. Wong 1 ; Cheuk Hang Lau 2 ; Xiaowen Mao 2 ; Judy WP YAM 1 1 The University of Hong Kong, Hong Kong, Hong Kong; 2 The University of Hong Kong, Hong Kong, USA Introduction : Our previous study suggested secretion of tumor‐derived small extracellular vesicles (T‐sEVs) elevate significantly in advanced hepatocellular carcinoma (HCC), but the exact mechanism remains unelucidated. Here, we aim to investigate key mediators involved in the biogenesis of HCC‐derived sEVs and their clinical significance. Methods : Bead‐based Amplified Luminescent Proximity Homogenous Assay (ALPHA) was performed on the conditioned medium of HCC cells after treatment of the Selleckchem kinase inhibitor library. Candidate inhibitors that displayed the strongest suppression effect on sEV secretion were identified. The targeted kinase was knocked down in HCC cells and their sEV were collected through ultracentrifugation for mass spectrometry analysis. Collected sEVs were functionally characterized by in vitro assays and animal models. Results : MRT68921, an ULK1 inhibitor, was found to be significantly involved in the inhibition of CD63+ and CD9+ sEV biogenesis through ALPHA. The involvement of ULK1 was corroborated by the significant reduction in sEV secretion by stable ULK1 knockdown cells compared to its respective control group established in metastatic MHCC97L cells. Mass spectrometry revealed a drastic shift in the protein cargos of sEV collected from ULK1 knockdown cells, notably a decreased level of sEV‐derived AE2. Functionally, the inducing ability of MHCC97L sEV on HCC proliferation and motility was significantly dampened compared to sEV collected from ULK1 knockdown cells. Intracellularly, AE2 was observed to be co‐localized with ULK1 whereas its expression was significantly upregulated in the endosomal fraction upon overexpression of ULK1, suggesting its regulatory role in sEV biogenesis and protein cargo sorting in HCC cells. Summary/Conclusion : This study unveils a bidirectional stimulation of ULK1 in terms of HCC‐sEV biogenesis and oncogenic signaling through sEV‐AE2 axis. This study also provides insights into targeting ULK1 as a new therapeutic strategy for HCC. Keywords : Hepatocellular carcinoma, small extracellular vesicles, EV secretion, ULK1, AE2

High

Po‐Chieh Chiang 1 ; Cheng‐Yu Chou 2 ; Andrew M. Wo 2 1 Reliance Bioscience, New Taipei City, Taiwan (Republic of China); 2 Institute of Applied Mechanics, National Taiwan University, New Taipei City, Taiwan (Republic of China) Introduction : Although many methods have been proposed, high purity and rapid isolation of EV from plasma have been challenging. For example, some polymer‐based methods, e.g. ExoQuick, would result in increased viscosity of the sample mixture raising the uncertainty in processing repeatability. Contaminants would also be co‐precipitated along with target EVs in some methods. This study presents an optimized polymer‐based approach (ExoRich, Reliance Biosciences) enabling high purity and rapid isolation of EV from minimal amount of plasma sample. The method* is based on a two‐part polymer recipe with simple centrifugation to isolate EV pellet. Methods : ExoRich was added to multiple plasma samples with volume from 50 uL to 250 uL. After well‐mixed by pipetting, the mixture was centrifuged at 1000 g for 10 minutes at 4‐degrees C. The supernatant was discarded and the pelleted EV was resuspended in PBS. The ExoRich‐treated samples were compared to that isolation from ultracentrifugation, SEC and a commercial EV isolation kit. Purified EV samples were characterized by TEM, NTA, nanoFCM and Western blot (WB). Results : TEM images show characteristic cup‐shaped features and intact EV morphology. NTA results of ExoRich‐treated sample show a distinct and clean peak at 69 nm with medium at 83 nm. Three repeated ExoRich treatments of the same plasma sample showed particle variation within 5%. WB revealed clear signal in EV markers (flotillin & CD81). Comparison of WB between exosomal fraction from SEC and ExoRich‐treated sample showed excellent agreement in exosomal protein expressions. Tests to minimize sample volume with 250 uL, 125 uL and 50 uL of plasma revealed only 50 uL is needed for consistent sandwich ELISA expression of CD81. Clinical sample of 50 uL was processed which verified that this low sample volume enabled consistent result with that from 250 uL sample volume. Summary/Conclusion : The new method enabled high purity isolation of EV in as low as 50 uL plasma in 10 mins. The approach should be suitable for clinical studies when plasma availability is challenging. *Provisional application Funding : Reliance Biosciences, Taiwan.

Ldha

Xin Zhang 1 ; Bodeng Wu 2 ; Anming Yang 3 ; Xiaoliu Liu 4 ; Quan Zhong 5 ; Minghui Wen 2 ; Liyi Ma 6 ; Bin Peng 4 ; Ya Gao 7 ; Bin Xu 5 ; Tianyu Wu 5 ; Jiaming Chen 8 ; Yu Wang 5 ; Lei Zheng 9 1 Nanfang Hospital, Southern Medical University, Guangzhou, China (People's Republic); 2 .Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, P. R. China, China (People's Republic); 3 Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, P. R. China, China (People's Republic); 4 Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, P. R. China, China (People's Republic); 5 Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China, China (People's Republic); 6 Department of Neurosurgery, Shunde Hospital, Southern Medical University (The First People's Hospital of Shunde Foshan), Foshan 528300, Guangdong, China, China (People's Republic); 7 Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, P. R. China, Guang zhou, China (People's Republic); 8 Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China, Guangzhou, China (People's Republic); 9 Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China (People's Republic) Introduction : GSCs (Glioma stem cells, GSCs) activation is a hallmark during Glioblastoma (GBM) recurrence and leading to poor outcome of GBM. However, there is no proper biomarker for GSCs activated and GBM recurrence warning at early stages.Here we demonstrated that plasma‐derived EVs (pl‐EVs) from recurrent GBM patient cohorts could activate GSCs phenotype of glioma cells. Methods : 265 cases of plasma from recurrent (n = 84) and non‐recurrent GBM patients (n = 181) post‐surgery and healthy donors (n = 50) were collectted and plasma derived EVs (plEVs) were concentrated.Using proteomics, “pyruvate‐glucose transforming” pathway was identified upregulated in the EV cargo and Lactate dehydrogenase A positive EVs (LDHA+EVs) numbers were detected in the plasma from recurrent GBM patients by eco‐counter.To study the mechanisms of exomal LDHA, LDHA upregulated giloma cells were constructed to obtain LDHA carried EVs (LDHA‐EVs),Glycolysis/glycogenesis activity were evalutaed by ECAR assay and lactate production were evaluated for EV uptaken glioma cells.Given the excellent clinical application prospects of EVs, the biodistributions of them were further evaluated in vivo. Besides,U87‐Luciferase (U87‐Luc) based glioma model was established, and LDHA‐EVs were administered intraperitoneally continued and tumor fluorescence was detected.Clinically, to determine whether LDHA+ pl‐EVs has the potential to predict GBM recurrence, we further compared the amount of LDHA+plEVs in the plasma of patients with recurrent GBM and their MRI‐detected images. Results : Mechanistically, we discovered that LDHA could be transferred via EVs into recipient cells, resulting in further enhancement of glycolysis and ATP production (Warburg effects) to maintain GSCs phenotype. Besides, LDHA‐carried EVs (LDHA‐EVs) were driven by HIF‐1α, also has more obviously homing effects entering to the intracranial tumor sites. Clinically, we found strong correlations of LDHA+plEV numbers and LDHA level in matched patient tumors, and LDHA+plEV was decreased post‐operatively and increased with GBM recurrence. Summary/Conclusion : Our study highlights the effects of increased circulating LDHA+EVs and its induced “Warburg effects” to maintain GSCs phenotypes may an important reason for GBM recurrence. Besides, this finding provides a potential molecular evidence for LDHA+EV‐mediated intracellular metabolisms and offers a candidate biomarker for GBM recurrence by liquid biopsy. Funding : This research was funded by grants from the National Key Research and Development Program (#2021YFA1300604), the National Science Fund for Distinguished Young Scholars (#82025024), National Natural Science Foundation of China (#81902147 and #82172966). Keywords : LDHA+EVs, GBM recurrence, glioma stem‐like cells, warburg effects, EVs homing, liquid biopsy

Long

Emily Mitsock 1 ; Kailey Babcock 2 ; Sudipto K. Chakrabortty 3 ; Shuran Xing 2 ; James McKiernan 4 ; Michael Donovan 5 ; Naveen Kella 6 ; James Eastham 7 ; Seth Yu 8 ; Johan Skog 9 1 Exosome Diagnostics, USA; 2 Bio‐techne, USA; 3 Bio‐techne, Waltham, USA; 4 Department of Urology, Columbia University, New York, USA; 5 Icahn School of Medicine at Mount Sinai, USA; 6 UT Health Science Center, USA; 7 Memorial Sloan‐Kettering Cancer Center, USA; 8 Exososome Diagnostics, Waltham, USA; 9 Exosome Diagnostics, Waltham, USA Introduction : Guideline‐recommended PSA screening for early detection of prostate cancer (PC) lacks specificity, especially for clinically significant (CS) disease, resulting in over‐diagnosis or over‐treatment of low‐grade PC, and exploration of additional non‐invasive biomarkers to improve CSPC prediction both at diagnosis and treatment decisions. Small extracellular vesicles (EVs) contain molecular constituents, such as RNA transcriptome, derived from their parent tumors and can be profiled from biofluids such as urine for these biomarkers. Methods : To identify transcripts originating from PC for biofluid‐based test development, we analyzed long RNAs in urinary EVs associated with CSPC. EVs were isolated using our clinical ExoLution™ platform from urine collected from prostate cancer patients with CS disease (≥GG2, the majority was GG3 or higher with extracapsular extension) pre‐ and post‐radical prostatectomy (RP), respectively. Over 60 RNA sequencing libraries were generated to profile long coding and non‐coding RNAs in whole transcriptome, whole exome, and a prostate cancer gene panel, respectively. Results : High‐quality QC metrics showed over 17,000 protein coding and long noncoding RNAs. About 456 differentially expressed long RNAs were found to be associated with CSPC from the matched pre‐ vs post‐RP urine samples, many of which have been implicated in PC. Further demonstrating biomarker specificity, the top 20 prostate‐specific genes predicted by the tissue GTEx database show higher abundance in pre‐ vs post‐RP, while the top 10 bladder‐specific genes, top 10 kidney‐specific genes, and house‐keeping genes exhibit little changes. Moreover, the top 3 pre‐RP upregulated pathways are shown to be associated with androgen response. Summary/Conclusion : This gene‐set provides a blue‐print for CSPC RNA biomarker targets in urine EVs and forms a foundation for robust biomarker discoveries, advancing the next generation of liquid biopsy‐based approaches in PC management. Funding : Exosome Diagnostics, a Bio‐Techne Brand. Keywords : clinically significant prostate cancer, radical prostatectomy, long RNAs, urinary EVs, biomarkers, RNA‐seq, transcriptome

Mass

Vendula Pospichalova 6 ; Anna Vyhlidalova Kotrbova 1 ; Kristina Gomoryova 1 ; Antonia Mikulova 1 ; Marek Kravec 1 ; David Potesil 2 ; Zbynek Zdrahal 2 ; Jan Kotoucek 3 ; Marketa Bednarikova 4 ; Jitka Hausnerova 5 ; Lubos Minar 5 ; Igor Crha 4 ; Eva Jandakova 5 ; Vit Weinberger 5 ; Vitezslav Bryja 6 1 Faculty of Science, Masaryk University, Czech Republic; 2 Central European Institute for Technology, Czech Republic; 3 Veterinary Research Institute, Brno, Czech Republic; 4 University Hospital Brno, Brno, Czech Republic; 5 University Hospital Brno, Czech Republic; 6 Faculty of Science, Masaryk University, Brno, Czech Republic Introduction : Ovarian cancer (OC) ranks among the deadliest cancers in women. Lack of symptoms, rapid metastases and common chemoresistance contribute to unfortunate fate of majority of OC patients, especially those having high‐grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common and most aggressive type of OC. Many HGSC patients have excess fluid in the peritoneum at the stage of diagnosis called ascites. Ascites is basically a tumor microenvironment (TME) containing various cells, proteins and also extracellular vesicles (EVs). Small size and polydispersity of EVs brings various challenges to their isolation and characterization, including method‐dependent enrichment of different EV subtypes as well as contaminants. Methods : Therefore we isolated EVs from ascites of 11 HGSC patients by two different methods: ultracentrifugation coupled to sucrose cushion and size‐exclusion chromatography (SEC) using qEV column, as well as the main protein fraction from SEC, serving as negative control for each patient; and analyzed all samples using tandem mass spectrometry. Results : We identified core ascitic EV proteins present in all patients that contain typical EV markers and are devoid of method‐dependent contaminants. To cover interpatient heterogeneity, we expanded these “core proteins” with proteins found in majority of patients. Next, we compared them with proteins of EVs from related control fluids and found proteins present only in/on EVs from HGSC patients. We believe this list of proteins contain both important players of HGSC progression as well as potential biomarkers. Using single cell RNA sequencing data we mapped the origin of EVs to different types of cells present in malignant ascites. Our results suggest that EVs in ascites do not come predominantly from tumor cells, but rather from variety of non‐malignant cell types including cancer‐associated fibroblasts and tumor‐associated macrophages, which presence in ascites we confirmed by flow cytometry. Summary/Conclusion : Our results emphasize the recently appreciated role of TME in the progression of HGSC. To conclude, this is the first study combining mass spectrometry and scRNA sequencing in an attempt to link EV composition to the cell types producing it. As such it opens numerous avenues both for better understanding of EV role in tumor promotion/prevention and for the improved HGSC diagnostics. Funding : This work was supported by the Grant Agency of Masaryk University (grant number MUNI/R/1225/2021) and Ministry of Health of the CR / Ministry of Health Research Programme 2020 – 2026 (grant number NU21‐03‐00306). Keywords : ovarian cancer (OC), high‐grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), extracellular vesicles (EV), ascites, mass spectrometry, tumor microenvironment, macrophage

Reck

Paula C. Mancilla‐González 1 ; Rodrigo Escalona 2 ; Delia Chiarello 2 ; Jaime Gutierrez 2 1 Universidad San Sebastián, santiago, Chile; 2 Universidad San Sebastián, Chile Introduction : Severe preeclampsia (sPE) is a pregnancy syndrome characterized by placental dysfunction and a generalized antiangiogenic state, associated to maternal hypertension and multisystemic damage. The number of placental derived small extracellular vesicles, recognized by PLAP expression (sEV PLAP) were found increased in maternal plasma (MP) of sPE compared to normal pregnancies. It is proposed that sEV PLAP are involved in sPE development. We evaluate the expression of RECK, a protein whit antiangiogenic effects and related to development of sPE, in sEV PLAP in the first trimester MP of mother that subsequently developed sPE and its angiogenesis effects. Methods : sEV PLAP from MP of sPE and normal pregnancies at 1° trimester, were isolated by ultracentrifugation and characterized by common features. The expression of RECK was measured by ELISA and immunoblotting. We determined the role of sEV PLAP in endothelial cells, the uptake of stained sEV, tube formation assay, and expression of angiogenic markers. Results : sEV from sPE and normal pregnancies present similar particle size, morphology, and exosome markers. The number of sEV PLAP are increased in sPE vs normal pregnancies. The expression levels of RECK are increased in sPE in relation to normal pregnancies. No differences in the uptake of sEV were observed. However, the sEV derived from sPE result in antiangiogenic effects. Summary/Conclusion : RECK expression and number of sEV PLAP are increased in first trimester MP from pregnancies that subsequently develop sPE. We demonstrate a role of RECK on sEV PLAP in the angiogenic process. This suggest that RECK in sEV play key roles in the origin and development of sPE and emerge as predictor biomarker in sEV PLAP. Funding : RECK expression and number of sEV PLAP are increased in first trimester MP from pregnancies that subsequently develop sPE. We demonstrate a role of RECK on sEV PLAP in the angiogenic process. This suggest that RECK in sEV play key roles in the origin and development of sPE and emerge as predictor biomarker in sEV PLAP. Keywords : preeclampsia, RECK, placental sEV, biomarker

Role

Hiu Yee Kwan 1 ; Minting Chen 2 ; Shilin Xiao 2 1 Hong Kong Baptist University, Hong Kong, Hong Kong; 2 Hong Kong Baptist University, Hong Kong Introduction : The packaging of exosomes may vary under diseased states or under different dietary interventions. Changes of the exosomal contents will lead to different pathological consequences. Under obesity conditions, whether exosomes play a role in the enhanced colorectal cancer (CRC) metastasis remains unknown. Methods : Obese mouse models were established by high‐fat diet (HFD) feeding. Serum exosomes (SExos) were purified from the mouse serum with differential ultracentrifugation method. Protein profiles of SExos were examined by iTRAQ‐based proteomic. Bioinformatics analysis was done to investigate the metastatic roles of the differentially expressed exosomal proteins. Results : In the proteomics study, a total of 2094 proteins were identified in SExos, and 479 of which showed significant difference between the obese and control mice. These differentially expressed proteins (DEPs) interacted with each other in a concert. Furthermore, KEGG enrichment analysis identified some DEPs with pro‐metastatic properties, suggesting a role of the serum exosomal proteins in mediating CRC metastasis under obesity conditions. Summary/Conclusion : Our data suggest that the serum exosomal protein profiles are different under obesity conditions, which may mediate the enhanced CRC metastasis under obesity conditions. Funding : This work partially supported by FNRA‐IG (RC‐FNRA‐IG/20‐21/SCM/01), GDNSF (2021A1515010655), Shenzhen Basic Research Program for Shenzhen Virtual University Park (2021Szvup131), and HMRF (08193596). Keywords : obesity, exosomal proteins

Sasp

Juan Antonio Fafián Labora 1 ; Sergio Lucio‐Gallego 2 ; Rocío Mato‐Basalo 2 ; Carmen Alarcón‐Veleiro 3 ; Mónica Carrera 4 ; Lola Gutiérrez 5 ; Concha Gil 5 ; Jesús Mateos 6 ; María C. Arufe 7 1 TCMR Group. CICA‐INIBIC. University of A Coruña. A Coruña. Spain, A Coruña, Spain; 2 TCMR Group. CICA‐INIBIC. University of A Coruña. A Coruña. Spain, A Coruña, USA; 3 TCMR Group. CICA‐INIBIC. University of A Coruña. A Coruña. Spain, USA; 4 IIM‐CSIC. Madrid. Spain., Vigo, USA; 5 Proteomics Facility UAM. Madrid. Spain, USA; 6 FarmaCHUS‐IDIS. Santiago de Compostela. Spain, USA; 7 TCMR Group. CICA‐INIBIC. University of A Coruña. A Coruña, Spain., A Coruña, USA Introduction : Cells have the capacity to modulate the microenvironment through secreted molecules and factors like interleukins, cytokines, chemokines, extracellular matrix proteins, etc. Pathological process like cancer or natural process like ageing can modify this microenvironment. The ageing modifications are related with the increasing number of senescent cells. Cellular senescence is a process characterized by a cell cycle arrest, an increased β‐galactosidase activity and a secretome that acquires a specific phenotype. This senescence‐associated secretory phenotype known as SASP leads the microenvironment to a more pro‐inflammatory state triggering with time age‐related diseases. SASP has the capacity of paracrine senescence transmission. Small extracellular vesicles (sEV) are an important part of SASP. The regulation of the sEV biogenesis has a high potential to develop senomorphics, drugs that modulate SASP, to treat age‐related diseases such as type II diabetes, cardiovascular diseases among others. Objetive. Find a proteomic signature of the SASP mediated by sEV to reveal pathways associated with the SASP senescence transmission through sEV. Methods : In this study we knock‐down in mesenchymal stem cells RELA or RAB27A, genes implicated in the paracrine senescence and sEV biogenesis respectively, using CRISPR‐Cas9 methodology. We compared the paracrine senescence transmission through sEV in these cells with proliferation and β‐galactosidase activity assays after the treatment with senescent or non‐senescent sEV. Finally, we perform the shot‐gun technique Tandem Mass Tag (TMT) Systems (10‐plex) to identify, quantify and compare the proteome of senescent cells with the knock‐down senescent ones. Results : The paracrine senescence transmission in the RELA and RAB27A knock‐downs was inhibited. The quantitative and comparative proteomic analysis identified 4099 proteins, which of 25 were differentially regulated by the SASP mediated by sEV. These proteins are involved in the Golgi traffic and network. Summary/Conclusion : This study provides evidence that Golgi traffic and transport are involved in the SASP mediated by sEV. This data will be useful to design new therapeutic strategies or support the actual ones against age‐related diseases. Funding : JFL was funded by Proof‐of‐concept from ProteoRed‐ISCIII (PPC2020) and Xunta de Galicia, Grant Number ED481D‐2021‐020 and MINECO (RYC2021‐032567‐I) for the funding and the InTalent program from UDC‐Inditex for the research grant. The proteomic analysis was performed in the Proteomics Unit of Complutense University of Madrid, a member of ProteoRed and is supported by grant PT17/0019, of the PE I+D+i 2013‐ 2016, funded by ISCIII and ERDF. Or Grant PRB3 (IPT17/0019 ‐ ISCIII‐SGEFI / ER. MCA. received a grant from the Spanish National Health Institute Carlos III (PI20/00497). Keywords : cellular senescence, SASP, senomorphics, omics

Sevs

Letícia Alves Fernandes 1 ; Gabriela Inhauser Riceti Magalhães 2 ; Mari Cleide Sogayar 3 ; Ana Claudia Oliveira Carreira 4 1 Surgery Department, Faculty of Veterinary and Animal Science, University of São Paulo, São Paulo, Brazil; 2 Faculty of Biotechnology, Federal University of Uberlandia, Brazil; 3 NUCEL, School of Medicine, University of São Paulo; Biochemistry Department, Chemistry Institute, University of São Paulo, Brazil; 4 Center of Natural and Human Sciences, Federal University of ABC; Surgery Department, Faculty of Veterinary and Animal Science, University of São Paulo; NUCEL, School of Medicine, University of São Paulo, Santo André, Brazil Introduction : Angiogenesis is one of the most challenging hallmarks of cancer due to its controversial molecular mechanisms. The CD90/Thy‐1 molecule has proven itself as a potential therapeutic target in triple‐negative breast cancer, the subtype responsible for the worst treatment and prognosis scenario. Considering the key molecular process mediated by sEV, this report aims to compare the angiogenic potential from sEV‐depleted conditioned medium derived from CD90 knockdown and parental cell line, to uncover the key role played by sEV in this molecular mechanism. Methods : Conditioned media (24h) were obtained from Hs578T triple‐negative breast cancer cell lines, both parental (Hs578T/WT) and CD90 knockdown (Hs578T/shCD90). EVs were depleted from the conditioned medium using PEG precipitation technique. We then studied the potential of complete conditioned medium (CM) and EV‐depleted CM (dEV‐CM) in a scratch wound healing assay using 3T3 fibroblasts and the angiogenesis potential using the embryo quail chorioallantoic membrane (quail‐CAM) in vivo assay. The evaluation was complemented with histological techniques to analyze blood vessel and angiogenesis biomarkers. All negative control and sample replicates were done (N = 3). Results : These data demonstrated that the sEV depletion from the Hs578T/shCD90 CM decreased the migration of 3T3 cells and blocks the angiogenesis in CAM assay, in contrast of the result seen using Hs578T/WT CM. Also, CD90 knockdown has an outstanding anti‐angiogenic performance together with sEV depletion. Summary/Conclusion : These findings evidenced that despite the CM present all molecular factors needed to promote angiogenesis, this mechanism occurs primarily in the sEV presence. Suggesting that sEV is the major intermediary in this tumor progression molecular process. Funding : Coordination for Improvement of Higher Education Personnel (CAPES), National Council for Scientific and Technological Development (CNPq), The State of São Paulo Research Foundation (FAPESP No. 2016/05311‐2). Keywords : triple‐negative breast cancer, target therapy, small extracellular vesicles, angiogenesis, CAM, CD90/Thy1

Size

Simonides Immanuel van de Wakker 1 ; Christian JB Snijders Blok 2 ; Julia Bauzá‐Martinez 3 ; Carla Rios Arceo 4 ; Herak Manjikian 2 ; Eduard F Willms 5 ; Olivier G de Jong 6 ; Renee GC Maas 2 ; Wei Wu 7 ; André Görgens 8 ; Samir EL Andaloussi 9 ; Joost PG Sluijter 4 ; Pieter Vader 10 1 Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht, Netherlands; 2 Department of Experimental Cardiology, University Medical Center Utrecht, USA; 3 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, USA; 4 Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht, USA; 5 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, USA; 6 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, Netherlands; 7 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht, USA; 8 1. Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden, 2. Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany, Stockholm, Sweden; 9 Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden, Huddinge, Sweden; 10 CDL Research, Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht, Netherlands Introduction : Increasing evidence indicates that small extracellular vesicles (sEVs) exist as heterogeneous populations. sEV heterogeneity and thereby reproducibility represents a major challenge in the field, in particular related to the understanding of differences in function. Progenitor cell‐derived sEVs have shown great potential to stimulate tissue repair. In this project, functional differences of progenitor cell‐derived EV subpopulations were studied to better understand functional EV heterogeneity and to provide new insights that will help with the development of regenerative EV therapeutics. Methods : Cardiac progenitor cell (CPC)‐ and mesenchymal stromal cell (MSC)‐derived sEVs were purified by binding chromatography, followed by size‐exclusion chromatography (SEC) for size‐based fractionation of different sEV‐subpopulations. sEVs were characterized using western blot, transmission electron microscopy, mass spectrometry and imaging flow cytometry (IFC). Functional differences were studied using multiple cellular assays on various cell types, including AKT phosphorylation, wound healing migration, angiogenesis, target cell EV uptake, fibroblast activation, cardiomyocyte survival and proteasome activity. Results : SEC was used to separate three distinct subpopulations of CPC and MSC‐derived sEVs, which were identified based on differential expression of sEV marker proteins. These sEV subpopulations differed in size, appearance and proteomic composition. Mass spectrometry and IFC analysis confirmed the differences in expression levels of sEV marker proteins. Furthermore, gene ontology cellular component analysis indicated differences in intracellular origin. sEV subpopulations exerted clear functional differences in different recipient cells. Smaller and middle‐sized sEV subpopulations were able to stimulate migration, activation and spheroid formation in endothelial cells, but no effect was observed for the larger sEVs. Only middle‐sized sEVs were able to stimulate cardiomyocyte survival whereas only the smallest subpopulation presented proteasomal activity. Furthermore, differences in subpopulation uptake were seen in endothelial cells, cardiomyocytes, fibroblasts and macrophages. Summary/Conclusion : SEC allows for isolation and in‐depth study of the functional heterogeneity of sEVs. In our study, we observed the existence of different subpopulations based on size which had a differential composition, origin and biological function. Increasing knowledge of sEV heterogeneity will contribute to a better understanding of the mechanisms of action of sEVs, thereby accelerating translation of EV therapeutics to the clinic. Funding : Van Herk Foundation, ERC EVICARE (725229), ESC FCIG, MDR Young Talent Incentives Program, NLSEV‐MOVE. Keywords : EV heterogeneity, subpopulations, tissue/cardiac repair

Stem

Yoon Kyoung Kim 1 ; Seohyun Kim 2 ; Gi Beom Kim 2 ; Seonghyun Kim 2 ; Gi‐Hoon Nam 2 ; In‐San Kim 3 1 Shiftbio, Seoul, Republic of Korea; 2 SHIFTBIO, Republic of Korea; 3 Korea Institute of Science and Technology, Seoul, Republic of Korea Introduction : Acute liver failure (ALF) is orphan disease that is hard to expect and does not have drugs. The only therapy is liver transplantation. To provide therapeutic options for ALF patients, we developed SBI‐102 which are mesenchymal stem cell (MSC) derived extracellular vesicles (EVs) expressing SIRPα (signal regulatory protein alpha) protein on their membrane. We expected that regenerative effect of MSC and enhancing efferocytosis by blocking CD47 would synergy in ALF. Methods : Protein, particle number, and SIRPa expression were confirmed. Two representative ALF models were used, LPS/D‐galN (LPS 10ug/kg and D‐galactosamine 700mg/kg) and acetaminophen (APAP, 300mg/kg). SBI‐102 were intravenously administered and therapeutic indexes were assessed. Results : ALF induced liver tissue showed increased expression level of CD47, and APAP treated mouse hepatocytes also overexpressed CD47. SBI‐102 exhibited higher CD47 binding effects to APAP treated hepatocytes, and pre‐blocking with anti‐CD47 antibody significantly reduced binding activity. The therapeutic effect of SBI‐102 showed prolonged survival and liver restoring effect against LPS/D‐galN induced ALF. Biochemical parameters and pro‐inflammatory cytokines (IL‐6, TNF‐α) were remarkably decreased and kidney, representative complication occurring tissue, structures were normal. Furthermore, SBI‐102 reduced liver damage and apoptotic scores in liver tissue when we applied APAP induced ALF model which has pathologically high similarity with human ALF. Also, when we analyzed liver single cell with flow cytometry, SBI‐102 alleviated infiltration of pro‐inflammatory monocytes, but promoted pro‐regenerative monocytes. Summary/Conclusion : We confirmed that SBI‐102 bound to CD47, effectively induced phagocytosis of damaged liver tissue and suppressed inflammation. Liver immune‐environmental change promoted regeneration in liver. Collectively, these data provided sufficient support for SBI‐102 as a promising ALF therapy. Keywords : acute liver failure, mesenchymal stem cell, regeneration, SIRPa, CD47

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. The society facilitates this mission through an array of initiatives, including educational offerings, 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 programs and services, ISEV plays an indispensable role in delivering vital training and research prospects for those immersed in the realm of EV research.

Acute

Ding‐Wen Chen 1 ; Jian‐Meng Fan 2 ; Julie Schrey 1 ; Seul Jung 2 ; Zhe Zhang 2 ; Dana V. Mitchell 2 ; Deanne M. Taylor 2 ; Peter Kurre 2 1 Children's Hospital of Philadelphia, Philadelphia, USA; 2 Children's Hospital of Philadelphia, USA Introduction : Patients with acute myeloid leukemia (AML) suffer frequent relapses and poor treatment outcomes. Acute inflammation in the bone marrow (BM) at diagnosis is propagated by leukemic blasts and contributes to therapy resistance. The source of chronic inflammation after initial remission remains unclear. BM hematopoietic stem and progenitor cells (HSPCs) are long‐lived contributors to innate immunity. We hypothesize that AML‐derived extracellular vesicles (EV‐AML) convert HSPCs into a key source of sustained inflammation in the BM. Methods : Two genetically different immune competent congenic murine models of AML (C1498 and dox‐inducible(i)MLL‐AF9) were utilized. EV‐AML from C1498 (C1498‐EV‐AML) and iMLL‐AF9 blasts (AF9‐EV‐AML) were characterized according to MISEV criteria. Gene expression was analyzed by single‐cell and bulk RNA sequencing as well as real‐time PCR. Results : In the C1498 AML model, we found elevated inflammatory cytokine secretion and upregulated inflammatory gene expression across HSPC subtypes at a low AML burden in the BM. To test for the involvement of EV‐AML, we challenged HSPCs with purified C1498‐EV‐AML in vitro and found highly upregulated inflammatory gene expression (e.g. Isg15) and chemokine secretion (e.g. Cxcl10) by HSPCs. We independently validated inflammatory signaling utilizing the translationally‐relevant AML model, iMLL‐AF9, in vivo. In a cell‐free approach, we demonstrated that injection of AF9‐EV‐AML alone can incite inflammatory activation in healthy BM‐HSPCs. Summary/Conclusion : Our results show for the first time that AML‐derived EV‐AML convert HSPCs to core constituents of the innate immune environment in the leukemic BM. While much recent work is focused on the possibility of redirecting adaptive immunity to treat AML, we propose to target innate immune components in an effort to improve therapeutic outcome in AML. Funding : Alex's Lemonade Stand Foundation Young Investigator Grant. Keywords : acute myeloid leukemia, inflammation, niche, hematopoietic stem and progenitor cells, innate immunity

Early

Erica Routila 1 ; Rufus Vinod 1 ; Marina Alexeeva 2 ; Kjetil Søreide 2 ; Janne Leivo 3 ; Kim Pettersson 3 1 Department of Life Technologies and inFLAMES Flagship Research Center, University of Turku, Turku, Finland, Turku, Finland; 2 Department of Gastrointestinal Surgery, Stavanger University Hospital, Norway, Norway; 3 Department of Life Technologies and inFLAMES Flagship Research Center, University of Turku, Turku, Finland., Turku, Finland Introduction : Colorectal cancer is a type of cancer that affects the colon and rectum. It is the third most common cancer in men and the second most common cancer in women. Early detection of colorectal cancer can significantly improve the chances for a successful treatment. Our technical approach is based on the use of fluorescent nanoparticles to detect specific glycoconjugates (sugar‐based molecules) associated with extracellular vesicles directly from patient serum. These glycoconjugates are thought to be associated with the development and progression of colorectal cancer, and their presence in the serum may be an early indication of the presence of the disease. Methods : EVs isolated with SEC (qEV, Izon) from cell culture and serum were immobilized using monoclonal antibodies specific to CD63 and CD151 and detected with the use of antibodies coated onto europium‐doped nanoparticles. The most promising subpopulations of EVs expressing tetraspanins (CD9, CD63, CD81, and CD151) found from the cell culture were clinically evaluated with a panel of serum samples including early‐stage CRC patients (n = 31), benign condition (n = 22), and healthy control (n = 18). Results : The majority of CRC cell lines expressed tetraspanin sub‐populations and also glycovariants of integrins and conventional tumor markers (CA19‐9, CEA). A subpopulation of CD151 in combination with CD63 (CD151CD63) was found to be significantly (p = 0.000054) elevated in CRC and benign patients and was able to significantly discriminate (p = 0.00011) between healthy controls and CRC patients. Summary/Conclusion : The use of nanoparticles for the detection of colorectal cancer‐associated EVs (tetraspanins) in human serum holds promise as a tool for the early detection and diagnosis of the disease.

Human

Sahithi Attaluri 1 ; Maheedhar Kodali 2 ; Leelavathi N. Madhu 3 ; R Upadhya 4 ; B Shuai 5 ; X rao 4 ; Ashok K. Shetty 6 1 Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, Texas, USA, College Station, USA; 2 Texas A&M University, College Station, USA; 3 Institute for Regenerative Medicine, Department of Molecular and Cellular Medicine, Texas A&M University College of Medicine, College Station, Texas, USA., College Station, USA; 4 Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, USA; 5 Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, USA; 6 Institute for Regenerative Medicine, Dept of Cell Biology and Genetics, Texas A&M Univ School of Medicine, College Station, USA Introduction : Repeated closed head injuries (rCHIs) typically lead to long‐term cognitive deficits linked with chronic neuroinflammation. Thus, biologics capable of restraining the progression of neuroinflammation after rCHI may prevent cognitive impairments. This study examined the ability of intranasally (IN) administered extracellular vesicles (EVs), purified through chromatographic methods from cultures of human induced pluripotent stem cell (hiPSC)‐derived neural stem cells (NSCs), to prevent cognitive impairments after rCHI. Methods : Adult mice subjected to rCHI for three days (one CHI/day) received IN administration of hiPSC‐NSC‐EVs or the vehicle at 60 minutes (70 × 109 EVs) and seven days (35 × 109 EVs) after the third CHI. Eight months later, the animals were tested for motor function, anxiety‐like behavior, and cognitive function, after which the brain tissues were examined for oxidative stress and neuroinflammatory markers. Results : rCHI mice receiving either vehicle or EVs displayed no motor impairments or anxiety‐like behavior. However, in objects‐based cognitive tests, vehicle‐treated rCHI mice displayed impairments in novel objection recognition, object location memory, and pattern separation. However, rCHI mice that received EVs displayed similar cognitive abilities as naïve control mice. Analysis of the affected cerebral cortical tissues revealed an increased concentration of oxidative stress and NOD‐, LRR‐ and pyrin domain‐containing protein 3 (NLRP3) inflammasome‐related markers and proinflammatory cytokines interleukin‐1 beta (IL‐1b), IL‐6, tumor necrosis factor‐alpha, interferon‐gamma, IL17‐alpha, and IL13. Notably, in rCHI mice receiving EVs, levels of most of these markers were reduced along with reduced microglial activation and astrocyte hypertrophy. Summary/Conclusion : IN administration of NSC‐EVs is a promising approach for maintaining better cognitive function after rCHI through modulation of neuroinflammation. Funding : Supported by a grant from the National Institute of Neurological Disorders and Stroke (1R01NS106907 to A.K.S). Keywords : neural stem cells, extra cellular vesicles, closed head injury, neuroinflammation, cognitive function

Islet

Diana Esparza 1 ; Jinhee Hwang 1 ; Eunjin Oh 1 ; Ima Ghaeli 2 ; Tijana Jovanovic‐Talisman 2 ; Debbie C. Thurmond 1 1 Beckman Research Institute at the City of Hope, Duarte, USA; 2 Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope, Duarte, USA Introduction : Loss of β‐cell function occurs early in type 1 diabetes (T1D). C‐peptide, the clinically standard biomarker, is insufficient to detect early pre‐type 1 diabetes‐related β‐cell dysfunction. Our recent publication demonstrates that circulating levels of exocytosis regulatory protein double C2‐containing protein β (DOC2B) abundance is closely related to β‐cell health and function. However, the mechanisms by which β‐cells export DOC2B into the circulation remain unknown. We hypothesize that DOC2B is uniquely sorted into β‐cell extracellular vesicles (EVs). Indeed, preliminary data show DOC2B is detected in human plasma EVs, and that DOC2B‐laden EVs can stem from β‐cells. Because DOC2B is a broadly expressed protein, we questioned whether DOC2B‐laden EVs are predominantly released by β‐cells, relative to that of other cell types known to express DOC2B protein. Methods : Cell/debris‐free conditioned media from DOC2B biosensor treated or untreated INS‐1 832/13 β‐cells, L6 myotubes, HEPG2 cells, and SH‐SY‐5Y cells were used to isolate EVs via size exclusion chromatography. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and immunoblots for DOC2B and EV marker proteins. Results : Biochemical analyses reveal that DOC2B largely exists as luminal cargo in β‐cell‐derived EVs and is largely absent from EVs released by liver, brain, and skeletal muscle cells. Mechanistically, the research has delineated that the structural domain enabling DOC2B protein sorting into β‐cell‐derived EVs is that which encompasses the tandem C2 domains at the C‐terminus of DOC2B. Summary/Conclusion : Taken together, these data indicate that DOC2B in circulation could originate from DOC2B‐laden EVs released from β‐cells, and that the mechanism by which DOC2B is sorted into those EVs involves one or both C2 domains of the DOC2B protein. Funding : National Institute of Diabetes and Digestive and Kidney Diseases ( DK067912 , DK112917 , and DK102233 ), the Wanek Family Project to Cure Type 1 Diabetes at the City of Hope (D.C.T.), and Ford Foundation Pre‐doctoral Fellowship (D.E.). Keywords : Islet β‐cells, DOC2B, diabetes

Large

Robert Myette 1 ; Janusz Feber 1 ; Pavel Geier 1 ; Christopher Kennedy 2 ; Dylan Burger 2 1 Children's Hospital of Eastern Ontario, Canada; 2 Ottawa Hospital Research Institute, Ottawa, Canada Introduction : Idiopathic Nephrotic Syndrome (iNS) is one of the most common causes of kidney injury in children. There has been an increased focus on reactive oxygen species (ROS) in podocytes as drivers of proteinuric disease. Our lab and others have suggested that large extracellular vesicles (LEVs) may have utility as biomarkers of podocyte injury. The aim of our study was to investigate the potential of urinary LEVs as biomarkers in iNS, and to characterize LEV release using cultured podocytes exposed to toxins in vitro. Methods : We analyzed urine samples from a prospective cohort enrolling children 1–18y with iNS. Podocyte specific LEV were quantified using flow cytometry and nanoparticle tracking (NTA). Human immortalized podocytes (hPod) were used in vitro. Puromycin aminonucleoside (PAN; 25 ug/mL; 24 hours) and lipopolysaccharide (LPS; 25 ug/mL; 24 hours) were used as podocyte toxins. Results : In paired relapse and remission samples from 14 patients the median [IQR] podocyte LEVs were significantly lower in remission (0 LEVs/mmol of Cr [0, 14.7 × 10^3]) vs. nephrosis 22.8 × 10^3 LEVs/mmol of Cr [IQR 1.11 × 10^3, 74.6 × 10^3] (p< 0.01). Urine Albumin to creatinine ratio was positively associated with elevated LEVs (p = 0.01). In cultured hPods, PAN treatment resulted in a 2.5‐fold increase in hPod LEVs (p = 0.03) while LPS caused a 3.5‐fold increase (p = 0.0004). This was abrogated with inhibitors of ROS generation (N‐acetyl cysteine,4‐OH‐Tempol and MITO‐Tempo). Summary/Conclusion : In summary, LEV appear to serve as a novel indicator of iNS relapse and their levels can differentiate disease status. hPods show similar characteristics when treated with common podocyte toxins, while protective antioxidant strategies also reduce LEV release. Funding : Canadian Institutes of Health Research, Kidney Foundation of Canada, Kidney Research Scientist Core Education and National Training Program. Keywords : kidney, nephrotic syndrome, podocyte, biomarker

Novel

Jugal Suthar 1 ; Esther Osarfo‐Mensah 2 ; Alberto Alvarez Fernandez 3 ; Beatriz Prieto‐Simon 4 ; Stefano Angioletti‐Uberti 5 ; Gareth R Williams 6 ; Stefan Guldin 1 1 University College London, United Kingdom; 2 University College London, London, United Kingdom; 3 Trinity College Dublin, Ireland; 4 Universitat Rovira i Virgili, USA; 5 Imperial College London, USA; 6 University College London, USA Introduction : Quartz crystal microbalance with dissipation monitoring (QCM‐D) has recently emerged as a powerful alternative for the phenotypic detection of EVs, offering multiple modes of analyte discrimination by frequency and dissipation. In this talk, I will present current activities in my group towards effective interfacing of QCM‐D‐based approaches for the immunosensing of EVs, including the use of additional electrochemical read‐out via impedance spectroscopy (eQCM‐D) and nanostructuring the biosensor surface to mirror lateral analyte feature sizes. Methods : Extracellular vesicles were obtained from human umbilical cord mesenchymal stem cell culture media and isolated using filtration, concentration by centrifugation and size‐exclusion chromatography. SEC fractions were assessed by nanoparticle tracking analysis, protein content analysis, western blot analysis and gold immuno‐electron microscopy. All QCM‐D measurements were carried out using a Q‐Sense E4 instrument (Biolin Scientific). For EQCM‐D measurements, an electrochemistry module was deployed in tandem with a potentiostat and a three‐electrode system. Nanostructuring of the sensor surface was conducted via diblock copolymer self‐assembly of polystyrene‐block‐poly(4‐vinylpyridine). Sensor functionalization was carried out using a mixture of SH‐PEG (2 kDa)‐Biotin and spacer molecule SH‐OEG (800 Da)‐COOH at a 1:9 mol/mol ratio, followed by exposure to streptavidin (SAv) and immobilization of mouse monoclonal biotinylated anti‐CD63 antibodies. Results : When comparing the various strategies, we found (A) a lowering of the detection limit by a factor of 2–4 when combining QCM‐D in tandem with in‐situ electrochemical impedance spectroscopy; (B) a higher degree of binding on nanostructured gold islands over flat surfaces, (C) a higher degree of binding when the nanostructured gold islands were dispersed on silica rather than on flat gold, and (D) a higher degree of binding when the nanostructured features were matched to the lateral dimensions of the EVs. Summary/Conclusion : In summary, we have investigated a range of strategies to enhance the capabilities of acoustic immunosensing of extracellular vesicles, including multimodal read‐out using changes in frequency, dissipation and electrochemical properties as well as nanostructuring the biosensor surface. Meanwhile, a limit of detection around 10^7 EV‐sized particles / ml can be routinely achieved. Crucially, this analytical platform provides novel opportunities to aid sensor development (e.g. when validating surface functionalization, biomarker recognition or calibrating alternative read‐out mechanisms) as well as for quality control. Funding : We are grateful to EPSRC (EP/L01646X, EP/R035105/1) and MRC (MR/R000328/1). Keywords : QCM‐D, immunosensor, biosensing, electrochemistry, nanofabrication

Plant

Fang Cheng 1 ; Rufan Huang 2 ; Hongbo Chen 3 1 Sun Yat‐sen University, Shenzhen, China (People's Republic); 2 Sun Yat‐sen University, China (People's Republic); 3 School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat‐sen University, Shenzhen 518107, China, Shenzhen, USA Introduction : Among autoimmune diseases, psoriasis and atopic dermatitis are two major skin disorders becoming the major issues threatening public health with increasing prevalence. However, Existing therapeutics for autoimmune skin diseases remain problematic due to low efficacy, severe side effects, and difficulties to reach target tissues. The shortcomings of mainstay treatments and surging cases of autoimmune skin disorders across the world meant safer and more effective therapeutic strategies were now urgently needed. we proposed a new approach to prepare engineered hybrid EVs by fusing plant‐derived EVs and engineered MSCs‐derived nanovesicles to take advantage of both types of nanovesicles and to generate personalized delivery nanovectors. Methods : We first investigated the literature as well as Chinese medicine prescriptions and determined ten promising tissues from edible plants to isolate plant‐derived extracellular vesicles (PLEVs) by differential ultracentrifugation. We assessed the quality of PLEVs by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), lipidomic analysis, miRNA sequencing, and analysis of their protein composition. We first screened out grapefruit‐derived exosomes (GEVs) with anti‐inflammatory and antioxidant effects, but they lacked immunosuppressive function. we then encapsulated CX5461 into GEVs by electroporation. By GEO data analysis, we found that chemokine CCL20 is elevated in the lesions of autoimmune diseases, so nanovesicles carrying CCR6 may target inflamed tissues by interacting with regional CCL20. However, genetic modification of EVs remains a big challenge in plant cells when compared to animal cells. Therefore, we proposed to fuse bioengineered cell membrane vesicles with the membrane of GEVs. Gingiva‐derived◻mesenchymal◻stem◻ cells (GMSCs) were selected for genetic engineering due to their significant immunosuppressive effect compared to other MSCs. Subsequently, we prepared CX5461‐loaded fusion vesicles (FV@CX5461) by hybriding GEVs with CCR6 enriched GMSCs membrane‐based nanovesicles (CCR6‐NVs) by extrusion. Results : 1. Grapefruit‐derived exosomes had anti‐inflammatory and antioxidant effects but lacked immunosuppressive function in vitro. 2. The anti‐oxidative, anti‐proliferative, anti‐inflammatory and immunosuppressive effects of FV@CX5461 were better than GEVs, CX5461, or CCR6‐NVs alone in vivo and in vitro. 3. In IMQ‐induced psoriasis mice and DNCB‐induced AD mice FV@CX5461 exhibited excellent immunomodulatory capabilities, including calm down Th17 cell activation, inhibition of intracellular ROS in macrophages, and induce Treg cell infiltration. Summary/Conclusion : A nanotherapeutic drug delivery strategy is developed using fusion nanovesicles derived from plant and animal cells with high clinical potential.

Raman

Matyas Bukva 1 ; Edina Gyukity‐Sebestyen 2 ; Timea Boroczky 2 ; Yasmin Ranjous 3 ; Maria Harmati 2 ; Gabriella Dobra 2 ; Laszlo Szivos 4 ; Katalin Hideghety 5 ; Krisztina Budai 6 ; Judit Olah 7 ; Peter Horvath 8 ; Gyorgy Lazar 6 ; Zoltan Konya 3 ; Pal Barzo 4 ; Almos Klekner 9 ; Krisztina Buzas 10 1 Department of Immunology, Faculty of Science and Informatics, University of Szeged, Szeged, Szeged, Hungary; 2 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eotvos Lorand Research Network, Szeged, Hungary, Szeged, Hungary; 3 Department of Applied and Environmental Chemistry, University of Szeged, Szeged, Hungary, Szeged, Hungary; 4 Department of Neurosurgery, University of Szeged, Szeged, Hungary, Szeged, Hungary; 5 ELI‐ALPS, ELI‐HU Non‐Profit Ltd., Szeged, Hungary, USA; 6 Department of Surgery, University of Szeged, Szeged, Hungary, Szeged, Hungary; 7 Department of Oncotherapy, University of Szeged, Szeged, Hungary, Szeged, Hungary; 8 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary, Szeged, Hungary; 9 Department of Neurosurgery, Clinical Centre, University of Debrecen, Debrecen, Hungary, Debrecen, Hungary; 10 Department of Immunology, University of Szeged, Szeged, Szeged, Hungary Introduction : Spectroscopic analysis of the molecular composition of small extracellular vesicles (sEVs) is a promising but underexplored method for diagnosing cancerous diseases, particularly central nervous system tumors. Using a sufficient number of clinical samples and Raman spectroscopic analyses, we attempt to elucidate the potential role of plasma‐derived sEVs in diagnosing seven distinct patient groups. Methods : The study is conducted in accordance with the Declaration of Helsinki, informed consent forms are collected and the study was approved by national ethics committee. Up to 490 plasma samples will be obtained from seven patient groups (glioblastoma multiforme, meningioma, melanoma and non‐melanoma brain metastasis, colorectal tumors, melanoma and a control group). SEV isolation is performed through differential centrifugation. The isolates are characterized by Western Blot, transmission electron microscopy and nanoparticle tracking analysis. Principal Component Analysis–Support Vector Machine algorithm is performed on the Raman spectra for classifications. Classification accuracy, sensitivity, specificity and the Area Under the Curve (AUC) value are used to evaluate the performance of classification. Results : According to preliminary results, the patient groups are distinguishable with 80–95% sensitivity and 80–90% specificity. AUC scores of 0.82–0.9 suggest excellent classification performance. Summary/Conclusion : Our findings indicate that Raman spectroscopic analysis of sEV‐enriched plasma isolates is a promising strategy for the development of noninvasive, cost‐effective methods for the clinical diagnosis of various cancers. Funding : GINOP‐2.2.1–15‐2017–00052; ÚNKP‐22‐3‐New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund; University of Szeged, Faculty of Medicine, Szent‐Györgyi Albert Research Fund (2021).

Rapid

Valeria Mantella 1 ; Vadim Krivitsky 2 ; Adva Krivitsky 3 ; Maya Ben‐Yehuda Greenwald 4 ; Devanarayanan Siva Sankar 5 ; Jil Betschmann 6 ; Johannes Bader 7 ; Nicole Zoratto 4 ; Jörn Dengjel 8 ; Sabine Werner 4 ; Jean‐Christophe Leroux 9 1 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, 8093 Switzerland., Zurich, Switzerland; 2 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, 8093 Switzerland., Zurich, Switzerland; 3 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, 8093 Switzerland, Zürich, Switzerland; 4 Institute of Molecular Health Sciences, Department of Biology, ETH Zürich, Zürich, 8093 Switzerland, Zürich, Switzerland; 5 Department of Biology, University of Fribourg, Fribourg, 1700 Switzerland., Switzerland; 6 Institute of Molecular Health Sciences, Department of Biology, ETH Zürich, Zürich, 8093 Switzerland, Switzerland; 7 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, 8093 Switzerland, Zürich, Switzerland; 8 Department of Biology, University of Fribourg, Fribourg, 1700 Switzerland, Switzerland; 9 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, 8093 Switzerland., Zürich, Switzerland Introduction : Extracellular vesicles (EVs) have recently emerged as versatile therapeutic agents, biomarkers, and potential drug carriers. Despite the growing interest in developing EVs for therapeutic and diagnostic usage, progress in this field is partly hindered by the lack of efficient isolation techniques. This work describes an electrochemically controlled device enabling the rapid capture and release of EVs, from various human biofluids and wounded skin tissue originating from healthy (WT) and diabetic mice. Methods : The device consists of a fluidic channel where EVs are immobilized by affinity interactions and controllably released by applying voltage. The median diameter and concentration of the isolated EVs were determined by nanoparticle tracking analysis and electron microscopy. Their protein composition was assessed by western blot and proteomics. A principal component analysis was used to reflect the variation between protein abundances of EV populations of diabetic and WT mice. Results : The investigated biosamples (i.e. serum, urine, cell culture supernatant, plasma) were injected into the fluidic channel and EVs were purified using two different antibody coatings. Isolated EVs displayed differences in their size distribution and concentration, indicating that the device could be further used for an estimation of the abundance of EV subpopulations, simply by changing the type of the attached antibody. EVs recovered from wounds in both WT and diabetic mice showed different protein composition. In particular, an increase in mitochondrial proteins in the EVs from wounds isolated from diabetic vs. healthy mice was observed. Summary/Conclusion : Our electrochemical device was used to isolate EVs from a number of physiological fluids. The selective isolation of different EV subpopulations could, in principle, be easily performed for more in‐depth vesicle characterization and diagnostic use, as suggested by the EVs wound data. Funding : This work was supported by the ETH Zurich – Open ETH project SKINTEGRITY.CH, the Swiss National Science Foundation (grant 31003B‐189364 to S.W.). Keywords : extracellular vesicles, immunoaffinity, electrochemical device, carbon microfibers, biosample, purification, preconcentration

Small

María Angélica Calderón‐Peláez 1 ; L. Johanna Madroñero 2 ; Jaime E. Castellanos 2 ; Myriam Lucía Velandia Romero 2 1 Virology lab, Universidad El Bosque, Bogotá, Bogotá, Colombia, Colombia; 2 Virology Lab, Universidad El Bosque, Bogotá, Bogotá, Colombia, Colombia Introduction : Neurons are the main targets for ZIKV infection. This can change mitochondrial morpho‐dynamics and induce apoptosis. However, it is unknown if sEVs transport elements that participate in these processes. This work aimed to characterize small EVs from ZIKV‐infected neurons (sEVIN) and determine the miRs and proteins that might regulate mitochondrial function. Methods : Neuron cultures from 1‐day‐old Balb/C mice purified by 2.5 μM AraC treatment were infected with ZIKV, MOI 0.1 for 1h, then, virus was removed, and cells were kept for 48h in neurobasal medium. Cell viability (Calcein‐AM and LDH), and viral infection (IFI and PCR for DENV C protein) were measured. After 48h post‐infection, EVs were isolated by ultracentrifugation and characterized by NTA, DLS, Western blot, LC/MS/MS, and small RNA sequencing. Protein files were analyzed, and enrichment and functional annotation were done (Panther and GORilla). Small RNAs were extracted (mirVana kit) and sequenced. Clean reads were aligned to Mus musculus genome (GRCm39), annotated (miRBase database) and expression levels were estimated. Results : Neuron infection did not affect cell viability but induced a high production of sEVs (4 × 10^8) that were slightly bigger than sEVs from non‐infected neurons (sEVNIN). Protein analysis of sEVs from infected neurons (sEVIN) showed 65 overexpressed proteins regarding sEVNIN, 5 of which were exclusive, including the 14‐3‐3 protein previously reported as modulator of ZIKV replication. sEVIN preliminary analysis showed 538 total miRs, 11 involved in mitochondrial regulation such as mmu‐miR‐22‐3p, 127, 181a/b‐5p, and 25b. Importantly, 9 miRs has been reported as axonal guidance regulators, impacting mitochondrial localization and ATP availability. Additionally, in silico analysis showed 18 miRs that might regulate viral protein function Summary/Conclusion : sEVs produced by ZIKV‐infected neurons carry proteins and miRs that may regulate mitochondrial function and distribution through neurons. These findings could help to explain the aggressive neurodegenerative outcome caused by ZIKV. Funding : Minciencias‐UEB Grant 130884467149, code 431–2020. ISLA Ltda.: Doctoral research grant, 2022. Keywords : ZIKV infection, neurons, sEVs, mitochondrial dynamics, axonal regulation

Study

Emeline COART 1 ; Hara Episkopou 2 ; Aurelie Christian 3 ; Valerie Lebrun 2 ; Kseniia Boriachek 4 ; Nicolas ROUSSEAU 5 ; Thibaut FOURNIOLS 5 ; Denis DUFRANE 6 ; Ingrid Struman 7 1 University of Liege, Liege, USA; 2 Novadip Biosciences, Discovery department, Watson & Crick Hill, Rue Granbonpré 11, 1435 Mont‐Saint‐Guibert, Belgium, Belgium; 3 University of Liege, USA; 4 Novadip Biosciences, Discovery department, Watson & Crick Hill, Rue Granbonpré 11, 1435 Mont‐Saint‐Guibert, Belgium, Brussels, Belgium; 5 Everzom, USA; 6 Novadip, USA; 7 ULiège (Université de Liège), Liege, Belgium Introduction : The tumor microenvironment plays a crucial role in tumor progression and relies on secreted factors, including extracellular vesicles. EVs secreted by cells from the osteosarcoma (OS) microenvironment participate directly in OS growth and invasion. Many studies have found that BMSC‐derived EVs can regulate cell proliferation, migration, survival, and OS drug resistance. Studies have highlighted that EVs from adipose‐derived stem cells (ASC) decrease OS cell proliferation. Our goal is to determine how ASC‐EVs affect OS growth and modify them to improve their therapeutic potential. Methods : The 3M 3 technology platform consists of a 3‐dimensional scaffold‐free extracellular matrix (MEC), utilizing differentiated ASC to generate exosomes. EVs were purified by differential ultracentrifugation. EVs and cellular miRNA (miR) content was determined using qRT‐PCR miR profiling. Functional tests were performed: scratch migration tests and MTT proliferation tests. Coculture was performed in a transwell system. Results : To investigate the therapeutic potential of miR encapsulated in the isolated EVs, we perform functional tests on OS cells transfected with the five miRs*. Our results show that the miR* could act as a tumor suppressor (impact migration and proliferation of OS cells). To investigate the impact on microenvironment cells, we made coculture with miR‐transfected OS cells and fibroblasts. Results show that this coculture impacts the migration of fibroblasts. Future work will evaluate the potential of miR‐encapsulated EVs on OS cells. *The names cannot be mentioned now because of patent/publication concerns. Summary/Conclusion : Overall results show that culturing ASCs in a 3‐dimensional scaffold‐free extracellular matrix leads to the enrichment of miRs in EVs with potential anti‐tumoral activities. Future work will determine if those EVs can be used to treat osteosarcoma. Funding : The authors declare no conflict of interest. This work has received funding from La Region wallonne and ULiege. Keywords : microRNA, homing, osteosarcoma

Tumor

Sara Nikseresht 1 ; Khairul Ansari 2 ; Ramin Khanabdali 3 ; Gregory E. Rice 4 1 Inoviq Limited, Notting Hill, Victoria, Australia., Melbourne, Australia; 2 Inoviq Limited, Notting Hill, Victoria, Australia., USA; 3 Inoviq Limited, Notting Hill, Australia., USA; 4 Inoviq Limited, Notting Hill, Victoria, Australia., Notting Hill, Australia Introduction : Despite emerging evidence of the surveillance and diagnostic potential of tumor‐derived extracellular vesicles (TEX), current extracellular vesicle (EV) isolation tools do not differentiate between tumor and non‐tumoral EVs. SubB2M 1, a site‐directed mutation of the B subunit of the subtilase cytotoxin (Sub2B), recognizes the tumor‐associated sialic acid, N‐Glycolylneuraminic acid (Neu5Gc). The aim of this study was to evaluate the utility of SubB2M immobilized on paramagnetic nanoparticles (TEXO‐NET) to isolate a Neu5Gc enriched subpopulation of tumor‐derived EVs for diagnostic application. Methods : SubB2M was covalently bound to paramagnetic nanobeads (∼140 nm diameter) using an amine‐reactive crosslinker. EV were isolated from both breast cancer plasma and normal human plasma using TEXO‐NET, EXO‐NET (a pan EV capture matrix) and two other commercial EV isolation kits. Captured EVs were quantified using Nanoparticle Tracking Analysis (ZetaView, Particle Metrix). Cancer known miRNAs and protein content of captured EVs were assessed by RT‐PCR and Western blotting. Results : ZetaView analysis showed TEXO‐NET captured a significantly higher number of particles ∼83% from breast cancer plasma compared to other two competitor kits which captured ∼66.5% and 68%. RT‐PCR analysis also demonstrated higher abundancy of tumor‐known miRNAs, miR1229 and Let7d, from TEXO‐NET‐captured EVs compared to other two kits. This data was supported by protein analysis data demonstrating a distinct protein profile for TEXO‐NET‐captured EVs compared to EXO‐NET‐captured EVs. Summary/Conclusion : The data obtained confirm that TEXO‐NET captures a subpopulation of EVs that is enriched in tumor‐specific biomarkers which can potentially be used for identification of diagnostics biomarkers of tumor onset, progression, triage to treatment, and treatment response. Keywords : extracellular vesicles, tumour derived EV, TEXO‐NET, EV isolation, cancer biomarker

Using

Brian S. Dobosh 1 ; Alexander Rojas 2 ; Rabindra Tirouvanziam 2 1 Emory University, Atlanta, USA; 2 Emory University, USA Introduction : Polymorphonuclear neutrophils (PMNs) are the most abundant white blood cell in circulation and respond to stress within minutes. We identified a key transition state (A0) during PMN activation that determines mobilization of the antimicrobial primary granules. The first fate of PMNs (A1), defined as HDAC11low (protein) and MALAT1low (lncRNA), has intracellular granules and are adept at clearing bacterial pathogens P. aeruginosa and S. aureus. The second fate (A2), HDAC11hi and MALAT1hi, puts granules to the surface of the cell and promotes clearance of viral pathogens such as influenza A, RSV, and SARS‐CoV‐2. Since PMNs are highly pinocytic, we hypothesized that engineered EVs could control the transition of A0 PMNs to the A1 or A2 fates and thus promote clearance of bacterial or viral pathogens, respectively. Methods : We used the designer EV toolkit (DEVkit), a modular cloning toolkit developed in our lab, to generate all EVs, which were purified by differential ultracentrifugation. Structure, size, concentration, contents and contamination were evaluated by electron microscopy, Nanosight NS300, western blot, luminescence and qRT‐PCR. Results : We validated A0 PMNs could use EVs with EGFP and nluc protein and mRNA. PMNs treated with HDAC11 or MALAT1 siRNA EVs differentiated into A1 PMNs and doubled bacterial pathogen clearance compared to no EV and scramble siRNA EV control conditions. HDAC11 or MALAT1 EVs caused conversion of A0 PMNs to the A2 fate, which then completely removed all viruses within 24 hours compared to the control groups that could only clear 30–60% of IAV or SARS‐CoV‐2 in 24 hours. Summary/Conclusion : Taken together, engineered EVs can control the fate and function of PMNs to clear bacterial and viral pathogens relevant in many inflammatory diseases Furthermore, this is another proof of concept of the utility of the DEVkit. We are very excited to unveil it via Addgene for broad use by the EV community to enable standardization and customization of a portion of the engineering process. Funding : Emory I3 Program, R01HL159058, CFF TIROUV19A0. Keywords : designer EVs, neutrophils, immunotherapy

Wound

Anu Sharma 1 ; Adam Anthony 2 ; Sashwati roy 3 ; David Clemmer 4 ; Chandan Sen 3 ; Subhadip Ghatak 3 1 Indiana University, Apt 42, USA; 2 Indiana University, USA; 3 Indiana University, Indianapolis, USA; 4 Indiana University, Bloomington, USA Introduction : Exosomes represent a major component of paracrine regulation in tissue repair. We tested the hypothesis that functional wound closure requires successful crosstalk between keratinocytes and wound macrophages (wmf) in vivo. Methods : Murine wmf‐derived exosomes were genetically labeled with RFP reporter (Exowmf) using tissue nanotransfection. Wound‐edge (WE) Exowmf were characterized per MISEV 2018 guidelines and reported in EV‐track (EV 220292; EV‐metric score 100%). Protein cargo in Exowmf was detected, validated, and quantified using LC‐MS/MS, dSTORM imaging, and flow cytometry. Functional wound closure was evaluated using analytical histology and Transepidermal Water Loss. Results : The Exowmf was localized at the leading‐edge keratinocytes post‐injury. LC‐MS/MS and dSTORM imaging identified presence of outer mitochondrial membrane (OMM) protein TOMM70 in Exowmf. At d5 post‐wounding, 92.65 ± 1.34% Exowmf were TOMM70+ (p < 0.001; n = 6). Such TOMM70‐enriched Exowmf increased keratinocyte migration by 72.05 ± 8.50%. Wound hypoxia significantly degraded TOMM70 in WE keratinocytes. Such hypoxic loss of keratinocyte TOMM70 inhibited cytosolic PTEN‐induced kinase 1 (PINK1) translocation to the inner mitochondrial membrane causing PINK1 oligomerization on OMM followed by mitophagy. Hypoxic loss of TOMM70 in keratinocytes was compensated by uptake of TOMM70+ Exowmf Downstream PINK1 oligomerization and mitophagy were thus spared in keratinocytes resulting in improved ATP pool necessary to support the metabolic cost of cell migration To block Exowmf uptake by WE keratinocytes, “eat me not” Exowmf were generated using a LysM promoter‐driven tetraspanins (CD9/CD63/CD81) plasmid connected via IRES element with “eat me not”‐CD47 sequence with in‐frame GFP reporter. Selective interruption of exosomal crosstalk between wmf and WE keratinocytes significantly delayed re‐epithelialization and impaired functional wound closure. Summary/Conclusion : This work lays the foundation for a novel paradigm that addresses the molecular bases of cell‐cell crosstalk in the wound microenvironment that has a direct bearing on wound tissue bioenergetics, a critical factor necessary to pay for the metabolic cost of wound closure. Funding : NIH R56DK129592 to SG. Keywords : macrophage‐derived exosomes, functional wound closure, mitochondria, inflammation

Atp1A3

Tsuneya Ikezu 1 ; Yang You 1 ; Zhengrong Zhang 1 ; Nadia Sultana 2 ; Maria Ericsson 3 ; Yuka Martens 4 ; Min Sun 5 ; Takahisa Kanekiyo 4 ; Seiko Ikezu 1 ; Scott Shaffer 6 1 Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL 32224, USA, Jacksonville, USA; 2 University of Massachusetts Chan Medical School, USA; 3 Harvard Medical School, Boston, USA; 4 Mayo Clinic Florida, USA; 5 Nanoview Biosciences, USA; 6 University of Massachusetts Chan Medical School, Worcester, USA Introduction : Background: Neuron‐derived extracellular vesicles (NDEVs) provide a reliable source for understanding the state of brain and discovering biomarkers of neurological diseases. Over the past several years, there has been a keen interest in capturing neuron‐specific EVs from patient‐derived biopsies or biofluids and characterizing their contents as a pathological reflection of the central nervous system (CNS). A reliable and reproducible NDEV markers are needed to isolate and characterize bona fide NDEVs from human samples. Our recent study identified ATPase Na+/K+ Transporting Subunit Alpha 3 (ATP1A3) as one of the abundant neuron‐specific EV markers. ATP1A3 is mostly enriched in brains, with some specific expression in heart muscles. Here we conduct systematic neuronal EV analyses to evaluate whether ATP1A3 is specific to NDEVs and a reliable marker for NDEV isolation from biofluids for disease monitoring. Methods : Methods: Immunoelectron microscopy was used to detect ATP1A3, L1 cell adhesion molecule (L1CAM) and neural cell adhesion molecule 1 (NCAM1) in EVs isolated from iPSC‐derived excitatory neurons, brain tissue, plasma and cerebrospinal fluid (CSF). Neuronal EV was enriched from isolated brain EVs using immunoaffinity isolation with either anti‐ATP1A3, L1CAM or NCAM1 antibodies, and the enrichment of neuronal population was evaluated by quantitative mass‐spectrometry and validated by immunoblotting. The enrichment of neuronal markers were compared among EVs isolated from CSF and plasma samples using ExoView and Nanoimager. We also tested if Alzheimer's disease‐related amyloid‐b peptide (Ab) are enriched in ATP1A3+ plasma EVs using Nanoimager and compared with the values of plasma AD biomarkers as determined by SIMOA. Results : Results: ATP1A3 is highly enriched in NDEVs isolated from induced human neurons, brain, cerebrospinal fluid, and plasma samples compared to NCAM1 or L1CAM as determined by quantitative proteomics, biochemistry, ExoView and NanoImager. Aβ+ population in ATP1A3+ EVs from plasma can distinguish Alzheimer's disease from mild cognitive impairment and control cases as determined by Nanoimager whereas the conventional quantification of Aβ in plasma by SIMOA show modest difference among groups. Summary/Conclusion : Conclusion: Our data demonstrate that ATP1A3 is a promising marker to isolate human NDEV from biofluids for diagnostic research in neurodegenerative diseases. Funding : Florida Department of Health Ed and Ethel Moore Alzheimer's Disease Research Program‐22A04 (YY), Alzheimer's Association AARF‐22‐918114 (YY), Cure Alzheimer's Fund (TI), NIH RF1 AG054199 (TI), R01AG054672, R01 AG066429 (TI), R01 AG067763 (TI), R01 AG072719 (TI). Keywords : neuron, brain, biomarker, induced pluripotent stem cells, cerebrospinal fluid, plasma, ExoView, nanoimager, proteomics

Breast

Yuima Sakamoto 1 ; Yusuke Yoshioka 2 ; Takahiro Ochiya 3 1 Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Japan; 2 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku‐Ku, Japan; 3 Department of Molecular and Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shimjuku‐ku, Japan Introduction : Cancer cells utilize extracellular vesicles (EVs) to promote cancer pathogenesis, including proliferation and metastasis. Our previous study showed that EVs from brain metastatic breast cancer cells break down the blood‐brain barrier (BBB) through the change in actin dynamics and promote brain metastasis in vivo (Nature Communications, 2015). These results indicate a novel mechanism of brain metastasis mediated by EVs that triggers the destruction of BBB. Therefore, we aimed to prevent brain metastasis of breast cancer by a new approach to inhibit EVs‐dependent metastatic pathways, especially focusing on interfering adhesion of cancer EVs to the recipient cells. Methods : For preparing EVs, the cultured medium was filtered with a 0.22‐μm and ultracentrifuged at 110,000g for 70 min at 4°C. Then the pellets were resuspended in PBS. BMD2a cells established from MDA‐MB‐231 cells were used as brain metastatic breast cancer cells, and mice were immunized with these cells‐derived EVs (BMD2a‐EVs). We obtained some monoclonal antibodies that specifically recognize BMD2a‐EVs and performed a screening for the antibodies to inhibit EV uptake. Inhibition of EV uptake by antibodies was assessed by the amount of adhesion of PKH‐labeled BMD2a‐EVs to the brain vascular endothelial cells. Results : As the screening result, we obtained two different EVs adhesion‐inhibitory antibodies. These antibodies suppressed the BMD2a‐EVs‐induced BBB destruction in an in vitro BBB model system. To further evaluate the effect on the prevention of BBB disruption in vivo, these antibodies and BMD2a‐EVs were co‐administered intravenously into Scid mice. The results showed that these antibodies also inhibited BMD2a‐EVs‐induced BBB destruction in vivo. Summary/Conclusion : This study provides a novel therapeutic strategy for preventing brain metastasis of breast cancer and other brain metastatic tumors, including lung cancer, by inhibiting adhesion between EVs and recipient cells using antibodies.

Cancer

Manideep Pachva 1 ; Peter Ruzanov 2 ; Valentina Evdokimova 2 ; Melanie Rouleau 3 ; Laszlo Radvanyi 2 ; Poul Sorensen 4 1 BC Cancer Research Center, Vancouver, Canada; 2 Ontario Institute for Cancer Research, Toronto, Canada; 3 British Columbia Cancer Research Center, Vancouver, Canada; 4 British Columbia Cancer Research Center, The University of British Columbia, Vancouver, Canada Introduction : Ewing sarcoma (EwS) is a highly aggressive cancer and the second most common malignant bone tumor in children and young adults with high propensity for metastasis. Patients with metastasis have a poor long‐term outcome. Novel targeted therapeutic strategies that are more efficacious and less toxic are therefore desperately needed. Intercellular communication within the tumor microenvironment (TME) is emerging as a crucial mechanism for cancer cells to establish immunosuppressive and cancer‐permissive environment. Extracellular vesicles (EVs) offer a candidate mechanism as they are actively released by tumor cells and enriched with proteins and RNAs to communicate with other cells in the TME. Methods : For EV purification, Conditioned medium (CM) was subjected to sequential centrifugation at 2000 g for 10 min and 10,000 g for 20 min. CM was then concentrated using the Tangential Flow Filtration Easy columns, passed through 0.22 μm filter, diluted with equal volumes of PBS and subjected to ultracentrifugation (UC) at 100,000 g for 4hrs. EV pellets were then re‐suspended in 3 ml of PBS and pelleted again by UC. Purified EVs were dissolved in 500 μl PBS. EVs were quantified using Nanoparticle Tracking Analysis. Purified EVs were tested for presence of exosome markers (CD63, CD9, and CD81) using western blot and using R‐PLEX Human CD63/CD81/CD9 (EV) Antibody Set. Results : In our recent study involving whole transcriptome RNA sequencing, it was found that EVs secreted by EwS cell lines as well as those detected in the plasma of EwS patients are selectively enriched with Adenosine to Inosine (A to I) edited RNAs. A high proportion of these A‐to‐I edited transcripts are derived from diverse long and short interspersed retrotransposon elements (LINEs and SINEs), human endogenous retroviral elements (HERVs) and pericentromeric genomic regions, where their abundance in plasma was associated with metastatic progression. A to I conversion is catalyzed by the ADAR1 enzyme. We therefore performed ADAR1 knock‐down (KD) in EwS cells, which accumulated cellular levels of these repeat RNAs in donor cells and limited their packaging into EVs. Moreover, we observed a significant reduction of pro inflammatory response in target cells treated with EwS ADAR1 KD EVs compared to the wild‐type cells derived EVs. Notably, ADAR1 KD in EwS cells decreased the potential of their EV mediated monocytes differentiation and T‐cell activation and priming. Summary/Conclusion : These results suggest that EwS cells secrete EVs enriched with A to I edited RNAs derived from various repeat elements to target the non‐tumor host cells, including stromal fibroblasts, monocytes, T cells for dampening and escaping the immune response against them. Keywords : ewing sarcoma, extracellular vesicles, repeat RNAs, tumor microenvironment, reprogramming

Design

Huarui Zhang 1 ; Yi Zuo 2 ; Zhanghao Li 3 ; Nanxi Li 4 ; Chuanxin Zhong 3 ; Baoting Zhang 5 ; Aiping Lyu 6 ; Jun Lu 2 ; Ge Zhang 3 ; Jin Liu 6 1 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong (CUHK), Hong Kong SAR, China, Hong Kong, Hong Kong; 2 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China, China (People's Republic); 3 Law Sau Fai Institute for Advancing Translational Medicine in Bone and Joint Diseases (TMBJ), School of Chinese Medicine, Hong Kong Baptist University (HKBU), Hong Kong SAR, China, China (People's Republic); 4 Law Sau Fai Institute for Advancing Translational Medicine in Bone and Joint Diseases (TMBJ), School of Chinese Medicine, Hong Kong Baptist University (HKBU), Hong Kong SAR, China, Hong Kong SAR, China (People's Republic); 5 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong (CUHK), Hong Kong SAR, China, China (People's Republic); 6 Law Sau Fai Institute for Advancing Translational Medicine in Bone and Joint Diseases (TMBJ), School of Chinese Medicine, Hong Kong Baptist University (HKBU), Hong Kong SAR, China/Guangdong‐Hong Kong‐Macau Joint Lab on Chinese Medicine and Immune Disease Research, China (People's Republic) Introduction : Exosomes play important roles in the tumor process. The tumor‐derived or tumor macrophage‐derived exosomes could promote tumor growth, immunosuppression and cancer cell dissemination. Therefore, inhibiting exosome release has therapeutic potential in cancer. However, most existing exosome inhibitors could either interrupt exosome biogenesis or block exosome release with high cytotoxicity, which may lead to undesired adverse effects on normal cells and thus limit their translation to anti‐cancer therapy. In this study, we redesigned a cytotoxic RAB27A inhibitor through chemical modification for developing novel exosome inhibitors with low cytotoxicity. Methods : Candidate compounds were synthesized by aldol reaction. The cytotoxicity of these compounds was evaluated by MTT assay on RAW264.7 cells. The exosome inhibitory potency of these compounds was evaluated on RAW264.7 cells, BMM cells, MDA‐MB‐231 cells and 4T1 cells through exosome isolation (ExoQuick‐TC, SBI) and exosome quantitation (FluoroCet Exosome Quantitation Kit, SBI). Results : The nitro group of the RAB27A inhibitor was replaced by other functional groups to reduce cytotoxicity and their chemical structures were confirmed by LC‐MS and NMR. The results showed that six candidates exhibit low cytotoxicity, among which LJ271, LJ272 and LJ245 showed strong exosome inhibitory potency on RAW264.7 cells, BMM cells, MDA‐MB‐231 cells and 4T1 cells. Summary/Conclusion : The results indicated that LJ271, LJ272 and LJ245 exhibited low cytotoxicity and exosome inhibitory potency on macrophage and breast cancer cells. Based on this, the drug candidates will be studied whether they could inhibit the macrophage‐derived or tumor‐derived exosome‐mediated modulation of the tumor microenvironment. Funding : Theme‐based Research Scheme (T12‐201/20‐R); General Research Fund (12136616 and 12103519) of the Research Grants Council of Hong Kong SAR; National Natural Science Foundation of China (No.82273812); The Sichuan Outstanding Youth Fund Project (No.23NSFJQ0099), The 2020 Guangdong Provincial Science and Technology Innovation Strategy Special Fund (Guangdong‐Hong Kong‐Macau Joint Lab, No: 2020B1212030006); Interdisciplinary Research Clusters Matching Scheme of Hong Kong Baptist University (RC‐IRCs/17‐18/02). Keywords : exosome inhibitors, cancers, drug development

Fungal

Tamires Bitencourt 1 ; Otavio Hatanaka 1 ; Fausto Almeida 2 1 Department of Biochemistry and Immunology, Ribeirao Preto Medical School, University of São Paulo, Ribeirao Preto, São Paulo, Brazil., Brazil; 2 Department of Biochemistry and Immunology, Ribeirao Preto Medical School, University of São Paulo, Ribeirao Preto, São Paulo, Brazil., Ribeirao Preto, Brazil Introduction : Fungal infections are responsible for over 2 million deaths per year. The diseases caused by Aspergillus spp., Candida spp., and the agents of mycoses such as Paracoccidioides species are among the deadliest mycoses. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungus‐host interaction process. We hypothesized that fungal EVs work as an additional element in the communication routes regulating fungal responses in intraspecies interaction systems. Methods : The aim of this study was to address the gene regulation profiles prompted by fungal EVs in intraspecies recipient cells. We sought to analyze the fungal cellular communication mediated by EVs using the fungal pathogens P. brasiliensis, A. fumigatus, and C. albicans using multiple approaches. The EVs isolation were obtained through ultracentrifugation.The size distribution and quantification of EVs isolated were obtained by nanoparticle‐tracking analysis. Results : Our data demonstrated the intraspecies uptake of EVs in pathogenic fungi, such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in the yeast‐to‐hypha transition, while in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and triggering phenotypic changes. The EVs treatment induced cellular proliferation and boosted the yeast to hyphal transition in C. albicans, while they enhanced stress responsiveness in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intraspecies communication. Summary/Conclusion : Our data demonstrate that fungal EVs mediate cellular communication by regulating the expression of target genes and by controlling cellular proliferation. Funding : FAPESP, CAPES, CNPq and FAEPA. Keywords : aspergillus fumigatus, candida albicans, paracoccidioides brasiliensis, EVs

Global

Britta A. Bettin 1 ; Edwin van der Pol 2 ; Rienk Nieuwland 3 1 Department of Clinical Chemistry, Amsterdam UMC location University of Amsterdam, Amsterdam, The Netherlands, Amsterdam, Netherlands; 2 (2) Laboratory of Experimental Clinical Chemistry and Vesicle Observation Center, Amsterdam; The Netherlands; (8) Biomedical Engineering and Physics, Amsterdam UMC, Amsterdam, The Netherlands, Amsterdam, Netherlands; 3 (2) Laboratory of Experimental Clinical Chemistry and Vesicle Observation Center, Amsterdam; The Netherlands;, Amsterdam, Netherlands Introduction : Extracellular vesicles (EVs) in body fluids are potential disease biomarkers. To measure EV concentrations, most laboratories use flow cytometers (FCMs), but concentrations are incomparable between FCMs. To improve comparability, the METVES II consortium developed reference materials and methods to calibrate FCMs. This developed infrastructure was tested in a global inter‐laboratory comparison study including 39 FCMs from 24 different laboratories. Methods : Concentrations of erythrocyte‐derived (CD235a‐PE) and platelet‐derived (CD61 ‐APC) EVs were measured in stabilized and pre‐labeled human plasma EV test samples (PEVTES). Flow rate was calibrated using solid silica beads, light scattering was calibrated using Rosetta calibration beads, and fluorescence was calibrated using MESF beads. EV concentrations were compared between FCMs within an EV size range of 215 – 1,000 nm and a fluorescence intensity >220 APC MESF, and >50 PE MESF. Results : Preliminary results from 9 FCMs show that FCM calibration is successful. For the platelet EV concentration, the coefficient of variation decreased from 72% without calibration to 40% after calibration. Summary/Conclusion : This is the first inter‐laboratory comparison study demonstrating that full flow cytometer calibration improves the comparability of EV concentration measurements between FCMs, thereby paving the road to clinically relevant multi‐center biomarker studies on EVs. Funding : This project has received funding from the EMPIR programme co‐financed by the Participating States and from the European Union's Horizon 2020 research and innovation program. Keywords : calibration, extracellular vesicles, flow cytometry, inter‐laboratory comparison study, standardization

Highly

Polina V. Shnaider 1 ; Andrey Pichugin 2 ; Ekaterina Grafskaia 1 ; Olga I. Aleshikova 3 ; Lev A. Ashrafyan 4 ; Victoria O. Shender 1 ; Elena Khomyakova 5 1 Lopukhin Federal Research and Clinical Center of Physical‐Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia; 2 Fabmid, EVRY‐COURCOURONNES, France; 3 National Medical Scientific Centre of Obstetrics, Gynaecology and Perinatal Medicine named after V.I. Kulakov, Moscow, Russia; 4 National Medical Scientific Centre of Obstetrics, Gynaecology and Perinatal Medicine named after V.I. Kulakov, Russia; 5 Exosome Analytics, EVRY‐COURCOURONNES, France Introduction : A liquid biopsy is a promising tool for non‐invasive cancer diagnosis, tumor profiling, evaluation of therapy response and post‐treatment follow‐up. Extracellular vesicles are promising for liquid biopsy tests since they are enriched with biomarkers reflecting the composition of the cell of origin. Majority of EV based liquid biopsy tests are based on analysis of RNA and DNA markers, however the gold standard of tumor profiling is analysis of protein markers on tumor cells. Detection of commonly used histological markers on EVs could provide more complete information about tumor phenotype and find the shorter root to the clinics. However, common ELISA which allows high throughput screening of protein markers is not enough sensitive to detect pathological markers on EVs isolated from clinical samples. We have developed innovative highly sensitive 3D ELISA techniques allowing profiling of low represented protein markers on EVs. The technique was validated by analysis of putative cancer stem cell markers CD166, CD117 and CD44 and EpCAM and onco‐marker CA125 on EVs isolated from cell cultures and clinical samples. Methods : EVs were isolated from HEK293t and SK‐OV‐3 cell culture supernatants and urine of ovarian cancer patients and cancer‐free individuals by differential centrifugation (500 g for 10 min, 3500 g for 25 min in an A‐4‐81 rotor at 20°C, EV size cutoff is 620 nm) followed by 100 kDa ultrafiltration. EV concentration was measured with NTA. Results : The sensitivity of the 3D ELISA technique was estimated for the mesenchymal markers CD9, CD81, CD166, CD44, CD117 and EpCam and the onco‐marker CA125. The detection limit (LOD) for CD9+/CD81+ EVs is 2 orders of magnitude higher than LOD of commercially available EV ELISA kits. The high sensitivity of the 3D ELISA technique allowed the detection of tumor markers on EVs isolated from the urine of patients with ovarian cancer. Summary/Conclusion : The highly sensitive 3D ELISA platform allows the profiling of histological and onco‐markers on EVs isolated from cell cultures and body fluids. Keywords : histological markers, highly sensitive protein detection

Impact

Juntaro Matsuzaki 1 ; Chihiro Oikawa 1 ; Takeshi Katsuda 2 ; Tomoko Yamaguchi 1 ; Rina Shibagaki 1 ; Kaito Yoshimizu 1 ; Takahiro Ochiya 3 ; Yoshimasa Saito 1 1 Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Japan; 2 Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA; 3 Department of Molecular and Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shimjuku‐ku, Japan Introduction : Circulating miRNAs are one of the promising biomarkers for cancer detection in the early stage. However, the alterations of extracellular vesicle miRNA (EV‐miRNA) profiles released from epithelial cells in the carcinogenic process have not yet been fully understood. This study aimed to compare EV‐miRNA profiles among pancreatic epithelial cells with a variety of drive gene mutations. Methods : We established a culture method of mouse pancreatic ductal progenitor cells (mPP) by modifying mature hepatocyte's chemical direct reprogramming technology (Cell Stem Cell 20:41, 2017). We introduced each driver gene mutation such as KRAS(G12D) and the loss of CDKN2A, TP53, SMAD4 into mPP by CRISPR‐Cas9. Small EVs (sEVs) released from each cell line were collected with ultracentrifugation. The quality of sEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting of EV‐positive and ‐negative marker proteins (CD9 and CANX). Results : Acquiring KRAS and TP53 mutations significantly enhanced the cell proliferation. sEV particle counts released from mPP lines were increased by the introduction of KRAS(G12D) and the deletion of TP53. miRNA‐seq analysis revealed that the levels of miR‐155‐5p, known as circulating biomarkers of pancreatic ductal carcinoma, in sEV were increased by KRAS(G12D). Summary/Conclusion : Extracellular miR‐155‐5p levels could be a biomarker of KRAS mutation in pancreatic ductal cells. In vitro culture techniques of organ progenitor cells and CRISPR‐Cas9 can be useful for elucidating the molecular mechanisms of the alteration of circulating miRNA profiles in cancer patients.

Mining

Tyler T. Cooper 1 ; Jaihui Liu 2 ; Yangxin Fu 2 ; Helen Steed 2 ; Gilles Lajoie 3 ; Lynne Postovit 4 1 Queens University, London, Canada; 2 University of Alberta, Canada; 3 Western University, Canada; 4 Queen's University, Canada Introduction : The proteome of extracellular vesicles (EVs) is comprised of a diverse range of protein ligands/receptors, enzymes, and cellular machinery that is often characterized by the identification of tryptic peptides during tandem mass spectrometry (MS/MS). As a result, proteomic depth (# of proteins and sequence coverage) is limited to peptide products from proteolysis at the C‐terminal of lysine or arginine. Thermolysin cleaves at the N‐terminal of aliphatic amino acids (FMVAIL) and our in silico analyses predict the parallel detection of these peptides will increase proteomic depth of EVs during exploratory MS/MS. Specifically, we hypothesized that thermolysin would provide complementary sequence coverage to TrypLysC, in return increasing proteomic depth. Methods : EVs were purified using ultracentrifugation from conditioned media of primary ascites cell lines or ascites fluid from donors with high‐grade serous carcinoma. EV number and size distribution were characterized by nanoscale flow cytometry using Apogee A60. EVs were lyophilized and 25μg of protein was digested either with TrypLysC at 37⁰C for 18hrs or Thermolysin at 75⁰C for 1hr followed by desalting using C18 stagetips. Offline high‐pH reverse phase or strong cation exchange in stagetips was used to decomplexify the peptide pool. 500ng of each peptide fraction was analyzed across 75 or 260min gradients using reverse‐phase ultraperformance liquid chromatography coupled to MS/MS on a QE Plus operating in data‐dependent acquisition. Peptide/protein identifications were compared between MaxQuant, FragPipe, and PEAKS de novo sequencing software. Results : Thermolysin proteolysis identified >100 additional proteins compared to TrypLysC alone. PEAKS] provided largest number of unique protein IDs for both TrypLysC and Thermolysin. Notably, Thermolysin provided complementary peptide identification, in return, a >2‐fold increase in mean sequence coverage was obtained. Limitations of Thermolysin proteolysis included a need for missed cleavages, an increased number of peptides per protein, and a narrow distribution of peptides based on length, charge, or hydrophobicity. As a result, we are currently investigating the use of high‐field asymmetric waveform ion mobility spectrometry to provide additional peptide fractionation and removal of low‐charge peptide species during MS/MS. Summary/Conclusion : Our data indicate that complementary proteolysis of the EV proteome with Thermolysin can be used as a tool to increase the proteomic depth obtained during MS/MS analyses. Funding : Canadian Institute of Health Research, NSERC, Canadian Cancer Society. Keywords : ovarian cancer, proteomics, mass spectrometry, sample preperation

Paving

Nikki Salmond 1 ; Wing Sum (Kelly) Tam 2 ; Karan Khanna 2 ; Renata Moravcova 2 ; Jason Rogalski 2 ; Karla Williams 2 1 University of British Columbia, Vancouver, Canada; 2 University of British Columbia, Canada Introduction : Breast cancer (BCa) screening uses mammography and ultrasound imaging, and suspicious lesions are investigated using an invasive needle biopsy. Mammograms and ultrasounds are unable to distinguish between benign and cancerous tumors. We need a non‐invasive blood test – a liquid biopsy – which can accurately identify BCa at its earliest stage‐ Stage I. Circulating tumor fragments (extracellular vesicles ‐ EVs) are a promising non‐invasive platform for the development of a novel BCa screening test. We isolated EVs from 86 BCa, Stage I, patients, 19 benign and 20 healthy individuals to identify new protein biomarkers unique to BCa. Methods : Different EV isolation methods were tested to determine the best platform for use with mass spectrometry: EQUltra, SEC, PEG‐SEC, EQUltra‐SEC, and precipitation. EVs were characterized by Western blot, protein concentration, electron microscopy and mass spectrometry. EVs were isolated by SEC from 86 Stage I BCa patients, 19 benign and 20 healthy (age‐matched controls); from 20 μg of processed EVs, 500 ng was spiked with 200 fm yeast glutathione reductase (internal standard) for unlabeled mass spectrometry. Results : SEC was used for EV isolation with downstream mass spectrometry analysis due to efficiency, purity, and quantity. Mass spectrometry identified 373 unique proteins. A Volcano plot identified 75 proteins with significantly higher expression in BCa relative to healthy/benign. Principal Component Analysis (PCA) and Multiple Logistic Regression analysis identified 26 proteins whose expression could correctly identify 96.5% of cancer patients and 89.7% of healthy/benign individuals. Summary/Conclusion : Our study has identified protein biomarkers found in the blood of BCa patients with potential utility to generate ‘fingerprints’ of healthy, benign, and early Stage I BCa. The detection of BCa cases at Stage I, would allow for rapid, curative, intervention reducing the mortality associated with a BCa diagnosis. Funding : Michael Smith Foundation for Health Research. Keywords : breast cancer, stage I, biomarker, diagnosis, circulating extracellular vesicles, liquid biopsy, mass spectrometry.

Plasma

Ursula S. Sandau 1 ; Erika Duggan 2 ; Sierra J. Smith 1 ; Marilyn Huckans 1 ; John P. Nolan 2 ; Jennifer M. Loftis 1 ; Julie A. Saugstad 3 1 Oregon Health & Science University, Portland, USA; 2 Scintillon Institute, San Diego, USA; 3 Department of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, PORTLAND, USA Introduction : The long‐term health effects of methamphetamine (MA) use include cognitive impairments, anxiety, and depression, which can persist during recovery and are associated with poor treatment outcomes. Thus, biosignatures based on objectively quantifiable blood biomarkers that relate to clinical features of MA use disorders could be used clinically to monitor recovery from addiction. Methods : Multiplexed bead‐based assays (Miltenyi) were performed on plasma extracellular vesicles (EVs) from humans with active MA use (MA‐ACT, n = 10) and controls (CTL, n = 10). EV subtypes were prioritized based on an F‐statistic ranking, large effect size, and area under the curve (AUC > 0.75) for classifying MA‐ACT vs. CTL. The normalized median fluorescence intensity (nMFI) values for prioritized EV subtypes were correlated to measures of i) MA use characteristics, ii) neuropsychiatric function, and iii) markers of inflammation and CNS injury. Next, the expression levels of 7 plasma EV miRNAs that are relevant to MA‐ACT were correlated to the aforementioned measures. Data analyzed by Pearson's correlations with false discovery rate corrections. Results : Plasma EVs positive for inflammatory markers significantly correlated to measures of craving in MA‐ACT as well as anxiety and memory impairments in MA‐ACT and CTL participants. Five EV miRNAs also significantly correlated to clinical features of MA use disorders including frequency of use, lifetime exposure, anxiety, memory, and pain. Plasma EV expression levels for 3 of these miRNAs also significantly correlated to ICAM‐1, S100β, and/or neurofilament in MA‐ACT. Relevant to these findings the predicted targets of the five miRNAs identified pathways associated with neuroinflammation, neuroplasticity, and neurodegeneration, which contribute to behavioral alterations that occur with MA dependency. Summary/Conclusion : These studies demonstrate the potential utility of plasma EVs to serve as metrics of recovery by relating EV markers and their miRNA cargo to clinical features of MA use disorders. Keywords : addiction, miRNAs, neuropsychiatric function, methamphetamine use disorders, inflammation

Retina

Seon Ok Kim ; Ji Eun Lee SungKyunKwan University, Republic of Korea Introduction : The wide heterogeneity of genetic causes reside in retinitis pigmentosa(RP). Most common form of disease is eys shut homolog(EYS) autosomal recessive mutation. zebrafish can be used as an ideal model to study eye disease. The zebrafish retina resembles human retina demonstrating similar arrangement of cells. Zebrafish has similar ocular development in humans and vertebrates. EYS mutation in zebrafish can address RP more critically, needs Crucial egineering. Here we present knock‐out system with exosomes. To avoide immune responses as Large size vector of CRISPR‐Cas9 in vivo and establish tissue specific knock out, we electroporated CRISPR‐Cas9 system in exosome. Exosomes extracted from tissue‐specific have different surface protein homologous to origin cell, which makes possible cell‐to‐cell communication, and does not induce immune responses. We established delivering Cas9 RNP system encapsulating into exosome for retina tissue specific knockout model Methods : For retinal pigment epithelial cell‐secreted exosome isolation, exosome‐depleted FBS was prepared by ultracentrifugation at 100,000g, 4°C for 20 hours. retinal pigment epithelial cells(RPE) were cultured in DMEM containing exosome‐depleted FBS. Then, exosomes were isolated from the culture media using differential centrifugation. The exosomal protein was quantified by BCA assay and the presence of exosome was determined by western blot analysis. Cas9 proteins were and dgRNA were thoroughly combined to create Cas9 RNP complexes. Then, RNP complexes were loaded onto exosome using electroporation. Exosome RNP complexes were added into RPE culture medium to investigate the delivery of exosomal RNP. After incubation, the effectiveness of gene targeting was confirmed by western blot. For tissue targeting, exosome RNP complexes were injected into retina tissue were collected for further gene targeting analysis. Results : We characterized the RPE‐derived exosomes expressing exosome specific markers but not the Golgi apparatus‐associated protein. The delivery of RNP targeted retina tissue resulted in tissue specific null phenotype. Targeting EYS gene with RNP encapsulated to exosome identified as reasonable model with higher effiency.Additionally EYS knock out caused a abnormal stricture in retina Summary/Conclusion : In this study, we established Retinitis Pigmentosa disease models with RNP‐based CRISPR‐Cas9. RNP encalsulated in exosomes secreted from RPE and transported to retina successfully Funding : This work has been done by Molecular & Medical Genomics lab of Ji Eun Lee in Sungkyunkwan University and supported by the National Research Foundation of Korea government's MSIP (2021R1A4A2001389 and 2021R1A2C3004572 to J.E.L).

Runner

Yogish Somayaji 1 ; Leelavathi N. Madhu 2 ; Maheedhar Kodali 3 ; Sahithi Attaluri 4 ; Shama Rao 5 ; B Shuai 6 ; Ashok K. Shetty 7 1 Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, Texas, USA, USA; 2 Institute for Regenerative Medicine, Department of Molecular and Cellular Medicine, Texas A&M University College of Medicine, College Station, Texas, USA., College Station, USA; 3 Texas A&M University, College Station, USA; 4 Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, Texas, USA, College Station, USA; 5 Texas A&M University, USA; 6 Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, USA; 7 Institute for Regenerative Medicine, Dept of Cell Biology and Genetics, Texas A&M Univ School of Medicine, College Station, USA Introduction : Gulf war illness (GWI), typified by persistent cognitive dysfunction, is linked to unrelenting neuroinflammation. Physical exercise is known to produce antiinflammatory cellular changes, which are potentially also reflected in the extracellular vesicles (EVs) released by multiple cell types into the circulating blood. This study tested the therapeutic effects of EVs isolated from the plasma of sedentary rats (naive EVs or nEVs) and rats that performed voluntary running (runner EVs or rEVs) in a rat model of GWI. Methods : nEVs were isolated from the plasma of 3‐month‐old sedentary rats, whereas rEVs were isolated from the plasma of age‐matched rats housed in cages fitted with voluntary running wheels for 28 days. Both nEVs and rEVs were isolated from the plasma through the size‐exclusion chromatographic method. The GWI rats were raised through daily exposure of two‐month‐old rats to GWI‐related chemicals pyridostigmine bromide, DEET, and permethrin and 15 min restraint stress for 28 days. Six months later, GWI rats received weekly intranasal nEVs or rEVs (1000 × 109 EVs/week) for 3 weeks or no treatment. The rats were subjected to behavioral studies to examine cognitive function, after which they were euthanized (∼3 months after the first dose of EV treatment). Brain tissues were processed for analyzing neuroinflammation. Results : rEV treatment improved spatial recognition memory and pattern separation in GWI rats, but nEV treatment showed no such improvements. Analysis of hippocampal tissues revealed decreased percentages of microglia displaying CD68 and diminished levels of markers of NOD‐, LRR‐ and pyrin domain‐containing protein 3 (NLRP3) inflammasome activation in GWI rats receiving either nEVs or rEVs. However, the suppression of inflammasome activation was more significant in GWI rats receiving rEVs. Summary/Conclusion : Intranasal administration of rEVs from the plasma of runners can improve cognitive function in GWI. Funding : Supported by grants from the NINDS and NIA (1R01NS106907 and 1RF1AG074256‐01A1 to A.K.S.) and Texas A&M University School of Medicine. Keywords : plasma ‐derived extracellular vesicles, gulf war illness, neuroinflammation, physical exercise

S100A9

Migmar Tsamchoe 1 ; Anthoula Lazaris 2 ; Diane Kim 3 ; lucyna Krzywon 4 ; Peter Metrakos 2 ; Janusz Rak 5 ; Kurt Dejgaard 6 ; Zu hua Gao 7 ; stephanie Petrillo 8 ; Jessica Bloom 7 1 McGill University, Montreal, Canada; 2 Research Institute of the McGill University Health Center, Montreal, Canada; 3 Research Institute of McGill University Health Centre, USA; 4 Research Institute of McGill University Health Centre, Montreal, USA; 5 Research Institute of McGill University Health Center, Montreal, QC H4A 3J1, Canada. Department of Pediatrics, McGill University, Montreal, QC H4A 3J1, Canada., Montreal, USA; 6 McGill University, Canada; 7 Research Institute of McGill University Health Centre, Canada; 8 Research Institute of McGill University Health Centre, Montreal, Canada Introduction : Colorectal cancer is the 3rd most common type of cancer and second leading cause of cancer death. The primary cause of death in colorectal cancer is due to metastasis with liver being the dominant metastatic site for colorectal cancer (CRC). Liver metastases present as two major histopathological growth patterns (HGP) 1. Desmoplastic Histological growth pattern (D‐HGP): Cancer cells are separated from hepatocytes by a desmoplastic ring, and it is angiogenic driven while 2. Replacement HGP (R‐HGP): cancer cells infiltrate the normal liver, and co‐opt pre‐existing blood vessels. CRCLM patients treated with chemotherapy and Bevacizumab have a worse five‐year overall survival when their lesions employ R‐HGP instead of D‐HGP. Thus, precisely defining the type of metastatic lesion that a cancer patient has is critical in selecting the appropriate treatment options. Methods : We have developed EV based signature using blood‐based biomarkers (Liquid Biopsy), which focuses on the protein cargo within extracellular vesicles (EVs) to stratify patients into D‐HGP or R‐HGP and guide a personalised treatment plan for CRCLM patients. We have collected plasma from 19 Chemonaive patients, isolated Extracellular vesicles using ultracentrifugation method, and characterised EV abiding to MISEV2022 guidelines. We performed Mass spectrometry in all the patient and analysis the data by performing GSEA for pathway and T‐sample test for biomarker detection. We used IHC for biomarker validation in CRCLM tissue, ExoView for biomarker localisation on EV and ELISA for EV biomarker validation. Results : GSEA analysis showed, all the pathways upregulated in R‐HGPs is either associated with cell proliferation or migration while D‐HGP showed increased adaptive immune response and B cell activation. Further, T sample test on the HGPs showed 22 differentially expressed significant gene and Unsupervised Principal component analysis showed clear segregation of both the HGPs suggesting potential signature. Interestingly, S100A9 protein was the top signature observed and the expression of the S100A9 in the EV correlated with the tumor tissue thus showing significant differences in expression at the tumor liver interphase in co‐opting lesions only. Further we observed that S100A9 proteins on the tissue were expressed by the macrophage and neutrophils which has been suggested to be involved in the inhibition of T‐Cell response. Summary/Conclusion : Our findings indicate that S100A9 protein from Plasma derived Extracellular Vesicles as a predictor of treatment response in CRCLM. Funding : MEDTEQ Innovation for Health and Fonds de recherche du Quebec Sante.

Serial

George Biouss 1 ; Lina Antounians 1 ; Agostino Pierro 2 ; Augusto Zani 1 1 The Hospital for Sick Children, Toronto, Canada; 2 The Hospital for Sick Children, Canada Introduction : Microglia are brain resident macrophages that once activated by neuroinflammatory signals undergo changes in morphology and surface marker expression. Little is known about microglia‐derived extracellular vesicles (M‐EVs) in the neonatal brain during sepsis. Herein, we separated and characterized M‐EVs in a neonatal sepsis model and evaluated their neuroinflammatory cargo. Methods : Animals: Brains of 9‐day‐old C57BL/6 mice were harvested from normal pups or pups with sepsis and brain neuroinflammation confirmed by high expression of inflammatory markers IL1β and NLRP3 (WB), and high density of ameboid IBA1+ microglia (immunofluorescence). M‐EVs: Brains were minced, digested with dispase, differentially centrifuged, and filtered (300g/1200g/100,000g). M‐EVs were enriched by serial immunocapture of canonical microglia receptors with a biotin‐streptavidin platform: ‐ EVs were incubated with P2Y12 antibody and biotin‐tagged secondary antibody ‐ Streptavidin coated agarose beads were added ‐ After washes, P2Y12+EVs were released by acid catalysis ‐ Incubation steps were repeated using TMEM119 antibody, and P2Y12+/TMEM119+ EVs were separated. M‐EVs were assessed for size (NTA), morphology (TEM), and CD63, TSG101, Histone‐H3, IL1β and NLRP3 protein expression (WB). Immunogold labelling was performed to confirm capture of P2Y12+/ TMEM119+ M‐EVs (TEM). Results : M‐EVs from control and inflamed brains had similar size distribution and were CD63+/TSG101+ and Histone‐H3‐. Immunogold labeling showed P2Y12 and TMEM119 localization on the M‐EV extracellular domain. M‐EVs from inflamed brains had higher P2Y12 (p = 0.03), NLRP3 (p = 0.002) and IL1β (p = 0.03), and lower TMEM119 (p = 0.03) protein levels compared to control M‐EVs. Summary/Conclusion : Our method was effective in separating M‐EVs from normal and inflamed neonatal brains. In an experimental model of neonatal sepsis, M‐EVs have a different signature during neuroinflammation. Funding : SickKids Foundation.

Single

Andras Saftics 1 ; Sarah Abuelreich 1 ; Eugenia Romano 1 ; Ima Ghaeli 2 ; Kathleen Lennon 1 ; Nan Jiang 1 ; Gagandeep Singh 3 ; Saumya Das 4 ; Kendall Van Keuren‐Jensen 5 ; Tijana Jovanovic‐Talisman 2 1 Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope, USA; 2 Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope, Duarte, USA; 3 Department of Surgery, City of Hope, Duarte, USA; 4 Cardiology Division and Corrigan Minehan Heart Center, Massachusetts General Hospital, Harvard Medical School, Boston, USA; 5 Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA Introduction : Extracellular vesicles (EVs) and their cargo constitute novel biomarkers. However, a significant current roadblock in EV‐based diagnostics is the lack of techniques with a wide dynamic range to visualize and rigorously quantify individual EVs within specific EV subpopulations. Methods : We combined affinity isolation with super‐resolution imaging to comprehensively assess individual EVs from human plasma. The analytical protocol was cost‐effective and could be easily customized. Our Single Extracellular VEsicle Nanoscopy (SEVEN) assay could be readily used to assess distinct EV subpopulations. In addition to quantifying the number of isolated EVs, SEVEN provided the size, shape, molecular content of specific markers, and overall heterogeneity of EV subpopulations. Results : We first assessed EVs enriched in abundant tetraspanins (CD9, CD81, CD63) isolated by size‐exclusion chromatography from pooled human plasma; the number of detected tetraspanin‐enriched EVs positively correlated with sample dilution in a 64‐fold range. We further characterized the size, shape, and molecular tetraspanin content (with corresponding heterogeneities) for CD9‐, CD63‐, and CD81‐enriched EVs. Importantly, when SEVEN was directly applied to crude plasma samples, it robustly detected EVs from ∼0.1 uL of plasma. Finally, we assessed EVs from the plasma of four pancreatic ductal adenocarcinoma patients with resectable disease. Compared to healthy plasma, their CD9‐enriched EVs were smaller with higher curvature; while their IGF1R‐enriched EVs were larger, rounder, and contained more tetraspanin molecules. The results suggest a unique pancreatic cancer‐enriched EV subpopulation. Summary/Conclusion : This study provides proof‐of‐concept for advancing SEVEN into a platform to characterize disease‐associated and organ‐associated EV subpopulations. We anticipate SEVEN could be implemented in a wide array of different biological contexts since super‐resolution imaging is becoming more widely available to the EV research community. Funding : National Institutes of Health grant UG3/UH3 TR002878; Dorrance Family Research Fund; Board of Governors of the City of Hope, Southwest Food Industries Circle, Circle 1500, Dancing with Chicago Celebrities, Bruce & Lyn Everette, Irell and Manella Graduate School of Biological Sciences at City of Hope. Research reported in this publication included work performed in the City of Hope Analytical Cytometry core supported by the National Cancer Institute of the National Institutes of Health under grant number P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Keywords : nanoscopy, imaging

Survey

Kotb Abdelmohsen 1 ; Alison Herman 2 ; Martina Rossi 2 ; Kenneth W. Witwer 3 ; Myriam Gorospe 2 1 NIH, Baltimore, USA; 2 NIH, USA; 3 Johns Hopkins University, Baltimore, USA Introduction : Extracellular vesicles (EVs) are secreted by all organs and enter teh bloodstream, and theirfore blood EVs, which comprise EVs secreted by blood cells and/or other organs, may reflect physiological and pathological conditions. Given dat blood EVs are extremely heterogeneous, it is challenging to identify teh specific organ of origin. Here, we set out to systematically identify tissue‐specific small (s)EV proteins secreted by six tissues (brain, liver, lung, heart, kidney, and fat) in blood serum using proteomic analysis. We identified a number of sEV proteins dat were specific to brain (68), liver (194), lung (39), heart (15), kidney (29), and fat (33). Validation by western blot analysis confirmed teh presence of tissue‐specific sEV proteins in sEVs isolated from serum, including brain (DPP6, SYT1, and DNM1L), liver (FABPL, ARG1, and ASGR1/2), lung (SFPTA1), heart (CPT1B), and fat (GDN). Finally, we applied these findings to an aging mouse model and discovered altered levels of these proteins in serum sEVs from old compared to young mice. In sum, we have identified and explored teh traceability of tissue‐specific sEV proteins in serum presenting potential non‐invasive biomarkers and/or therapeutic targets. Methods : Tissue dissociation and sEV isolation sEV isolation from serum Nanoparticle tracking analysis Transmission Electron Microscopy Mass spectrometry‐based proteomics of EVs Western blot analysis Results : sEV protein contents reflect teh parent tissue organ of origin Proteome analysis reveals common and unique sEV proteins for each organ Identification of Organ‐specific sEV proteins Detection and validation of organ‐specific sEV proteins in serum Summary/Conclusion : Here, we surveyed sEV proteins from mouse organs: brain, liver, lung, heart, kidney, and fat. Given dat organ‐specific sEVs retain their protein identity as they travel into the bloodstream, we performed proteomic analysis using sEVs isolated from these tissues and serum. We identified sEV proteins dat were both (me) exclusively or highly specific to a single organ wifin these limited comparisons, and (ii) detectable in serum sEVs, in order to create a protein catalog dat may serve for noninvasive, rapid detection of diagnostic biomarkers of various organ EVs present in the blood. Funding : NIH. Keywords : extracellular vesicles, exosomes

Tissue

Alicja Głuszko 1 ; Mirosław Szczepański 2 ; Andrzej Ciechanowicz 3 ; Nils Ludwig 4 1 Medical University of Warsaw, Warsaw, Poland; 2 Medical University of Warrsaw, Poland; 3 Medical University of Warsaw, Poland; 4 University Hospital Regensburg, Regensburg, Germany Introduction : Tissue hypoxia, present in solid tumors, including head and neck squamous cell carcinomas (HNSCCs), alters molecular and functional activity of cancer cells. Our previous experiments demonstrated that hypoxia increased the release of tumor‐derived small extracellular vesicles (sEVs) and influenced their proteomic profile. In this study we aimed to characterize the lipidomic profile of sEVs released from cells cultured in normoxic and hypoxic conditions. Methods : HNSCC cells (PCI‐30) and normal control cells (HaCaT keratinocytes) were exposed to 21 % (normoxia) and 1 % (hypoxia) oxygen supply. sEVs were isolated from supernatants using size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis, electron microscopy, immunoblotting, and high‐resolution mass spectrometry. Gene expression levels based on RNA‐seq data from HNSCC patients and clinical characteristics were obtained from the Cancer Genome Atlas (TCGA). Expression profiles of lipidomic signatures were compared between a total of 522 cases of primary HNSCC and 44 normal control samples. Results : Isolated sEVs ranged in size from 125–135 nm and carried CD63 and CD9 but not Grp94. We detected 6176 lipids and glycerolipids were among the most abundant lipid classes in sEVs. We found almost 1000 lipids exclusively carried by tumor‐derived sEVs in comparison to normal sEVs from keratinocytes. Hypoxia triggered a major switch of the lipid profile with ∼1000 lipids being exclusively detected in hypoxia‐derived sEVs and 706 lipids being significantly upregulated under hypoxic conditions. In terms of numbers, fatty acids were the most significantly hypoxia‐induced lipid class, in terms of abundance the most significant changes were observed for glycerophospholipids. This data was validated on the transcriptome level using the TCGA HNSCC cohort. The expression levels of genes involved in the biosynthesis of glycerophospholipids significantly correlated with hypoxia‐, angiogenesis‐ as well as sEV secretion‐related genes in the TCGA HNSCC cohort. Also, the expression levels of genes involved in catabolism of glycerophospholipids significantly correlated with improved survival. Summary/Conclusion : Lipid profiles in HNSCC‐derived sEVs are characterized by remarkable plasticity and are modulated by environmental factors such as hypoxia. Thus, sEV‐associated lipids may emerge as clinical biomarkers for tumor progression or tissue hypoxia in HNSCC. Funding : MB/M/48(79)# to A.G./Medical University of Warsaw.

Vision

Our vision is to be the leading advocate and guide of extracellular vesicle research and to advance the understanding of extracellular vesicle biology.

Annexin

Rucha Trivedi ; Jamboor K. Vishwanatha University of North Texas Health Science Center, Fort Worth, USA Introduction : Tumor‐derived extracellular vesicles (TEVs) are highly implicated in tissue‐specific metastasis. Additionally, TEVs interacts with the distant microenvironment to shape a pre‐metastatic niche (PMN) for homing the tumor cells. Annexin A2 (AnxA2) is a plasma and endosomal membrane‐associated protein. Its high levels have been correlated with poor distant metastasis‐free survival and poor overall survival in triple negative breast cancer (TNBC) patients. It is also abundantly present in TEVs and recruits TEV‐associated cargo such as proteins and microRNAs. Our lab reported that in vivo education with AnxA2 depleted EVs led to reduced TNBC metastasis to lungs and brain suggesting a key role in the formation of a PMN. While the presence of AnxA2 in EVs has been reported, its contribution in the formation and development of PMN via EVs is still unexplored. We aim to evaluate the implications of AnxA2 in EVs and elucidate the mechanisms promoting TNBC metastasis. Methods : We used RNA interference‐mediated gene silencing to stably downregulate AnxA2 in organotropic TNBC cell lines derived from the parent MDA MB 231 cells, LM2 and BrM2. Differential ultracentrifugation was used to isolate EVs from cell culture supernatant and size analysis was done using Nanoparticle Tracking Analyzer. Biological characterization was done in concordance with MISEV 2018 guidelines using immunoblotting. Additionally, the EVs will be subjected to quantitative proteomic analysis to identify differentially expressed proteins upon loss of AnxA2. We will further carry out transcriptome profiling of the AnxA2 depleted TNBC cells and ‐derived EVs to identify the differentially expressed genes. Results : Upon depletion of AnxA2 protein, we observed a significant effect of AnxA2 depletion on its physiological role in plasmin generation. We observed a size distribution of the isolated EVs between 30–300 nm. Using immunoblotting we confirmed reduced levels of AnxA2 in EVs derived from AnxA2 depleted TNBC cells. We verified their purity using EV enriched markers ‐ ESCRT, Heat shock proteins and tetraspanins such as CD81, CD9, CD63 and confirmed the absence of negative markers ‐ GM130, calnexin and cytochrome c. Interestingly, we observed a reduced yield of EVs with AnxA2 depletion indicating a potential effect on EV biogenesis and release. Summary/Conclusion : The role of AnxA2 in TEVs biogenesis, release and selective cargo loading will lead to potential identification and understanding of the novel secretory and EV protein that may act as a functional regulator in promoting advanced metastasis in TNBC. Funding : This research is supported by the NCI of National Institute of Health under Award Number R01CA220273 (JKV), awarded to Dr. Jamboor K. Vishwanatha. Keywords : Annexin A2, TNBC, extracellular vesicles.

Aqueous

Boyang Su 1 ; Morteza Jeyhani 2 ; Jenie Marian Cruz Burgos 3 ; Gobi Thillainadesan 4 ; Dennis K. Lee 4 ; Thamara Dayarathna 5 ; Scott S.H. Tsai 2 ; Hon S. Leong 1 1 Department of Medical Biophysics, University of Toronto, Biological Sciences Platform, Sunnybrook Research Institute, Toronto, Canada; 2 Department of Mechanical and Industrial Engineering, Toronto Metropolitan University, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Institute for Biomedical Engineering, Science and Technology (iBEST), Toronto, Canada; 3 Instituto Nacional de Medicina Genómica, Cuautitlán Izcalli, Mexico; 4 Biological Sciences Platform, Sunnybrook Research Institute, Toronto, Canada; 5 Center for Clinical and Translational Research, Nationwide Children's Hospital, London, USA Introduction : Development of extracellular vesicle (EV)‐based liquid biopsies and EV‐based nanomedicines requires isolation of ultra‐pure EVs with an intact vesicular structure. Ultracentrifugation (UC) does not satisfy these requirements because of excessive damage to EVs, length of time required to pellet EVs, and difficulties in identifying or locating the pellet. Aqueous two‐phase separation (ATPS) may be a more effective EV isolation approach. Methods : Enrichment and recovery efficiency of multiple EV sources (human and mouse cell culture conditioned media, human plasma, etc.) before and after ATPS were measured using nanoscale flow cytometry (nFC). Canonical EV biomarkers were detected using nFC with pre‐conjugated antibodies specific for CD9, CD63, and CD81. EV vesicular structure was analyzed using transmission electron microscopy (TEM). EVs were co‐cultured with human recipient cells and internalization rate was measured with confocal microscopy. “omics” studies were performed to determine EV molecular cargo profile. Results : ATPS has greater EV enrichment capability (21.4 × vs. 10.9 × times fold enrichment) and higher EV recovery efficiency (97.6% vs. 69.3% recovery) than UC. ATPS improves EV‐antibody labelling efficiency as determined by flow cytometry analysis, possibly by reducing the protein corona on the surface of EVs. EVs isolated by ATPS are monodispersed and exhibit higher circularity than EVs isolated by UC. Maintenance of EV vesicular integrity via ATPS led to higher internalization rates by recipient cells. Transcriptomics and proteomics analyses revealed a strong overlap, thus validating ATPS for effective EV isolation. Summary/Conclusion : Our study demonstrates ATPS as a more attractive EV isolation method that produces canonical EVs. ATPS isolated EVs show greater vesicular integrity, reduced protein corona, and relatively homogenous and monodispersed qualities. Lastly, the proteome and transcriptome were consistent between the two methods.

Cardiac

Tal Caller 1 ; Olga Shaihov–Teper 2 ; Daria Lendengolts 2 ; Itai Rotem 2 ; Yeshai Schary 2 ; Ruty Shai 3 ; Efrat Glick‐Saar 4 ; Dan Dominissini 4 ; Alex Boomgarden 5 ; Crislyn D'Souza‐Schorey 5 ; Nili Naftali‐Shani 2 ; Jonathan Leor 2 1 Neufeld and Tamman Cardiovascular Research Institutes, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Israel; 2 Neufeld and Tamman Cardiovascular Research Institutes, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, USA; 3 Pediatric Hemato‐Oncology, Edmond and Lilly Safra Children's Hospital, and Cancer Research Center, Sheba Medical Center, Tel Hashomer, USA; 4 Cancer Research Center and Wohl Centre for Translational Medicine, Chaim Sheba Medical Center, Tel‐Hashomer, USA; 5 Department of Biological Sciences, University of Notre Dame, IN, USA Introduction : Heart failure (HF) is associated with increased incidence of cancer. However, the mechanisms that link heart failure to cancer remain unclear, and specific therapies are limited. We hypothesized that the failing heart secrets small extracellular vesicles (cEVs) that carry and disseminate pro‐tumorigenic factors. Methods : To determine the role of cEVs in tumor growth, we focused on cardiac mesenchymal stromal cells (cMSCs), which play a central role in cardiac repair and remodeling. Using size exclusion chromatography, we isolated cMSCs‐EVs from hearts of mice, 10 days after myocardial infarction (MI) and HF or sham‐MI. We characterized cEVs by nanoparticle tracking analysis, cryo‐electron microscopy, and western blot. Results : cMSCs after MI secreted twice more EVs than cMSCs from sham‐MI. Proteomic analysis revealed a distinct profile of cMSC‐EVs after MI and HF. cMSC‐EVs from the failing heart harbored more tumor‐promoting proteins, cytokines, and microRNA (miR), such as Periostin, Osteopontin, VEGF, IL‐6, TNFα, miR 221, miR 21, miR 24 and miR 214. Next, lung cancer cells were inoculated into the hind limb of mice. While MI and HF accelerated tumor growth, EV depletion by GW4869 markedly attenuated this effect (n = 28). In addition, we found that labeled cEVs targeted lung cancer tumors. Moreover, adoptive transfer of cMSC‐EVs from failing hearts accelerated tumor growth compared to sham‐MI EVs (n = 27). Finally, we found that Spironolactone, a renin‐angiotensin‐aldosterone inhibitor, mitigated the neoplastic effects of HF, attenuated tumor growth, and reduced the secretion of cMSC‐EVs by 26% (n = 38). Summary/Conclusion : We show, for the first time, that mesenchymal stromal cells from the failing heart secret small EVs that carry neoplastic mediators that target and accelerate tumor growth. Anti‐heart failure therapy attenuates the tumor‐promoting effects of cardiac extracellular vesicles. Keywords : heart failure, reverse cardio‐oncology, inflammation

Changes

Yi Zhang 1 ; Yunhui Tang 2 ; Ye Shen 3 ; Xinyi Sun 4 ; Min Zhao 3 ; Larry w. Chamley 5 ; Qi Chen 6 1 Department of Obstetrics and Gynaecology, The University of Auckland, auckland, New Zealand; 2 The Hospital of Obstetrics and Gynaecology, Fudan University, China (People's Republic); 3 Wuxi Women's Hospital, Wuxi China, China (People's Republic); 4 Department of Obstetrics and Gynaecology, The University of Auckland, New Zealand; 5 Department of Obstetrics and Gynaecology, Room 201J Building 502 FMHS,University of Auckland, Auckland, New Zealand; 6 Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand Introduction : Placental extracellular vesicles (pEVs) may be involved in the coordinated regulation of fetal development and maternal adaptation during pregnancy. In this study, we compared the miRNA profiles of pEVs derived from first trimester and term placentae to understand the changes in the miRNA profiles between two gestational ages and whether they may be involved in the regulation of fetal development or the dynamic changes in maternal adaptation to pregnancy. Methods : Large and small pEVs were collected by differential centrifugation from first trimester and term placentae (n = 5 each). Small RNA was sequenced and significantly differentially abundant miRNAs were identified. Target gene enrichment analysis identified differentially abundant miRNAs that are associated with maternal adaptation and fetal development. Results : In total there were 823 or 918 miRNAs present in large and small EVs, respectively. Of these miRNAs, 123 were more abundant and 177 were less abundant in term than first trimester large pEVs. In small pEVs, 70 miRNAs were more, and 138 miRNAs were less abundant in first trimester than in term pEVs. GO and KEGG analyses showed that these more or less abundant miRNAs may participate in processes required for maternal vascular adaptation including: angiogenesis, vascular genesis, regulation of blood vessels, endothelial cell migration, and blood vessel morphogenesis. Analysis using Placentacellenrich showed that fetal fibroblasts, decidual perivascular cells, and maternal endothelial cells are enriched for genes that are targets of the differentially abundant miRNAs in both large EVs and small EVs. Analysis using Funrich confirmed the interaction of the differentially abundant miRNAs with these target genes. Summary/Conclusion : The miRNA content of small and large pEVs changes with increasing gestations. The changes in miRNA content of these EVs may reflect developmental changes in the fetus/placenta and/or regulate fetal development and maternal adaptation during pregnancy. Keywords : placental extracellular vesicles, miRNAs, fetal development, maternal adaptation

Curcuma

Audrey Jalabert 1 ; Emmanuelle Berger 2 ; César Cotte 3 ; Elizabeth Errazurig‐Cerda 4 ; Valérie Bardot 5 ; Anne Leblanc 5 ; Lucile Berthomier 6 ; Michel Dubourdeaux 7 ; Sophie Rome 8 1 CarMeN Laboratory (UMR INSERM 1060/INRA 1397), Lyon‐Sud Faculty of Medicine, University of Lyon, Pierre‐Bénite, France, Lyon, France; 2 Laboratoire Ecologie Microbienne (LEM) (UMR CNRS 5557, INRAE 1418), VetAgroSup, University of Lyon, 69622 Villeurbanne cedex, France, Pierre Benite, USA; 3 Groupe Pileje, Paris, France, France; 4 University Lyon 1, Center for Imagery, Ciqle, Rockfeller Faculty of Medicine, 69008 Lyon, France, Lyon, France; 5 Groupe Pileje, Paris, France, Paris, France; 6 Groupe Pileje, Paris, France, France; 7 Groupe Pileje, Paris, France, Paris, France; 8 CarMen Laboratory, INSERM 1060/INRAE 1397, LYON‐SUD Hospital, 69152 Pierre‐Bénite, France, Lyon, France Introduction : It has been demonstrated that plants contain microvesicles with anti‐inflammatory properties which participate in intestinal tissue renewal process and modulation of gut microbiota. Here we determined whether curcuma (Curcuma longa L.) roots also contain microvesicles with immunomodulation properties on macrophages Methods : As we did not know the physicochemical properties of curcuma microvesicles (CuMVs), the use of commercial kits has been banned and the method of differential centrifugations/ultracentrifugations and filtrations at 0.45 um was preferred to isolate CuMVs. CuMV pellet was characterized by transmission electron microscopy, Zetasizer, and metabolomics. THP‐1 macrophages were treated with different concentrations of CuMVs (1  to 5 ug/ml). ROS production was detected by FACS. Results : CuMVs were heterogeneous in size (50–250nm). Interestingly, they carried curcumin, the main active molecule in curcuma, and its derivatives. At low concentration, CuMVs induced ROS production in THP‐1 macrophages and consequently, their polarization into a population of anti‐inflammatory macrophages (decreased CD86 expression and increased CD163 expression). However, these CD163+ macrophages retained their anti‐tumor IL‐10 and TGF‐beta secretory properties. In addition, CuMVs stimulated the expression of anti‐bacterial cytokines (Il1‐beta, Il‐6, TNF‐alpha). Summary/Conclusion : By affecting the ROS levels of macrophages, curcuma vesicles prevent the passage of M2 into tumor‐associated macrophages (TAM) and thus reduce the pro‐tumor effect of M2. CuMVs are involved in the anti‐inflammatory properties of turmeric because they carry curcumin. It is therefore important that the industrial processes developed to commercialize curcuma keep high concentrations of CuMVs as they participate in the passage of curcumin, which is insoluble in aqueous solution, across cell membranes Keywords : inflammation, macrophages, curcuma‐derived microvesicles

Dipsaci

Yue Cao 1 ; Fubing Liu 2 ; Kewei Zhao 3 1 The Third Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, China, guangzhou, China (People's Republic); 2 The Third Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, China, guangzhou, USA; 3 Guangzhou Key Laboratory of Chinese Medicine Research on Prevention and Treatment of Osteoporosis, The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, No.261 and 263, Longxi Avenue, Guangzhou, China, China (People's Republic) Introduction : Dipsaci Radix has been clinically used for strengthening muscles and bones. However, the efficacy of Dipsaci Radix derived extracellular vesicle‐like nanoparticles (DREVNs) on relieving osteoporosis in mice models and its underlying mechanism remain poorly understood. Methods : DREVNs were isolated and characterized from Dipsaci Radix. Using DREVNs to treat bone marrow mesenchymal stem cells (BMSCs) and the postmenopausal osteoporosis (PMOP) mouse models, we analyzed associated genes and poteins of BMSCs and targeting, skeletal structure parameters and proteins of PMOP mouse models. Results : In vitro experiments showed that DREVNs upregulated the osteogenic‐related factors and activate the BMP‐2/Smads signalling pathway to promote osteogenic differentiation of BMSCs. In vivo, DREVNs had definite femur and BMSCs targeting. Micro‐CT suggested that BMD, BV, BV/TV, BS/TV and Tb.N were significantly increased in the DREVNs treatment group, whereas BS/BV, Tb.Sp., SMI and Tb.pf. were significantly reduced, and the increased levels of BMP2, RUNX2, p‐Smad1/5/9 and Smad1/5/9 proteins in femurs reconfirmed the importance of the BMP2/Smads signaling pathway in the progress of the anti‐osteoporosis mediated by DREVNs. Summary/Conclusion : Taken together, our work demonstrates that DREVNs can effectively promote osteogenic differentiation of BMSCs by activating the BMP2/Smads signaling pathway to prevent osteoporotic bone loss in PMOP mouse models. Funding : DREVNs may represent a new class of nano‐drugs for the prevention of PMOP. Keywords : dipsaci radix, extracellular vesicle‐like nanoparticles, osteoporosis, osteogenic differentiation, BMSCs

Dynamic

Dominic Guanzon 1 ; Andrew Lai 2 ; Carlos Palma 3 ; Ramin Khanabdali 4 ; Aase Handberg 5 ; Lewis Perrin 6 ; John Hooper 6 ; Jim Coward 7 ; Terry K. Morgan 8 ; Gregory E. Rice 9 ; Carlos Salomon 10 1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, QLD 4029, Brisbane, Australia; 2 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia, Brisbane, Australia; 3 Inoviq Limited, Notting Hill, Australia., Brisbane, USA; 4 Inoviq Limited, Notting Hill, Australia., USA; 5 Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark., Aalborg, Denmark; 6 Mater Research Institute‐University of Queensland, Translational Research Institute, Woolloongabba, Australia., Brisbane, USA; 7 Mater Research Institute‐University of Queensland, Translational Research Institute, Woolloongabba, Australia., USA; 8 Department of Obstetrics and Gynecology, OHSU, Portland, OR, USA., Oregon, USA; 9 Inoviq Limited, Notting Hill, Victoria, Australia., Notting Hill, Australia; 10 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia., Brisbane, Australia Introduction : Circulating changes in the content and bioactivity of extracellular vesicles (EVs) has been associated with oncogenic transformation of ovarian cancer (OVCA). Thus, characterisation of EV protein and nucleic acids is an essential step for understanding changes to predict cancer outcome. The objective of this study was to describe changes in the EV‐associated proteins and miRNAs that change with the progression of the disease and determine biological processes that are disturbed in OVCA. Methods : A cohort of 97 patients were included in this study from healthy controls (n = 20), benign (n = 20), high grade epithelial ovarian cancer stage I (n = 20), stage II (n = 20), stage III (n = 20), and stage IV (n = 20). EV were isolated using EXO‐NET (INOVIQ LTDA, Australia) and characterised by Nanoparticle tracking analysis, protein abundance (CD63, CD9, Alix, TSG101 and CD81) and morphology using NanoSight, Western blot and electron microscopy, respectively. In addition, a Targeted Multiple Reaction Monitoring proteomic approach was designed to evaluate the top 20 proteins associated with EV (exocarta) in our preparations. EV‐associated miRNA and protein profile was determined by small RNA sequencing and Mass Spectrometry SWATH Analysis, respectively. Generalised additive modelling was used to model protein and miRNAs abundance as a function of progression of OVCA, while pathway analysis was performed using Ingenuity. Results : Using an EV capture technology (EXO‐NET) around 20% of the total circulating particles was isolated, and an enrichment of proteins CD63, CD9, Alix, TSG101 and CD81 compared to total plasma was observed. A total of 18 out of the top 20 proteins associated with EVs in the Exocarta were identified in our preparations. Of the total proteins identified (1517) within EV, 33% (599) changed in abundance as function of the progression of OVCA (p< 0.05). Our modelling analysis identified a total of 20 clusters with different trends, in which 116 proteins (including Carboxypeptidase E, Adenosine deaminase, and Sex hormone‐binding globulin) increased with the progression of ovarian cancer. We identified 703 miRNAs within EV, and 24% (171) changed with the progression of OVCA. A total of 10 clusters with different trends were identified, in which 7 miRNAs (miR‐503‐5p miR‐181d‐5p, miR‐548ay‐5p, miR‐548ad‐5p, miR‐3157‐5p, miR‐135a‐5p, miR‐6815‐5p) continuously increased with the progression of OVCA. Bioinformatic analysis showed that the top functions associated with the proteins and miRNAs with EV across progression of OVCA are inflammation, lipid metabolism, transport, and binding of tumour cells. Summary/Conclusion : The EV proteome and miRNA profile across OVCA demonstrates dramatic changes associated with the progression of the disease. Such information is important to understand the physiology of OVCA and the development of biomarkers to differentiate women with early stages of OVCA and determine the response to chemotherapy. Funding : NHMRC, MRFF, INOVIQ.

Effects

Nicholy Lozano 1 ; Paula Meneghetti 1 ; Gabrieli Camossa Armagni 1 ; Sergio Schenkman 1 ; Ana Claudia Torrecilhas 2 1 UNIFESP, Sao Paulo, USA; 2 Federal University of Sao Paulo, Sao Paulo, Brazil Introduction : Extracellular vesicles (EVs) released from trypomastigote forms of Trypanosoma cruzi modulate the invasion and host immune response. The parasite can be maintained in tissue culture infected cells but loses mice virulence. We have regenerated virulence by 20 serial passages in mice. Therefore, we compared the effects of EVs released by non‐virulent trypomastigotes maintained in vitro (P0), with virulent trypomastigotes maintained in vivo (P20). Methods : BALB/c mice were pre‐treated with 108 total EVs 3 days before infection with 500 either P0 or P20 trypomastigotes forms. Parasitemia was counted at 5, 7, 9, 11, and 13‐days post infection (dpi). Mortality was monitored until 45 dpi. For histopathology, the organs (heart, lungs, spleen, rim, liver, bladder, skeletal muscle, and intestine) were collected at 15 dpi to count amastigote nests Results : Pre‐treatment with EVs released by virulent trypomastigote (P20) increased blood parasitemia and anticipated mortality when compared with non‐virulent (P0) and control groups (without EVs). Animals pre‐treated with P0 EVs did not show significant increase in the quantity of parasites in peripheral blood compared with animals only infected with P0 parasites. Besides that, animals pre‐treated with P20 EVs have doubled the number of amastigote nests in heart and bladder, followed by significant difference in intestine and lungs when compared to other groups. Summary/Conclusion : We show for the first time that T. cruzi virulence is related to EVs released by the parasite. It increases parasitism in heart, followed by bladder, intestine, and lungs. Funding : FAPESP, CAPES and CNPq.

Exhaled

Rashmi Bhardwaj 1 ; Afsareen Bano 2 1 Maharshi Dayanand University, India, Rohtak, India; 2 Maharshi Dayanand University, India, India Introduction : Being the leading cause of worldwide mortalities, the burden of early diagnosis of lung cancer is the utmost requirement of the present research. Due to the invasive nature of Blood and Biopsy samples, noninvasive sample sources such as Exhaled breath condensate (EBC) can be screened for early predictive lung cancer biomarkers. Methods : Human EBC (HE) and Sputum (HS) samples were correlated with invasive sample sources (human plasma (HP) and tissue biopsy) to check their efficacy as biomarker sources. Samples were collected after permission from the institute's human ethical committee and with the consent of patients. Exosomes were isolated from all the samples and characterized for their size, concentration, and surface markers. Exosomal protein was also quantified to check the downstream reliability of noninvasive exosomes as compared to invasive sources. Results : Exosomal size analysis through DLS (HE 61.08 ± 24.12 nm, HS 50.32 ± 6.20 nm, HP 85.92 ± 25.45 nm) and NTA (HE 129.2 ± 10.91 nm, HS 114.3 11.09 nm, HP 105.0 3.92 nm) results showed that EBC and Sputum exosomal dimension range was similar as that of plasma exosomes i.e., 30–150 nm. TEM showed characteristic morphology of EBC and sputum exosomes. NTA concentration results provided valuable data that along with plasma samples (HP1.2E+11‐8.69E+09 particles/mL), EBC (HE 2.6E+09‐2.64E+08 particles/mL) and sputum samples (HS6.44E+09‐4E+09 particles/mL) can also provide adequate exosome quantity to proceed for downstream processing. Bradford assay confirms that EBC (31.75 ± 8.09 μg/20μl) and sputum (27.91 ± 2.30 μg/20μl) exosomes have a quantifiable amount of protein. Summary/Conclusion : Results conclude that EBC and sputum could be novel and efficient sample sources for exosomes to be screened for early lung cancer biomarkers. We can avoid invasive samples such as blood and tissue biopsies and proceed with these noninvasive samples to effectively diagnose lung cancer Funding : Indian Council of Medical Research, India. Keywords : exosomes, exhaled breath condensate, plasma, lung cancer

Exosome

Bong Hwan Sung 1 ; Samantha Beck 2 ; Alissa M. Weaver 1 1 Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN, USA, Nashville, USA; 2 Vanderbilt University School of Medicine, USA Introduction : Exosomes are a type of small extracellular vesicle (EV) that are actively secreted from cells and promote cancer cell motility and metastasis. Previous work has determined that exosome‐type small EVs are critical for both directional sensing and cell speed during cancer cell migration in vitro and in vivo. Along with chemical cues, cancer cells also sense and migrate along extracellular matrix (ECM) fibers. The goal of this project is to identify the role of exosomes in driving cancer cell motility along topologically defined nanofibers that mimic in vivo ECM fibers and the role of ECM in that process. Methods : Small and large EVs were isolated by differential centrifugation. The secretion rate was quantitated using nanoparticle tracking analysis to test the effect of Rab27a expression on exosomes secretion. Random single cell migration and cell migration on the nanofibers were recorded by using a temperature‐controlled wide‐field microscope. The motility aspect was analyzed by ImageJ. Results : Here, we show several aspects of cancer cell motility affected by exosome secretion. Cancer cells with a defect of exosome secretion show defects of speed and distance of migration in random single cell migration assay. These findings are complemented by experiments showing that exosome secretion drives directionally persistent migration of cancer cells in a topologically defined nanofiber environment. The speed, total distance, displacement, and directionality of cancer cells on the nanofibers are affected by exosome secretion. The defects of cell migration on the nanofibers are fully rescued by extra ECM protein coating on the nanofibers. Summary/Conclusion : Overall, we find that dynamic exosome secretion drives directionally persistent migration and quorum sensing behavior of cancer cells. Funding : R01CA206458, R01CA249684, R01CA249424. Keywords : cancer cell migration, persistent migration, extracellular matrix, topologically defined nanofibers

Genetic

Astrid Digruber 1 ; Tanja Edelbacher 2 ; Masoumeh Alinaghi 2 ; Monika Ehling‐Schulz 1 1 Institute of Microbiology, Department of Pathobiology, Vetmeduni Vienna, Vienna, Austria; 2 Institute of Microbiology, Department of Pathobiology, Vetmeduni Vienna, Austria Introduction : The food‐pathogen Bacillus cereus is a Gram‐positive endospore‐forming rod causing emesis and diarrhea. Although studies on EVs deriving from gram‐positive bacteria are on the rise, the exact secretion mechanisms are still unknown. By using gene deletion mutants of the emetic B. cereus F4810/72, the influence of seven well‐known transcription factors and virulence regulators on EV secretion was studied. Furthermore, three different growth media were used to investigate the consequences of external factors influencing EV secretion. Methods : EVs were isolated from bacterial cultures grown in different media using differential centrifugation. EV numbers and sizes were measured by NTA, bacterial growth was monitored spectrophotometrically, and protein concentration was determined with the aid of a colorimetric assay. The peptide‐to‐lipid ratio was calculated by means of Fourier‐Transform‐Infrared (FTIR) spectroscopy. The impact of intrinsic as well as extrinsic factors on EV compositions was analyzed by transmission electron microscopy (TEM), FTIR spectroscopy, and lipidomics. Results : Our study revealed that global transcriptional regulators, as well as virulence‐associated transcription factors, have a significant influence on EV secretion and vesiculogenesis in B. cereus. Furthermore, growth media significantly impacted EV secretion as well as EV composition in a genotypic manner, indicating a tight interplay of intrinsic and extrinsic factors in vesiculogenesis. Summary/Conclusion : This study shows that the choice of the media considerably affects vesiculogenesis, and thus, establishing suitable culturing conditions is crucial to studying EV secretion dynamics. Furthermore, we show that knock‐out mutants are a valuable tool to gain insights into the mechanisms of EV secretion and pave the way for deciphering EV vesiculogenesis in gram‐positive bacteria. Funding : The project is partially funded by the city of Vienna through the HJS program (409332/2021).

Hovenia

Quan Zhong 1 ; Jiaming Chen 2 ; Gaofeng Liu 3 ; Bodeng Wu 4 ; Bin Xu 5 ; Zhenxun Wang 5 ; Xiaoqing Jiang 6 ; Xin Zhang 7 ; Lei Zheng 8 ; Yu Wang 1 1 Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China, China (People's Republic); 2 Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China, Guangzhou, China (People's Republic); 3 School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong, P.R. China, Guangzhou, China (People's Republic); 4 .Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, P. R. China, China (People's Republic); 5 Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China, China (People's Republic); 6 Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China, China (People's Republic); 7 Laboratory Department, Nanfang Hospital, Southern Medical University, Guangzhou, China, China (People's Republic); 8 Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China (People's Republic) Introduction : By previous exploration, Extracellular vesicles (EVs) derived from plants have been proved to have great bioactivity. However, there is no specific study on whether Evs can maintain their morphology and bioactivity and exert biological effects under special circumstances. Hovenia dulcis Thunb is a plant with medicinal value, and the discovery of its effects originated in ancient China. In this study, we explore those questions, to study whether the Hovenia dulcis Thunb still exists Evs with normal morphology and bioactivity after being decocting at high temperature, and whether the decocted halelns‐EVs can play an active role in the treatment of alcoholic liver disease (ALD). Methods : After decocting Hovenia dulcis Thunb at 100°C for 30 minutes, Evs were isolated by ultracentrifugation from decoction. Evs were identified and characterized by TEM, NTA and BCA. The differential expression of micro‐RNA in Hovenia dulcis Thunb‐derived exosome‐like nanovesicles (HALELNs) were identified by RNA sequencing, and the constant expression of miRNA in HALELNs were detected by RT‐PCR. By confocal, HALELNs labeled with PKH6, were performed to confirm the uptake of HALELNs by hepatocytes. In order to explore the biosafety and therapeutic effect of HALELNs on alcoholic injury, we designed an in vitro cell model of alcohol injury, and then verified the effect of HALELNs on Alpha Mouse Liver 12 cell (AML‐12) by CCK‐8. Results : The HALELNs isolated from the decoction still showed vesicles in a bilayer membrane structure with an average diameter of 135nm under TEM. Moreover, Let‐7b‐5p was stably expressed in HALELNs. HALELNs uptake was enhanced in alcohol‐treated AML‐12 cells compared with normal AML‐12 cells. In mice, HALELNs administered by intraperitoneal injection were mainly enriched in the stomach and intestine after 24 hours. In particular, HALELNs appear to have a therapeutic effect on alcoholic injury. When AML‐12 cells were treated with 4μg/mL HALELNs for 48 hours and then treated with 400 mM ethanol, the decrease of cell viability was significantly reduced compared with those treated without HALELNs. Compared to alcohol gavage alone, interval intraperitoneal injection of HALELNs reduced serum glutathione transaminase (AST) concentrations in mice. Summary/Conclusion : The decoction of Hovenia dulcis Thunnb was found to contain Evs, which maintain normal morphology and bioactivity, after high temperature decoction. Most surprisingly, we found that decoction‐Evs still had stably expressed micro‐RNAs. Besides, decoction‐Evs were indicated to play a positive role in the treatment of alcoholic liver disease (ALD). Funding : This research was funded by grants from National Natural Science Foundation of China (#81902147 and #82172966). Keywords : plant‐derived Evs, micro‐RNA, alcohol stress

Hypoxic

Irene Bertolini 1 ; Michela Perego 1 ; Andrew Milcarek 2 ; Jagadish Ghosh 1 ; Dario Altieri 2 1 The Wistar Institute, Philadelphia, USA; 2 The Wistar Institute, USA Introduction : Extracellular vesicles (EV) have emerging roles in cell‐to‐cell communication. Cancer cells produce higher levels of EV compared to normal cells, and this response is augmented in hypoxia. Intra‐tumoral hypoxia is a common occurrence in breast cancer, correlating with increased risk of metastasis and poor prognosis. We have demonstrated that EV produced by cancer cells in hypoxia induce oncogenic changes in normal epithelial cells in vitro. Therefore, we have now examined the effect of hypoxic‐EV in vivo. Methods : We isolated EV from four different mouse mammary carcinoma cell lines (two metastatic and two non‐metastatic) in hypoxic (EV‐HYP) or normoxic (EV‐NORM) conditions (1% or 5% O2 for 24 h). EV were isolated using differential centrifugation followed by SEC (Izon). EV characterization was carried out according to MISEV guidelines. EV were quantified after each isolation using ZetaView. 6 × 10^9 EV were injected in the abdominal mammary gland of C57BL/6 mice and sample tissues were harvested after 3–6 and 18 weeks. Results : Our data show that EV released by invasive breast cancer cells in hypoxic conditions disrupt the differentiation hierarchy of the normal mammary epithelium, with expansion of stem and luminal progenitor cells, and induction of atypical ductal hyperplasia, and intra‐epithelial neoplasia. Moreover, EV‐HYP induce sustained angiogenesis and resistance to cell death. Mechanistically, we demonstrate that EV released by metastatic cell lines in hypoxic condition contain HIF1α, which is required for downstream responses of aberrant mammary epithelium developmental morphogenesis. Summary/Conclusion : Invasive breast cancer cells in hypoxic condition produce HIF1α‐containing EV. These EV can induce critical steps of mammary epithelium transformation and sustained angiogenesis, in vivo. Taken together, these results identify a novel pathway of local breast cancer recurrences. Keywords : breast cancer, hypoxia, HIF1a, angiogenesis, TME

Imaging

Allaura Cone 1 ; Yijun Zhou 2 ; Meredith Chambers 3 ; Justin Landis 3 ; Ryan McNamara 4 ; Dirk Dittmer 5 1 UNC‐Chapel Hill Lineberger Cancer Center, Durham, USA; 2 UNC‐Chapel Hill Lineberger Cancer Center, Chapel Hill, USA; 3 UNC‐Chapel Hill Lineberger Cancer Center, USA; 4 Ragon Institute of MGH, USA; 5 [email protected], Chapel Hill, USA Introduction : EVs, including exosomes, are too small to be seen by conventional microscopy. Direct stochastic reconstruction microscopy (dSTORM) bypasses the diffraction limit of light. This allows us to not only to visualize individual EVs, but also tetraspanin (CD81, CD63, CD9) complexes on their surface. Previous research found that EVs are enriched in tetraspanins and that tetraspanin enriched microdomains (TEMs) are essential for EV biogenesis and cargo sorting. Methods : EVs were purified using tangential flow filtration and chromatography. We then stained the EVs using a membrane dye, seeded the EVs on slides or in glass‐bottom wells, and incubated with fluorescent‐conjugated tetraspanin antibodies. EVs were then placed in an oxygen‐scavenging buffer and imaged by dSTORM. Images were reconstructed and analyzed using R. Results : Through dSTORM, we were able to see tetraspanin clusters on the surface of EVs in 3D. We were able to characterize EV heterogeneity and to compute signal clusters on individual EVs. Finally, we used a variety of methods for staining to determine what works well. Summary/Conclusion : Single particle EV detection of tetraspanins supports the existence of TEM on EVs under physiological conditions. Funding : This work was supported by R01‐ CA228172 , P01‐ CA019014 to DPD, and UNC at Chapel Hill. AC and RM received funding through T32 5T32AI007151. DPD has received material support from ONI and Cytiva Inc. Keywords : tetraspanin, microscopy, dSTORM

Journal

Jan Lötvall (Sweden)

Mapping

Laura Perin 1 ; Charmi Dedhia 2 ; Astgik Petrosyan 2 ; Benedetta Bussolati 3 ; Sargis Sedrakyan 4 1 Children's Hospital Los Angeles‐University of Southern California, Los Angeles, USA; 2 Children's Hospital Los Angeles, Los Angeles, USA; 3 2Department of Molecular Biotechnology and Health Sciences, Torino, Italy; 4 Children's Hospital Los Angeles ‐ University of Southern California, Los Angeles, USA Introduction : Transcriptomic studies have improved our understanding of the mechanism of action of different therapeutic approaches aimed at slowing down/reversing chronic kidney disease (CKD). Using Spatial Transcriptomics (ST) Visium 10x Genomics, we describe for the first time the spatial kidney transcriptomic map that characterizes the molecular mechanisms underlying the protective effect of extracellular vesicles in a mouse model of CKD characterized by mutation of the col4a5 gene, Alport Syndrome (AS). Methods : Extracellular vesicles (2.8 × 1010) derived from amniotic fluid stem cells (hAFSC‐EVs) were injected in AS mice at the early CKD stage and sacrificed 2.5m after injections. Frozen kidney sessions from injected mice, age‐matched non‐injected AS mice, 2.5m old AS (early CKD stage), and WT mice were collected and processed for ST following the Visium protocols. Imaging data were collected, and analysis was performed using Space Ranger software v1.0.0, Seurat v3.2 in Rv3.31, IPA, Panther, and Loupe Browser 6.0.0. Results : First, using our ST integration data, we determined that the alteration of metabolic pathways in the early AS stage (2.5m) and alteration of the ECM components are the significant glomerular transcriptomics changes that characterize AS progression. Second, our data clearly show that EVs can restore to normal important pathways for glomerular homeostasis like integrin binding, actin filament binding, laminin‐binding, collagen binding, fibronectin binding, vasculature development, AGE‐RAGE signaling, PI3K‐Akt signaling pathway, extracellular matrix organization. ST also allowed the identification of specific genes (Igfbp2, Nupr1, Serpine1) that are modulated by EVs in the AS glomeruli. Summary/Conclusion : Our ST analysis reveals that hAFSC‐EVs are reno‐protective since they can stimulate glomerular cell survival and remodeling. We also identified some specific‐EV targets that can be used as therapeutics for slowing down disease progression in AS. Funding : NIH R01:R01DK121037. Keywords : spatial transcriptomics, kidney disease

Massive

Cristian‐Tudor Matea 1 ; Eva Klinglmayr 2 ; Heloisa Melo‐Benirschke 2 ; Melanie Schürz 2 ; Andreas Marl 3 ; Vesna Stanojlovic 2 ; Anna Müller 2 ; Tanja Plank 2 ; Maria Jaritsch 2 ; Constantin Blöchl 4 ; Christian Huber 5 ; Christof Regl 5 ; Thomas Felder 6 ; Julia Tevini 6 ; Mario Gimona 7 ; Claudia Arzt 8 ; Juan Manuel M. Falcon 9 ; Oihane Elena Albóniga Díez 10 ; Ancuela Andosch 3 ; Astrid Obermayer 11 ; Gregor Zickler 12 ; Carola Alampi 13 ; André Görgens 14 ; Angelika Sales 15 ; Martin Hintersteiner 16 ; Nicole Meisner‐Kober 1 1 Chemical Biology and Biological Therapeutics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, Salzburg, Austria; 2 Chemical Biology and Biological Therapeutics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, Austria; 3 Chemical Biology and Biological Therapeutics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, USA; 4 Bioanalytics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, USA; 5 Bioanalytics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, Salzburg, USA; 6 Department of Laboratory Medicine, Paracelsus Medical University, Salzburg, Austria., USA; 7 Spinal Cord Injury and Tissue Regeneration Center Salzburg, GMP Lab, Paracelsus Medical University, Salzburg, Austria, Salzburg, Austria; 8 Spinal Cord Injury and Tissue Regeneration Center Salzburg, GMP Lab, Paracelsus Medical University, Salzburg, Austria, USA; 9 1. Exosomes Laboratory, CIC bioGUNE‐BRTA, CIBERehd, Bizkaia Technology Park, Derio, 48160 Bizkaia, Spain. 2. Metabolomics Platform, CICbioGUNE‐BRTA, CIBERehd, Bizkaia Technology Park, Derio, 48160 Bizkaia, Spain.bioGUNE, Derio, Spain. 3. IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain., Derio, Spain; 10 Metabolomics Platform, CICbioGUNE‐BRTA, CIBERehd, Bizkaia Technology Park, Derio, 48160 Bizkaia, Spain.bioGUNE, Derio, Spain, USA; 11 Department of Environment and Biodiversity, University of Salzburg, Austria, Salzburg, USA; 12 Department of Chemistry and Physics of Materials, Faculty of Natural and Life Sciences, University of Salzburg, Austria, USA; 13 BioEM Lab, Biocenter, University of Basel, Switzerland, USA; 14 1. Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden, 2. Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg‐Essen, Essen, Germany, Stockholm, Sweden; 15 Central Animal Laboratory, University of Salzburg, Austria, Austria; 16 EvoBiotiX SA, Lugano, Switzerland, USA Introduction : Cow milk derived extracellular vesicles have recently gained momentum for pharmaceutical and nutraceutical applications. Their accessibility at scale and intrinsic potential for oral bioavailability make them particularly attractive as drug delivery vehicles. Industrial implementation of milk EV isolation is still challenging since current state of the art methods are largely based on ultracentrifugation or require final chromatographic steps. Methods : For the development of an industrially scalable process for milk EV isolation we (1) evaluated the suitability of different side streams of cheese manufacturing and dairy processing, (2) iteratively optimised the isolation and quality control processes between the lab and the plant and (3) systematically benchmarked the industrially isolated EVs against corresponding samples obtained by standard lab scale isolation of EVs from raw milk. Results : An industrial, proprietary process was developed for massive scale isolation of EVs from a waste stream of the dairy industry which showed increased yields of EVs, reduced levels of copurified protein particles as compared to standard lab scale protocols. A systematic comparison of the physicochemical properties (NTA, TEM, 3D cryo‐TEM, IR ‘fingerprint’, IFCM, ζ‐potential), molecular composition (protein/particle ratios, proteomics, lipidomics, glycomics, metabolomics) and biological function revealed that industrial milk EVs retain the physico‐chemical properties, composition, morphology, as well as cell and tissue uptake activity in vitro and in vivo models. Summary/Conclusion : We present a scalable process for functional milk EV isolation, that uses the available waste streams from the dairy industry, is directly transferable to the x100,000 L scale and is fully compatible with industrial milk processing. Funding : EV‐TT BPro (County of Salzburg, WISS2025, P1812596), EVTT (European Union, EFRE/IWB 20102‐F1900731‐KZP), CONSONANT (County of Salzburg, WISS2025, F2200397‐KZP).

Mission

Advancing extracellular vesicle research globally.

Mutated

Yuji Hakozaki 1 ; Yuta Yamada 2 ; Haruki Kume 2 ; Koji Ueda 3 1 Cancer Precision Medicine Center, Japanese Foundation For Cancer Research, Japan; 2 Department of Urology, Graduate School of Medicine The University of Tokyo, Japan; 3 Cancer Precision Medicine Center, Japanese Foundation For Cancer Research, Koto‐ku, Japan Introduction : Cancer cell‐derived EVs are considered to have a great potential as biomarker carriers. Since no effective biomarker is available for diagnosis of clear cell renal cell carcinoma (ccRCC) in clinical use, we aimed to develop a novel liquid biopsy method for ccRCC by targeting mutated proteins encapsulated in circulating EVs. Methods : The ccRCC and matched normal tissues were collected from 11 patients who received partial or radical nephrectomy at the University of Tokyo Hospital. Whole exome sequence analysis was performed using tissue samples to construct personalized amino acid sequence databases containing somatic mutations (neo‐sequences) by the R package, Neoantimon. The tissues were then analyzed by Orbitrap Fusion Lumos‐FAIMS Pro LC/MS system to identify mutated proteins. We also constructed the multiple reaction monitoring (MRM)‐based absolute quantification method for detection of the ccRCC mutated protein panel and analyzed EVs isolated from plasma samples collected from each patient before and 2–4 months after surgery. Results : Whole exome sequence analysis of cancer tissues and matched normal samples identified 63.5 nonsynonymous and 16.0 frameshift mutations per sample on average. Subsequent mutated proteome analysis identified 11,417 proteins (FDR < 0.01), in which, importantly, 3 mutated proteins were included. Further absolute quantification measurement of the mutated protein panel for pre/post operative plasma EV samples showed that drastic reduction or complete disappearance of EV mutated proteins were observed in all post‐operative cases. Summary/Conclusion : Cancer‐specific mutated proteins were detectable in plasma or urine EVs, suggesting that our circulating mutated protein‐based liquid biopsy could serve as an effective tool for diagnosis of kidney cancer detection or monitoring.

Nsmase2

meixiang huang 1 ; Barbara Slusher 2 ; Carolyn Tallon 3 ; benjamin Bell 2 ; Xiaolei Zhu 2 ; Medhinee Malvankar 2 ; Ajit Thomas 2 ; Angela Rubin 3 ; Arindom Pal 2 ; Kristen Hollinger 2 ; Erden Eren 2 ; Carlos J. Nogueras‐Ortiz 2 ; Rangaramanujam Kannan 2 ; Rana Rais 2 ; Norman Haughey 2 ; Dimitrios Kapogiannis 3 1 Johns Hopkins Univeristy, baltimore, USA; 2 Johns Hopkins University, Baltimore, USA; 3 JHDD, Baltimore, USA Introduction : Mounting evidence correlates the propagation of hyperphosphorylated tau (pTau) along synaptically connected networks in the brain with progressive cognitive decline in Alzheimer's Disease (AD). Recent findings have highlighted extracellular vesicle (EV)s in enabling transcellular transmission of pathological tau and identified the partial inhibition of EV biogenesis via small‐molecule inhibitors of nSMase2 as a potential therapeutic avenue. However, there are no suitable compounds for clinical development so far. Methods : Through high‐throughput screening and subsequent chemistry, our lab identified PDDC, a highly selective and potent nSMase2 inhibitor with excellent brain penetration and oral bioavailability. To characterize the potential therapeutic effect of PDDC in vivo, we administered PDDC‐containing chow to both PS19 transgenic mice and to wild‐type mice stereotaxically injected with an AAV vector encoding for P301L mutant human tau into their hippocampus (AAV‐hTau seeded model). After chronic dosing, we quantified tau levels in the hippocampus of PS19 mice and the contralateral dentate gyrus (DG) of the AAV‐hTau mice. Neuronally‐derived EVs (NEV)s from plasma were isolated via immunocapture against L1CAM/CD171. Intact NEVs were used to determine particle concentration and diameter using nanoparticle tracking analysis (NTA). Total plasma EVs isolated via Size Exclusion Chromatography were subjected to flow cytometry analysis (FCA) with labeling for p262Tau and b‐III‐tubulin. Results : PS19 mice exhibited robust elevation of multiple ceramide species and enhanced brain nSMase2 enzymatic activity, both of which were normalized by PDDC treatment. PS19 mice treated with PDDC had significantly reduced total tau and pTau, reduced gliosis, protected synapses, and increased neuronal counts. Plasma NEVs of treated mice were fewer in number, greater in size, and had lower p181‐Tau levels than the untreated group; FCA confirmed the decrease of NEVs carrying p262‐Tau at the single EV level. Similarly, the AAV‐hTau‐seeded mice treated with PDDC had reduced tau staining intensity in the contralateral DG. Summary/Conclusion : Data in two AD models using PDDC provides strong preclinical support for using nSMase2 inhibition as a therapeutic strategy to slow tau propagation in AD. Funding : NIH R01 AG063831, R01 AG059799, P30 MH075673, R25GM109441 (Hopkins PREP), Tau Consortium (T‐PEP‐18‐579974C), the Maryland Innovation Initiative award (135726), and the Intramural Research Program of the National Institute on Aging, NIH. Keywords : alzheimer's disease, EV, nSMase2, tau

Optical

Jaena Park 1 ; Katerina Baxter 2 ; Sarah Samuels 2 ; Edita Aksamitiene 1 ; Marina Marjanovic 1 ; Eric Chaney 2 ; Kimberly Selting 2 ; Stephen A. Boppart 1 1 University of Illinois at Urbana‐Champaign, Urbana, USA; 2 University of Illinois at Urbana‐Champaign, USA Introduction : Radiation therapy is commonly used for treating many types of solid tumors. The radiation affects the metabolism in cells which subsequently alters the concentration of autofluorescent biomolecules. These changes can be identified by analyzing their autofluorescence by label‐free multimodal multiphoton microscopy. Because extracellular vesicles (EVs) reflect the metabolic state of their parent cells, this phenomenon could be used to determine the efficacy or dosimetry of radiation therapy. In this study, we investigated the effects of non‐lethal radiation on the optical signatures of live canine tumor cells, their EVs, and urinary EVs (uEV) of tumor‐bearing dogs. Methods : Large EVs were isolated using differential velocity centrifugation at 4°C from either control or linear accelerator irradiated canine transitional cell carcinoma (TCC) cell‐conditioned serum‐free media, or from the clean‐catch urine collected from both healthy and radiotherapy‐treated urogenital cancer‐bearing dogs under an IACUC‐approved protocol. Samples were pre‐cleared at 800 × g for 10 min and 2,000 × g for 30 min. 34 ml of supernatant was spun in a swinging bucket rotor at 12,000 × g for 1 h. The resulting pellet was resuspended in 100 μl of PBS supplemented with 25 mM trehalose. Concentration and size of EVs was measured by NTA. Fresh EVs were imaged with a custom‐built multimodal microscope capable of detecting two‐ and three‐photon autofluorescence, and second and third harmonic generation. Results : EVs isolated from the media of irradiated TCC cells and EVs from urine of radiotherapy‐treated dogs had a greater number of EVs that showed stronger autofluorescence from FAD and NAD(P)H. Furthermore, we were able to distinguish between radiotherapy‐treated dogs and untreated control animals by combining optical characteristics of uEVs, such as the optical redox ratio, the number of uEVs, and the intensity of the third harmonic generation. Lastly, the cumulative radiation dose given to the dogs was reflected in the optical properties of the uEVs. Summary/Conclusion : The effect of non‐lethal radiation treatment on the metabolites of canine TCC cell‐derived EVs and uEVs was measured. The irradiation dose was reflected in the label‐free optical characteristics of metabolites in EVs. This study demonstrated the potential of using uEVs and label‐free multimodal multiphoton microscopy to develop a noninvasive, label‐free method for dosimetry following radiotherapy in pre‐clinical animal models. Keywords : transitional cell carcinoma, bladder cancer, prostate cancer, dog urine, radiotherapy, linear accelerator, dosimetry, nonlinear optics, two photon autofluorescence, three photon autofluorescence, second harmonic generation, third harmonic generation

Pooling

Rebecca L. Davies 1 ; Claire Mennan 2 ; Mark Platt 3 ; Karina Wright 2 ; Oksana Kehoe 2 1 Keele University, Shropshire, United Kingdom; 2 Keele University, Oswestry, United Kingdom; 3 Loughborough University, Loughborough, United Kingdom Introduction : Mesenchymal stromal cells (MSCs) are immunomodulatory, meaning they can be effective at treating autoimmune disorders, such as rheumatoid arthritis (RA). This is replicated using their extracellular vesicles (EVs), yet we are limited by our ability to yield enough EVs for therapeutic application. Hence, we explored the effect of pooling on MSC EV harvest and therapeutic potential, in comparison to single donors. Methods : Sucrose cushion ultracentrifugation isolated umbilical cord MSC EVs were characterised by particle concentration and sizing, cryo‐EM, nano flow cytometry (NanoFCM) and mass spectrometry. Therapeutic potential was assessed by reduction of swelling and improvement of histological parameters in an antigen‐induced arthritis (AIA) model. Here, the ‘best donor’ was compared with pooled MSCs, and their derived EVs, against control mice injected with serum free DMEM. Results : Pooled MSCs yielded significantly more particles than single donors (p< 0.001). These were of EV nature due to characteristic morphology, which showed a lipid membrane bilayer, and mode size, 114 ± 3.4nm. Higher particle yield encouraged the increase in EV associated tetraspanins (CD9, CD63, CD81) positive particles, as shown by nano flow cytometry, but there was no real trend in MSC positive particles (CD73, CD90, CD105), perhaps reflecting MSC heterogeneity. All conditions contained proteins common to EV preparations, but some differences were identified. When injected into our AIA model, swelling was significantly reduced for both single donor (p< 0.05) and pooled EVs (p< 0.0001) 48‐hours after peak swelling. Yet, all conditions were significant at 72 hours, MSCs (p< 0.05) and EVs (p< 0.001 and p< 0.0001), in comparison to control. Still, only EVs significantly reduced arthritis index (p< 0.05), and associated RA inflammatory phenotype, with pooled MSC EVs appearing superior (p< 0.001). Summary/Conclusion : Pooling MSCs is an effective strategy to increase EV yields for therapeutic application and may be more beneficial when treating inflammatory arthritis. Funding : EPSRC/MRC DTC in Regenerative Medicine, The James Richardson Studentship, Institute of Orthopaedics Ltd., Oswestry, ISTM and ACORN funding, Keele University. Keywords : mesenchymal stromal cells, extracellular vesicles, inflammatory arthritis

Protein

Karen Lahme 1 ; Sarah Frömbling 2 ; Johannes Brand 2 ; Stephanie Zielinski 1 ; Catherine Meyer‐Schwesinger 2 1 University medical center Hamburg Eppendorf, Hamburg, Germany; 2 University medical center Hamburg Eppendorf, Germany Introduction : Enyzmatic distrubances of the ubiquitin proteasomal system (UPS) are a hallmark for progressive kidney cell injury. The assessment of UPS enzyme activities is not feasible in archived kidney biopsies from patients. Extracellular vesicles (EVs) provide opportunities in biomarker discovery for diagnosis, prognosis as well as therapy monitoring. Here we focus on the potential of EVs from human podocytes as a source for protein‐biochemical assessment of proteostasis disbalances. Podocytes are specialized cells of the kidney blood filter, which are challenged during autoimmune diseases such as membranous nephropathy (MN) resulting in breakdown of the renal filter. In MN the UPS is impaired in podocytes. Methods : Human cultured podocytes exposed to autoantibodies and isolated podocyte‐derived urinary EVs from patients with MN or other kidney diseases were analyzed. From cultured podocytes, EVs were collected in exosome depleted medium and isolated by differential ultracentrifugation. EVs were isolated from healthy control and patient urine with kidney diseases using a combination of liquid size exclusion chromatography and differential ultracentrifugation. The amount of EVs was measured by image stream. Next to total protein abundance, the activity of DUBs and of proteolytic subunits of the proteasome were examined by activity‐based protein profiling. Results : Upon autoimmune injury the amount of podocyte‐derived EVs from cultured podocytes and from patient urine increased. Protein biochemical analysis unraveled the presence of mainly inactive enzymes of the UPS within released EVs from cultured podocytes after exposure to autoantibodies. Content of inactive enzymes of the UPS was highly abundant in urinary EVs from patients with MN compared to other kidney diseases. Summary/Conclusion : Podocyte‐derived EVs are a suitable source for protein biochemical analyses of the functional state of the UPS. Keywords : biomarker, protein, kidney

Reduced

Naoya Kuriyama 1 ; Yusuke Yoshioka 2 ; Shinsuke Kikuchi 3 ; Nobuyoshi Azuma 3 ; Takahiro Ochiya 4 1 Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku‐ku, Japan; 2 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku‐Ku, Japan; 3 Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan; 4 Department of Molecular and Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shimjuku‐ku, Japan Introduction : There is limited research on the role of extracellular vesicles (EVs) derived from atherosclerotic plaques, which are composed of various cell types including endothelial cells, smooth muscle cells (SMCs), and macrophages and may contribute to the progression of atherosclerosis. To investigate the roles of EVs from atherosclerotic plaques, we compared the function of EVs derived from plaques removed by femoral endarterectomy (FEA) to EVs derived from healthy arteries in vitro. Methods : Plaques and healthy samples (iliac arteries) were collected from 12 patients undergoing FEA and from 5 patients with abdominal aortic aneurysm undergoing open surgical repair, respectively. The sampling was approved by the local ethics committee. Single cell RNA sequencing was performed on 3 plaques and 1 healthy sample. 4 samples from each group were incubated in serum‐free medium with 10% oxygen for 4 hours. EVs were isolated from the supernatants by ultracentrifugation (210,000 g, 70 min) and characterized by Nanosight and Western blotting (WB). EVs from both groups were added to SMCs, and their RNAs were analyzed by qPCR. The chemokine profiles were analyzed using a Human chemokine array kit, which can profile 31 chemokines. The adhesion ability of THP‐1 cells to SMCs treated with EVs was evaluated using a THP‐1 cell adhesion assay. Proteome analysis of EVs derived from 5 plaque samples was performed. Results : Single‐cell RNA sequencing showed that the proportion of CD8 T cells and pro‐inflammatory macrophages tends to be higher in plaques. Several chemokines that induce immune cell migration were significantly upregulated in SMCs treated with plaque‐derived extracellular vesicles (P‐EVs) as determined by qPCR and chemokine array. In addition, the mRNA level of ICAM‐1, which is involved in immune cell adhesion, was upregulated in the P‐EVs group. Functionally, P‐EVs increased the ability of SMCs to adhere to THP‐1 cells. Proteome analysis revealed that P‐EVs carried several types of annexins. The expression level of Annexin A1, which has anti‐inflammatory effects, was significantly lower in P‐EVs by WB. Summary/Conclusion : The amount of Annexin A1 in P‐EVs was decreased, indicating that the anti‐inflammatory effects of arteries may be attenuated via the upregulation of ICAM‐1 and several chemokines.

Removal

Cheng‐Yu Chou 1 ; Po‐Chieh Chiang 2 ; Andrew Man‐Chung Wo 3 1 Institute of Applied Mechanics, National Taiwan University, New Taipei City, Taiwan (Republic of China); 2 Reliance Bioscience, New Taipei City, Taiwan (Republic of China); 3 Institute of Applied Mechanics, National Taiwan University; Reliance Biosciences, Taiwan (Republic of China) Introduction : Lipoprotein (LP) in plasma is five‐order of magnitude more than that of EV, rendering high purity EV challenging. Much progress has been made with a sequential size‐ and density‐based process, e.g. size exclusion chromatography (SEC) followed by density gradient (DG). The two‐step process is required due to the EV‐fractions from SEC still contain substantial EV‐sized LP, however, DG requires ultracentrifugation (UC) hence time‐consuming. Further, stand‐alone UC process to isolate EV would co‐isolate LP with similar density. Available LP removal reagents mostly use antibody with some success. This study presents a glycan‐based approach for high affinity LP removal resulting in purified EV from SEC‐or UC‐treated plasma samples. Methods : LipoMin (Reliance Biosciences) with functional magnetic beads was loaded into UC‐ or SEC‐treated samples. After mixing for 10 minutes for LP capture, the supernatant containing purified EV was collected while on a magnetic separator. The purified EV sample was characterized with ApoA1, ApoB and CD81 sandwich ELISA, as well as TEM, NTA and Western blot (WB) Results : TEM images of UC‐LipoMin‐ and SEC‐LipoMin‐treated samples showed characteristic cup‐shaped and intact EV morphology. NTA results showed particles concentration and distribution comparable with that of SEC‐DG‐treated samples. WB results revealed significant removal of both ApoA1 and ApoB for both UC‐LipoMin‐ and SEC‐LipoMin‐treated expressions while substantially preserving EV flotillin and CD81 signatures. Sandwich ELISA results showed SEC‐LipoMin‐treated ApoB expression decreased by 72% compared to that without LipoMin treatment while CD81 signal is essentially unchanged (within 8%). WB of LipoMin‐treated colorectal cancer (CRC) and Alzheimer's disease (AD) plasma samples verified the corresponding EV and clinical markers (CDX2 & CD66a for CRC; ptau217 for AD) are clearly expressed. Summary/Conclusion : This novel approach requires 10‐mins processing for removal of lipoproteins in UC‐ or SEC‐treated plasma samples. LipoMin‐treated samples showed robust EV and clinical expressions and should be viable for clinical studies. Funding : Reliance Biosciences, Taiwan.

Section

Yuichiro Iwamoto 1 ; Benjamin Salmon 2 ; Miu Tamamitsu 3 ; Yusuke Yoshioka 4 ; Alexander Krull 2 ; Sadao Ota 5 1 University of Tokyo, Meguro‐ku Komaba, Japan; 2 University of Birmingham, United Kingdom; 3 Research Center for Advanced Science and Technology, The University of Tokyo, USA; 4 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku‐Ku, Japan; 5 Research Center for Advanced Science and Technology, The University of Tokyo, Japan Introduction : Large‐scale optical analysis of extracellular vesicle(EVs) properties is essential to understand the characteristics of EV populations and holds potential to realize EV‐based clinical diagnosis in practice.However, no method simultaneously satisfies the requirements as an ideal EV analyzer: enough sensitivity for detecting individual nanoparticles(< 100 nm) with sizing capability, fluorescence sensitivity, a high throughput and a large scalability.Here we realized a high throughput (96,000 events/sec) and sensitive (< 40 nm) nanoparticle analyzer by integrating a unsupervised learning‐based denoising method for detecting weak scattering detection with a home‐built optofluidic set‐up. Methods : We performed all experiments using a home‐built optical and fluidic analyzer system. First, we measured the scattering of polystyrene particles of known refractive index and size to evaluate the performance of our system. Next, we demonstrated large‐scale EV analysis. Scattering and fluorescence of antibody‐stained EVs of HCT(human undifferentiated thyroid cancer cell) were measured. Results : Using the analyzer developed, we demonstrated the detection of polystyrene particles (< 40 nm) at a detection throughput of 98,000 particles/s.We show that applying a machine learning‐based denoising method significantly improves the measurement system's sensitivity. Experiments with polystyrene particles show that this method accurately removed noise and detected nanoparticles even though it does not require training data‐set. Finally, we performed an experiment of detecting EVs which were labeled with CD9 and CD147 with a detection throughput of 5,000 particles/s. As a result of analyzing 100,000 particle detections in total, 85% of the detected EVs were observed to be CD9‐positive, and 10% of them were CD9/147 double positive. Summary/Conclusion : We developed a high throughput and high sensitivity nanoparticle analyzer by combining a machine learning‐based denoising method and an optofluidic system for detecting weak scattering.Furthermore, we demonstrated large‐scale EV analysis using EVs labeled with CD9 and CD147.Our system potentially becomes a powerful tool for large‐scale individual EV analysis. Funding : This work was sup‐ported by JST, CREST grant number JPMJCR19H1, Japan. Keywords : flow cytometry, deep learning

Shining

Anabel Silva 1 ; Melanie Schoppet 2 ; Natasha Pereira 1 ; Sam Law 2 ; Tahnee Kennedy 1 1 Exopharm Ltd, Australia; 2 Exopharm Ltd, Melbourne, Australia Introduction : Extracellular vesicles (EVs) are lipid bilayer‐delimited nanoparticles released by all cells. Given their potential for diagnostic and therapeutic applications, numerous approaches have been utilised for labelling EVs to assess their biodistribution, cell uptake and trafficking, as well as to determine their size and abundance in a biofluid. Specific and complete labelling of EVs is complicated by their heterogeneity and the unavoidable presence of non‐vesicular material in EV preparations. These limitations generate a high level of false positive signal, often leading to inaccurate conclusions. There is a need for more robust labelling techniques. In this work we developed and established different techniques to label EVs – including a novel lipophilic dye, ExoriaTM, and a scalable approach to fluorescently engineer EVs – that overcome current limitations. Methods : EVs from engineered or naïve human embryonic kidney (HEK) 293 cells were produced using Exopharm capabilities, including Hexocollect for collection and LEAP for purification. A range of analytical techniques such as Asymmetric Flow‐Field Flow Fractionation, flow cytometry and Western blotting were performed to characterize the labeled EVs. Results : In‐house experiments have shown that Exoria is less prone to forming dye aggregates and that Exoria‐labelled EVs showed an increased florescence signal than all controls, with a reduced background signal as opposed to other commercially available dyes. Using Exopharm's manufacturing capabilities, we have demonstrated the scale‐up production of fluorescently engineered EVs from HEK293 cells. Over 90% of LEAP‐purified engineered EVs were mGL positive and exhibited stable fluorescence. Summary/Conclusion : Both techniques enable successful tracking of EV cell uptake as well as assay development, calibration and validation. The study also highlights the importance of fluorescently labelling EVs to comprehensively analyse their properties.

Silicon

Siddharth S. Sahu 1 ; Moein Talebian Gevari 2 ; Ábel Nagy 3 ; Maxime Gestin 3 ; Petra Hååg 4 ; Amelie E. Karlström 5 ; Kristina Viktorsson 6 ; Rolf Lewensohn 7 ; Apurba Dev 8 1 Department of Applied Physics, KTH Royal Institute of Technology, 10691 Stockholm, Sweden, Sweden; 2 Division of Solid‐State Electronics, Department of Electrical Engineering, Uppsala University, 75121 Uppsala, Sweden, Uppsala, Sweden; 3 Department of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden, Sweden; 4 Department of Oncology‐Pathology, Karolinska Institutet, 171 64 Solna, Sweden, Stockholm, USA; 5 Department of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden, Stockholm, Sweden; 6 Department of Oncology‐Pathology, Karolinska Institutet, 171 64 Solna, Sweden., Solna, USA; 7 1. Department of Oncology‐Pathology, Karolinska Institutet, 171 64 Solna, Sweden; 2. Theme Cancer, Medical Unit head and neck, lung, and skin tumors, Thoracic Oncology Center, Karolinska University Hospital, SE‐171 64 Solna, Sweden, Stockholm, USA; 8 Division of Solid‐State Electronics, Department of Electrical Engineering, Uppsala University, 75121 Uppsala, Sweden, Department of Applied Physics, School of Engineering Sciences, KTH Royal Institute of Technology, 10691 Stockholm, Sweden., Stockholm, USA Introduction : The heterogeneity of extracellular vesicles (EVs) in terms of their of surface proteins means that their immunocapture may result in a bias towards EVs with high expression of the target protein. This may affect the protein profiles of the EVs measured by surface‐based bulk sensors. However, non‐immunocapture methods need labelling which is mostly limited to fluorescence‐based methods. Besides, measuring the relative abundance of EV‐subpopulations is becoming vital even for bulk analyses. Here we show a microchip‐based electrical method that can analyze EV surface proteins related to lung cancer and EV subpopulations without requiring immunocapture. Methods : Cell culture medium of non‐small cell lung cancer (NSCLC) cells H1975 was used as a source of EVs. The EVs were isolated by size exclusion chromatography (on 70 nm qEVoriginal columns) and characterized for size and amount by nanoparticle tracking analysis and for EV surface CD9 and TSG101 expression by western blot. Streaming current was used for sensing and surface protein analysis of EVs. Capturing was done by either antibodies (biased) or non‐specifically (unbiased), followed by surface protein analysis using antibodies conjugated to charge labels. Antibodies targeting CD9, EGFR and CD73 were used. These labels comprised positively charged poly‐L‐lysine peptides or negatively charged DNA. Single‐EV fluorescence microscopy was used to verify the results. Results : Clear differences in the surface protein profiles were observed when the EVs were captured biasedly and unbiasedly. Biased capture led to higher expression level of the target protein, but the EVs were fewer in number vs. unbiased capture. Further, the possibility to perform correlation analysis between the surface proteins, and carry out sequential profiling of up to three such proteins (CD9, EGFR and CD73) on the captured EVs using the method was also shown. Summary/Conclusion : Using a combination of single and bulk EV analyses, we show that immunocapture may render bulk analysis less reliable. We then developed an electrical approach for unbiased analysis of EV surface proteins and various EV subpopulations of relevance for lung cancer. Funding : Erling Persson Family Foundation, Swedish Research Council and Swedish‐ and Stockholm Cancer Societies, Stockholm County Council. Keywords : streaming current, charge label, electrical sensor

Sorting

Dongsic Choi 1 ; Chul Won Seo 2 ; Chaeyeong Jung 2 ; Yoon‐Jin Lee 3 1 Department of Biochemistry, Soonchunhyang University, College of Medicine, Cheonan, Republic of Korea; 2 Department of Biochemistry, Soonchunhyang University, college of Medicine, Chenonan, Chungcheongnam,31151, Repubilic of Korea, Cheonan‐si, Republic of Korea; 3 Department of Biochemistry, Soonchunhyang University, College of Medicine, USA Introduction : Extracellular vesicles (EVs) are umbrella term to cover the whole lipid bilayer membrane particles. Classically, exosomes or ectosomes with canonical EV marker protein such as tetraspanins, i.e. CD63, are main subsets of EVs but recent single EV detection technologies such as single EV imaging and cytometry have been revealed the complex and heterogeneous nature of EVs with different size and molecular composition. Although it is postulated that EV subsets have different functionality, the research has been hampered due to the lack of efficient EV subset sorting technology. In this study, we developed the efficient sorting procedure of EV with different antigen‐positive, DNA‐positive, and different size by nano‐flow cytometry. Methods : CD63/GFP‐containing EVs were isolated by size exclusion chromatography. Fluorescent dye‐labeling methods were applied with CFSE or Far Red dyes for total EVs, anti‐CD147 for CD147‐positive EVs, and PicoGreen for DNA‐containing EVs. EV subsets were sorted by optimized multicolor nano‐flow cytometry with sorter, CytoFLEX SRT. Results : We established the EV sorting by nano‐flow cytometry according to different size of CD63/GFP EVs with diverse parameters. Comparing with different flow rate, low flow rate (10,000 events/sec) showed the considerable sorting of single CD63/GFP EV from other EVs. Moreover, we applied the different sorting modes including single, purity, and enrich but there is no significant difference of sorting purity at optimal flow rate. Further, we mixed the CFSE‐ and Far Red‐ labeled EVs with one‐to‐one ratio and then each subpopulation was sorted again. Sorter effectively isolated each fluorescent‐positive population without contamination of other population. We further applied EV sorting of CD147‐positive and ‐negative subpopulation by antibody labeling and we established the sorting of DNA‐positive subset among total EVs from Docetaxel‐treated cells. Summary/Conclusion : In this study, we isolated the EV subsets by nano‐flow cytometry. This approach illustrates the potential of nano‐flow cytometry coupled sorter is unique method to isolate the specific subset of EVs. Keywords : exosome, sorting, nano‐flow cytometry, DNA

Spatial

Akira Yokoi 1 ; Kosuke Yoshida 2 ; Masami Kitagawa 2 ; Hiroaki Kajiyama 1 ; Yusuke Yamamoto 3 ; Takao Yasui 2 1 Nagoya University, Nagoya, Japan; 2 Nagoya University, Japan; 3 Laboratory of Integrative Oncology, National Cancer Center Research Institute, Chuo‐Ku, Japan Introduction : Extracellular vesicles (EVs) including exosomes are recognized as promising targets of disease mechanisms. However, one of the limitations of EV analysis is the requirement for a certain volume of bio‐fluids. In addition, the location EV heterogeneity in cancer patients is largely unknown. Methods : We developed a unique nanofiber‐based sheet, named EV sheet for resolving these challenges. EV sheets successfully captured small‐EVs (sEVs) from around 10 micro litters of bio‐fluids and enabled to analyze proteins or RNAs which are included in sEVs. Results : By attaching the EV sheet to the moistened organs in vivo, the sEVs from a tiny amount of ascites were collected and miRNA sequencing for those sEVs successfully worked. In the ovarian cancer mouse model, the EV sheet revealed that cancer‐related miRNAs were detected from the very early phase when the mice did not have apparent ascites. EV sheet analysis in ovarian cancer patients, it is revealed that the tumor surface sEVs had distinct profiles from whole ascites. Furthermore, the direct EV sheet attaching method during the surgery of ovarian cancer patients identified the location‐based unique sEV miRNA profile, and it can contribute to revealing unknown mechanisms of cancer progression. Comparing advanced‐stage cases, the trajectory analysis revealed that the pattern of connection was different in patients with localized disease. Regarding biomarker application, the EV sheet has innovative properties that allow sEVs to be stored for one week in a dry condition. Tumor‐derived sEV miRNAs on tumor surfaces were also detectable in serum, urine, or saliva those sEVs were captured by EV sheet, and reflected patient conditions. Summary/Conclusion : EV sheet analysis can provide a whole new concept that the tiny amount of ascites‐sEVs from ovarian cancer patients had location heterogeneity, and can create a new biomarker strategy, contributing to cancer diagnosis, staging evaluation, and therapy planning. Funding : The Fusion Oriented Research for disruptive Science and Technology (FOREST; JPMJFR204J) from Japan Science and Technology Agency (JST) and the Project for Cancer Research and Therapeutic Evolution (P‐PROMOTE) grant number: 22ama221407h0001 from the Japan Agency for Medical Research and Development (AMED).

Surface

Kyunghwan Moon 1 ; Kangwon Lee 2 ; Jiyoon Kang 3 1 Korea Institute of Science and Technology, Seoul, Republic of Korea; 2 Seoul National University, Seoul, USA; 3 Korea Institute of Science and Technology, Seoul, USA Introduction : Recently, membrane‐modified mammalian exosome is considered as a novel nanovesicle carrier for targeted drug delivery owing to their biocompatibility, biodistribution and low immune response. There are, however, still several challenging issues with mammalian exosomes including stability and homogeneity as well as large‐scale manufacturing. To address these challenges, artificial extracellular vesicles(EVs) like nanovesicles obtained from cell membranes or liposomes from various lipids have been studied for targeted drug delivery systems, but most of them do not meet the demands of efficacy, efficiency, and less side effects. We studied on the feasibility of plant‐derived extracellular vesicles(pEVs) as targeted drug delivery carriers replacing mammalian cell exosomes and liposomes, since pEVs are non‐toxic and stable nanovesicles that can be easily internalized into mammalian cells. Moreover, pEV is suitable for mass‐production with high yield and stability. Methods : Grapefruit was used as an origin of pEVs isolated by ultracentrifugation and size exclusion chromatography. Their sizes, concentrations and shapes were characterized using DLS(Dynamic Light Scattering), NTA(Nanoparticle Tracking Analysis), and Cryo‐EM(Cryogenic Electron Microscopy), Confocal microscopy. To impart targeting ability to pEVs, a functionalizable lipid moiety with maleimide group for drug targeting was inserted in the membrane of pEVs. Results : The diameter of pEVs from grapefruit was 175 ± 12 nm and the concentrations were 1.96 × 1012 particles/ml obtained from 3 ml juice of a grapefruit. The size distribution of pEV was maintained stable at 4°C for 4 weeks. Cellular uptake of pEV was studied using U87MG and hCMEC/D3 cell line. The DiO labelled pEVs was visualized in the inside of cells by confocal microscopy. To impart targeting ability to pEVs, a functionalizable lipid moiety with maleimide group for drug targeting was inserted in the membrane of pEVs. The moiety at pEV membrane was confirmed by colocalized fluorescence of DiO and Cy5‐labelled lipids. The targeting function can be easily augmented to the moiety with click chemistry. The cellular (hCMEC/D3 and U87MG) uptake of pEV whose surface was attached by aptamer were observed using of DiO dye by confocal microscopy. The result confirmed that aptamer enhances selective cellular uptake of pEV by the increase of fluorescence intensity. Summary/Conclusion : We expect the functionalization of pEV membrane would be helpful to provide low‐cost target drug delivery carriers with no less efficacy than mammalian exosomes or liposomes with high stability and mass productivity. Keywords : Plant‐derived Extracellular Vesicle, targeted drug delivery, drug carrier

Towards

Sofie Van Dorpe 1 ; Nele De Langhe 1 ; Sarah Deville 1 ; Sandor Dedeyne 2 ; Quentin Roux 1 ; Niké Guilbert 1 ; Amélie Vander Cruyssen 1 ; Olivier De Wever 1 ; An Hendrix 3 1 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium, Gent, Belgium; 2 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium, Gent, USA; 3 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium, Ghent, Belgium Introduction : Gram‐negative and Gram‐positive bacteria release nanometer‐sized membrane vesicles enriched with bioactive proteins, lipids, nucleic acids, metabolites and virulence factors into the extracellular environment. The International Society for Extracellular Vesicles (ISEV) has previously issued consensus guidelines on EV (Théry et al., JEV, 2018). While the general principles of these MISEV guidelines are applicable, research on bacterial extracellular vesicles (BEV) encounters specific challenges and requires tailored recommendations to ensure rigor and standardization. Methods : We conducted a systematic analysis following the PRISMA guidelines of all manuscripts (n = 885), published between 2015 and 2021, preparing and analysing BEV from biological samples. We submitted experimental parameters to the EV‐TRACK knowledgebase (http://evtrack.org). Publications that included multiple sample types or EV preparation protocols were divided into multiple entries, resulting in over 3000 experiments. To assess current practice and rigor in BEV experiments, we performed an in‐depth analysis of recorded experimental parameters related to sample type, EV separation and characterization methods. Results : We revealed a high diversity in nomenclature, study aim, species of origin, sample types, separation methods, characterization methods, and storage protocols used for BEV research. Cell culture supernatant was mostly used as BEV source, while only in few experiments BEV were harvested from body fluids. Different separation methods enrich for various BEV subtypes with diverse composition and purity. In most of the experiments (differential) (ultra)centrifugation was included in the separation protocol, but with variable parameters and in combination with different techniques. Characterization of BEV was often limited to particle analysis or not provided. Based on this analysis, we identified BEV‐specific experimental parameters for inclusion and centralized the knowledge on BEV biology and methodology in the EV‐TRACK knowledgebase to support data queries and coach BEV researchers. Summary/Conclusion : The high heterogeneity in BEV research revealed by this analysis demonstrates the need for experimental and reporting guidelines to facilitate interpretation and reproducibility of BEV experiments. Funding : This work was supported by FWO, ERC, and Ghent University. Keywords : extracellular vesicles, bacteria, outer membrane vesicles, systematic review, rigor, standardization, databases

Urinary

Mei He 1 ; Kiley Graim 2 ; Padraic O'Malley 2 1 University of Florida, Gainesville, USA; 2 University of Florida, USA Introduction : The molecular mechanisms of muscle invasive bladder cancer (MIBC) phenotypes and their progression are poorly understood. Current MIBC patients with residual tumor or node positive disease have only 25% 5‐year disease‐free survival after treatment of radical cystectomy and pelvic lymph node dissection (RC‐PLND). Even though, the benefit of neoadjuvant chemotherapy (NAC) is underutilized to overcome the complication and morbidity from RC‐PLND, due to the lack of robust response prediction. Extracellular vesicles, particularly nano‐sized exosomes, have gained tremendous attention in bladder cancer therapy and diagnosis recently. The ability to pinpoint tumor markers enriched from exosomes could greatly aid our understanding of molecular mechanisms driving the initiation, progression, and malignancy of bladder cancer. Methods : Our recent studies also found the unique bladder cancer biomarkers derived from urinary exosomes with improved specificity and sensitivity for detecting bladder cancer. In this presentation, we will report a deep machine learning bioinformatic approach for multi‐omic molecular information extraction from exosome subtypes,enabling precisely definition of MIBC for therapy response prediction, which is building upon our developed NanoPoms capture‐release exosome isolation approach. The prepared exosomes are highly specific to enrich tumor markers in multi‐tissue fluids, including urine and patient plasma, which is unique biomarker sources for studying multi‐omic profiles. Mapping the multi‐omic profiles from exosome subtypes across multiple body fluids has not been explored, yet is an emerging approach for reliably and precisely modeling MIBC tumor biology. Results : The developed machine learning bioinformatic approach for spatial multi‐omic exosome analysis overcomes the challenges caused by data heterogeneity and complexity, and enables simultaneously elucidation of tissue‐specific and cross‐tissue cancer biomarkers from NGS total RNA sequencing data. The patient urine samples from healthy, non invasive stage, MIBC, and after therapeutic treatment were tested by Nanopom exosome preparation and NGS total RNA seq, which exhibited unique differentiating profile for patient therapy selection. Summary/Conclusion : This study would provide a platform to develop accurate and reliable prognostic and therapeutic predictors as the fingerprint to drive individual patient therapy selection and improve clinical and functional outcomes. Instead of relying on one single genomic or cluster of genomic markers, this technology would allow for a multi‐tissue multi‐omic modelling of a MIBC patient's cancer biology. Funding : NIH 1R35GM133794 UFHCC GU pilot.

Adapting

Meghan E. Muse 1 ; Anne G. Hoen 1 ; David A. Armstrong 2 ; Diane Gilbert‐Diamond 1 ; Jiang Gui 1 ; Tom J. Palys 1 ; Brock C. Christensen 3 ; Margaret R. Karagas 1 ; Caitlin G. Howe 1 1 Geisel School of Medicine at Dartmouth, USA; 2 Dartmouth‐Hitchcock Medical Center, Lebanon, USA; 3 Geisel School of Medicine at Dartmouth, Lebanon, USA Introduction : MicroRNAs (miRNAs) carried by extracellular vesicles and particles (EVPs) in breast milk are hypothesized to facilitate maternal‐offspring communication. The study of EVP miRNAs is complicated by the zero‐inflated nature of the data, which is not addressed by standard methods for the analysis of miRNA expression data. Here, we employ methods developed for the study of ecological and microbiome data to characterize EVP miRNA levels in a pilot study of breast milk samples from the New Hampshire Birth Cohort Study. Methods : EVP miRNAs were extracted from the supernatant of breast milk samples (n = 54) collected approximately 6 weeks postpartum using the Norgen Urine Exosome RNA Isolation kit. Extraction efficiency was monitored using a synthetic miRNA spike‐in from Oryza sativa (osa‐miR‐414). Eluted miRNAs were further purified using Amicon Ultra Centrifugal Filters and concentrated with a speed vacuum concentrator. 798 miRNAs were profiled using the Nanostring nCounter platform. We characterized and compared the total EVP miRNA transcripts measured in each sample, the number of unique miRNA transcripts present, and sample evenness, calculated using the Shannon Diversity Index, and examined relationships between these measures and maternal pre‐pregnancy BMI. Informed consent was provided by all participants. Results : Total EVP miRNA counts were negatively correlated with sample evenness (r = ‐0.37, p = 0.006) and demonstrated no correlation with the number of unique miRNAs detected (r = 0.05, p = 0.722). The five most abundant miRNAs (hsa‐miR4454+hsa‐miR‐795, hsa‐miR‐148a‐3p, hsa‐miR‐320e, hsa‐miR‐4488, and hsa‐miR‐494‐3p) accounted for a larger proportion of total miRNA counts for samples with higher overall miRNA levels. Participants with a pre‐pregnancy BMI > 25 kg/m2 had both lower total miRNA counts (p = 0.018) and a smaller number of unique miRNAs present (p = 0.026) relative to participants with a pre‐pregnancy BMI between 18.5 and 25 kg/m2. Summary/Conclusion : The application of diversity measures developed for ecological and microbiome data may be useful for characterizing EVP miRNA composition. Using these measures, we found that maternal pre‐pregnancy weight status may influence the total quantity and diversity of miRNAs in breast milk. Funding : This work was supported by NIH grants 5P20GM104416, 5UH3OD023275, and 5T32CA134286‐13. Keywords : miRNA, pregnancy

Advanced

Hyungsoon Im 1 ; Mi Ho Jeong 2 ; Taehwang Son 2 ; Jae‐Sang Hong 1 1 Massachusetts General Hospital, Boston, USA; 2 Massachusetts General Hospital, USA Introduction : Recent studies have suggested the importance of single extracellular vesicle (EV) profiling for early cancer detection, accurate quantification of cell‐specific EVs and their cargos for longitudinal monitoring, and better understanding of EV subpopulations and heterogeneity. However, multiplexed molecular characterization of single EVs is technically challenging, mainly due to weak optical signals from EVs in small sizes. My lab has developed advanced nanoplasmonic technologies for reliable and robust multiplexed single EV analysis powered by novel nanostructures and plasmon‐enhanced signal amplification. Methods : We have designed plasmonic nanostructures that exhibit strong resonances and thus significantly amplify EVs’ weak optical signals. The nanoplasmonic sensor chips were fabricated on a wafer scale for low‐cost, high‐throughput chip production. We have characterized and quantified signal amplification of fluorescently labeled EVs and particles in four fluorescence channels. We compared the system's performance with the existing standard EV analysis methods. Finally, we tested the system for cancer detection and longitudinal treatment monitoring using EVs from cell lines and human clinical samples. EVs were isolated by size‐exclusion chromatography and characterized according to MISEV2018 guidelines, including nanoparticle analysis tracking, electron micrographs, and western blot analysis. Results : We demonstrated robust signal enhancements in multiple optical channels. This allows us to significantly improve the detection sensitivity (x1,000) by capturing EVs on our plasmonic sensor chip compared to bead‐flow cytometry. More importantly, we could quantify total EVs, cell‐specific or marker‐specific subpopulations, and protein and RNAs at a single EV level for cancer detection and monitoring. Using the single EV sensing platform, we demonstrated the sensitive detection of ovarian cancer and cholangiocarcinoma from EV analysis in human clinical samples and the potential of EV analysis to evaluate patients' responses to therapy from longitudinal tumor‐derived EV quantification. Summary/Conclusion : The simple, robust, and sensitive multiplexed single EV assay could improve our understanding of EV biology and accelerate clinical translation for cancers and other diseases Funding : NIH R01GM138779 and R21CA217662. Keywords : nanoplasmonics, sensing, multiplexing, technology, cancer

Analysis

Martin E. van Royen 1 ; Natasja Dits 2 ; Gerald Verhaegh 3 ; Volkert van Steijn 4 ; Irene Bijnsdorp 5 ; Elena S. Martens‐Uzunova 2 ; Connie Jimenez 6 ; Jack Schalken 3 ; Guido W. Jenster 2 1 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands, Rotterdam, Netherlands; 2 Department of Urology, Erasmus MC, Rotterdam, The Netherlands, Rotterdam, Netherlands; 3 Department of Urology, Radboud University Medical Center, Nijmegen, The Netherlands, Nijmegen, Netherlands; 4 Department of Chemical Engineering, Delft University of Technology, Delft, The Netherlands, Delft, Netherlands; 5 Department of Urology, Cancer Center Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands, Amsterdam, Netherlands; 6 Department of Medical Oncology, Cancer Center Amsterdam, OncoProteomics Laboratory, Amsterdam UMC, Amsterdam, The Netherlands, Amsterdam, Netherlands Introduction : Urinary extracellular vesicles (uEVs) are a promising source of biomarkers for detection of prostate cancer (PCa). As a proof of concept for detection of PCa EV surface markers, we quantified uEVs in a large cohort of urines from PCa patients and analyzed CD9/CD63 status of individual uEVs and compared outcome with clinical information. Methods : Clinical urine samples were collected from men after Digital Rectal Examination (DRE) and stored at ‐80˚C. EVs in minimally processed urines of men without PCa, with indolent PCa and with significant, aggressive PCa were analyzed in three independent pre‐validation and a validation cohort. We used two complementary assays that allow high‐throughput quantification and characterization of (individual) EVs. Time Resolved‐Fluorescence Immuno Assay (TR‐FIA) measures in bulk surface marker levels of captured EVs. Immune‐EVQuant assay uses a unique in‐gel EV immobilization and fluorescent labeling to identify EV subpopulations based on their surface markers. EV concentrations and CD9/CD63 EV marker status were related to relevant clinical information (serum and urinary PSA, age at diagnosis and prostate volume). Results : A positive correlation is found between uEV concentration and uPSA, indicating that more prostate fluid in the urine is accompanied with a larger release of prostate EVs. However, total uEV concentration, uEV marker (CD9/CD63) status of individual markers in TR‐FIA analysis and combined in EVQuant did not consistently provided enrichment in urines of men with PCa. Interestingly, when corrected uPSA levels as measure for prostatic fluid in the urine samples, several marker combinations in uEVs were consistently higher for men with PCa in both assays. This indicates uPSA as strong correction factor for uEV analysis, reducing the large variability of the measured markers among urine samples. Summary/Conclusion : The complementary TR‐FIA and EVQuant assays have shown to be able to rapidly quantify uEVs in minimally processed clinical urine samples, and characterize their marker status with high sensitivity and in high throughput. However, EV markers CD9 and CD63 alone do not show diagnostic value. Only after correction for prostatic fluid levels in urine, several (CD9/CD63) marker combinations correlate with presence of (significant) PCa. Funding : Supported by IMMPROVE Alpe d'HuZes grant of the Dutch Cancer Society (EMCR2015‐8022). Keywords : urinary EVs, prostate cancer, biomarkers, CD9, CD63

Cellular

Bahnisikha Barman 1 ; Elizabeth Semler 1 ; Kasey Vickers 2 ; Alissa M. Weaver 3 1 Vanderbilt University, Nashville, USA; 2 Vanderbilt University Medical Center, Nashville, USA; 3 Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN, USA, Nashville, USA Introduction : RNA carried by extracellular vesicles (EVs) has emerged as a novel mechanism for cell‐to‐cell communication and drives many physiological and pathological processes, including cancer. Previously, we identified the conserved endoplasmic reticulum membrane contact site (ER MCS) linker proteins VAP‐A and the ceramide transfer protein CERT as significant regulators of the RNA and RNA‐binding protein content of a subpopulation of small EVs. Since VAP‐A also binds the cholesterol transporter Oxysterol binding protein‐related protein 1L (ORP1L) and cholesterol may contribute to EV biogenesis, here we explored the role of cholesterol and ORP1L in the regulation of RNA‐containing EVs. Methods : For cholesterol depletion conditions, DKs‐8 cells were cultured for 96h in 10% lipoprotein‐depleted serum (LDS) supplemented with 250 μM mevalonate and 10 μM mevastatin for 96h. Small EVs were purified by cushion density gradient from control and cholesterol‐depleted colon cancer cells. We used confocal, electron microscopy, and various biochemical techniques to analyze EV biogenesis and cargo content. Results : We observed a substantial alteration of EVs secreted from colon cancer cells cultured in lipoprotein‐depleted growth media and inhibited for cholesterol synthesis. QRT‐PCR for candidate microRNAs showed a significant alteration in the RNA content of EVs purified from cholesterol‐depleted cells. Knockdown of the VAP‐A‐binding cholesterol transfer protein ORP1L led to a substantial alteration in the RNA contents of small and large EVs. We propose that cholesterol binding and/or transfer via ORP1L at MCS affects the biogenesis of RNA‐containing small and large EVs. Summary/Conclusion : Altogether, these data suggest cholesterol transfer and/or sensing at ER MCS regulates RNA trafficking into small and large EVs. Funding : Funding was provided by NIH grants U19CA179514 and PO1CA229123. Keywords : extracellular vesicles, cholesterol sensing, membrane contact sites, extracellular RNA

Clinical

Matthew H. Forsberg 1 ; John A. Kink 1 ; Michael A. Bellio 2 ; Alexandra Lobo 1 ; Anna S. Thickens 1 ; Bryson M. Lewis 1 ; Irene M. Ong 1 ; Aisha Khan 2 ; Christian M. Capitini 3 ; Peiman Hematti 1 1 University of Wisconsin School of Medicine and Public Health, USA; 2 University of Miami Miller School of Medicine, USA; 3 University of Wisconsin School of Medicine and Public Health, Madison, USA Introduction : Ionizing radiation can lead to hematopoietic acute radiation syndrome (H‐ARS). As a potential countermeasure, we developed a bioreactor‐based, large‐scale production of clinical‐grade exosomes derived from lipopolysaccharide (LPS) primed bone marrow (BM)‐mesenchymal stromal cells (MSCs) employing good manufacturing practice (GMP) standards. Methods : Exosomes isolated by ultracentrifugation from unprimed or LPS‐primed BM MSCs were grown in a Quantum hollow‐fiber bioreactor and compared to gold standard, flask‐scale exosomes. The physical identity of exosomes included a time course assessment of particle diameter, yield, protein content and surface marker profile by flow cytometry. Comparison of the RNA cargo in exosomes was determined by bulk RNA‐seq. Capacity of exosomes to generate exosome educated monocytes (EEMos) was determined by qPCR and flow cytometry, and potency was assessed using a lethal H‐ARS model with NSG mice. Results : MSC‐exosomes produced by flask and bioreactor are similar in terms of purity by particle size/protein content and identity by flow cytometry. RNA–Seq comparison of the cargo within MSC‐exosomes indicate flask‐derived exosomes upregulate micro‐RNAs including the let‐7 family and miR‐143. MSC‐exosomes generated by bioreactor produces much higher yields (up to 50‐fold) from relatively small media volumes. Comparing the ability to educate monocytes, MSC‐exosomes generated by either platform increase the gene expression of IL‐6, IDO and FGF‐2. Significant increases in M2 markers like PD‐L1 (p< 0.0001) and CD163 (p< 0.0001) and significant decreases in M1 markers CD86 (p< 0.0001) and CD16 (p< 0.0001) are present in monocytes by flow cytometry after education using flask or bioreactor MSC‐exosomes. Bioreactor MSC‐exosomes are as effective as flask MSC‐exosomes as treatment in a lethal H‐ARS mouse model, significantly extending survival and improving clinical scores (p< 0.05). Induction of hematopoiesis is observed in peripheral blood from recipients of bioreactor MSC‐exosomes, with enhanced absolute neutrophil and lymphocyte counts at Day 30–31 post‐treatment. Summary/Conclusion : Overall, we describe a GMP‐compliant, clinical scale biomanufacturing platform for the production and characterization of therapeutic MSC‐exosomes as an “off the shelf” countermeasure for H‐ARS. Funding : Production Assistance for Cellular Therapies (PACT) program from the NIH/NHLBI at the University of Miami (PACT Contract No. HHSN268201600012I. (J.A.K, P.H.). Bioinformatics analysis by A.L. was supported through the National Library of Medicine (5T15LM007359). This work was also supported by the Don Anderson GVHD fund and Crystal Carney Fund for Leukemia Research (P.H.), St. Baldrick's‐Stand Up to Cancer Pediatric Dream Team Translational Research Grant SU2C‐AACR‐DT‐27‐17 and NIH/NHLBI R01 HL153721 (P.H. and C.M.C.). Keywords : exosomes, GMP, mesenchymal stromal cells, TLR4, acute radiation syndrome

Delivery

Xinyi Sun 1 ; Yi Zhang 2 ; Ye Shen 3 ; Yunhui Tang 4 ; Larry w. Chamley 5 ; Min Zhao 3 ; Qi Chen 6 1 Department of Obstetrics and Gynaecology, The University of Auckland, New Zealand; 2 Department of Obstetrics and Gynaecology, The University of Auckland, auckland, New Zealand; 3 Wuxi Women's Hospital, Wuxi China, China (People's Republic); 4 The Hospital of Obstetrics and Gynaecology, Fudan University, China (People's Republic); 5 Department of Obstetrics and Gynaecology, Room 201J Building 502 FMHS,University of Auckland, Auckland, New Zealand; 6 Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand Introduction : Ovarian cancer is a leading cause of death in gynaecological cancers, and current treatment has limited efficacy. There is a similarity between the human placenta and tumours, as the placenta is invasive and develops its own vasculature. Unlike tumorus, the development of the placenta is strictly controlled. One control mechanism is placental extracellular vesicles (EVs). Placental EVs carry many functional proteins, regulatory RNAs, DNA, and lipids. Previous studies have shown that placental EVs significantly reduced the growth of ovarian cancer cells. However, the mechanism underlying this inhibitory effect is unclear. In this study, we investigated the levels of miRNAs and their target mRNAs in ovarian cancer cells (SKOV‐3 cells) after exposure to placental EVs. Methods : Placental EVs (n = 8) were collected from the first trimester placentae by differential centrifugation. SKOV‐3 cells were cultured with or without placental EVs for 24 hours and total RNA was then collected for miRNA and mRNA sequencing and quantification. Results : There was a significant increase in the level of miRNA‐519a‐5p and miRNA‐143‐3p in SKOV‐3 cells after exposure to placental EVs. miRNA sequencing showed high levels of miRNA‐519a‐5p and miRNA‐143‐3p in placental EVs. The mRNA levels of Ras‐GTPase‐activating protein binding protein 1 (G3BP1) and E3 ubiquitin‐protein ligase CBL‐B (CBLB) which are target genes of these two miRNAs were significantly reduced in SKOV‐3 cells after exposure to placental EVs. Both G3BP1 and CBLB are associated with the development of ovarian cancer. Transfection of SKOV‐3 cells with mimics of miRNA‐519a‐5p or miRNA‐143‐3p significantly reduced the viability of SKOV‐3 cells. Summary/Conclusion : Our data demonstrate that the delivery of miRNAs that are associated with the promotion of cancer cell death by placental EVs could contribute to the reduction of ovarian cancer cell growth. Our findings provide a novel pathway for therapeutic intervention in ovarian cancer.

Dynamics

Maria Harmati 1 ; Akos Diosdi 2 ; Ede Migh 2 ; Gabriella Dobra 1 ; Matyas Bukva 3 ; Timea Boroczky 1 ; Edina Gyukity‐Sebestyen 1 ; Ferenc Kovács 4 ; Peter Horvath 2 ; Krisztina Buzas 5 1 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eotvos Lorand Research Network, Szeged, Hungary, Szeged, Hungary; 2 Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, Eotvos Lorand Research Network, Szeged, Hungary, Hungary; 3 Department of Immunology, Faculty of Science and Informatics, University of Szeged, Szeged, Szeged, Hungary; 4 Single‐Cell Technologies Ltd., Szeged, Hungary, Hungary; 5 Department of Immunology, University of Szeged, Szeged, Szeged, Hungary Introduction : Evolutionary process of solid tumors highly relies on the extracellular vesicle (EV)‐mediated cross‐talk between malignant cells and stromal cells in the tumor microenvironment (TME). In this study, we aimed to establish a multicellular three‐dimensional (3D) tumor model system for tracking the EV communication network of different tumor tissues under physiological conditions and cytostatic treatments. Methods : Human ductal carcinoma, melanoma and osteosarcoma models were established via co‐culturing the respective tumor cell line (T‐47D/A375/MG‐63) with MRC‐5 fibroblasts and EA.hy926 endothelial cells on flat‐ or U‐bottom plates after staining with CellTracker dyes (Orange CMTMR, Deep Red, Green CMFDA). To mimic chemotherapeutic stress, low dose doxorubicin were used and the 2D and 3D cultures were imaged daily by a PerkinElmer Operetta High Content Screening Sytem and a Leica SP8 Digital LightSheet microscope, respectively. Results : We showed that CellTracker dyes can be used for in‐cell labelling of EVs, allowing the quantitative monitoring of EV cross‐talk, i.e. EV routes between each cell type and in both directions. The three types of tumor models showed differences in their 3D structure, EV cross‐talk activity and drug‐induced effects as well. We could observe distinct temporal kinetics in the development of the EV communication network in 2D and 3D, also priorities of the investigated EV routes varied between the two co‐culture systems. Summary/Conclusion : The developed 3D model system is suitable for live tracking of EV cross‐talk in the TME, which can dinamically change depending on the microenvironmental conditions. Further data will help (i) to identify potential targets of EV‐blocking therapies, which may increase the efficacy of chemotherapies, and (ii) to predict the drug‐induced changes of the communication activity in different tumor tissues. Funding : TKP2021‐EGA09, Szent‐Györgyi Albert Research Fund (University of Szeged), OTKA‐K143255. Keywords : 3D tumor model, EV cross‐talk, EV tracking, tumor microenvironment

Enabling

Katalin Korpany 1 ; Lucia Vojtech 2 ; Alexis Stamatikos 3 ; James J. Lai 4 1 Bioengineering, University of Washington, USA; 2 Obstetrics & Gynecology, University of Washington, Seattle, USA; 3 Department of Food, Nutrition, and Packaging Sciences, Clemson University, USA; 4 Materials Science and Engineering, National Taiwan University of Science and Technology, Taiwan (Republic of China) Introduction : The utility of extracellular vesicles (EV) for biomedical applications have been demonstrated. However, their clinical translation is hindered by technical issues—mostly related to isolation. Common processing methods result in variable yield and purity, are time consuming, require complex procedures, or are difficult to scale up. To address these challenges, temperature‐responsive (smart) polymeric reagents were developed to enable rapid and specific EV isolation. The reagents diffuse rapidly at room temperature to promote EV binding, and form aggregate at 40°C to facilitate separation via benchtop centrifugation. Methods : The polymer‐antibody (Ab) conjugates were synthesized by covalently linking azido‐polymer(N‐isopropylacrylamide) to dibenzocyclooctyne modified anti‐mouse IgG Ab using click chemistry. Raw cow milk, processed to remove cream, was used as the specimen. Isolation of EVs from milk is done by adding a transmembrane immunoglobin and mucin domain‐4 (TIM‐4):mouse Fc construct, which targets phosphatidylserine (PS) expressed by all EV. Then, the polymer‐Ab conjugates were added to recognize mouse Fc, bound on PS. The EV were separated by centrifuging the solution at 12000 rpm (12,386 × g) and 40°C for 5 minutes. After the supernatant was removed, the captured EV were characterized and quantified by RT‐qPCR. Results : To evaluate the isolation process, microRNA let‐7b, found in high abundance in bovine milk EV, was used. An equivalent volume of unprocessed milk was also analyzed to estimate capture efficiency. Ct values for let7‐b were normalized to the miR‐cel‐39 spike‐in control, which was added before RNA extraction of the collected EVs. Compared to the control group, mouse IgG1 kappa isotype, isolation via the TIM‐4 construct led to a 4–13 fold increase in let‐7b. The EV isolation efficiency was estimated to be 51–77%. The total process duration is 75 min and consists only of 4 steps. Summary/Conclusion : The smart polymeric reagents have demonstrated EV isolation from raw, unpasteurized bovine milk by targeting the lipid PS, expressed on EV membranes. Compared to the common EV isolation approaches, the new method is rapid and can potentially be scaled to larger processing volumes.

Enhanced

Meishan Wu ; Angela C. Brown Lehigh University, Bethlehem, USA Introduction : Outer membrane vesicles (OMVs) are spherical proteoliposomes derived from the outer membrane of Gram‐negative bacteria that are capable of encapsulating and delivering biomolecules, such as toxins and genetic material. Inspired by their ability to cross the complex Gram‐negative outer membrane, our research focuses on utilizing OMVs as a delivery vehicle for antibiotics. Several passive and active methods have been chosen to test loading optimization. We hypothesize that loading efficiency is based on the hydrophobicity of the antibiotics, and that active methods are more efficient than passive methods. Methods : OMVs were harvested from the hypervesiculating E. coli JC8031 strain and loaded with ciprofloxacin, norfloxacin, levofloxacin, or moxifloxacin. The two passive loading methods were incubation of free‐antibiotics with purified OMVs, and direct incubation, in which free‐antibiotics were incubated in the E. coli culture and aOMVs were harvested. The two active loading methods were electroporation and sonication. The encapsulation efficiency was determined by quantifying the antibiotic concentrations before and after loading using UV‐vis spectroscopy. Results : Direct incubation resulted in minimal/no encapsulation of any antibiotic. Of all the loading methods, electroporation provided the highest encapsulation efficiency for all antibiotics except levofloxacin. For levofloxacin, simple mixing with purified OMVs was the most effect method. The active loading methods were more effective than the passive loading methods in encapsulating norfloxacin and ciprofloxacin. Moxifloxacin showed the overall highest encapsulation efficiency, supporting our hypothesis that hydrophobic antibiotics gain entry into OMVs more effectively than hydrophilic ones. Summary/Conclusion : These results demonstrate that fluoroquinolone antibiotics can be readily incorporated into OMVs to create novel delivery vehicles. Funding : National Institutes of Health ( DE027769 ). Keywords : outer membrane vesicles, loading, antibiotics

Ev‐Dna

Inbal Wortzel 1 ; Han Sang Kim 2 ; David C. Lyden 3 1 Weill Cornell Medicine, New York, USA; 2 Yonsei Cancer Center, USA; 3 Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA, New York, USA Introduction : Cells actively secrete extracellular vesicles and particles (EVs), transporting selectively packaged functional biomolecules to mediate intercellular communication in normal physiology and pathology. We previously showed that EVs contain DNA (EV‐DNA) representing the entire genome and reflecting the mutational status of parental cells. However, the underlying mechanisms of DNA packaging into EVs and their functional consequence in human diseases including malignancy are still elusive. Methods : Here, via super‐resolution imaging of single EVs and biochemical assay, we show that only 30 % of vesicles are DNA positive, and the majority of EV‐DNA is presented on the surface of EVs. Histones associated with EVs are remarkably distinct from their cellular counterparts in their variants, size and post‐translational modifications, indicating/suggesting unique chromatinization of EV‐DNA. To identify factors essential for DNA loading on EVs, we designed a novel strategy of genome‐wide CRISPR knockout screening in multiple cancer types. And performed in‐vivo experiments to study how EV‐DNA affects cancer progression. Results : The screen uncovered several immune‐developmental pathways and genes (e.g., APAF1, NCF1) that play essential roles in EV‐DNA packaging. Furthermore, using colorectal cancer as a model system, we found that the amount of EV‐DNA is inversely correlated with tumor metastatic potential in animal models. We show that tumor‐derived EVs are taken up by Kupffer cells (KCs) in the liver and activates DNA damage response signaling in an EV‐DNA‐dependent manner, and stimulate the secretion of anti‐tumor cytokines from KCs. Finally, analysis of patient‐derived tissue EVs showed that the amount of EV‐DNA can serve as a predictive biomarker for metastasis. Summary/Conclusion : Our work suggests that EV‐DNA induces immune surveillance in the pre‐metastatic organ, and provides molecular insight that tumor‐derived EV‐DNA activates anti‐tumor immunity and prevents metastatic progression. Funding : This work is supported by the ‘Research Assistance for Primary Parents’ grant from the Mastercard Diversity‐Mentorship Collaborative at Weill Cornell Medicine, and by the ‘Worldwide Cancer Research Foundation’ (23‐0105). keywords : cancer, metastasis

Exomicro

SeHyun Shin 1 ; MinJu Bae 2 ; JunSoo Park 3 ; HyeonAh Seong 3 1 Korea University (Microgentas), USA; 2 Korea University, Seoul, Republic of Korea; 3 Korea University, USA Introduction : Extracellular vesicles (EVs) are receiving great attention as biomarkers in liquid biopsies since they are involved in physiological and pathological processes. It is important to obtain a high‐yield of nucleic acids because it is possible to prediagnose through the exosome. Methods : Here, we report an efficient one‐step method for extraction of miRNAs by clustering EVs. When cationic salt is added to samples, anionic EVs tend to form clusters and precipitate within a short time. After centrifugation and adding lysis buffer, miRNA can be extracted with a spin column. Commercial methods were compared to determine the yield of the precipitation method using cationic salt. In addition, we compared with two reference methods to determine the miRNA extraction efficiency. The first one is consisted of two‐step method of extracting miRNA from isolated exosome (2 step), which is a general method, where as the second reference method is a commercial product consisting of 1‐step extracting miRNA from biofluids. Results : Cluster precipitation efficiency using cationic salt was identified through cluster image, SEM, and Western blot. The present method for extract exosomal miRNA has an EV isolation efficiency of more than 3 times compared to the commercial EV isolation methods. In addition, it has excellent exosomal miRNA extraction efficiency from various biofluids such as Saliva, Urine, and Plasma, and can be used as a sample of breast cancer patients. Summary/Conclusion : These results might be contributed by the sequence of precipitation of exosomes, exosome lysis, and nuleic acid extraction. The rapid and efficient method for extracting exosomal nucleic acids will make an innovative contribution to clinical applications. Funding : This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government, MSIP (2016R1A5A1010148). Keywords : exosome, exosome isolation, exosomal miRNA, precipitation, Cationic salt

Exophers

Ibrar A. Siddique 1 ; Jing Di 2 ; Harry K. Vinters 1 ; Daniela Markovic 3 ; Williams K. Christopher 1 ; Saumya Tawakley 1 ; Joanna Zhao 1 ; Kylie McCauley 1 ; Gal Bitan 1 1 UCLA, Los Angeles, USA; 2 U Kentucky Medical Center, Los Angeles, USA; 3 UCLA David Geffen School of Medicine Department of Internal Medicine., USA Introduction : We have discovered that exophers, which previously had been described in C. elegans, exist in mammalian neurons. Exophers are thought to be a mechanism for non‐autonomous degradation of unwanted cellular material. Unlike other extracellular vesicles, exophers can be as large as the cell itself. They may remain connected to the cell via a nanotube for many hours and continue to receive cellular material. Proteostatic stress has been shown to increase exopher number in C. elegans yet whether this is also the case in mammalian neurons is not known. It is also unknown if the size of exophers is responsive to the stress level. Methods : Exophers were analyzed using light and fluorescence microscopy in cell culture systems, including human iPSC‐derived neurons, primary mouse neurons from three different lines, and in human and mouse brain sections. Human brains included patients with Alzheimer's disease (AD), other tauopathies, and controls. They were stained, as appropriate, for markers of the neuronal cell body, hyperphosphorylated tau, Aβ42, and nuclei. Results : In cell culture, the number of exophers increases over time and in response to tauopathy, yet the response differs in different cellular models and in neurons from different mouse models. Interestingly, in human brain, although both Aβ and tau pathologies correlate with increased exopher number, only tau pathology appears to increase exopher size, a finding that is recapitulated in cell‐culture models. Initial co‐culture experiments suggest that neuronal exophers are taken up by microglia for processing of their content. Summary/Conclusion : Exophers are a newly discovered type of extracellular vesicles, apparently playing important roles in non‐autonomous degradation of unwanted cellular material. In the context of AD and other tauopathies, they may be a double‐edged sword – helping clear proteotoxic aggregates, but also facilitating cell‐to‐cell spread of the pathologic protein aggregates. Keywords : exopher, tauopathy, alzheimer's disease.

Exosomal

Disha Nagesh Moholkar 1 ; Raghuram Kandimalla 2 ; Jeyaprakash Jeyabalan 3 ; Ramesh Gupta 4 ; Farrukh Aqil 5 1 Department of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40202, Louisville, USA; 2 Brown Cancer Center, Department of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40202, Louisville, USA; 3 3P Biotechnologies, Louisville, KY 40202, Louisville, USA; 4 Brown Cancer Center, Department of Pharmacology & Toxicology, University of Louisville, 3P Biotechnologies, Louisville, KY 40202, Louisville, USA; 5 Brown Cancer Center, Department of Medicine, University of Louisville, Louisville, KY 40202, Louisville, USA Introduction : The current cancer therapies are limited by lack of specificity and the numerous adverse effects. Cannabidiol (CBD) is garnering attention in the scientific field due to its potential as an anti‐cancer therapeutic agent. However, its application is limited due to low oral bioavailability. Here, we show that CBD can be administered orally in folic acid (FA) functionalized tumor targeted exosomal formulation to enhance its efficacy and minimize toxicity. Methods : Exosomal formulation of CBD (ExoCBD), prepared by incubation of CBD with bovine colostrum exosomes, were characterized for size, charge and hallmark protein markers. Cell culture assays (MTT, colony forming) were performed to analyze the anti‐cancer activity of the CBD and ExoCBD against drug sensitive and resistant breast cancer cells. In the ongoing in vivo study, free CBD and ExoCBD formulations, with and without a targeting ligand, FA are being tested for efficacy against orthotopic breast cancer xenograft in a murine model. Results : Size of exosomes (78 ± 2.4 nm) somewhat increased in ExoCBD (123 ± 1.4 nm) and FA‐ExoCBD (122 ± 1.3 nm) formulations. Drug load, as measured by UPLC, was 15% and 25% with Exo and FA‐Exo, respectively. ExoCBD showed enhanced antiproliferative activity vs. free CBD against MCF7 and MDA‐MB‐231; this finding was supported with colony forming assay. CBD and exosomal CBD chemosensitized the drug‐resistant MCF7‐TR and MDA‐MB‐231‐TR cells to paclitaxel. Analysis of cell lysates by Western blot showed downregulation of CB2 and MDR‐1 receptors. We hypothesize that FA‐ExoCBD would enhance the anti‐cancer activity of CBD by enhancing oral bioavailability and targeting breast tumors in the murine model. Summary/Conclusion : The ExoCBD showed enhanced antiproliferative activity in breast cancer cells accompanied by downregulation of CB2 and MDR‐1 receptors. Funding : Supported from Duggan Endowment: RCG and University's IPIBS fellowship: DNM. Keywords : colostrum exosomes, breast cancer, CBD, folic acid functionalization

Exosomes

Justin B. Nice 1 ; Mafalda Cacciottolo 1 ; Yujia Li 2 ; Michael LeClaire 2 ; Ryan Twaddle 2 ; Ciana Mora 2 ; Stephanie Adachi 2 ; Esther Chin 2 ; Meredith Young 2 ; Jenna Angeles 2 ; Kristi Elliott 2 ; Minghao Sun 2 1 Capricor Therapeutics, San Diego, USA; 2 Capricor Therapeutics, USA Introduction : Here we designed an exosome‐based, bivalent vaccine with the goal of producing broader immunity against SARS‐CoV‐2 using Capricor's Stealth X technology. Spike, in its naturally folded structure presented on the surface of exosomes, was designed to protect against newer spike mutations. In addition, nucleocapsid is a more conserved SARS‐Cov‐2 protein than spike. Both cell lines produced exosomes highly enriched in the desired protein, and when combined in a in vivo model produced a strong immune response and T‐cell memory with only nanogram levels of protein presented on the surface of exosome. Methods : Cells were engineered using lentiviral transduction and expanded for exosome production and the resulting cell‐free supernatant was concentrated using a tangential‐flow filter (TFF), proteins were then removed using Sephacryl size exclusion chromatography, and the resulting exosome‐containing fractions concentrated again using TFF. Exosome size and concentration was measured using ParticleMetrix ZetaView. Spike or nucleocapsid protein concentration was measured using an ELISA assay. Mice were injected with two rounds of spike‐expressing and nucleocapsid‐expressing exosomes (day 0 and 21) and immune response was measured by ELISAs for IgG against spike or nucleocapsid at days 14 and 35. Results : Results showed that IgG production against spike increased up to 1,500‐fold while IgG against nucleocapsid increased up to 7‐fold. Neutralizing of different VOCs of SARS‐CoV‐2 was carried out by Retrovirox, Inc. The results showed that mouse plasma could neutralize delta variant at 100%, as well as partial protection against omicron BA.1 and BA.5.2.1 variants. Summary/Conclusion : This study demonstrates that exosomes can be engineered with viral antigens to be used for rapid vaccine development with implications beyond COVID‐19 and to other vaccines that combine the power of Stealth X targeting, multiplexing, low dosage, better safety profile and short turnaround time. Keywords : COVID‐19, SARS‐CoV2, engineered‐exosomes

Function

Soon‐Kyung Hwang 1 ; Myung‐Haing Cho 2 1 RNABIO, Inc,, Seongnam‐si, Republic of Korea; 2 RNA BIO, Inc., Republic of Korea Introduction : The midbody some (MBsome), once thought to be a remnant of cell division, is now known to play a key role in cytokinesis. It was thought that the MBsome is either released into the extracellular space or autophagically degraded by one of its daughter cells. However, recent studies have revealed that MBsomes can be maintained by cells even after cell division is complete, and that they accumulate in the cytoplasm and regulate cell proliferation and survival through integrin and epidermal growth factor receptor‐dependent pathways. Methods : we examined the ability of MBsomes to act as carriers of mRNAs, a novel function that has not been studied. Results : We found that MBsomes isolated from human lung cancer and stem cells via sucrose cushion ultracentrifugation were 300–400 nm in size and stable for up to 4 days when stored at 4°C. In addition, we confirmed successful expression of the EGFP protein following incubation of the isolated MBsomes with the EGFP mRNA at room temperature. Summary/Conclusion : These results suggest that MBsomes have the potential to serve as mRNA carriers and therapeutic agents capable of delivering a gene‐of‐interest. Funding : This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF‐2022R1A2C100780111). Keywords : MBsome, mRNA carrier, sucrose cushion ultracentrifugation

Isev2023

IOC Chairs: Dolores Di Vizio (Italy), Uta Erdbruegger (Germany) IOC Members: Dylan Burger (Canada), Tom Driedonks (Netherlands), Muller Fabbri (Italy), Sai Lim Kiang (Singapore), Metka Lenassi (Slovenia), Ursula Sandau (USA), Edwin Van Der Pol (Netherlands) and Sara Veiga (USA)

Magnetic

Wenshen Wang 1 ; Safiya Aafreen 2 ; Olesia Gololobova 3 ; Kenneth W. Witwer 4 ; Jeff Bulte 5 ; Ethel Ngen 5 ; Guanshu Liu 6 1 F.M Kirby Center, Kennedy Krieger Institute; Department of Radiology, Johns Hopkins University,, USA; 2 F.M Kirby Center, Kennedy Krieger Institute; Department of Radiology, Johns Hopkins University; Department of Biomedical Engineering, Johns Hopkins Univeristy;, Baltimore, USA; 3 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, USA; 4 Johns Hopkins University, Baltimore, USA; 5 Department of Radiology, Johns Hopkins University, USA; 6 F.M Kirby Center, Kennedy Krieger Institute; Department of Radiology, Johns Hopkins University, Baltimore, USA Introduction : Up to 90% of brain tumor patients treated with radiotherapy will suffer from radiotherapy‐induced brain injury (RIBI) and there is no effective treatment or prevention yet. Stem cell‐derived extracellular vesicles (EVs) are emerging as a new regenerative approach for RIBI. EVs show superior properties compared to synthetic nanoparticles for drug delivery. In the context of these developments, imaging guidance plays a vital role by visualizing the temporal and spatial distribution of EVs and encapsulated drugs. Here, we aimed to develop a magnetic resonance imaging (MRI)/ magnetic particle imaging (MPI) trackable, liposome‐EV biohybrid system for image‐guided, effective treatment for RIBI. Methods : Induced pluripotent stem cell (iPSC)‐derived EVs were isolated from conditioned media of iPSC culture using size exclusion chromatography (SEC). Liposomes (PC:PS = 2.5:1) were prepared using the thin‐film hydration method and loaded with superparamagnetic paramagnetic iron oxide nanoparticles (SPIONs, 20 nm) as imaging agent and IL‐10 as therapeutic agent, and then fused with EVs by PEG‐mediated fusion. The RIBI mouse model was induced by X‐ray irradiation (80 Gy) under CT guidance. Two days post radiation, 108 EVs (2 μL in PBS) were stereotactically injected into the irradiated brain hemisphere, and MRI and MPI were performed over a period of 4 weeks. Results : We have prepared biohybrid EVs that share similar characteristics (i.e., size and cell internalization) as native iPSC‐EVs whilst loaded with therapeutic and imaging agents. Moreover, biohybrid EVs could be readily detected by T2*‐weighted MRI and MPI, and the MRI/MPI signal persisted for up to one month. Summary/Conclusion : Our study demonstrates the feasibility of developing MRI and MPI‐guided EV‐based therapy or drug delivery systems to ameliorate RIBI. Funding : NIH R33 HL161756, R01CA262887 and S10 OD026740 . Keywords : radiotherapy‐induced brain injury, magnetic resonance imaging, magnetic particle imaging, iPSC EV, biohybrid AND IL‐10

Microrna

Islam M. Saadeldin 1 ; Bereket Molla Tanga 1 ; Seonggyu Bang 1 ; Heejae Kang 2 ; Dabin Cha 2 ; Sanghoon Lee 1 ; Jongki Cho 1 1 Chungnam National University, Daejeon, Republic of Korea; 2 Chungnam National University, Republic of Korea Introduction : MiRNAs are small non‐coding RNA molecules that play important regulatory roles in diverse biological processes. Royal jelly, a milky‐white substance produced by nurse bees, is the primary food of queen bees and plays a crucial role in their development. However, little is known about the microRNAs (miRNAs) content of royal jelly and their potential functions. Anastasis is the mechanism by which cells can recover from apoptotic lesions and revert to their previous functioning state from the brink of cell death. Methods : In this study, we isolated nanovesicles from the royal jelly of 60 samples through sequential centrifugation and targeted nanofiltration, and performed high‐throughput sequencing to identify and quantify the miRNA content of honeybee Apis mellifera royal jelly extracellular vesicles (RJEVs). RJEVs were visualized through cryogenic transmission electron microscopy, and the size and concentration were evaluated through nanoparticle tracking analysis. To investigate the potential roles of RJEVs in cell viability, RJEVs were supplemented to apoptotic cells induced by ethanol 6% exposure for 30 min, and cell migration and viability were also checked. Results : We found a total of 27,134 mapped sequences and 29 known mature miRNAs and 17 novel miRNAs. Through bioinformatic analysis, we identified several potential target genes of the miRNAs present in royal jelly, including those involved in developmental processes and the cell differentiation. TUNEL assay showed a significant reduction in the apoptosis percentage when apoptotic cells supplemented with RJEVs when compared with the control no‐supplemented group. Moreover, wound healing assay performed on the apoptotic cells showed a rapid healing capacity of RJEVs‐supplemented cells when compared with the control no‐supplemented group. We observed a significant reduction in the expression of target genes such as FAM131B, ZEB1, COL5A1, TRIB2, YBX3, MAP2, CTNNA1, and ADAMTS9 suggesting that RJEVs may play a role in the regulation of gene expression of cellular motility and cell viability. Moreover, RJEVs reduced the expression of apoptotic genes (CASP3, TP53, BAX and BAK), while significantly increased the expression of anti‐apoptotic genes (BCL2 and BCL‐XL). Summary/Conclusion : Our findings provide the first comprehensive analysis of the miRNA content of RJEVs and suggest a potential role for these vesicles in the regulation of gene expression and cell survival as well as augmenting cell resurrection or anastasis. Funding : NRF Grants 2021R1A2C2009294 and 2022R1I1A1A01065412, and Brain Pool program Grant 2021H1D3A2A02040098. Keywords : royal jelly, cell viability, anastasis, apoptosis, cryo‐TEM, miRNAome

Modeling

Ruben R. Lopez 1 ; Chaymaa Zouggari 2 ; Thupten Tsering 3 ; Yunxi Chen 4 ; Prisca Bustamante 1 ; Vahe Nerguizian 5 ; Julia V. Burnier 6 1 McGill University, Verdun, Canada; 2 École de technologie supérieure, USA; 3 Research Institute of the McGill University Health Centre, Montreal, USA; 4 Research Institute of the McGill University Health Centre, USA; 5 École de technologie supérieure ÉTS, Montreal, Canada; 6 Research Institute of the McGill University Health Centre, montreal, Canada Introduction : Extracellular vesicles (EVs) play a crucial role in disseminating cancer to distant organs, communicating with the tumor microenvironment (ME) through cellular uptake by recipient cells. EV parameters influencing cellular uptake include surface proteins, lipid profile, and physicochemical properties such as size and zeta potential. However, EVs are heterogeneous, making it difficult to study single EV variables. Moreover, EV isolation is a lengthy and laborious process with an extremely low yield. Liposomes are synthetic vesicles that share properties with EVs, such as a lipid bilayer and the capability to encapsulate biomolecules. Our aim was to develop a synthetic system to mimic EVs by producing “EV‐like liposomes”. Methods : Our approach was to “progressively mimic” cancer EVs. To do this, we aimed to: 1) characterize the physicochemical and lipidomic profile of EVs isolated from a panel of cancer cells; 2) produce EV‐like liposomes through microfluidics; and 3) assess their uptake by ME cells in vitro and 4) in vivo. 1) EVs were isolated using ultracentrifugation (UC) and characterized using dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy (TEM). Lipids were extracted for high‐throughput lipidomics by high‐resolution ‘shotgun’ mass spectrometry. 2) EV‐like liposome formulations were produced using the periodic disturbance mixer (nanoprecipitation). 3) Cellular uptake of labeled EV‐like liposomes was assessed in human hepatocytes and fibroblasts through microscopy and Incucyte. Finally, we assessed the biodistribution of EV‐like liposomes in SCID mice after tail vein injection. Results : Using surface response methodology, we created a model to predict size and zeta potential. We produced liposomes ranging in zeta potential (0‐∼‐30 mV) and size (100∼200 nm), mimicking the characterized cancer EVs. TEM demonstrated EV‐like morphology. We identified four lipid species that segregated EV subpopulations. Our data confirmed uptake of EV‐like liposomes by ME cells within 12 hours. Modulating size, zeta potential and lipid profile had significant impact on cellular internalization. In‐vivo imaging indicated high accumulation of EV‐like liposomes in the kidney and liver, with no indication of organ damage. Summary/Conclusion : We describe a novel approach using synthetic biology to produce EV‐like liposomes. Our data show that liposomes are a feasible tool to study EV variables individually, thereby addressing the high heterogeneity of biological samples and producing high liposome yield. This model is therefore valuable to accelerate research on the contribution of EVs in disease, such as cancer metastasis. Funding : New Frontiers in Research Fund Exploration grant (to JVB). Keywords : EVs, metastasis, liposomes, biomedical engineering, synthetic EVs

Negative

Yonghyun Kwon 1 ; Sunyoung Park 2 ; Kyung‐A Hyun 2 ; Hyo‐Il Jung 3 1 Laboratory of Biochip Technology, School of Mechanical Engineering,Yonsei University, Republic of Korea; 2 School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea, Seoul, Republic of Korea; 3 Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea Introduction : Purification of extracellular vesicles(EVs) from blood plasma is essential to increase the accuracy of the assay in the use of EVs as a diagnostic biomarker. However, lipoproteins, which are lipid and protein complexes that transport lipid molecules in the blood, are not only 104 times more abundant than EVs but also overlap the size and density properties with those of EVs, making it difficult to remove lipoproteins from EVs. Methods : The EVs were isolated by size‐based ultrafiltration and characterized by using NTA. In this work, we proposed a high‐resolution spiral microfluidic chip for the negative enrichment of EVs by selectively eliminating lipoproteins. First, before the plasma sample was injected into the microfluidic chip, the size difference between EVs and lipoproteins was created by immobilizing lipoproteins onto 7 μm beads using an immunoaffinity method. Then this pretreated sample was loaded onto the chip. In the microfluidic channel, nanometer‐sized EVs were focused on the inner wall due to high‐resolution dean flow fractionation force while the lipoprotein‐associated 7 μm beads were moved to the outer wall, resulting in separation of pure EVs from lipoproteins. Results : Based on our chip, over 97% of lipoproteins conjugated 7 μm beads were removed and over 93% of 100 nm beads, representing the average size of EVs, were recovered with a purity of 95% at 53.2: 531.8 μ/min (sample: sheath) flow rates. Based on these experimental settings, the performance of the spiral chip was explored by using A549 cells derived EVs and commercialized lipoproteins, individually. More than 88% of EVs were recovered, and the remaining LDL(low density lipoprotein) levels using the high‐resolution spiral microfluidic chip were decreased to 5% of the initial loading while the HDL(high density lipoprotein) content was further lowered to 1%. Summary/Conclusion : The proposed technique has the potential to improve the accuracy of EVs analysis because the signals from purified EVs are high while the background signal is low. Funding : This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 22021R1C1C2007646, No. 2021R1A2C3011254) and the Technology Innovation Program (20008829) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).

Presence

Jaques Franco Novaes de Carvalho 1 ; Gabriela Rodrigues Barbosa 1 ; Nancy Cristina Junqueira Bellei 2 ; Flávio Freitas 3 ; Ana Claudia Torrecilhas 4 ; Reinaldo Salomão 1 1 Universidade Federal de São Paulo ‐SP, São Paulo, Brazil; 2 Universidade Federal de São Paulo ‐SP, Brazil; 3 Hospital SEPACO, Universidade Federal de São Paulo ‐SP, Sao Paulo, Brazil; 4 Federal University of Sao Paulo, Sao Paulo, Brazil Introduction : The extracellular vesicles (EVs) have been related with the transfer of viral components, contributing to the spread of the virus, such as HIV and HTLV. Our objective is to verify the presence of viral particles and to characterize the SARS‐CoV‐2 variants in EVs isolated from patients with COVID‐19. Methods : We selected plasma samples fro patients with COVID‐19 (N = 20) who were admitted between 03/2021 and 08/2021 to Hospital Sepaco, São Paulo, and samples from healthy individuals (N = 5). We isolated EVs by UC at 100,000 x g for 16 hours and the size and concentrations (particles/mL) were perfomed by NTA. The presence of SARS‐CoV‐2 viral particles was verified in EVs using the RT‐qPCR kit (GeneFinder Kit; OSANG Healthcare) targeting the genes RdRp,Envelope (E) and Nucleocapsid (N). To verify the variants in EVs we used the RT‐qPCR molecular 4Plex SC2/VOC (Bio‐Manguinhos), which allows the screening of variants Alpha, Beta, Gamma, Delta and Omicron. Positive result was considered Cycle threshold ≤ 40. Results : The EVs of patients and controls had an average size of 183.7nm and 233.3nm, respectively. Patients with COVID‐19 had a higher concentration of EVs (2.3e+10 particles/mL) than the healthy controls (4.0e+08 particles/mL) (P< 0.05). RT‐qPCR analysis showed amplification in 14 samples of EVs from patients and none of the controls. Gene amplification was detected for RdRp (4 samples), E (5 samples) and N (11 samples) virus particles of SARS‐CoV‐2. Regarding the screening of variants by RT‐qPCR, two samples were able to confirm the Gamma variant. Summary/Conclusion : EVs can bring a new perspective as potential carriers of SARS‐CoV‐2 viral particles, contributing to its propagation, making healthy cells more susceptible to infection. Funding : FAPESP, CAPES and CNPq.

Proteome

Amir wahid 1 ; Amir sohail 2 1 Shanghai Jiao Tong university, buner, Pakistan; 2 Shanghai Jiao Tong university, shanghai, China (People's Republic) Introduction : Human salivary extracellular vesicles (sEVs) have emerged as a potentially rich source of disease‐related molecules for biomarker discovery and disease diagnosis. In the context of lung cancer (LC), in‐depth analysis of sEVs could potentially yield robust and specific biomarkers that are critically needed to improve diagnostic routines and clinical outcomes. The current clinical assessment of lung cancer relies heavily on a few clinical biomarkers (EGFR, ALK, KRAS) determined through tumor tissue examinations. Quantitative comparison of changes in sEVs proteins of the lung cancer and control group could help to discover protein candidates related to lung cancer. Methods : In this work, we collected clinical saliva samples from seven normal subjects and seven lung cancer patients. EVs were isolated from the saliva samples through high‐speed ultracentrifugation at 110,000 g for 2.5 hours at 4C. The sEVs size and distribution were analyzed through NTA analysis, while the morphology of the EVs was evaluated with TEM. Results : The nanoparticle tracking analysis (NTA) revealed the mean size of sEVs was 197.9 nm and concentration 2.31e+009+/‐9.45e+007, respectively. Transmission electron microscopy (TEM) analysis revealed the size of sEVs was 105 and 145 nm, correspondingly. The label‐free quantification analysis revealed the identification of 64 up‐regulated (FC>1.5) and 174 down‐regulated (FC< 0.66) proteins in normal and lung cancer groups. Moreover, the sEVs proteomic data revealed the identification of 47 unique sEVs proteins by comparing them against Vesiclepedia and Exocarta databases.sEVs biomarkers (CD9, TSG101, and CD63) were verified by western blot (WB). It was observed that the up‐regulated proteins were involved in signal transduction, organismal death, and morbidity. In contrast, down‐regulated sEVs proteins were involved in tumor cell proliferation, cell death of tumor cell lines, and apoptosis. Qualitative analysis revealed that the lung cancer sEVs protein cluster pattern was different from the normal. Summary/Conclusion : In summary,the work demonstrates the great potential of using mass spectrometry (MS) based quantitative proteomics approaches to study and describe the role of the sEVs protein in signaling networks in cancer research. In particular, using LC as a model system, we demonstrate that sEVs‐proteomics is a very powerful technology offering great prospects to discover novel protein candidates, molecular mechanisms, and protein‐protein interaction networks involved in cancer progression, metastasis, and treatment. It is a step forward in the field of liquid biopsy for the non‐invasive study of biomarkers. Funding : This work is supported by the Key Scientific Project of Shanghai Jiao Tong University (No. TMSK‐2020‐130, No. YG2017MS80). Keywords : saliva, extracellular vesicles, proteins, lung cancer

Reversal

Sicheng Wen 1 ; Elaine Papa 2 ; Mark Dooner 2 ; Mandy Pereira 2 ; Michael Del Tatto 2 ; Peter Quesenberry 2 1 Rhode Island Hospital/ Brown University, providence, USA; 2 Rhode Island Hospital, Providence, USA Introduction : The current therapies for patients exposed to a lethal dose of radiation are limited. We have previously shown that human mesenchymal stromal cell (MSC) derived vesicles would completely or partially reverse the bone marrow injury elicited by exposure to mild or moderate radiation in vitro or in vivo. Methods : We now evaluated the effect of MSC‐EVs on the reversal of radiation damage after 950 cGy of whole‐body lethal irradiation. Mice received an IV infusion of 1 × 109 hMSC EVs daily for three days, after 24 hours of irradiation. Results : The MSC‐EV untreated mice were dead between 12–27 days post‐radiation, but MSC‐EV treated mice maintained a 70% survival rate in 120 days post‐radiation. The peripheral blood counts significantly reduced, at ten days after radiation with/without EV treatment and recovered after three months of EV treatment although WBCs were still lower than normal control, suggesting that MSC‐EV treatment could significantly extend the survival rate of mice and enhance the recovery of blood cells after exposure to lethal radiation. We further evaluated the 30‐marrow stem cell‐related gene expression in WBMCs from these mice with/without EV treatment at 14 and 120 days post‐exposed radiation by real‐time PCR analysis. We identified the differential expression genes (DEGs) between radiation with/without EV treatment and healthy controls according to the cutoff values of | log2FC| ≥ 1.0 and p‐values < 0.05 (t‐test), and the 14 DEGs were accordingly identified between groups. Within the 14 DEGs, the gene expression of CXCL12, CD90, and Nestin were upregulated, and another 10 genes including SATB1, RUNX1, PECAM, PBX1, CKIT, CD34, CD33, MPO, SPI1, and S100A9 were downregulated in mice 14 days post radiation with/without EV treatment compared to the non‐radiation control mice. However, the alterations of gene expression in these genes were rescued to normal levels at day 120 post‐EV treatment. No DEGs were found between mice with and without EV treatment at 14 days post‐radiation except the gene CD3E was upregulated by radiation but came down to normal on day 14 with EV treatment. We didn't observe DEGs between the non‐radiation control mice group and the radiation with 120 days post EV treatment. This suggests that the change of gene profile induced by radiation is not rescued by EV treatment at day 14 post‐radiation but significantly recovered at day 120 post‐radiation. Summary/Conclusion : Our data suggest that EV treatment could significantly extend the survival rate of mice and help bone marrow cells recover from radiation injury. Funding : NIH P20GM119943. Keywords : mesenchymal stromal cell, extracellular vesicles, radiation

Salivary

Saroj Kumar 1 ; Komal Rani 2 ; Simran Rastogi 3 ; Fredrik Nikolajeff 4 1 Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India, New Delhi, India; 2 All India Institute of Medical Sciences, Hyderabad, NEW DELHI, India; 3 UT Southwestern Medical center, New Delhi, USA; 4 Lulea University of Technology, Lulea, Sweden Introduction : Parkinson's disease is generally asymptomatic at earlier stages. At an early stage, there is an extensive progression in the neuropathological hallmarks, although, at this stage, diagnosis is not possible with currently available diagnostic methods. Therefore, the pressing need is for susceptibility risk biomarkers that can aid in better diagnosis and therapeutics as well can objectively serve to measure the endpoint of disease progression. The role of small extracellular vesicles (sEV) in the progression of neurodegenerative diseases could be potent in playing a revolutionary role in biomarker discovery. Methods : The salivary sEVs of the subjects (PD = 70, healthy controls = 26 and prodromal = 08) were isolated by chemical precipitation followed by antibody‐based validation through CD63, flotilin, CD9 (universal surface marker) and confirmed neuronal origin by CD171 as well morphologically characterized through cryo‐EM. The sEVs quantification via fluorescence‐tagged sEVs NTA and antibody‐based NTA using CD63. The α‐syntotal in sEVs cargo was determined by ELISA. The confirmation of the disease severity staging was done by 99mTc‐TRODAT‐SPECT. Results : We observed a significant increase of fluorescence‐tagged sEVs in PD (p< 0.0001) than the HC via NTA (sensitivity of 94.34%) as well it was in line with the antibody‐tagged sEV p = 0.006 (sensitivity of 94.12%). A significant increase of α‐syntotal concentration in the sEVs of PD when compared to HC (sensitivity of 88.24%). The fluorescence‐tagged sEV depicted a positive correlation with the hallmark protein of PD α‐syntotal r = 0.4709, p = 0.0486. The striatal binding ratios in the 99mTc‐TRODAT‐SPECT shown to have a positive correlation with the fluorescent sEVs concentration r = 0.3000, α‐syntotal concentration r = 0.8000. Based on our analysis, we were speculating that some of the recruited healthy controls could be cases of prodromal Parkinson's disease. We osbserved the increased concentration of sEV (particle/ml) in these prodromal compared to HC but less than PD patients and that further validated by the α‐syn protein. Summary/Conclusion : This study is the first to address that the sEVs can screen the early as well progression of the disease with clinically acceptable sensitivity and can be a potent early detection method for PD. Funding : The funding provided by Department of Health Research (DHR), Indian Council of Medical Research (ICMR), India and Lulea University of Technology, Sweden. Keywords : Screening method, neurodegenerative disease, extracellular vesicle

Serpina3

Fumihiko Urabe 1 ; Kagenori Ito 2 ; Tomofumi Yamamoto 3 ; Shun Sato 4 ; Jun Nakayama 5 ; Shin Egawa 6 ; Takahiro Ochiya 7 ; Yusuke Yamamoto 8 1 Department of Urology, The Jikei University School of Medicine, Tokyo, Japan, Nishi‐Shimbashi, Japan; 2 Department of Urology, The Jikei University School of Medicine, Japan; 3 Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjuku‐ku, Japan; 4 Department of Pathology, The Jikei University School of Medicine, Japan; 5 Laboratory of Integrative Oncology, National Cancer Center Research Institute, Tokyo, Japan; 6 Department of Urology, The Jikei University School of Medicine, Tokyo, Japan; 7 Department of Molecular and Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shimjuku‐ku, Japan; 8 Laboratory of Integrative Oncology, National Cancer Center Research Institute, Chuo‐Ku, Japan Introduction : Prostate cancer (PCa) exhibits two primary features: the frequent occurrence of osteogenic bone metastases and a favorable survival rate. PCa boasts a 10‐year survival rate of 45%, even for patients with metastatic disease, which may be due to the characteristics of osteoblastic bone metastasis. In this study, we hypothesized that understanding the molecular mechanisms of osteoblastic bone metastasis could clarify the pathogenesis of PCa. Methods : To assess the impact of osteoblasts on PCa cells, we employed a horizontal co‐culture model with osteoblasts (OBs) and either osteolytic PCa (LPCa) or osteoblastic PCa (BPCa). RNA sequencing of these PCa cells revealed SERPINA3 and LCN2 as key players in BPCa. To evaluate the effect of SERPINA3 and LCN2 on osteogenesis, we generated an overexpression model using LPCa cells. Furthermore, we collected conditioned medium from SERPINA3‐ or LCN2‐overexpressing HEK293T cells, administered the medium to BPCa cells, and evaluated their roles in BPCa itself. We also examined the feasibility of SERPINA3 and LCN2 as biomarkers using TCGA data from PCa patients. Results : In a co‐culture of OBs and BPCa cells, SERPINA3 and LCN2 were significantly upregulated in BPCa via OB‐derived extracellular vesicles, while they were not in the co‐culture of OBs and LPCa cells. In both the co‐culture system and mouse xenograft experiments with intracaudal injection, enhanced expression of SERPINA3 and LCN2 in PCa led to osteogenesis. Additionally, the addition of SERPINA3 or LCN2 to BPCa cells significantly suppressed proliferative potential. Retrospective analysis also confirmed that high expression levels of SERPINA3 and LCN2 were significantly correlated with a better prognosis. Summary/Conclusion : Our results may partially explain why the prognosis for PCa forming osteoblastic bone metastasis is relatively better compared to osteolytic bone metastasis. Funding : JSPS KAKENHI grant numbers 21H02721, 19K07652, and the Jikei University Research Fund for Graduate Students. Keywords : prostate cancer, osteoblastic bone metastasis

Shedding

Christopher C. Reimann 1 ; Tomas Koudelka 2 ; Michaela Schweizer 3 ; Andreas Tholey 2 ; Lesley Cheng 1 ; Markus Glatzel 4 ; Marina Mikhaylova 5 ; Andrew F. Hill 6 1 La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia; 2 Institute of Experimental Medicine, Christian‐Albrechts‐Universität zu Kiel, Kiel, Germany; 3 Center for Molecular Neurobiology, University Medical Center Hamburg‐Eppendorf (UKE), Hamburg, Germany; 4 Institute of Neuropathology, University Medical Center Hamburg‐Eppendorf (UKE), Hamburg, Germany; 5 Institute of Biology, Humboldt Universität zu Berlin, Berlin, Germany; 6 Institute for Health and Sport, Victoria University, Melbourne, Australia Introduction : Proteases are a common cargo of extracellular vesicles (EVs), however, the effects of their activity towards surface proteins on EVs and the impact on EV signaling are mostly unexplored. Here, we focus on the metalloprotease ADAM10 in cortex cells as it is a key player of brain function and a master regulator of neuronal proteins and adhesion molecules. Continuous activity of EV‐associated ADAM10 (EV‐ADAM10) could release bioactive fragments away from the sending cell or alter the specificity of EV‐cell interaction, and investigating EV‐ADAM10's processing capacity will allow for assessment of its functional significance in brain cell communication. Methods : Primary cortex cells from E18 rat embryos were cultured for two weeks and small EVs were isolated from the cell supernatant by serial centrifugation and characterized by NTA, western blot, and EM. Activity assays with isolated EVs were performed over 24h in presence or absence of an ADAM10 inhibitor and a fluorogenic substrate. To assess processing of EV proteins, assayed EVs were subjected to N‐terminal proteomics by HYdrophobic Tagging‐Assisted N‐termini Enrichment (HYTANE) followed by bioinformatic analysis. Results : EV‐ADAM10 from cortex cells demonstrated activity towards an exogenous fluorogenic substrate as well as EV endogenous proteins over 24h. N‐terminomics identified 16 membrane‐associated or secreted proteins with reduced numbers of N‐termini following inhibition of EV‐ADAM10 over 24h compared to control (>2‐fold, p< 0.05). The affected proteins include cadherins, chaperones, and membrane traffic proteins with roles in cell adhesion, β‐amyloid binding, and nervous system development. Summary/Conclusion : Our findings provide novel insights into EVs as platform of proteolytic processing and the role of ADAM10 as potential modulator of EV function in the brain. Funding : This work was supported by a La Trobe University Graduate Research Scholarship and Full Fee Research Scholarship, the German Research Foundation (DFG) and by grants from the NHMRC and ARC. Keywords : ADAM10, brain cell communication, EV proteolysis, metalloprotease, N‐terminal proteomics

Syntenin

Rui zhang 1 ; Zhiwei Wu 2 1 Nanjing Drum Tower Hospital, China (People's Republic); 2 Nanjing university, Nanjing, USA Introduction : ZIKV (Zika virus) were emerging from 1947. Since its outbreak, ZIKV has infected countless people with fever, rash, joint pain and Guillain‐Barré syndrome, including severe microcephaly in newborns from infected pregnant women. Small extracellular vesicles (sEVs) are small, nanoscale, natural phospholipid bilayer vesicles secreted by various cells and are widely found in fluids such as blood, urine et al. Large of studies have shown that sEVs are capable of transport ting a range of bioactive molecules that mediate cellular and inter‐tissue communication and participate in processes such as viral infection,immune regulation and tumor therapy. It has been found that sEVs derived from virus‐infected cells can encapsulate viral components and host factors, and that such sEVs can act on adjacent cells or enter the bodily circulatory system to effect distal target cells, thereby modulating viral infection. For example, sEVs released from host cells infected with hepatitis C virus (HCV) can encapsulate the complete viral genome and can fuse with distal dendritic cells and establish effective infection. We found the sEVs released by ZIKV‐infected cells contained viral genomes and proteins and was capable of establishing productive infection in non‐receptor expressed cells. The biogenesis of sEVs involves a complex series of formation mechanisms. Viruses may hijack vesicle sorting pathways to alter sEVs secretion and contents. In this research, we found ZIKV infection affected the expression of syntenin, a cytosolic adapter interacted directly with Alix, and supported the intraluminal budding of endosomal membranes. Methods : Transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA), Exosome isolation, purification., in Vivo Imaging, Flow Cytometry Results : 1: ZIKV infection increased the production of sEVs. 2: sEVs derived from ZIKV‐infected cells contain viral components. 3: sEVs‐ZIKV could establish productive infections in recipient cells 4: ZIKV promoted sEVs production by upregulating syntenin expression. 5: Syntenin regulated sEVs uptake through affecting SDC2 and GPC1. Summary/Conclusion : We found that ZIKV infection result in packaging of the viral genomic RNA and partial viral proteins into sEVs and upregulates sEVs secretion, and demonstrated that the viral genomic RNA in sEVs could be transferred to and establish productive infection in a new target cell. Importantly, our study reveals that ZIKV‐induced syntenin expression enhances sEVs secretion and sEVs‐mediated virus transmission. Additionally, our data show that syntenin may be involved in the uptake of the infectious‐sEVs to facilitate ZIKV transmission through the vesicles. Taken together, our results suggest a novel mechanism involving syntenin and sEVs‐mediated ZIKV intercellular transmission.

Targeted

Raghuram Kandimalla 1 ; Disha Nagesh Moholkar 2 ; Jeyaprakash Jeyabalan 3 ; Jun Yan 4 ; Zhao‐Hui Song 5 ; Farrukh Aqil 6 ; Ramesh Gupta 7 1 Brown Cancer Center, Department of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40202, Louisville, USA; 2 Department of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40202, Louisville, USA; 3 3P Biotechnologies, Louisville, KY 40202, Louisville, USA; 4 Brown Cancer Center, Department of Surgery, University of Louisville, Louisville, KY 40202, Louisville, USA; 5 Department of Pharmacology & Toxicology, University of Louisville, KY 40202, Louisville, USA; 6 Brown Cancer Center, Department of Medicine, University of Louisville, Louisville, KY 40202, Louisville, USA; 7 Brown Cancer Center, Department of Pharmacology & Toxicology, University of Louisville, 3P Biotechnologies, Louisville, KY 40202, Louisville, USA Introduction : Cannabidiol (CBD) is a potential anticancer plant bioactive. However, low oral bioavailability and rapid hepatic metabolism limits its application. Bovine colostrum has been discovered to be an abundant source of exosomes and can be used for drug delivery. In this study, we report a targeted oral delivery system for CBD as an effective cancer therapeutic. Methods : Exosomes were isolated from bovine colostrum powder by rehydration and ultracentrifugation. Exosomes were functionalized with the targeting ligand, folic acid, and loaded with CBD. Exosomes were characterized for size and charge and hallmark proteins and the UPLC was used to determine CBD loading. The antiproliferative and colony formation inhibitory activity of CBD and exosomal CBD (ExoCBD) was tested against drug‐sensitive (A549 and H1299) and drug‐resistant (A549TR) lung cancer cells. The molecular mechanism was assessed by Western blot and the anti‐cancer effects were determined against A549 orthotopic lung tumor xenografts in mice. Results : Colostrum exosomes exhibited size (107 ± 3 nm), charge (‐21 ± 0.7) and carried typical surface proteins. High drug load was observed (∼20%) on exosomes and FA‐functionalized exosomes. Particle size of ExoCBD (127 nm) and FA‐ExoCBD (134 nm) was somewhat increased vs. exosomes. CBD and its exosomal formulations showed dose‐dependent antiproliferative and colony formation inhibitory activities. Orally delivered FA‐ExoCBD (≈80%; p< 0.001) showed higher tumor inhibition vs. the free CBD (≈60%; p< 0.01) at half the CBD dose. Mechanistically, CBD demonstrated dose‐dependent downregulation of MDR‐1, NFκB and oncogenic CB2, and upregulation of the anti‐proliferative GPR3 in A549 and A549‐TR cells. Summary/Conclusion : Targeted oral exosomal formulation of CBD inhibits lung cancer via favorable modulation of GPR3, NFκB, oncogenic CB2 and multi‐drug resistance (MDR) pathways. Funding : Supported from 3P Biotechnologies and Duggan Endowment. Keywords : colostrum exosomes, lung cancer, CBD, oral delivery, tumor targeting

Timegate

jacopo zini 1 ; Petr Nickl 2 ; Saara Laitinen 3 ; Amuthachelvi Daniel 1 ; Mari tenhunen 1 1 Timegate Instruments Oy, Finland; 2 Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the Czech Academy of Sciences, Czech Republic; 3 Finnish Red Cross Blood Service, Helsinki, Finland Introduction : Raman spectroscopy (RS) is a fast and non‐destructive method able to reveal the biochemical composition of complex samples. RS is gaining interest in the extracellular vesicle (EV) field as a quality control tool. Here, we demonstrate the potential application of a timegated (TG) Raman spectrometer in EV characterization. TG spectrometers, in contrast to the traditional ones, suppress fluorescence interferences, thus, improving the signal‐to‐noise ratio and enabling the characterization of EVs labelled with fluorophores such as green fluorescent protein (GFP). Here, we highlight the capacity of TG‐Raman to discriminate between EV preparations with different degrees of purity and between EV obtained from healthy cells and cells infected by adeno‐associated viruses. Additionally, we show that TG Raman is effectively able to suppress fluorescence in EVs expressing GFP and allows measuring the Raman spectra of such samples. Methods : Platelet derived EVs were provided by the Finnish Red Cross Blood Service and purified by ultracentrifugation (UC) and 2xUC for higher purity. EVs‐AAV1, EVs‐AAV9 and GFP EVs, were purified by differential centrifugation from cell conditioned media derived respectively from HEK293 cells, HEK293 cells infected with adeno‐associated virus serotype 2/1 (both GFP positive and negative or 2/9). Raman measurements were performed by timegate Raman Microprobe (Timegate Instruments Oy), with a 532 nm pulsed laser. EVs were dried on CaF2 substrate prior to measurement. Each sample was measured 30 times. Results : Raman spectra of platelet derived EVs purified with different protocol show clear differences: preparation with more thorough purification protocol display a reduction in intensity of peaks associated to proteins and amino acids, indicating less contaminating free proteins. In addition, principal component analysis (PCA) cluster the spectra based on the purification method used. Regarding the EVs, CD(?)9‐GFP‐EVs, EVs‐AAV1 ‐AAV9, PCA display three major clusters: one associated to EVs‐AAV9, one to EV and one to EV‐AAV1 which include the GFP positive EV‐AAV1. Summary/Conclusion : These findings indicate that TG Raman is a suitable tool for EV quality control since it discriminates EV preparations based on the degree of purity and distinguishes between healthy EV preparations and preparations infected by AAV. In addition, TG Raman highlights the differences between the diverse AAV found in the EV preparations. Lastly, TG Raman can overcome the fluorescence interference given by GFP. This allows the acquisition or Raman spectra of GFP‐EVs, which display no remarkable difference compared to the unmarked EVs. Funding : Timegate Instruments Oy Finnish Red Cross Blood Service. Keywords : raman, timegate raman

Tracking

Anthony Yan‐Tang Y. Wu 1 ; Wendy Wan‐Ting Wong 1 ; Shannon Yu‐Hsuan Yeh 2 ; Angela Yun‐Fei Zhang 2 ; Charles Pin‐Kuang Lai 1 1 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan (Republic of China); 2 Institute of Atomic and Molecular Sciences, Academia Sinica, Taiwan (Republic of China) Introduction : Accurate and precise tracking nano‐sized extracellular vesicles (EVs) remain as a major challenge in EV research in vivo due to the inverse correlation between signal intensity and penetration depth. We previous developed a multi‐resolution, bioluminescence resonance energy transfer (BRET)‐based PalmGRET reporter with pan‐EV labeling ability, which emits signals at blue‐to‐green spectra (∼450‐520nm). In an effort to increase in vivo imaging capability of EV subpopulations including small EV (sEV; 1,000 nm), we developed a near‐infrared (NIR)‐BRET EV reporter, PalmSORET. Methods : We molecularly fused palmitoylation signal of growth cone associated protein 43 to the N‐terminus of iRFP‐Rluc8.6‐535SG (Nishihara and colleagues) to create PalmSORET. PalmSORET emits bioluminescence (BL) and BRET‐excited fluorescence (BRET‐FL) for dual reporter outputs. The palmitoylation signal enables EV inner membrane labeling through S‐palmitoylation of cysteine which minimizes PalmSORET from being exposed to the EV surface, thereby mitigating enzymatic degradation and/or surface ligand‐receptor hindrance. PalmSORET catalyzes BBlue2.3 substrate to yield peak BL and BRET‐FL signals at 413 nm and 713 nm (via Soret band excitation of the iRFP713), respectively. Results : Upon stable expression in HEK293T cell (293T‐PalmSORET), PalmSORET localized to the membranes and enabled tracking of EVs under super‐resolution at near‐infrared range (> 700 nm). PalmSORET labels the inner membrane of both bEV and sEV without significantly affecting their respective sizes. In addition, bEV/sEV‐PalmSORET emitted robust BL (413/ 10 nm) and BRET‐FL (700/ 50 nm) signals with a positive correlation to EV amount (R2 value > 0.98) and high sensitivity (100 ng EV), which could be rapidly detected in vitro with 1 sec of integration time. Summary/Conclusion : We successfully established a NIR‐BRET EV reporter, PalmSORET, with pan‐EV labeling and multi‐resolution imaging functions. Efforts are currently underway in applying this powerful and sensitive imaging system to track bEVs and sEVs in vitro and in vivo. Funding : National Science and Technology Council (NSTC) grants NSTC 111‐2628‐B‐001‐022 (C.P.L.), Academia Sinica Innovative Materials and Analysis Technology Exploration (i‐MATE) Program AS‐iMATE‐107‐33 (C.P.L.), Institute of Atomic and Molecular Sciences grants IAMS 30‐08 (C.P.L.), and Academia Sinica Career Development Award 109‐M04 (C.P.L.). Keywords : small EV, big EV, near‐infrared bioluminescence resonance energy transfer, Palmitoylation

Vascular

Sandrine Juillard 1 ; Annie Karakeussian Rimbaud 2 ; Marie‐Hélène Normand 3 ; Julie Turgeon 2 ; Nathalie Grandvaux 2 ; Alexa C. Robitaille 2 ; Isabelle Allay 4 ; Éric Boilard 5 ; Marie‐Josée Hébert 2 ; Mélanie Dieudé 6 1 Université de Montréal, CHUM Research Center (CRCHUM), Canada; 2 Research Centre, Centre hospitalier de l'Université de Montréal (CRCHUM), USA; 3 Université de Montréal, CHUM Research Center, Canada; 4 5Centre de Recherche du CHU de Québec, Université Laval, Québec, USA; 5 University Laval, Faculty of Medecine, CHU de Québec – CHUL, Québec, Canada; 6 University of Montreal, CHUM Research Center, Héma‐Québec, Affaires Médicales et Innovation, Canada Introduction : Apoptotic exosomes (ApoExo) are vascular injury derived extracellular vesicles (EVs) released by apoptotic endothelial cells (EC). Their comprehensive characterization showed distinct size, protein, ARN profile and enzymatic activity from classical apoptotic bodies. We have shown that ApoExo accelerated rejection in association with circulating anti‐LG3/perlecan autoantibodies (AutoAb). We hypothesize that ApoExo stimulate specific B cells that exist in the normal immune repertoire to secrete AutoAb and that pro‐inflammatory condition prevalent in the context of organ transplantation and autoimmune diseases can amplify this response. Methods : B cells from spleen or peritoneal cavity (pc) were isolated from C57Bl/6 mice. After in vitro stimulation with Toll‐Like‐Receptor (TLR) agonists, anti‐LG3 levels were assessed in supernatants by ELISA. Serum‐free medium conditioned by apoptotic murine EC from C57Bl/6 mice is fractionated based on sequential centrifugations up to 200 000g and ApoExo are isolated and quantified according to MISEV2018 requirements. C57Bl/6 mice were infused with ApoExo or vehicle every second day for 3 weeks. At sacrifice, splenocytes and pc lymphocytes were analyzed as above and by flow cytometry. Circulating AutoAb were quantified by ELISA /microarray and cytokine levels were evaluated by multiplex. Results : B cells specific to LG3 were found in the pc of C57Bl/6 mice and produced anti‐LG3 AutoAb when stimulated with TLR 1/2, 4, 7 and 9 agonists. Interestingly, these cells disappeared from the pc of mice infused with ApoExo. ApoExo infusion also triggered circulating IL‐23 –IL17 autoimmune axis, increased splenic Germinal center B cell, increased total circulating IgG and significant production of classical AutoAb such as anti‐dsDNA, anti‐SSA‐B anti‐Sm/nRNPand anti‐LG3. Summary/Conclusion : These observations suggest a specific role for vascular injury derived EVs ApoExo in modulating the production of AutoAb of importance in autoimmune diseases and transplant rejection. Funding : Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC).

Activated

Kaitlyn E. Bunn 1 ; Brenna Giese 2 ; Cherie Saffold 1 ; Heather H. Pua 3 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA, Nashville, USA; 2 Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA, USA; 3 Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville TN, USA, Nashville, USA Introduction : Extracellular vesicles (EVs) are small, secreted membrane particles that mediate intercellular communication by delivering cell‐derived cargoes. We previously found that immune cells secrete EVs into the airways during allergic lung inflammation in mice. The goal of this study was to determine the contribution of T cells to allergic airway EVs, identify T cell EV protein cargoes, and assess the effects of T cell EVs on eosinophils, important cellular mediators of allergic inflammation. Methods : To determine the contribution of T cells to airway EVs, allergic airway inflammation was induced in mice with T cell membrane labeling, and bronchoalveolar lavage fluid (BALF) was collected and subjected to single EV flow cytometry. To identify T cell EV protein cargoes, EVs were purified from primary mouse T cell culture media by size exclusion chromatography (SEC), ultrafiltration, and density gradient flotation for mass spectrometry. To assess effects of T cell EVs on eosinophils, bone marrow‐derived eosinophils were treated with EVs purified from resting and activated T cell culture by SEC and ultrafiltration, and eosinophil viability and degranulation were assessed. Results : We found that 1% of EVs (6 million EVs per airway) in BALF of mice with induced allergic airway inflammation were of T cell origin, with 75 EVs detected per airway T cell. We identified a total of 957 T cell EV proteins by mass spectrometry, including cell membrane proteins known to be involved in eosinophil survival, such as CD40L, CD47, and CD22. Finally, we found that EVs secreted by activated T cells, but not resting T cells, enhance eosinophil viability with no effect on degranulation. This effect on viability is EV concentration‐dependent and can be abrogated with surface protein shaving. Summary/Conclusion : These results provide evidence that T cells secrete EVs carrying protein cargoes into the allergic airway and that T cell EVs may play a role in allergic airway pathology by improving eosinophil survival through a mechanism that involves EV surface protein(s). T cell EVs may be important and potentially targetable components of allergic and other immune‐mediated reactions. Funding : NIH DP2‐ HL152426 (HHP), NIH T32GM008554 (KB). Keywords : T cells, eosinophils, allergic inflammation, asthma,

Advancing

Marta Costa 1 ; Beatriz Painho 2 ; Margarida Costa 2 ; Carolina Sousa 2 ; Inês Carrondo 2 ; Enrique Oltra 3 ; Beatriz Pelacho 3 ; Felipe Prosper 3 ; Inês Isidro 2 ; Paula Alves 4 ; Margarida Serra 5 1 iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; 2 iBET, Instituto de Biologia Experimental e Tecnológica, USA; 3 Regenerative Medicine Department, Center for Applied Medical Research, University of Navarra, USA; 4 iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, USA; 5 iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal, Lisboa, Portugal Introduction : Clinical translation of mesenchymal stromal cells (MSC)‐derived extracellular vesicles (EV) is currently limited by their lack of scalability. Although most methods focus on maximizing EV production by increasing the cell culture surface area, besides integrating cell expansion in stirred‐tank bioreactors (STB) with scalable downstream EV processing protocols, cell preconditioning strategies could further contribute to boost EV yields. Methods : In this work, we have evaluated the effect of tailoring glucose concentration throughout MSC culture in STB on EV secretion yields, isolated by tangential flow filtration followed by size exclusion chromatography. Additionally, since the cell microenvironment can influence the properties of their derived EVs, Process Analytical Tools (PAT) tools were integrated in EV manufacturing workflows to monitor critical process parameters in real‐time, therefore contributing to ensure that EV‐based products retain their potency while manufactured at a clinically‐relevant scale. Results : Cell culture at low glucose concentration levels can maximize EV yields in a scalable bioprocess supported by cell expansion in STB, showing 1.4‐fold increase in EVs secreted per cell relatively to MSC cultured at higher glucose concentration. Nonetheless, the distinct glucose levels observed on the harvest day highlight the need to standardize cell culture towards the implementation of more reproducible bioprocesses. To this purpose, we have shown that Raman Spectroscopy can be used to continuously monitor glucose levels in STB as an alternative to off‐line glucose concentration measurements (R2 = 0.923). Summary/Conclusion : MSC culture at low glucose concentration can improve their ability to secrete EVs, shedding light on the importance of standardizing cell culture conditions and, particularly, controlling metabolites concentration through the implementation of Raman Spectroscopy tools to develop more robust platforms for EV production. Funding : This work was performed under the scope of the CardioPatch project (SOE4/P1/E1063) and iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), a program financially supported by FCT/Ministério da Educação e Ciência, through national funds. Keywords : extracellular vesicles (EVs), mesenchymal stem/stromal cells (MSCs), stirred‐tank bioreactors, metabolic preconditioning, Raman spectroscopy

Alexander

Konstantin Glebov 1 ; Daniel Lashley 2 ; Caitlin O'Shea 3 ; Anis Sahoo 3 ; Rupert Noad 2 1 University of Plymouth, United Kingdom; 2 University Hospitals Plymouth NHS Trust, USA; 3 University of Plymouth, USA Introduction : Alexander disease (AxD) is a rare and fatal neurological disorder that affects the central nervous system. A broad set of symptoms and the presence of Rosenthal fibres characterize AxD. The disorder is caused by mutations in the glial fibrillary acidic protein (GFAP), a protein found in the cells that support and protect nerve cells in the brain. Studies have shown that mutations in GFAP can lead to the formation of abnormal protein aggregates, which contribute to the development of AxD. The specific mutations that cause AxD have been identified, and research is ongoing to understand the underlying mechanisms of the disease. Methods : Cell culture. COS7 cells were a gift from Prof Michael Schrader, University of Exeter. COS7 cells were grown in T75 flasks in DMEM with the addition of foetal bovine serum (FBS) 10%v/v and penicillin streptomycin 1%v/v. Protein detection using Western Blot. Running buffers were prepared using NuPAGE MES gels. Immunocytochemistry. COS7 cells were grown on coverslips in 6 well plates at a density of 200,000 cells per well and transfected with GFAP wild types and mutants and later fixed and stained with anti‐GFAP primary and fluorescent Alexa secondary ABs. Microscopy & Image analysis. A Leica TCS SP8 microscope was used to obtain Z‐stacks at a magnification of 63x. Results : The patient, a 40‐year‐old male, presented with a one‐week history of anterograde amnesia and apathy and was found to have subclinical corticospinal tract dysfunction. MRI imaging showed progressive volume loss in the medulla, upper cervical spinal cord, and supratentorial brain. Neuropsychological testing indicated persistent deficits in learning and memory likely caused by sub‐cortical cognitive impairment. Genetic analysis confirmed a GFAPG301D mutation, and in vitro experiments showed that the mutation resulted in large aggregates and changes in protein conformation or post‐translational modifications. The study highlights the need for further research on the link between GFAP mutations and the unconventional secretory pathway in AxD. We utilized the Tunable Resistive Pulse Sensing measurement using a qNanoGold device to analyze the effects of GFAP variants on EVs release. Concentration of EVs were significantly reduced in the case of GFAP AxD‐causing variants in comparison to the GFAPwt. AxD mutants also affected size of secreted vesicles in comparison to the GFAPwt, suggesting that AxD‐causing mutations may reduce release of EVs from cells and influence size of secreted EVs Summary/Conclusion : In this study, we present a case of Alexander disease (AxD) characterized by erectile dysfunction and memory impairment as the main clinical symptoms. We provide the first evidence that mutations in the glial fibrillary acidic protein (GFAP) can impair the unconventional secretory pathway.

Automated

Mandy Chan 1 ; Jason Dixon 2 ; Allen C. Eaves 3 ; Sharon A. Louis 4 ; Karina L. McQueen 4 1 STEMCELL Technologies Inc., Vancouver, Canada; 2 STEMCELL Technologies Inc., USA; 3 STEMCELL Technologies Inc.; Terry Fox Laboratory, Vancouver, USA; 4 STEMCELL Technologies Inc., Vancouver, USA 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 based on the tetraspanins or user‐defined surface markers. In this study, we have developed fully automated EV enrichment protocols on the RoboSep™‐16 platform, which can process up to 16 samples with volumes ranging from 0.5 ‐ 2 mL. Methods : EVs were either labelled directly with a tetraspanin (CD9, CD63, and/or CD81)‐specific antibody cocktail or indirectly with a PE‐conjugated antibody, followed by a PE‐specific antibody cocktail. The labeled EVs were bound to magnetic particles and separated from unwanted EVs using an EasySep™ magnet. Automated isolation was performed as follows: guided by the on‐screen prompts, plasma, EasySep™ reagents, tips, tubes, and wash solutions were loaded onto RoboSep™‐16; the instrument automatically labeled EVs with antibody cocktails and particles, and transferred the sample to an EasySep™ magnet on the instrument deck for magnetic separations. EVs isolated manually or via RoboSep™‐16 were analyzed by western blot to assess recovery. Results : With EasySep™, EV subtypes from human and non‐human (e.g. mouse) species were isolated in less than 30 minutes. The manual EasySep™ pan‐EV kit recovered 1‐ to 2‐fold more EVs than UC (n = 6). The fully automated RoboSep™‐16 pan‐EV and CD63+ EV protocols recovered 89 ‐ 98% EVs with lower albumin contamination relative to the manual protocols (n = 6 for pan‐EV; n = 6 for CD63+ EV). A full capacity run of 16 CD63+ EV enrichments took approximately 1 hour and 40 minutes with variance in recovery of 9.3%. Summary/Conclusion : EasySep™ allows for fast and easy immunomagnetic enrichment of EVs. Fully automated EV isolation on RoboSep™‐16 further reduces impurities and user variability, offering practical solutions for clinical laboratories using EVs as biomarkers of disease. Funding : N/A. Keywords : EV separation, immunomagnetic isolation, automation, high‐throughput

Bacterial

Irma Schabussova Medical University of Vienna, Vienna, Austria Introduction : Probiotic bacteria such as E. coli O83 (EcO83) have been shown to reduce the development of allergies. Oral administration of EcO83 reduced allergic sensitisation in children but not allergic lung disease. We showed that intranasal administration of EcO83 reduced allergic airway inflammation in mice in a TLR4‐dependent manner. Bacteria produce vesicles that mediate the functions of the microbiota by delivering effector molecules into host cells and modulating host signalling pathways in health and disease. The potential of outer membrane vesicles (OMVs) produced by bacteria for the prevention or treatment of allergy is unclear. Methods : We isolated OMVs from EcO83 (EcO83‐OMVs) by ultracentrifugation and investigated their effect in a model of ovalbumin‐induced allergic airway inflammation (AAI). The OMVs were analysed by TEM and NTA. HEK293 cells expressing NOD1, NOD2, TLR2 and TLR4 and bone marrow‐derived dendritic cells (BMDC) from wild‐type (WT) and TLR4KO‐BALB/c mice were stimulated with EcO83‐OMVs. Cytokines were measured by ELISA. Results : Stimulation of HEK293 NOD1, NOD2, TLR2 and TLR4 cells with EcO83‐OMVs increased the production of IL ‐8, indicating the involvement of these receptors in signal transduction by EcO83‐OMVs. Stimulation of WT BMDC with EcO83‐OMVs increased the production of IL‐23, IL‐12, TNFα, IL‐1β and IL‐6, while BMDC from TLR4KO mice showed decreased production of these cytokines. Intranasal administration of EcO83‐OMVs reduced allergic airway hyperresponsiveness and lung eosinophil counts compared to sham‐treated controls and increased pulmonary neutrophil counts. Summary/Conclusion : Here, we have shown that i) EcO83‐OMVs are recognised by NOD1, NOD2, TLR2 and TLR4, ii) EcO83‐OMVs induce cytokine production in a TLR4‐dependent manner, and iii) intranasal administration of ECO83‐OMVs reduces the development of experimental allergy. Our research suggests that probiotic‐derived OMVs could be a novel treatment option for allergic diseases in humans. Funding : Danube Allergy Research Cluster; FWF, OEAD. Keywords : microbiota, probiotic, allergy, bacterial extracellular membrane vesicles

Biomarker

Hash Brown Taha 1 ; Simon Hornung 2 ; Suman Dutta 3 ; Leony Fenwick 4 ; Otmane Lahgui 5 ; Karl E. Biggs 6 ; Carter Lantz 7 ; Kathryn Howe 3 ; Nour Elabed 3 ; Irish Del Rosario 8 ; Darice Y. Wong 9 ; Aline Duarte Folle 8 ; Daniela Markovic 10 ; Jose‐Alberto Palma 11 ; Un J. kang 12 ; Roy N. Alcalay 13 ; Miriam Sklerov 14 ; Horacio Kaufmann 11 ; Nadia Stefanova 15 ; Brent L. Fogel 16 ; Jeff M. M. Bronstein 17 ; Joseph A. Loo 7 ; Beate Ritz 18 ; Gal Bitan 19 1 Department of Integrative Biology & Physiology, University of California Los Angeles, Boulder, USA; 2 Department of Neurology, University of California Los Angeles, Freising, Germany; 3 Department of Neurology, University of California Los Angeles, USA; 4 Department of Neurology, University of California Los Angeles, Appeltern, Netherlands; 5 UCLA Department of Neurology, Gal Bitan's Lab, USA; 6 University of California at Los Angeles, USA; 7 Department of Chemistry and Biochemistry, University of California Los Angeles, USA; 8 University of California Los Angeles, Department of Epidemiology, USA; 9 Department of Neurology, and Clinical Neurogenomics Research Center, David Geffen School of Medicine at UCLA, USA; 10 UCLA David Geffen School of Medicine Department of Internal Medicine., USA; 11 Department of Neurology, New York University Grossman School of Medicine, USA; 12 NYU Grossman School of Medicine, New York, USA; 13 columbia university, USA; 14 University of North Carolina School of Medicine, Chapel Hill, USA; 15 Division of Neurobiology, Department of Neurology, Medical University of Innsbruck, Austria; 16 UCLA, USA; 17 David Geffen School of Medicine at UCLA, USA; 18 FSPH, UCLA, USA; 19 UCLA, Los Angeles, USA Introduction : CNS‐originating EVs cross the blood‐brain barrier and circulate in the blood thus providing a rich source of minimally invasive biomarkers for CNS disorders. Differential diagnosis of Parkinson's disease (PD) and multiple system atrophy (MSA) is challenging due to symptom overlap, especially in the early stages. Current diagnostic tests do not achieve high sensitivity or specificity. Because α‐synuclein (α‐syn) deposition occurs in neurons in PD and oligodendrocytes in MSA, we reasoned that comparing the concentration of α‐syn in neuronal (nEVs) and oligodendroglial EVs (oEVs) could help distinguish between these diseases. Indeed, the total α‐syn concentration together with the oEV:nEV α‐syn ratio separated MSA from PD with high sensitivity and specificity. Here, we tested whether pS129‐α‐syn, a pathological form of α‐syn, as well as tau, and neurofilament light (NfL) would improve the model's diagnostic accuracy. Methods : CNS‐originating EVs were isolated using a 2‐step process from remaining samples in the cohort originally used for measurement of α‐syn. EV enrichment was validated using TRPS, western blots, FACS analysis, and TEM. pS129‐α‐syn was measured using an in‐house electrochemiluminescence ELISA (ECLIA). Tau and NfL were measured using commercial ECLIA kits. Multinomial logistic regression was used for ROC analyses. Results : pS129‐α‐syn increased significantly in the order healthy control (HC) < PD < MSA in oEVs only. The addition of oEV‐pS129‐α‐syn to the statistical model increased the separation between these groups. Total tau was significantly lower in nEVs and oEVs in MSA compared to HC and PD but did not increase the model's diagnostic accuracy. NfL was statistically significantly reduced in PD. The inclusion of α‐syn in nEVs, oEV:nEV α‐syn ratio, pS129‐α‐syn in oEVs, and total EV concentration into the prediction model achieved an accuracy of ∼80% for separating PD from HC, ∼99% for MSA and HC, and ∼94% for PD and MSA. Summary/Conclusion : A blood‐based biomarker panel can achieve high separation of HC, PD, and MSA. Funding : Multiple system atrophy coalition (20170367 & 2017‐10‐007), California Department of Public Health (18‐10926), Michael J. Fox Foundation for Parkinson's Research (17990 & 18303), CurePSP (665‐2019‐07), National Ataxia Foundation (20201551), Cure Sanfilippo Foundation (20215318), Parkinson/Parkinson Alliance, The Alzheimer's Association, Weston Brain Institute & Alzheimer's Research UK Biomarker Across Neurodegenerative Diseases (BAND 3, 17990; PI Dr. GB) NIH/NIEHS (ES10544; PI Dr. BR) NIH (R35GM145286 & S10RR028893) & US Department of Energy (DE‐FC02‐02ER63421; PI Dr. JAL) Ruth L. Kirschtein National Research Service Award Program (GM007185; PI Dr. CL) NIH P50AG16573 & P30AG066519. Keywords : synucleinopathies, parkinsonism, phosphorylated α‐synuclein, tau, neurofilament light

Capturing

Sena Yaman 1 ; Tessa devoe 2 ; Gozde Durmus 3 1 Stanford University, Palo Alto, USA; 2 Brown University, USA; 3 Stanford University, USA Introduction : Extracellular vesicles, in the size of 30–150 nm, can carry molecules including DNA, RNA, and metabolites, which are essential for cell‐to‐cell communication. Tumor cells are thought to secrete extracellular vesicles into bodily fluids such as plasma to facilitate angiogenesis and metastasis, to make space for migration and proliferation while deactivating tumor suppressors. However, their size at the nanoscale makes the isolation and subtyping extremely challenging. A simple method of isolating and characterizing extracellular vesicles based on protein markers such as CD9, CD63, CD81 would facilitate the research on chemoresistance, metastasis, and disease progression. Methods : Here, we introduced Exo‐Lev, an extracellular vesicle isolation and subtyping method using microfluidic magnetic levitation. In ExoLev, polymer beads of distinct densities, i.e.,1.05 and 1.18 g/mL, act as selective capture surfaces for different sub‐types of extracellular vesicles. For this, the beads are first decorated with either anti‐CD63 or anti‐CD81 antibodies and incubated with filtered plasma samples. Second, the beads mixed with paramagnetic media are fed through the inlet and levitate to a specific equilibrium height within the microfluidic channel of the Exo‐Lev. Then, the beads are collected at either top or bottom outlets of the Exo‐Lev depending on their density. Third, captured extracellular vesicles are eluted from the collected beads and subjected to downstream analyses. Results : Beads flowed into the correct output channel with a 100% purity when a flow rate of 7.5 mL/h was applied. According to the Nanoparticle Tracking Analysis (NTA), the size of CD63‐positive vesicles captured by Exo‐LEV were 59.3 ± 4.7 nm and the size of CD81‐positive vesicles captured by Exo‐LEV was 86.5 ± 14.0 nm. Total RNA extraction showed that these particles contained biological material (ranging from 0.088 to 0.11 ng/μL). Summary/Conclusion : In this work, we developed a rapid, cost‐effective, and straightforward method to capture and subtype extracellular vesicles from filtered plasma. We anticipate that the ExoLev protocol can be applied to isolate any other subtypes of extracellular vesicles from whole plasma. Funding : We thank Stanford Department of Radiology for funding our work.

Compounds

Sofia Oliveira Miguel 1 ; Paula Meneghetti 2 ; Thaís Fernanda Amorim Pavani1 3 ; Daniela Gonçales Galasse Rando1 3 ; Ana Claudia Torrecilhas 4 1 UNIFESP, São Paulo, USA; 2 UNIFESP, Sao Paulo, USA; 3 UNIFESP, USA; 4 Federal University of Sao Paulo, Sao Paulo, Brazil Introduction : Trypanosoma cruzi, the etiologic agent of Chagas disease, releases EVs and containing surface molecules represented by glycoproteins anchored to the membrane via GPI (glycosylphosphatidylinositol). Our group previously showed that EVs released by infective T. cruzi forms modulate inflammatory responses in host cells and during in vivo infection. However, the biogenesis and mechanism of shedding of the epimastigotes EVs is poorly understood. Aiming to better understand EV release in this parasite, we studied the effects of GPQF‐815 and GPQF‐817, which are derivatives of N‐acylhydrazones. GPQF‐815 and 817 are synthetic compounds that do not yet have well‐determined biological activities, but which have already shown anti‐prion and antimicrobial actions. Methods : Epimastigotes forms of the Y strain of T. cruzi were incubated with different concentrations of the compounds (250, 125 and 62.5, 31.25, 15.625 and 7.8125 μg/mL) during 2 h, at 28oC. Released EVs were characterized in size and concentration by NTA. In parallel, we measure the protein content. Results : The results showed that released EVs concentration and size were not affected in any case. However, the amount of protein released with EVs increased for both compounds relative to the control, which indicated that the number of released particles is not dependent on the protein concentration. Summary/Conclusion : Compounds GPQF‐815 and 817 could have a possible regulatory role in the EVs formation in T. cruzi and consequently be used to control the inflammatory response during parasite infection. Funding : FAPESP, CNPq and CAPES.

Conferred

Viral D. Oza 1 ; Shilpa Sampathi 2 ; Yelena Chernyavskava 2 ; Kenan Flores 2 ; Simone Crivelli 3 ; Erhard Bieberich 1 ; Colin L. Hisey 4 ; Jessica Blackburn 2 1 University of Kentucky, Lexington, USA; 2 University of Kentucky, USA; 3 University of Kentucky, LEXINGTON, USA; 4 The Ohio State University, Columbus, USA Introduction : H3K27M‐driven Diffuse Midline Gliomas (DMG) are a subset of uncurable malignant pediatric gliomas. In all cases, patient tumors evolve to be resistant to radiation therapy, the current standard of care. DMGs are multi‐clonal, and each subclone has distinct genetic and transcriptional profiles. Data suggest that activation of oncogenic pathways through sub‐clonal communication, specifically extracellular signaling, impacts how tumors respond to therapy and evolve. Secreted extracellular vesicles (EVs) are a mode of intratumor communication. However, EVs in H3K27M‐DMG have not been extensively characterized, and their role in multi‐clonal tumor evolution and development of radioresistance is unknown. Methods : We isolated 35–350nm EVs from a panel of primary and relapsed patient‐derived H3K27M‐DMG cells and individual sub‐clones from a primary patient‐derived cell line. We utilized Next‐Gen sequencing and mass spectrometry to profile miRNA, proteins, and metabolites in H3K27M‐DMG EVs. Additionally, we identified H3K27M‐DMG EV membrane signatures using surface‐enhanced Raman spectroscopy. We are using DMG cells that express a genetically encoded death indicator (GEDI) and a cell cycle indicator (FUCCI) for longitudinal imaging to assess the functional impact of EVs on radiation‐induced cell death and cell senescence, two dynamic processes. Results : EVs isolated from radioresistant H3K27M‐DMG conferred radioprotective effects to radio‐sensitive DMG cells given 8 Gy ionizing radiation, preventing apoptosis. These EVs also induce enhanced glycolysis in radiosensitive H3K27M‐DMG, compared to treatment with autologous EVs. Summary/Conclusion : Pediatric DMG‐derived EVs induce functional changes in radiation‐naive DMG cells and confer radioresistant phenotypes.

Dendritic

Xiao‐Qing Liu ; Qingling Fu The First Affiliated Hospital, Sun Yat‐sen University, Guangzhou, China (People's Republic) Introduction : Mesenchymal stromal cells‐derived small extracellular vesicles (MSC‐sEVs) have recently attracted considerable attention because of their therapeutic potential in various immune diseases. We previously reported that MSC‐sEVs could exert immunomodulatory roles in allergic airway inflammation by regulating group 2 innate lymphoid cell (ILC2) and dendritic cell (DC) functions. Therefore, this study aimed to investigate the therapeutic effects of MSC‐sEVs on mature DC (mDC)‐ILC2 interplay in allergic rhinitis (AR). Methods : Here, we isolated MSC‐sEVs from induced pluripotent stem cells (iPSC)‐MSCs using anion‐exchange chromatography for the generation of sEV‐mDCs. sEV‐mDCs were co‐cultured with peripheral blood mononuclear cells (PBMCs) from patients with AR or purified ILC2s. The levels of IL‐13 and GATA3 in ILC2s were examined by flow cytometry. Bulk RNA‐sequence for mDCs and sEV‐mDCs was employed to further probe the potential mechanisms, which were then validated in the co‐culture systems. Results : sEV‐mDCs showed weaker capacity in priming the levels of IL‐13 and GATA3 in ILC2s when compared with mDCs. Furthermore, there was higher PGE2 and IL‐10 production from sEV‐mDCs, and the blockade of them especially the former one could reverse the inhibitory effects of sEV‐mDCs. Summary/Conclusion : We demonstrated that MSC‐sEVs were able to dampen the activating effects of mDCs on ILC2s in patients with AR. Mechanismly, the PGE2‐EP2/4 axis played an essential role in the immunomodulatory effects of sEV‐mDCs on ILC2s. Herein, we provided new insights into the mechanism underlying the therapeutic effects of MSC‐sEVs in allergic airway inflammation. Funding : National Key R&D Program of China (2022YFA1104900). Keywords : Small extracellular vesicles, mesenchymal stromal cells, dendritic cells, group 2 innate lymphoid cells, prostaglandin E2, allergic rhinitis

Detection

Mariani Farias Fiorenza 1 ; Alessandra Bridi 1 ; Gislaine Dos Santos 2 ; Paola Rosa 1 ; Luana Alves 1 ; Priscila Assis Ferraz 3 ; Matheus A. Chaves 3 ; Guilherme Pugliesi 3 ; Felipe Perecin 4 ; Flávio Meirelles 4 ; Juliano C. Da Silveira 2 1 University of São Paulo, Brazil; 2 University of São Paulo, PIRASSUNUNGA, Brazil; 3 University of Sao Paulo, Brazil; 4 University of São Paulo, 13635900, Brazil Introduction : Embryo‐maternal communication is essential for pregnancy establishment. Extracellular vesicles (EVs) are significant molecules in intercellular communication required during early pregnancy. The EVs can directly or indirectly trigger an endocrine response. However, at this stage of pregnancy, the uterine epithelium has a stable adhesion that could alter the exchange of molecules. Accordingly, the present study aims to investigate whether stained EVs leave the uterine environment. Methods : The experiment used six Nellore heifers. The heifers received an intrauterine infusion of either PBS+PKH26 (control group) or follicular fluid (FF)‐derived EVs, isolated by ultracentrifugation twice at 120000xg and stained with PKH26 (treatment group). To isolate EVs, plasma samples were taken from the jugular vein at pre‐determined periods (10 min, 30 min, 1 h, and 3h). EVs were isolated by size exclusion chromatography (Izon qEV35) and evaluated by nanoparticle tracking analysis for particle size and concentration and flow cytometry for positive events for PKH26. Controls were performed to discriminate other nanoparticles in flow cytometry, and samples were labeled with CD9‐FITC and CD63‐FITC. Data were compared by ANOVA followed by Tukey. Results : Evs from the treatment group had higher concentration and particle size than those from the control group (P< 0.05). Considering only the treatment group, size and concentration increased over time (P< 0.05). The PKH26‐positive events differed by time and group, although the number of events was relatively small (P< 0.05). The detection of EVs in the systemic circulation was relatively rapid, peaking at 30 min, and after a longer period, the detection decreased, suggesting that the effect and migration of EVs are timely. The detection of PKH26 in the control animals could also represent the autofluorescence of particles, one of the limitations of the present experiment; however, the values in the treatment group were always numerically higher when subtracted from the negative samples. Summary/Conclusion : Although this experiment has some pitfalls, our data suggest that stained EVs leave the uterine environment based on their detection in the bloodstream and elicit an endocrine response in as short as 30 minutes. Funding : FAPESP #2022/01235‐0; #2021/06645‐0; #22/01505‐8; #2015/21829‐9. Keywords : reproduction, pregnancy, extracellular vesicles

Ectosomes

Hui Su Jeong 1 ; Seon Ok Kim 2 ; Ji Eun Lee 2 1 Sungkyunkwan University, Seoul, Republic of Korea; 2 SungKyunKwan University, Republic of Korea Introduction : Neuropathy occurs when the peripheral nervous system (PNS) loses its ability to repair nerves by aging. While neurons are regenerating, Schwann cells work with immune cells to aid neurons. Inflammatory factors accumulate with age, causing chronic inflammation and interfering with nerve regeneration. Although Schwann cells have been suggested as a key player in PNS regeneration, how Schwann cells regulate the PNS in chronic inflammatory conditions is poorly understood. In the present study, we hypothesize ectosomes of Schwann cells are influenced by inflammation, which affects nerve regeneration. Neuromuscular junction (NMJ) is the synapse between motor neuron and muscle fiber in the PNS. Recently, interest has shifted from the research of neuronal regeneration to the study of the NMJ recovery mechanism in order to overcome peripheral neuropathy. Thus, we investigated the role of Schwann cells in the formation of NMJ and the effect of chronic inflammation on Schwann cells‐dependent NMJ function. Methods : S16 Schwann cell line and mouse primary Schwann cell were treated with 2,4,6‐Trinitrobenzenesulfonic acid(TNBS) to induce inflammation and Mice were treated with dextran sulfate sodium (DSS) and cardiotoxin (CTX) to induce both inflammation and neuromuscular junction regeneration. We observed NMJ defects with abnormal ectosome‐related gene expression in aged mice. Schwann cell media were collected for ectosome extraction using ultracentrifuge and identified their composition. Results : We treated mice and S16 Schwann cell line with chemicals and examined the levels of inflammatory cytokines to determine whether chronic inflammation was induced. Intriguingly, we found that in chronic inflammatory conditions, Schwann cells cannot make amount of ectosomes comparing with normal condition. We used Schwann cell ectosomes to rescue neuromuscular junction defects in zebrafish and found there were enhanced synaptic regeneration in zebrafish trunk. Summary/Conclusion : In summary, we showed Schwann cell‐derived ectosomes can rescue the defects of neuromuscular junction and can be negatively affected by chronic inflammation. Funding : The work has been done by Molecular & Medical Genomics lab of Ji Eun Lee in SungKyunKWan University and supported by the National Research Foundation, funded by the Korean government's MSIP (#2021R1A2C3004572, #2021R1A4A2001389 to J.E.L., and #2021R1A6A3A13041249, #2022K1A3A1A12079560 to H.S.J.). Keywords : schwann cell, ectosome, neuromuscular junction, chronic inflammation

Effective

Jacki Kornbluth 1 ; Emily Matchett 2 ; Natalie Sutton 2 1 Saint Louis University, St. Louis, USA; 2 Saint Louis University, USA Introduction : NK3.3 is a human NK cell line derived from the blood of a healthy donor; it has the same strong anti‐tumor activity as primary NK cells. We demonstrated that EVs derived from NK3.3 cells kill leukemia, lymphoma, myeloma, breast cancer and glioblastoma cells without harming healthy cells. This makes NK3.3 EVs a potential new treatment for cancer. However, as a normal cell line, NK3.3 eventually undergoes growth arrest. Another challenge to clinical development is large scale production and processing of EVs. To begin addressing these issues, we immortalized NK3.3 and isolated EVs using polyethylene glycol (PEG)‐acetate precipitation to simplify processing and increase yield. Methods : NK3.3 cells were immortalized by transduction with htert lentivirus (NK3.3‐LTV). EVs were isolated from culture supernatants by differential high‐speed centrifugation, sterile filtration, followed by precipitation using either a commercial kit (ExoQuick‐TC) or homemade PEG‐acetate. EVs were characterized by nanoparticle tracking analysis, protein composition and yield. Viability and proliferation of tumor and normal cells after NK EV treatment were evaluated by cell counts, cytotoxicity assays, and flow cytometry. Results : NK3.3 EVs from all preparations ranged in size from 100–200nm and had identical protein profiles. PEG isolation resulted in a higher protein and particle concentration than ExoQuick isolation. EVs from NK3.3‐LTV cells had similar protein and particle concentration as EVs from non‐immortalized cells. All NK3.3 EVs decreased tumor growth and viability equally and none affected healthy cells. Summary/Conclusion : EVs from immortalized NK3.3, isolated by centrifugation, filtration, and precipitation with PEG‐acetate, displayed the same strong anti‐tumor activity as EVs from non‐immortalized cells, with no toxicity to normal cells. This study is the first step towards large‐scale production of NK3.3 EVs for clinical use. Funding : VA and NIH grants to JK. Keywords : natural killer, cancer, cytotoxicity, tumor

Efficient

Lucia Catani 7 ; Ghazal Narimanfar 1 ; Valentina Marassi 2 ; Dorian Forte 1 ; Martina Barone 3 ; Giuseppe Auteri 4 ; Valentina Papa 5 ; Stefano Giordani 2 ; Camilla Mazzoni 4 ; Filippo Maltoni 6 ; Nicola Vianelli 4 ; Giovanna Cenacchi 5 ; Michele Cavo 7 ; Francesca Palandri 8 ; Lucia Catani 7 1 Department of Medical and Surgical Sciences, Institute of Hematology “L. and A. Seràgnoli”, University of Bologna, Bologna, Italy, Italy; 2 Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy, USA; 3 Department of Surgical and Medical Sciences, University of Bologna, Bologna, Italy, USA; 4 Department of Medical and Surgical Sciences, Institute of Hematology “ L. and A. Seràgnoli”, University of Bologna, Bologna, Italy, USA; 5 Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, Bologna, Italy, USA; 6 Department of Medical and Surgical Sciences, Institute of Hematology “L. and A. Seràgnoli”, University of Bologna, Bologna, Italy, Bologna, Italy; 7 1. Department of Medical and Surgical Sciences, Institute of Hematology “L. and A. Seràgnoli”, University of Bologna, Bologna, Italy; 2. IRCCS Azienda Ospedaliero‐Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy, Italy; 8 IRCCS Azienda Ospedaliero‐Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy, USA Introduction : The role of Extracellular Vehicles (EVs) in orchestrating the complex interplay between tumor cells and the inflammatory and senescent microenvironment is poorly understood. New method to obtain EVs from low amount of blood are urgently needed. Here, based on a novel and customized flow field fractionation (FFF) method, we isolated EVs from low amount of plasma from patients with Polycythemia Vera (PV). PV is a clonal disorder of hemopoietic stem cell characterized by chronic inflammation. Functional characterization of EVs in PV with a special focus on the inflammatory and senescence pathways is still elusive. Methods : To isolate EVs, platelet‐free plasma samples (40 μl/subject) were collected from PV patients (n = 3) and healthy donors (HD; n = 3). Plasma was size‐fractionated with FFF method. EV‐enriched fractions were concentrated by ultrafiltration (100 kDa ultrafilters) and characterized by transmission electron microscopy, Tunable Resistive Pulse Sensing, western blotting (according to MISEV 2018), and MACSPlex. Then, we functionally investigated in vitro effects of EVs on senescence phenotype (β‐Galactosidase expression) and intracytoplasmic production ability of Interleukin‐1β (IL‐1β) and IL‐6 in primary normal circulating mononuclear cells by Flow Cytometry. Results : The isolated EVs from PV patients were 95–117 nm in size, spherical, and expressed EV‐specific markers including CD9, CD63, CD81, FLOT‐1 and TSG101. Western blotting ruled out also the presence of contaminants, such as albumin. Interestingly, PV‐EVs were enriched in immune, tumor, and senescence markers including CD25, CD24, ROR1, CD29, and β‐Galactosidase. Based on EV functionality, EV fractions from both PV patients or HD promoted the senescent phenotype, but reduced the intracytoplasmic production of IL‐6 by CD14+ monocytes. Notably, no effects were observed for IL‐1β. Summary/Conclusion : The newly customized method was convenient (20 minutes isolation time) and effective in the isolation of biologically active EVs directly from plasma of blood cancer patients to cast light on the role of EVs in senescence and inflammation. Funding : Italian Ministry of Research through the PhD scholarship (PNRR PON project‐University of Bologna), the FIN‐RER Bottom up project and Italian Ministry of Health, RC‐2022‐ 2773402 project. Keywords : senescence, inflammation, blood cancer, flow field fractionation

Embryonic

Hernán González‐King Garibotti 1 ; Patricia Rodrigues 2 ; Tamsin Albery 2 ; Karin Åvall 2 ; Gentian Musa 2 ; Dominika Kardasz 2 ; Tania Incitti 3 ; Cecilia Graneli 3 ; Kasparas Petkevicius 2 ; Miguel Carracedo 2 ; Sandra Tejedor 2 ; Sepideh Hagvall 2 ; Alena Ivanova 4 ; Philippe Menasche 5 ; Niek Dekker 6 ; Qing‐Dong Wang 1 ; Karin Jennbacken 2 1 1. Bioscience Cardiovascular, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Mölndal, Sweden; 2 1. Bioscience Cardiovascular, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Sweden; 3 2. Cell Therapy, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Sweden; 4 3. Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Mölndal, Sweden; 5 4. Université de Paris, PARCC, Inserm, F‐75015 Paris, France., paris, France; 6 3. Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden, Molndal, Sweden Introduction : Heart failure is a leading cause of morbidity and mortality, affecting over 60 million patients worldwide. Although several drugs and mechanical devices can improve cardiac function, they do not stop disease progression or regenerate new cardiac tissue. During the past two decades, several clinical trials have shown stem cells as a promising way to repair damaged myocardium and small extracellular vesicles (SEV) have been identified as one of the major mediators of stem cell‐induced effects. Previous studies showed that EV isolated from different cell sources improve cardiac function through cardiac repair after myocardial infarction (MI) in rodents. The SEV components responsible for the effects observed, as well as a comparison of EV from different cell sources, are still unclear. In this work, we seek to understand which of these sources of SEV is the most suitable to be used for cardiac repair. Methods : We isolated the SEV from the conditioned media of primary bone marrow mesenchymal stromal cells (BM‐MSC), an immortalized line of MSC (hTERT‐MSC), human embryonic stem cells (ESC), cardiac progenitor cells (CPC), human cardiomyocytes (CM) and human ventricular cardiac fibroblasts (VCF) by a serial ultracentrifugation protocol. This was followed by a characterization of our isolates by western blot, nanoparticle tracking analysis, and electron transmission microscopy. Then, we evaluated the functionality of the different types of SEV in in vitro assays of cardio‐protection, angiogenesis, cardiac fibrosis, cardiomyocyte proliferation, and immunomodulation. Furthermore, we evaluated the functionality in vivo in a rodent model of myocardial ischemia‐reperfusion injury (IRI) and characterized the composition of the best SEV candidates. Results : ESC‐SEV showed stronger pro‐regenerative effects in vitro compared to the other SEV types evaluated. Furthermore, ESC‐SEV dampened the increase in the left ventricular end‐diastolic volume (LVEDV) in the post‐ischemic heart. These effects correlated with reduced fibrosis and increased angiogenesis detected by histological analysis of the post‐ischemic heart sections. Summary/Conclusion : In summary, our data show that ESC‐SEV significantly reduced adverse cardiac remodeling after MI by promoting a reduction in fibrosis and an increase in angiogenesis in the post‐ischemic heart, representing a promising treatment to promote cardiac repair post‐MI. Funding : AstraZeneca. Keywords : cardiac repair, small extracellular vesicles, regeneration

Evolution

Kenneth W. Witwer Johns Hopkins University, Baltimore, USA Introduction : The “extracellular vesicle club”, or EVClub, is an education, networking, and post‐publication peer review suite consisting of a weekly virtual journal club event, regular social media and email communications, and video archiving. Established in March, 2020, as many laboratories around the world began to be shut down by the COVID‐19 pandemic, EVClub continued to amass followers even after the lifting of COVID restrictions. Methods : EVClub uses a combination of video conferencing software, data collection by survey, email and social media marketing, and video archiving on YouTube and other platforms. Results : EVClub achieved its initial objective of bringing EV researchers together during the COVID pandemic restrictions of 2020 and 2021 but has continued to serve the community. Today, EVClub has >4000 email subscribers and >3000 YouTube subscribers. Although average attendance at the live events has declined with the lifting of COVID restrictions, from >120 participants per session (2020‐2021) to just under 80 (2022‐2023), views of video archives have increased strongly, to more than 100,000 total. In addition to scientific presentations, EVClub features collaborations with allied societies including the Student Network on EVs and SOCRATES, and videos advertising national or regional EV societies and the ISEV journals, JEV and JExBio. Not everything has “worked”: for example, a straight‐to‐video format called “EVTrailers” was first offered in 2022 but received only one submission. Plans for the future of EVClub and its relationship with ISEV will be presented here, including ideas for deeper integration with society journals and rigor initiatives. Summary/Conclusion : The EVClub will continue to serve the EV community. Feedback and suggestions are encouraged.

Exogenous

Johannes Bader 1 ; Elita Montanari 2 ; Valeria Mantella 3 ; Finn Brigger 2 ; Jean‐Christophe Leroux 4 1 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, 8093 Switzerland, Zürich, Switzerland; 2 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, 8093 Switzerland, Switzerland; 3 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, 8093 Switzerland., Zurich, Switzerland; 4 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, 8093 Switzerland, Zurich, Switzerland Introduction : Extracellular vesicles (EVs) are important intercellular communicators that might be used as delivery vehicles for macromolecular drugs. Yet, challenges remain in manufacturing EV‐based drug carriers. Presently, a versatile and efficient drug loading strategy, especially for hydrophilic macromolecular drugs, such as nucleic acids, is still missing. In this work, various exogenous loading strategies were compared head‐to‐head in terms of loading efficiency and vesicle integrity to find the optimal one. Methods : EVs were isolated by ultracentrifugation from 3D cultures of mesenchymal stem cells (MSCs). A small (69 nts) RNA aptameric probe and fluorescently labeled small interfering RNA (21 bp) were used as model compounds and were subjected to different exogenous loading methods (e.g., extrusion, electroporation and vesicle hybridization). Unloaded RNA aptamer was removed by degradation with RNase, and loading efficiencies were subsequently analyzed by fluorescence read‐out or nano flow cytometry (nanoFCM). Physicochemical characteristics of MSC‐EVs, as well as the biological activity of two membrane enzymes, CD73 and DPP4, were assessed after loading. Results : Most of the investigated loading methods did not lead to efficient incorporation of the RNA aptamer in EVs, except for the hybridization‐based approach. However, nanoFCM experiments revealed that loading efficiencies of commercially available EV hybridization reagents still remained relatively low (< 5 %). Further, we showed that the enzymatic activity of the MSC‐EV membrane proteins was downregulated after the hybridization process and that the polydispersity increased, suggesting the formation of aggregates. Summary/Conclusion : Efficient loading of nucleic acids into EVs remains challenging, with the hybridization‐based approach being the most promising one. However, novel formulations with minimal impact on the EV structural and functional characteristics need to be developed. Funding : This work was supported by the ETH Zurich – Open ETH project SKINTEGRITY.CH. Keywords : extracellular vesicles, drug delivery, drug loading, method comparison, hybridization, nucleic acids, gene therapy

Exploring

Gaelle M. Vanderstichelen 1 ; Florian Müller 2 ; Claire Remacle 3 ; Ingrid Struman 4 1 University of Liège, Jambes, Belgium; 2 Université de Liège, Belgium; 3 University of Liège, Belgium; 4 ULiège (Université de Liège), Liege, Belgium Introduction : Studying extracellular vesicles (EVs) in the tumor microenvironment (TME) remains challenging because one needs help to distinguish EVs between cell origins easily. In this project, we studied surface proteins in EVs in an in‐vitro and in‐vivo model of pancreatic cancer. The objective is to characterize the different EV populations and select specific EV populations based on their surface markers, determine their role in pancreas cancer pathogenicity, and conduct longitudinal studies to assess the evolution of the EVs landscape during pancreatic cancer progression. Methods : EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, dynamic light scattering, and Western blot. The surface marker profile for the exosomes isolated from different cell models or that compose the TME in liquid biopsy was obtained using the Luminex MAGPIX (Luminex Corp., Texas, USA). This multiplex bead methodology allows the detection of up to 50 surface markers simultaneously. We also generated murine pancreatic cell lines carrying a CD9‐HA TAG that we could detect on EVs. These cells were injected into mice to follow the tagged EVs during pancreatic cancer progression. The other EVs originating from other cells that compose the TME were also analyzed. Results : We showed that EVs isolated from different cell lines expressed different markers and that the EVs showed the same pattern of expression for the cell markers of the cells of origin. Hence, we found similar markers specific to the cell of origin, and we established a distinct signature for different EVs that compose the TME. We also demonstrated that the MAGPIX protocol could detect EV surface markers in mice plasma samples. Summary/Conclusion : We showed that EVs isolated from different cell types expressed distinct markers and that MagPix could detect specifically surface markers on EVs in vitro and plasma samples. Funding : Fonds de la Recherche Scientifique (F.R.S.‐FNRS), ULiège. Keywords : Biomarkers, MAGPIX, pancreatic cancer

Fastev™

Maija Puhka 1 ; jaana Kekkonen 2 ; Rikke Baek 3 ; Malene Møller Jørgensen 4 ; Pia RM Siljander 5 1 EV Core and Institute for Molecular Medicine Finland FIMM, University of Helsinki, Finland, Helsinki, Finland; 2 EV Core and Institute for Molecular Medicine Finland FIMM, University of Helsinki, Finland, Finland; 3 Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark, Aalborg, USA; 4 Department of Clinical Immunology and Medicine, Aalborg University Hospital, Aalborg, Denmark, Aalborg, Denmark; 5 EV group & core, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Finland, Helsinki, Finland Introduction : Interest in the EV biomarker discovery is constantly expanding. To this end, tens of different EV isolation methods have been developed generating different EV populations, yields, purities, stability, volumes, molecular detectability and downstream applicability. Usually, it is not known which is the best EV isolation method in each individual case and finding a suitable one can take years. Furthermore, as most of the methods are of low‐to‐medium throughput or expensive, high impact studies and clinical feasibility remain out of reach. Here we introduce a novel high throughput platform, FastEV™, which enables fast screening of various EV enriching precipitation conditions from blood samples. Methods : Healthy donor plasma and serum samples (∼0.1‐1 ml) were subjected to EV enrichment with different proprietary FastEV™ conditions, ultracentrifugation or precipitation using polyethylene glycol (PEG). Then, the isolates or plasma/serum controls were analysed for their protein, RNA and cfDNA pattern, or using EV Array, ApoB ELISA, and small RNA and mRNA sequencing. Results : FastEV™ workflow in 96‐well plates took < 1h. FastEV™ isolates presented variable quantities of proteins, lipoproteins, small RNA, mRNA, cfDNA and EV protein markers depending on the FastEV™ condition used. EVArray showed that reproducibility of isolation between days was up to R2 = 0.99 for the 14 EV proteins tested. When comparing the results among the different FastEV™ conditions or conditions vs controls, we observed both similarities and differences in individual parameters and also unique multiparametric profiles. Summary/Conclusion : This work provides a catalogue of isolate compositions obtained from each FastEV™ condition. With this knowledge, the best isolation condition can be selected if the desired biomarker target is known. Additionally, FastEV™ platform can be used to screen for the optimal condition to separate cases from controls. As the isolates were suitable for many downstream analytics, FastEV™ enables a wide range of high throughput biomarker discovery studies. Funding : This work has received funding from Business Finland and University of Helsinki. Keywords : biomarker discovery, high throughput, plasma, serum, extracellular vesicles, exosomes, isolation, RNA, miRNA, protein, cfDNA

Hif‐1Α

Orazio Fortunato 1 ; Ilaria Petraroia 2 ; Francesca Pontis 2 ; Patrizia Ghidotti 2 ; Luca Roz 2 ; Paola Suatoni 2 ; Ugo Pastorino 2 ; Gabriella Sozzi 3 ; Giulia Bertolini 4 1 Fondazione IRCCS Istituto Tumori Milano, Milan, Italy; 2 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 3 Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy, Milan, Italy; 4 Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy, Italy Introduction : Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) or the onset of lung cancer. Reduced air flow in COPD patients promotes the activation of HIF‐1α hypoxic signaling pathway that was associated to a reduction in lungs functionality and increased risk to develop lung cancer. The subset of CD133+ cancer stem cells (CSC) was demonstrated to be responsible for lung cancer initiation. Cells co‐expressing CD133 and CXCR4 are defined as the subpopulation of metastasis initiating cells (MIC). Recently, the involvement of extracellular vesicles in the pathogenesis of lung diseases gained attention. The aim of this project is to evaluate the pro‐tumorigenic role of EVs from COPD patients in the development of lung cancer, exerted through the modulation of CSC subsets properties Methods : EVs were isolated by ultracentrifugation from plasma of heavy smoker volunteers with (COPD‐EVs) or without (HS‐EV) COPD enrolled in a LDCT screening trial. EVs characterization was performed accordingly to the MISEV guidelines (Flow cytometry, NTA, TEM and WB). The protumorigenic role of EVs was evaluated on transformed Epithelial Cells (HBEC) with different oncogenic manipulation (P53 and/or KRAS) using 2D and 3D in vitro assays. Results : Circulating COPD‐EVs mainly derived from immune and endothelial cells as indicated by high levels of CD31, CD146, CD45 and HLA. COPD‐EVs increased the subset of CD133+CXCR4+ MICs along with the stimulation of 3D growth and acquisition of mesenchymal phenotype. Notably this effect was observed only in HBEC‐ shP53‐KRASV12high cells but not in non malignant parental HBEC‐1, single mutated HBEC‐KRASV12 or HBEC‐shP53 cells. Mechanistically, HIF‐1α transferred from COPD‐EVs triggers CXCR4 activation that in turn mediates MICs expansion and acquisition of pro‐tumorigenic effects. Hypoxia induced a 4‐fold increase of MIC cells as observed after COPD‐EVs. HIF‐1α inhibitor or CXCR4 silencing prevents acquisition of malignant traits induced by COPD‐EVs or hypoxia. Interestingly, we found higher levels of HIF‐1α inside EVs isolated from COPD individuals that develop lung cancer compared to cancer‐free individuals Summary/Conclusion : Our findings demonstrated that in a context of transformed but not fully tumorigenic lung epithelial cells, COPD‐EVs can promote the expansion of MICs through HIF‐1α‐CXCR4 axis activation, providing those malignant properties that might sustain lung cancer initiation. Funding : The study was supported by grants from the Italian Association for Cancer Research [Investigator Grant Nos. 18812 and 23244 to G.S.], Italian Ministry of Health (RF‐2018‐12367824 to G.S.; GR‐2019‐12369047 to O.F), Fondazione Regionale per la Ricerca Biomedica (Regione Lombardia) (1731093 to G.B.).

Increased

Giulia Marostica 1 ; Alessandra Mandelli 2 ; Annamaria Finardi 3 ; Susanna Manenti 2 ; Valeria Berno 2 ; Alessia Loffreda 2 ; Roberto Furlan 4 1 Ospedale San Raffaele‐ IRCCS San Raffaele Scientific Institute, PONZANO VENETO, Italy; 2 IRCCS San Raffaele Scientific Institute, Italy; 3 IRCCS San Raffaele Scientific Institute, milan, Italy; 4 IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction : The role of extracellular vesicles (EVs) in several oncological and neurological diseases is widely studied. Their use in clinical practice is still delayed due to some difficulties in tailoring their cargo and tissue tropism. Their use as a delivery vector for several types of molecules could be a very interesting tool to exploit in several pathological conditions. In our system, we loaded EVs with IL4 (IL4 EVs) and used them to mediate microglia phenotype shift towards an activated state. Methods : We engineered a murine microglia cell line modified with a lentivirus overexpressing IL4, or with CRISPR Cas9 to display IL4R tagged with eGFP. We observed that the use of IL4 EVs elicits a faster and more potent effect on recipient microglia when compared to a similar concentration of recombinant IL4 (rIL4), administered in soluble form. Results : EVs use endocytosis as a general mechanism to enter recipient cells and release part of their cargo by endosomal escape in the cell cytosol. So, we studied EVs internalization by electron microscopy and immunofluorescence, measuring the colocalization of specific organelles belonging to the endocytic pathway. We hypothesized that the difference in the signaling efficiency between the treatment could be related to their different interaction with IL4R, in terms of cellular localization and receptor engagement. We finally studied in live imaging receptor clusters formation mediated by short‐term administration of IL4 EVs and rIL4. Our experiments indicate that IL4 EVs and rIL4 have different effects on recipient cells. In our model, IL4 EVs change microglia phenotype after 3h post‐administration. The kinetic of the process mediated by EVs is faster than the one mediated by rIL4. Such difference suggests that the process, or some component of it, is different between IL4 EVs and rIL4. Receptor clustering on plasma and endosomal membranes can mediate signal boosting, so cluster formation mediated by the two treatments can be different. Summary/Conclusion : In our system, it is clear that EVs stabilize receptor clustering formation keeping their number stable, with respect to rIL4, and increasing receptor quantity over time. Although EV use in clinical practice is distant, we think that understanding EVs signaling mechanisms and biology is a key point in moving forward the research on their use as a delivery tool. The advantage given by their use in vitro, if translated in vivo, could be greatly exploited and open a very new way of delivering therapeutic molecules. Keywords : uptake, internalization, signaling, receptor clustering

Inducible

Xinyu Niu 1 ; Zhifeng Deng 2 ; Yang Wang 3 ; Qing Li 3 1 Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, China (People's Republic); 2 Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, USA; 3 Institute of Microsurgery on Extremities, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China (People's Republic) Introduction : The polarization of microglia plays an important role in the outcome of ischemic stroke. In the aged population, senescent microglia show a predominant pro‐inflammatory phenotype, which leads to worse outcomes in aged ischemic stroke compared to young ischemic stroke. We hypothesized that iPSC‐sEVs could alleviate microglia senescence to regulate microglia polarization in aged ischemic stroke. Methods : iPSC‐sEVs were pre‐administrated to mice once a week for 2 months. Ischemic stroke operation was performed after the last administration of sEVs followed by the detection of polarized microglia. The infarct volume and behavior tests were examined to assess the protective function of iPSC‐sEVs on aged ischemic stroke. Proteomics combined IPA was used to explore the functional proteins carried by iPSC‐sEVs. Results : We showed that treatment with iPSC‐sEVs significantly alleviated microglia senescence and inhibited pro‐inflammatory activation of microglia both in vivo and in vitro. Furthermore, iPSC‐sEVs shifted microglia from pro‐inflammatory phenotype to anti‐inflammatory phenotype, which reduced the apoptosis of neurons, and improved the outcome of aged stroke mice. Mechanism studies showed that iPSC‐sEVs reversed the loss of Rictor and downstream p‐AKT (s473) in senescent microglia by transferring TGF‐β1, which was involved in the senescence and pro‐inflammatory phenotype regulation of microglia. Taken together, our work demonstrates iPSC‐sEVs reverse the senescent characteristic of microglia in the aged brains and therefore improve the outcome after stroke. Summary/Conclusion : This study demonstrated that iPSCs‐sEVs improved the inflammatory microenvironment and facilitated the shift of microglia polarization from pro‐inflammatory phenotype to anti‐inflammatory phenotype through alleviating microglia senescence, therefore protecting neurons from death and improved outcome of aged ischemic stroke. As the anti‐ageing nano‐vesicles, iPSCs‐sEVs provide a novelty therapeutic strategy for aged ischemic stroke and other ageing‐associated neurodegenerative disease. Funding : This study was kindly funded by the National Natural Science Foundation of China (Grant No. 82071371, 82172421 and 82201543). Keywords : induced pluripotent stem cell‐derived small extracellular vesicles, senescent microglia, ischemic stroke, aging

Isolation

Ji Yeong Yang 1 ; Jianning Yu 2 ; Hyorim Jeong 3 ; Kyung‐A Hyun 1 ; SeHee Cho 4 ; Juhee Jeon 4 ; Yoonah Kim 4 ; Sunyoung Park 1 ; Hyo‐Il Jung 5 1 School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea, Seoul, Republic of Korea; 2 Department of Biomedical Laboratory Science, College of Software and Digital Healthcare Convergence, Yonsei University, Republic of Korea; 3 The DABOM Inc., Seoul, South Korea, Republic of Korea; 4 ACTIVON Co., Ltd, Cheongju, South Korea, Republic of Korea; 5 Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea Introduction : Plant‐derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as cosmetic and therapeutic materials. It has been known for a long time that Centella asiatica extracts exhibit lots of biological activity such as anti‐aging, anti‐oxidant, collagen enhancement, and moisturizing. In order to isolate EVs from Centella asiatica, it is necessary to develop a new method because the widely used method for the isolation of EVs, such as ultracentrifugation, precipitation, and filtration, have shown a low yield and difficulty in mass production. Methods : Here, we developed an EV extraction method to effectively separate the specific EVs from Fresh Centella asiatica. First, total EVs from fresh Centella asiatica (CEVs) were extracted by using tangential flow filtration (TFF). Specific EVs from fresh Centella asiatica (spCEVs) were sequentially isolated by using the microfluidic chip with specific target‐biotinylated streptavidin beads. Then, the biological properties of CDEVs and spCDEVs were evaluated in comparison with EVs from commercialized Centrella asiatica extracts (exCEVs). Results : Our results showed that spCEVs were isolated at higher concentrations and had faster separation time than the other three different conventional methods. In addition, the spCEVs had better uptake efficiency to the target cells than CEVs and exCEVs, resulting in the promotion of skin regeneration. Also, spCEVs showed significant improvement in skin hydration. Summary/Conclusion : These results provide a foundation for the development of plant‐derived therapeutic agents as well as the potential industrial applications of various PDEVs. Funding : This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2021R1A2C3011254), Korea Medical Device Development Fund grant funded by the Korea government (MSIT,MOTIE, MOHW,MFDS) (Project Number: 1711139115, KMDF_PR_20210527_0008), and Korea Environment Industry & Technology Institute (KEITI) through Aquatic Ecosystem Conservation Research Program, funded by Korea Ministry of Environment (MOE) (2020003030007).

Linearity

Jeff Bodycomb 1 ; Julie Chen Nguyen 2 1 HORIBA Instruments, Piscataway, USA; 2 HORIBA Scientific, Irvine, USA Introduction : EV concentration measurement results are routinely reported. For NTA analysis many exosome samples need to be diluted before measurement. Naturally, the degree of required dilution needs to be known and that is coupled to trade‐off's in measurement performance. One issue is the linearity of the measurement results. That is how do determine the range of concentrations for which results are sufficiently reliable. Methods : Lyophilized exosomes were obtained from Abcam (Lyophilized MCF7 Exosome Standards, lot GR3401014‐1). These are derived from MCF7 a human cancer cell line and characterized by Abcam. Exosomes were reconstituted following manufacturer instructions, and diluted in Dulbecco's phosphate buffered saline (dPBS). NTA was performed with a HORIBA ViewSizer 3000 multi‐laser NTA instrument. Multiple lasers were used to ensure analysis of all EV sizes in the sample. Laser power settings were 210 mW blue, 12 mW green, 8 mW red and camera gain was 30 dB. Results : A single aliquot of reconstituted exosomes was diluted to varying degrees and the concentration of each resulting sample was determined by NTA. Dilutions ranged from 30,000 times to 1000 times and the resulting measured concentrations ranged from ∼5e6 p/mL to 6e7 p/mL. The lowest measured concentrations showed distortion due to background particle concentration while the highest measured concentration showed an overly low concentration. Summary/Conclusion : A linearity study is important to determine the acceptable range of measured concentrations. There is a trade‐off between linearity (underreporting concentration) and uncertainty/measurement duration (measuring too few particles). Keywords : concentration, NTA, nanoparticle tracking analysis, viewsizer, uncertainty, error, linearity

Mammalian

Gal Bitan 1 ; Ibrar A. Siddique 1 ; Jing Di 2 ; Saumya Tawakley 1 ; Joanna Zhao 1 ; Kylie McCauley 1 1 UCLA, Los Angeles, USA; 2 U Kentucky Medical Center, Los Angeles, USA Introduction : Proteostasis dysfunction and accumulation of protein aggregates occur under many cellular stress conditions. Cellular mechanisms that remove misfolded proteins and damaged organelles have mostly been studied in the context of cell‐autonomous housekeeping mechanisms, such as the autophagy‐lysosomal pathway. Exophers are new cellular structures, first discovered in C. elegans light‐touch neurons in 2017, and hypothesized to be a heretofore unknown component of neuronal proteostasis. It has not been known whether they exist in higher‐order organisms and what roles they might serve if they do. We discovered exophers in mammalian neurons of mice and humans. Methods : Exophers were analyzed in multiple cell culture systems and brain sections using light and fluorescence microscopy. They were recognized morphologically and by the absence of a nucleus. Results : Similar to nematode exophers, mammalian neuronal exophers vary widely in size from ∼1.5 μM in diameter to structures that are as large as the originating cell itself. Accordingly, they can be substantially larger than those in C. elegans. They contain multiple types of cellular organelles and cytoskeletal proteins. Exopher number increases adaptively in response to cell stress but falls sharply under over‐stress conditions. In addition to their adaptive role in the removal of unwanted cellular material and protein aggregates, innate exophers were observed that presumably assist in sharing resources among neighbor cells. Summary/Conclusion : Exophers are newly discovered extracellular vesicles that exist not only in lower organisms but also in mammals, including humans. There appear to be two types of exophers – innate and adaptive – that participate in the transfer and removal of cell content throughout the neuron life and may be important in both health and disease. Funding : The work was supported by NIH/NIA grants R01AG050721 and RF1AG054000. Keywords : exopher, neuron, glia, non‐autonomous, proteotoxicity, clearance, intercellular

Metabolic

Janet E. Sorrells 1 ; Jaena Park 2 ; Edita Aksamitiene 2 ; Marina Marjanovic 2 ; Elisabeth Martin 3 ; Eric Chaney 3 ; Anna Higham 4 ; Kimberly Cradock 4 ; Zheng Liu 4 ; Stephen A. Boppart 2 1 University of Illinois at Urbana‐Champaign, Champaign, USA; 2 University of Illinois at Urbana‐Champaign, Urbana, USA; 3 University of Illinois at Urbana‐Champaign, USA; 4 Carle Foundation Hospital, USA Introduction : Label‐free nonlinear optical microscopy can image and characterize the autofluorescent metabolic cofactors NAD(P)H and FAD in vivo, ex vivo, and in vitro to make inferences about cellular metabolism. One signature of cancer cells is dysregulated metabolism, which can be quantified by NAD(P)H and FAD autofluorescence. Here, we show a multivariate analysis of the optical metabolic signatures of EVs in breast cancer by examining EVs in ex vivo tissue samples as well as EVs isolated from patient serum and urine to determine the potential for using optical metabolic signatures of EVs as biomarkers of cancer. Methods : EVs were isolated from fresh urine and blood serum samples from human breast cancer patients (n = 31; 18 with invasive tumors, 7 with DCIS, 6 with benign tumors) or breast reduction surgery patients (n = 5). Large EVs (LEVs) were isolated by differential ultracentrifugation: samples were spun at 800 × g for 10 min and 2000 × g for 30 min and pellets discarded. The supernatant was spun at 12000 × g for 60 min and the LEV pellet was resuspended in sterile PBS with 25 mM trehalose. Isolated LEVs and ex vivo tissue (received post‐surgery) were imaged with label‐free nonlinear optical microscopy. Informed consent was obtained from participants; the study was approved by the Institutional Review Boards at Carle Foundation Hospital and the University of Illinois at Urbana‐Champaign. Results : LEVs showed optical metabolic differences by cancer status and EV origin. In urinary LEVs, cancer samples showed significantly higher NAD(P)H fluorescence lifetime, indicative of more NAD(P)H‐related protein activity, however this trend was not seen in serum EVs. Summary/Conclusion : The optical signatures of urinary LEVs and ex vivo tissue EVs show potential as biomarkers. Funding : JES was supported by UIUC Department of Bioengineering and an NIH/NIBIB training grant (T32EB019944). This work was supported in part by NIH (R01CA213149, P41EB031772). Keywords : multiphoton microscopy, breast cancer, metabolism, single‐EV analysis, biofluids

Modelling

Giulia Cricrì 1 ; Stefania Bruno 2 ; Linda Bellucci 3 ; Andrea Gobbini 4 ; Federico Caicci 5 ; Stefano Turolo 6 ; Renata Grifantini 4 ; William Morello 6 ; Giovanni Montini 7 ; Federica Collino 8 1 Laboratory of Translational Research in Paediatric Nephro‐urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy, Italy; 2 Department of Medical Sciences, University of Turin, Turin, Italy, Turin, Italy; 3 Laboratory of Translational Research in Paediatric Nephro‐urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy, Milano, Italy; 4 Istituto Nazionale Genetica Molecolare (INGM), Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milan, Italy, Italy; 5 Department of Biomedical Sciences, University of Padova, Padua, Italy, Padua, Italy; 6 Pediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy, Milano, Italy; 7 Laboratory of Translational Research in Paediatric Nephro‐urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; Pediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy, Milano, Italy; 8 Laboratory of Translational Research in Paediatric Nephro‐urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; Pediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy, Milan, Italy Introduction : Idiopathic Nephrotic Syndrome (INS) is a paediatric glomerular disease characterized by immune dysfunction and lipid dysregulation. INS frequently responds to corticosteroids, showing possible adverse effects. Biofluid‐extracellular vesicles (EVs) may represent a source of INS diagnostic biomarkers. Methods : EV surface characterization was analyzed on urine and serum from 80 patients compared to control children (approved informed consent). EV structure, size, and concentration were measured by NTA, TEM, and STORM analysis; expression of EV surface proteins was analyzed by beads‐based cytometry; profiling of lipids was realized by GC on ultracentrifuged EVs. Results : Urine‐EVs showed typical cup‐shaped structure and different tetraspanins spatial distribution, related to the disease. NTA analysis showed a higher urine‐EVs number in INS children, that positively correlated with urinary creatinine levels. A positive correlation was also found between CD9/CD63/CD81 expression and pathological proteinuria levels. Principal component analysis clearly separated patients in active phase of disease from remission. Unsupervised clustering study revealed a superimposable pattern between different disease stages and clearly distinguished INS patients from controls, mainly based on immune/platelet/cell adhesion markers. A cluster of four markers (CD24, HLA‐DR, CD142, SSEA‐4) separated patients in the active phase from remission by univariate analysis. The concomitant serum EVs evaluation allowed the generation of a biofluid matrix protein profile able to better distinguish different disease subtypes. Urine‐EVs lipid profile showed an unbalance in saturated and polyunsaturated fatty acids, as well as a significant decrease of omega‐3/omega‐6 ratio, associated with an inflammatory and pro‐oxidant milieu in INS. Summary/Conclusion : Biofluid‐EV surface signature, by representing immune dysfunction and inflammatory processes, may complement the stratification of different INS subtypes. Keywords : extracellular vesicles, nephrotic syndrome, immune dysfunction, lipid dysregulation

Molecular

Lifang Yang 1 ; Morgan Rouse 2 ; Caleb Smack 2 ; Eric Feliberti 2 1 Eastern Virginia Medical School, Norfolk, USA; 2 Eastern Virginia Medical School, USA Introduction : Breast cancer (BC) is a highly heterogeneous disease with various subtypes that differ in pathological features, prognosis, and therapeutic responses. Current tissue‐based molecular subtyping presents several disadvantages including invasive nature, a snapshot of tumor heterogeneity, and inability to monitor cancer evolution and subtype interconversion. While tumor‐shed extracellular vesicles (EVs) have been shown to play important roles in cancer and glycans are major components of EVs, little is known about the structures of EV glycosylated cargos and their diagnostic value in BC. Here, we characterized the sialylated molecules in EVs derived from different BC subtypes to define subtype‐specific EV glycosylation. Methods : BC cells representing 4 different subtypes (Luminal A, Luminal B, HER2‐enriched, Triple Negative) were cultured in the presence of azido‐modified sialic acid biosynthetic precursor. EVs were isolated from the conditioned media using a differential ultracentrifugation approach and characterized by Nanoparticle Tracking Analysis, transmission electron microscopy, and Western blot (WB) with a panel of EV markers. Surface azidosialic acid in parent cells and EVs were further tagged by biotin via copper‐free click chemistry. Immunofluerecence (IF), WB, and Norther Blot (NB) were performed to evaluate labelled sialoglycans, sialoglycoproteins, and glycoRNAs, respectively. Results : IF results showed that azidosialic acid was efficiently incorporated into cellular nascent sialylatedglycoconjugates, mainly located at cell surface and the Golgi complex. A large range of tagged cell‐surface sialoglycoproteins demonstrated marked difference across BC subtypes and were correlated with BC aggressiveness. Small RNAs bearing sialyloglycans were detected on the cell surface. Collected EVs, mainly between 50–150 nm, harbored membrane‐encapsulated vesicular structure and presented classical EV markers. Importantly, they were heavily sialylated and retained similar surface sialoglycoprotein patterns as their parent cells. Summary/Conclusion : EVs are enriched with BC subtype‐specific silaylated molecules, with the potential in the discovery of novel glycosylated biomarkers to improve BC stratification and diagnosis in a non‐invasive way. Keywords : breast cancer, extracellular vesicles, glycosylation

Nanoscale

Justus Ndukaife 1 ; Theodore Anyika 2 ; Chuchuan Hong 2 1 Electrical Engineering, Center for EV Research, Nashville, USA; 2 Electrical Engineering, Vanderbilt University, USA Introduction : Extracellular vesicles are heterogeneous particles released by cells and comprise a bonafide means for cellular communication. In addition to EVs, recently new kinds of extracellular particles known as exomeres and supermeres have been discovered. The heterogeneity of extracellular vesicles and particles (EVPs) have been identified as a key factor limiting an enhanced understanding of EVPs. Most of the EV analysis techniques such as western‐blotting work at the EV population level and unable to analyze EVs on an individual particle level. Recently, laser trapping Raman spectroscopy (LTRS) has become an emerging approach for EV analysis. In this approach, EVs are trapped within a laser focus using an optical tweezer and the Raman signal is acquired, which provides information on the nature of the biomolecules present in EVs such as proteins, lipids, and nucleic acids. This approach could be harnessed to discriminate EVs from contaminating lipoproteins. However, LTRS are not applicable for analyzing nanosized EVs because the nanosized EVs are too small to be trapped in an optical tweezer due to the diffraction limit of light. In this work, we report a novel approach for trapping nanoscale EVPs by coupling light to optimized gold nanostructures that generate stable optical force due to the excitation of plasmonic waves. We report the stable trapping of nanoscale EVs using the plasmonic cavities for the first time. Methods : The plasmonic cavities for trapping EVs were fabricated by using focused ion billing machine to mill nanoscale features on a 100 nm thick gold film, and packaged into a microfluidic chip. Experimental testing of stable EV trapping at the plasmonic cavities was performed using commercially available EVs obtained from Creative Diagnostics. The EV solution was injected into the microfluidic chip containing the plasmonic cavities. One of the plasmonic cavities was illuminated with a laser beam to trap the EV at the plasmonic cavity. The EV was released by turning off the laser beam. To confirm the size of the trapped EV, we applied a low frequency alternating current field to pattern the trapped EV and image with an SEM. Results : Our results show that nanoscale EVs can be trapped with a low optical power of 2 mW using the plasmonic cavity. In comparison with the conventional laser tweezers, the trapping would have required about 70 to 100 mW optical power. Furthermore, our results show that the EVs can be reversibly released from the trap by turning off the laser power or moving the laser away from the plasmonic cavities. Summary/Conclusion : We have reported a novel nanotweezer approach based on plasmonic cavities for the stable trapping of nanoscale EVs, which are too small to be trapped and analyzed using the conventional laser tweezer. Our proposed plasmonic cavity nanotweezer system paves the way for tether‐free stable trapping of nanoscale EVs and Raman spectroscopy of trapped EVs to understand their heterogeneity. Keywords : laser tweezers, nanoplasmonics

Placental

Saravanakumar Murugesan 1 ; Lakshmi Saravanakumar 2 ; Hanna Hussey 2 ; Rachel G Sinkey 3 ; Adam B Sturdivant 2 ; James A Mobley 2 ; Mark F Powell 2 ; Michelle Tubinis 2 ; Alan Tita 3 ; Tamas Jilling 4 ; Dan E Berkowitz 2 1 Department of Anesthesiology and Perioperative Medicine, Division of Molecular and Translational Biomedicine, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA, Birmingham, USA; 2 Department of Anesthesiology and Perioperative Medicine, Division of Molecular and Translational Biomedicine, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA, USA; 3 Department of Obstetrics and Gynecology, Division of Maternal‐Fetal Medicine, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA, USA; 4 Department of Pediatrics, Division of Neonatology, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL, 35294, USA, USA Introduction : Women with preeclampsia (PE) are at increased risk of cardiovascular disease (CVD) during pregnancy and postpartum. The mechanisms of heightened risk for CVD are poorly understood. Consequently, approaches for prevention and treatment are unknown. Preliminary data indicates that the protein vasorin (VASN) was highly down regulated in extracellular vesicles (EV) isolated from patients with severe PE (sPE‐EV), as compared to normotensive‐EV (NTP‐EV). VASN has been implicated in regulating calcium homeostasis and calcium dynamics in cardiomyocytes. Therefore, we hypothesized that the down regulation of VASN protein in EV in sPE may play a role in PE‐associated cardiac dysfunction via altered regulation of intracellular calcium dynamics. Methods : EV from pregnant women with sPE (n = 15) and NTP (n = 15) were separated from plasma using a precipitation‐based kit. We used an unbiased proteomic approach to compare the EV protein cargo profile from women with sPE and NTP using a tandem mass spectrometry approach. In adult murine cardiomyocytes (mCM), calcium dynamics were assessed using fura‐2AM imaging in an Ion Optics instrument and VASN and calcium sensor proteins were quantified by western blot. Results : We verified VASN down regulation in maternal plasma EV, and in placental tissue in sPE as compared to NTP. We also found that the levels of stromal interaction molecule 1 (STIM 1) were decreased in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CM) and in mCM treated with sPE‐EV. Treatment of contractile mCM with sPE‐EV that have decreased VASN content inhibited levels of STIM 1 while mCM isolated from pregnant mice injected with AD‐sFLT‐1 (a model of PE) exhibited altered dynamics of calcium signaling as compared to mCM from untreated pregnant mice (UT). Likewise, treatment of hiPSC‐CM results in the decrease of STIM1, phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) levels. We also found that in CM isolated from AD‐hsFLT‐1 injected mice had elevated peak Ca2+ ratio when compared to mCM from UT. Summary/Conclusion : Our data indicate that levels of VASN, a TGF‐beta signaling inhibitor, in EV correlate with the effects of EV on expression levels of proteins with roles in Ca2+ signaling and alterations of Ca2+ dynamics in CM. We speculate that that loss of VASN in sPE‐EV contribute to the mechanisms of pathological cardiac remodeling, by decreased inhibition of deleterious TGF‐beta signaling. Funding : This study was supported by seed funds from the Department of Anesthesiology and Perioperative Medicine (D.E.B.) and a REINVENT grant (S.M. and T.J). Keywords : preeclampsia, cardiac dysfunction, extracellular vesicles, vasorin, calcium signalling

Polarized

Dylan T. Krajewski 1 ; Kalpani Dona 2 ; Allison Andrews 2 ; Shujun Ge 1 ; Evan jellison 1 ; Servio Ramirez 2 ; Joel Pachter 3 1 UConn Health Center, Farmington, USA; 2 Lewis Katz School of Medicine at Temple University, USA; 3 UConn Health Center, USA Introduction : Our lab previously showed EVs from brain microvascular endothelial cells (BMECs) contain the tight junction protein CLN‐5, can bind to leukocytes in vitro and, during neuroinflammation, varied types of circulating leukocytes acquire CLN‐5. This led us to consider EV‐mediated acquisition of CLN‐5 might be coupled to leukocyte transendothelial migration (TEM) via a zipper mechanism. An extension of this hypothesis is that BMEC‐EVs are released in a polarized fashion, wherein EVs derived from the apical plasma membrane would be released luminally, while EVs originating from the basolateral plasma membrane would be discharged abluminally. This would allow apical‐derived EVs to more efficiently interact with blood‐born cells, and basolateral‐derived EVs to target elements in the vascular adventitia or parenchyma. Methods : Apical vs basolateral membranes of BMECs grown on a Transwell filter were differentially labeled with fluorescent dyes. EVs isolated from top and bottom chambers by differential ultracentrifugation were assessed by flow cytometry, NTA & transmission electron microscopy. For TEM experiments, splenocytes were added to the top chamber and collected from both chambers at 24 hours. Results : EVs from either chamber were mostly labeled with that dye associated with the respective membrane surface. This was the case for all‐sized EVs assessed. Significantly, multivesicular bodies largely contained dye derived from only one membrane surface, suggesting that exosomes may remain segregated from their incipient state till release. Transmigrated leukocytes of all subtypes analyzed preferentially acquired apical‐derived EVs. Summary/Conclusion : BMEC‐EVs are released in a polar manner in vitro and may be predestined for interaction with specific targets. Future investigation of the proteomic cargo of apical vs basolateral‐derived EVs will highlight distinctive roles of EVs in neuroinflammation. Analogous experiments are being conducted in a flow‐based system to better recapitulate the in vivo circulatory environment. Funding : This work was funded by NIH R0‐1 NS099855‐02 and NIH R21NS113593‐01. Keywords : neuroinflammation, blood‐brain barrier, leukocytes, transendothelial migration

Potential

Jana Bzdilova ; Jana Matejova; Lucia Slovinska; Livia K. Fecskeova; Denisa Harvanova; Timea Spakova Associated Tissue Bank, Faculty of Medicine, P. J. Safarik University in Kosice, Slovakia, Kosice, Slovakia Introduction : Extracellular vesicles (EVs) represent paracrine factors secreted by almost all cell types. Both, normal and pathological cells are able to release various types of EVs with different physiological properties, functions and compositions. EVs play an important role in intercellular communication, mechanism and tissue repair. Moreover, EVs could help not only in the treatment of diseases but also in their diagnostics. This work aimed to evaluate the potential of EVs used as biomarkers for the diagnosis of osteoarthritis (OA) by comparing the surface markers expression of EVs isolated from platelet‐poor plasma (PPP) of healthy donors and patients at different stages of OA. OA is a chronic disease of musculoskeletal system that mainly affects the elderly. Nowadays, there is still no drug for the treatment of OA, patients are either treated symptomatically or in the end‐stage of the disease endoprothesis is done on severely damaged joints. For this reason, there is a high interest in trying to find biomarkers through which it would be possible to diagnose OA in the early stages and thus improve the quality of life of affected patients by slowing down the progression of OA. Methods : Collection of the blood samples was in accordance with ethical approval and realized after obtaining informed consent from healthy volunteers and OA patients. PPP samples were prepared from whole blood. PPP‐EVs were separated from 3 groups of donors ‐ healthy control (Kellgren‐Lawrence grade (KL) = 0), early stage OA (KL = I‐II), end‐stage OA (KL = III‐IV), and their content was compared and correlated. EVs from PPP were separated by SEC using qEVoriginal ∼35nm columns and characterized in terms of their size, yield and surface marker expression by NTA, western blotting and flow cytometry. Results : Our study confirmed that PPP‐EVs were positive to the well‐established EVs surface markers CD9, CD63 and CD81 in each donor group, with highest expression of CD81. There were found also significant differences between EVs surface markers of patients and healthy controls correlating with the age of donor (CD31 and ROR1) and early stage of OA (CD45, CD326 and CD56), respectively. Summary/Conclusion : As potential targets for biomarker discovery, circulating EVs have been extensively under investigation for their capability to predict OA pathology diagnosis. Obtained results suggested potential usage of PPP‐EVs surface markers in the OA early diagnosis. Funding : This publication is the result of the project implementation: OPENMED, ITMS2014+: 313011V455 supported by the Operational Programme Integrated Infrastructure, funded by the ERDF. This research was also supported by the Slovak Research and Development Agency under the contract No. APVV‐17‐0118. Keywords : extracellular vesicles, platelet‐poor plasma, osteoarthritis, diagnosis

Profiling

Joseph B. Hwang ; Laura Marquardt; Osama Sait; Brooke Gilliam; Qiang Xiao MilliporeSigma, St. Louis, USA Introduction : The study of exosomes has attracted worldwide research due to its ability to modulate cellular activities, especially in the progression of cancer. Exosomes are membrane vesicles that can deliver lipids, proteins, and nucleic acids from one cell to another through membrane fusion, providing a unique form of cell‐to‐cell communication. Exosomes have been isolated from bodily fluids including blood, saliva, urine, milk, and cell supernatant using various isolation methods. Our goal for this study was to examine protein content in exosomes isolated from various cancer cell line supernatant. Methods : To this end, we at MilliporeSigma, the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany, developed MILLIPLEX® immunoassays to detect cell signaling proteins as well as the exosome markers (CD9, CD63 and CD81), using Luminex® xMAP® technology which allows multiplex detection of proteins in a small volume of sample. Exosomes were isolated from various cancer derived cell lines, including A549 (lung), MCF‐7 (breast), COLO 201 (colon), PANC‐1 (pancreas) and AsPC‐1 (pancreas), using Qiagen exoEasy Maxi kit according to the kit protocol. Results : The most abundant cell signaling proteins found in these isolated exosomes were ERK and p70S6K, which are important kinases for mitogenesis. Less abundant cell signaling proteins included Ras, JNK and p38, which are also part of the MAPK signaling pathway. In addition, all the isolated exosomes were enriched in CD63 marker, whereas both CD9 and CD81 were less abundant, with highest expression in exosomes isolated from MCF‐7 supernatant. As a control, all analytes tested were expressed at various levels in all cancer cell line lysates. Summary/Conclusion : Using MILLIPLEX® multiplex immunoassays, our data supports research findings that exosomes contain mitogenic signaling components that may enhance progression of cancer. Funding : salary from MilliporeSigma company. Keywords : cancer cell supernatant, multiplexing immunoassays, profiling exosome cargo proteins

Prolonged

P. Debishree Subudhi Institute of Liver and Biliary Sciences, New Delhi, India Introduction : The prolonged presence of SARS CoV‐2 virus found in the respiratory tract especially in patients with underlying co‐morbidities. We aim to investigate the presence of virus inside Extracellular vesicles (EV) in patients with active and recovered COVID‐19 infection, also with underlying chronic liver diseases. Methods : SARS CoV2 RT‐PCR positive n = 78 {n = 24(66.6%) chronic liver disease (CLD); n = 52 (81.3%) non‐liver disease} were studied. SARS CoV2 patients were also followed up on day (d) 7, 14 and 28 posts RT‐PCR positivity. EVs were isolated using differential ultracentrifugation from both plasma and respiratory tract followed by detection of SARS CoV2 RNA Transmission was assessed in Vero cells. Results : In baseline RT‐PCR positive patients, SARS‐CoV2 RNA inside the EV was present in 64/74 (82%) patients with comparable viral load between nasopharyngeal swab (NP) and EV (mean 1CT – 0.033 ± 0.005 vs. 1CT – 0.029 ± 0.014, p = ns). The sequencing data shows 92–95% similarity in SARS CoV2 in nasal swab and EV isolated SARS Cov2 RNA. On follow‐up at day 7, of the 24 patients negative for COVID19, 10 (41%) had persistence of virus in the EV (1CT – 0.028 ± 0.004) and on day 14, 14 of 40 (35%) negative RT‐PCR had EVs with SARS CoV2 RNA (1CT – 0.028 ± 0.06). The mean viral load decreased at day7 and day14 in NP from baseline (p = 0.001) but not in EV. SARS‐CoV2 RNA otherwise undetectable in plasma was found to be positive in EV in 12.5% of COVID19 positive patients. Interestingly, significantly prolonged and high viral load was found in EV at day 14 in CLD‐COVID19 patients compared to COVID19 alone (p = 0.002). Summary/Conclusion : Identification of SARS‐CoV2 RNA in EV, in RT‐PCR negative patients indicates persistence of infection for and likely recurrence of the infection. EV associated RNA might play a role in the clinical course of the disease especially in patients with underlying co‐morbidities like CLD. Funding : This project was funded by SERB Power. Keywords : SARS CoV2, extracellular vesicles

Promotion

Sunyoung Park 1 ; Yeo‐Jun Yoon 2 ; Jae‐Min Cho 2 ; Yongpyo Hong 2 ; Haeun Yu 3 ; Jianning Yu 4 ; Kyung‐A Hyun 1 ; Hyo‐Il Jung 5 ; Jae‐Yol Lim 2 1 School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea, Seoul, Republic of Korea; 2 Yonsei University College of Medicine, Republic of Korea; 3 Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, USA; 4 Department of Biomedical Laboratory Science, College of Software and Digital Healthcare Convergence, Yonsei University, Republic of Korea; 5 Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea Introduction : Stem cell therapy has been suggested for anti‐inflammatory, antifibrotic, and regenerative purposes. For successful therapeutic efficacy and reduced side effect, cell‐free therapeutic approaches such as extracellular vesicles (EVs) have emerged containing growth factors, cytokines, miRNAs, and lipids. Despite the increased number of recent studies on cell‐free therapy and their promising results, it is still necessary to optimize massive purification protocols and evaluation of the purified EVs to determine the treatment dose, frequency, and delivery routes of the EVs. Methods : We applied to a modular microfluidic technology that enables us to enrich CD9‐specific EVs (CD9+ EVs) within 10 minutes. Modular microfluidic platform can selectively capture the specific EVs by CD9 antibody‐coated beads in a horseshoe‐shaped orifice micromixer (HOMM) chip. Sequentially, a fish‐trap‐shaped microfilter unit contained the microbeads into weir structure and released the captured EVs from the microbeads. Then, we harnessed the retroductal delivery route for the EV administration as it is a direct, non‐invasive approach to the salivary gland for the first time. Results : In the wound healing assay using epithelial cells, all the experimental groups treated with the EVs showed faster growth than the control group. Especially, the group treated with CD9+ EVs was superior than those with total EVs. In the mouse duct ligation model (sialoadenitis), the group treated with PBS as a control showed very severe tissue damage, decreased mucus, and increased fibrosis through H&E, PAS, MTC, and sirius red staining. However, the experimental group treated with CD9+ EVs reduced the degree of tissue damage, reduction of mucus, and fibrosis. The proteomic and miRNA profile of CD9+ EVs revealed the regenerative properties and anti‐fibrotic effect for epithelial cells. Summary/Conclusion : The modular microfluidic device successfully enriched the CD9+ EVs and direct administration of the EVs through the retroductal route can open the gate to potential therapeutic strategy for treating salivary gland dysfunction. Funding : This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2021R1A2C3011254), Korea Medical Device Development Fund grant funded by the Korea government (MSIT,MOTIE, MOHW,MFDS) (Project Number: 1711139115, KMDF_PR_20210527_0008), and Korea Environment Industry & Technology Institute (KEITI) through Aquatic Ecosystem Conservation Research Program, funded by Korea Ministry of Environment (MOE) (2020003030007).

Proteomic

siqi ying 1 ; yuan cao 2 ; jiyu qiu 2 ; Bing‐Dong Sui 3 ; Yan Jin 3 1 The Fourth Military Medical University, Xi'an, China (People's Republic); 2 The Fourth Military Medical University, China (People's Republic); 3 Fourth Military Medical University: Air Force Medical University, Xi'an, China (People's Republic) Introduction : Psychological stress emerges as a common health burden in the current society due to its highly correlated risk of mental and physical disease outcomes. However, it is still unclear how fast adaptive stress response processes are connected to long‐observed changes in organisms. Here, we investigated the profile of circulating extracellular vesicles (EVs) after acute stress (AS) of restraint mice by phenotypic and proteomic analyses. Methods : Here, we investigated the profile of circulating extracellular vesicles (EVs) after acute stress (AS) of restraint mice by phenotypic and proteomic analyses. And circulating EVs were separated by sequential centrifuged 3,500r for 15 min, 2,500g for 10 min and 16,800 g for 30 min. Results : We surprisingly discovered that AS‐EVs demonstrated significant changes in size distribution and plasma concentration compared to control group (CN) EVs. AS‐EVs were further characterized by various differentially expressed proteins (DEPs) in close relationships with biological, metabolic and immune regulations and were functionally important in potentially underlying multiple diseases. Notably, we first identified the lipid raft protein Stomatin as an essential biomarker expressed on the surface of AS‐EVs. Summary/Conclusion : These findings collectively reveal that EVs are a significant function‐related liquid biopsy indicator that mediate circulation alterations impinged by psychological stress, while also supporting the idea that psychological stress‐associated EV‐stomatin can be used as a biomarker for potentially predicting acute stress responses and monitoring psychological status. Our study will pave an avenue for implementing routine plasma EV‐based theranostics in the clinic. Funding : This work is supported by grants from the National Natural Science Foundation of China (82170988 to F.J., 81930025 to Y.J., 32101096 to X.Q., 32000974 to B.S.) and the China Postdoctoral Science Foundation (2019M663986 and BX20190380 to B.S.). Keywords : psychological stress, extracellular vesicles, proteomic analysis, Stomatin, plasma

Puerariae

Yang Xu 1 ; Ge Yan 1 ; Qiyao Xiao 1 ; Jingyu Zhao 1 ; Lihua Peng 2 1 Zhejiang University, China (People's Republic); 2 Zhejiang University, Hangzhou, China (People's Republic) Introduction : The treatment of Parkinson's disease (PD) is hindered by the complex pathology and the blood brain barrier (BBB), which forms a big obstacle for the delivery of most drugs into brain. Recently, exosomes are emerging as promising nanoplatforms in treating brain disorders with enhanced efficacy in transferring therapeutic nucleic acids through BBB to regulate the brain pathological process. Methods : Herein, the medicinal plant, Puerariae Lobatae Radix derived exosomes (Pu‐Exos) were prepared and characterized for the contained small molecules, nucleic acids, proteins and lipids qualitatively and quantitatively. Further, Pu‐Exos were modified by the functional target ligand (named as Pu‐Exos‐RVG) and investigated for their capabilities in BBB penetration, miRNAs delivery and therapeutic potential in PD treatment both in vitro and in vivo. Results : And for the first time, we demonstrate that Pu‐Exos‐RVG are efficient in delivering nerve‐regulating plant microRNAs with efficacy in modulating the mitochondrial dysfunction of SH‐SY5Y cells through intervening ubiquitin and PINK1‐Parkin mediated mitophagy and recovering the function of mitochondrial respiratory chain, as well as reversing the deficiency in ATP production and apoptosis. In vivo, Pu‐Exos‐RVG are shown for the efficiency in delivering the incorporated plant microRNAs to brain lesion site that significantly affords neuroprotective effects in reducing DA neurons loss, promoting neural function recovery and improving movement coordination/psychiatric symptoms. Summary/Conclusion : Pu‐Exos‐RVG were shown as a promising nanoplatform with excellent efficacy in delivering functional plant miRNAs to brain with obvious therapeutic capacity for PD treatment that possesses great potential in treating brain neuron degenerative diseases. Funding : The study was supported by National Key Research and Development Program of China (2022YFC3501904, 2021YFC1712805), Zhejiang province com monweal projects (LG F22H280001), the Key Project at Central Government Level (2060302), and the Macau Science and Technology Development Fund, Macau Special Administrative Region, China. Keywords : parkinson's disease, blood brain barrier, Puerariae Lobatae Radix derived exosomes, mitochondrial dysfunction, plant microRNAs

Reduction

María Angélica Calderón‐Peláez 1 ; L. Johana Madroñero 2 ; Jaime E. Castellanos 3 ; Myriam Lucía Velandia Romero 3 1 Virology lab, Universidad El Bosque, Bogotá, Bogotá, Colombia, Colombia; 2 Virology lab, Universidad El Bosque, Colombia; 3 Virology Lab, Universidad El Bosque, Bogotá, Bogotá, Colombia, Colombia Introduction : DENV infection can cause endothelial cell (EC) hyperpermeability and vascular leakage. Small EV of infected EC (sEVIC) may participate in the viral spread and regulate EC response to DENV. Still, there is no information on sEVIC cargo and its effect on non‐infected EC. This work aimed to characterize the sEVIC and evaluate their impact on polarized EC. Methods : EA.hy 926 were infected with DENV‐2, MOI 1. The virus was removed, and serum‐free medium was added. Cell viability (Resazurin, LDH) and viral infection (IFI and PCR for DENV E protein) were confirmed. After 48h post‐infection, sEV were isolated (ultracentrifugation) and characterized by NTA, DLS, Western blot, LC/MS/MS, and small RNA sequencing. For function assay, sEVIC were pretreated with neutralizing antibody 4G2 or UV exposed after pretreatment. Later, polarized EC were exposed to the different sEV for 24h and then infected. TEER and permeability (Dextran‐blue 2KDa) were measured at different times, and IFI for ICAM, E‐sele, and actin were made along with qPCR for DENV detection. sEV non‐infected cells (sEVNIC) were used as control. Results : EC infection induced a high concentration of EV (2 × 109) with low ALIX expression. Compared to EVNIC, EVIC had 129 increased proteins (mainly of immune response ‐IR‐), 206 were downregulated (cellular adhesion (CA) and developmental processes), and a viral NS5 peptide was found along with sncRNA like YRNA and miRs that mainly regulate CA and IR genes. Interestingly, miRs found exclusively in EVIC may have a proviral effect. Functionally, EVIC induced ICAM and E‐sele expression and a protective effect in the polarized EC that maintained a stable TEER (45Ω) and reduced viral replication. Summary/Conclusion : EVIC transported DENV elements and sncRNA that may regulate viral response. Our model suggests that EVIC induced a protection response during infection, reducing viral replication and activating the EC in the first 24h. Funding : MinCiencias – U Bosque: Grant 130884467149, Contract 431–2020. Keywords : DENV, sEVs, endothelial cells, viral replication

Screening

Hash Brown Taha 1 ; Lynda Polgreen 2 ; Lea M. Chamoun 3 ; Alanna Morris 4 ; Anna Luzzi 5 ; Jonathan Acevedo 5 ; Adolfo Morales 6 ; Nour Elabed 7 ; Leony Fenwick 8 ; Giselle Perez de la Garza 9 ; Lillianne Bakhshi 7 ; Cara O'Neill 10 ; Agnes Chen 11 ; Michelina Iacovino 9 ; Julie B. Eisengart 12 ; Gal Bitan 13 1 Department of Integrative Biology & Physiology, University of California Los Angeles, Boulder, USA; 2 The Lundquist institute at Harbor‐UCLA, USA; 3 University of California Los Angeles, USA; 4 UCLA, USA; 5 The Lundquist Institute, Torrance, USA; 6 The Lundquist Institute for Biomedical Innovation, USA; 7 Department of Neurology, University of California Los Angeles, USA; 8 Department of Neurology, University of California Los Angeles, Appeltern, Netherlands; 9 Lundquist institute, USA; 10 Cure Sanfilippo Foundation, Columbia, USA; 11 Lundquist Institute at Harbor‐UCLA, USA; 12 University of Minnesota, Minneapolis, USA; 13 UCLA, Los Angeles, USA Introduction : Extracellular vesicles (EVs) are released by all cells and contain cargo reflecting the status of the original cell. EVs in the central nervous system (CNS) can cross the blood‐brain‐barrier into the blood, providing a window into the CNS's biochemistry. Mucopolysaccharidosis III (MPSIII) is a genetic neurodegenerative disease characterized by neurocognitive and neurobehavioral abnormalities due to defects in lysosomal enzymes required for heparan sulfate (HS) catabolism. In addition to the primary storage of HS, lysosomes of patients with MPS III and animal models of the disease accumulate large amounts of amyloid proteins as secondary storage, including α‐synuclein (α‐syn), tau, and amyloid β‐protein (Aβ). In neurodegenerative diseases, autophagy‐lysosomal pathway dysfunction leads to increased secretion of these proteins in EVs. Thus, we hypothesized that measuring them in EVs of patients with MPSIII and healthy controls (HC) could lead to sensitive, minimally invasive biomarkers for MPSIII. Methods : 24 patients with MPSIII (20 MPSIIIA, 3 MPSIIIB, 1 MPSIIIC) and 15 HCs were enrolled. EVs were isolated from plasma by an ExoQuick kit followed by immunoprecipitation of nEVs using an anti‐L1CAM antibody. EVs were analyzed using TRPS, western blots, and TEM and nEV enrichment was assessed by flow cytometry. α‐Syn, tau and Aβ were measured in nEVs. In addition, IL‐10, IL‐2Rα, and neurofilament light (NfL) were measured in plasma using ECLIA. Statistical significance was assessed using independent t‐tests. Effect sizes were assessed using Hedge's g (Hg). Results : Patient EVs had significantly higher levels of α‐syn compared to HCs with a large effect size (Hg = 1.1). Tau was detected in 23/24 patient samples and 0/15 HC samples. Aβ was found in 3 patient samples only. NfL, a general marker of neurodegeneration, and IL‐10 were significantly higher in patients compared to HC with large effect sizes (Hg = 1.2 and 0.9). IL‐2Rα did not differ between the groups. Summary/Conclusion : nEVs α‐Syn and tau, and plasma NfL and IL‐10 are significantly higher in patients with MPSIII than in HC suggesting that these biomarkers may be useful for monitoring disease progression and assessing treatment effects. Funding : Cure Sanfilippo Foundation 20215318 (GB) Cure Sanfilippo Foundation ( NCT04018755 ) (LP). Keywords : biomarker, diagnostics, mucopolysaccharidosis, Sanfilippo syndrome, α‐synuclein, tau, amyloid β‐protein, neurofilament light, cytokines, lysosomal‐storage disorders

Selection

Lei He 1 ; zhiyang li 2 1 Department of Clinical Laboratory, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, P. R. China, China (People's Republic); 2 Department of Clinical Laboratory, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, P. R. China., Nanjing, China (People's Republic) Introduction : Extracellular vesicles (EVs) are cell‐derived vesicles showing various proteins on their membrane surfaces and they have the potential to provide novel biomarkers to diagnose cancer. Aptamers are short single stranded nucleic acid molecules which can bind to the target. At present, there are few reports on direct aptamer screening with EVs as target. Methods : Here, we applied a newly EVs‐SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method to select aptamers against A549‐EVs based on immunomagnetic beads. Results : Through multiple rounds of screening, we have identified one aptamer (Ap3) that can bind to A549‐EVs with high affinity and specificity. The Kd of Ap3 is 2.59 ± 3.42 nM. Under different temperatures (4°C, 25°C and 37°C), Ap3 can bind to A549‐EVs with the similar affinity. Then, the detection efficiency of the aptamer for lung cancer (LC) EVs was evaluated by quantitative polymerase chain reaction (qPCR). The detection limitation was 1.6 × 105 particles/mL and a linear range was 1.6 × 105‐1.63 × 1011 particles/mL. Summary/Conclusion : The method would be used in clinic for early diagnosis. Funding : This study was supported by the National Natural Science Foundation of China (61971216 and 82002242), the Key Research and Development Project of Jiangsu Province (BE2022692 and BE2020768), Nanjing Important Science & Technology Specific Projects (2021‐11005), Nanjing Science and Technology Development Plan Project (202205066). The Fellowship of China Postdoctoral Science Foundation (2022M711580). Keywords : lung cancer, extracellular vesicles, exosomes, aptamer, SELEX, qPCR

Selective

Hannah M. Nelson 1 ; Sherman Qu 2 ; Sydney Chapman 1 ; James G. Patton 1 1 Vanderbilt University, Nashville, USA; 2 Vanderbilt University, USA Introduction : We previously showed that miR‐100 and miR‐125b are selectively exported from KRAS mutant colorectal cancer cells. We also generated a Cetuximab resistant cell line (CC‐CR) that dramatically overexpresses and selectively secretes miR‐100 and miR‐125b. These two miRNAs are encoded within an intron of the long noncoding RNA MIR100HG. Initial analysis suggested that miR‐100 and miR‐125b target negative regulators of Wnt signaling. Here, we used RNA sequencing, bioinformatic analyses, and knockout cell lines to identify the full range of mRNA targets for miR‐100 and miR‐125b. Methods : NAseq was performed on Cetuximab resistant CC‐CR cells, Cetuximab sensistive CC cells, and cells lacking miR‐100, miR‐125b, or MIR100HG. We used bioinformatic approaches and miRNA prediction algorithms to identify putative mRNA targets for miR‐100 and miR‐125b. To better identify such targets, we also performed immunoprecipitation of Ago2 complexes and conducted small and long RNAseq. The combined data sets allowed for the identification of mRNA targets for miR‐100 and miR‐125b. Validation of these targets is being performed using luciferase assays in which the 3’ UTR sequences from predicted targets are fused to luciferase and the extent of silencing determined in transfected cells. Testing of the ability of these miRNAs to undergo transfer from donor to recipient cells is being performed using Transwell assays. Results : miR‐100 and miR‐125b are selectively exported from colorectal cancer cells and can be transferred to recipient cells. The use of knockout recipient cells to validate transfer of these miRNAs has shown the level of mIR‐100 and miR‐125b can be increased from 70‐150‐fold in Transwell assays. RNAseq and Ago2 immunoprecipitation experiments allowed us to develop extensive lists of mRNA targets for miR‐100 and miR‐125 including IGF2BP2, CGN, and RASGRP3. Direct testing of these targets is underway. Summary/Conclusion : miR‐100 and miR‐125b target many mRNAs that are involved in colorectal cancer progression and metastasis, through both cell‐autonomous and non‐cell‐autonomous function. How these miRNAs are transferred between cells, whether by extracellular vesicles or other mechanisms is under active investigation. Funding : Funding for this work is PO1CA229123. Keywords : miRNA

Sensitive

Moein Talebian Gevari 1 ; Siddharth S. Sahu 2 ; Dhrubaditya Mitra 3 ; Petra Hååg 4 ; Kristina Viktorsson 5 ; Jan Linnros 6 ; Apurba Dev 7 1 Division of Solid‐State Electronics, Department of Electrical Engineering, Uppsala University, 75121 Uppsala, Sweden, Uppsala, Sweden; 2 Department of Applied Physics, KTH Royal Institute of Technology, 10691 Stockholm, Sweden, Sweden; 3 Nordita, KTH Royal Institute of Technology and Stockholm University, Roslagstullsbacken 23, 10691 Stockholm, Sweden, USA; 4 Department of Oncology‐Pathology, Karolinska Institutet, 171 64 Solna, Sweden, Stockholm, USA; 5 Department of Oncology‐Pathology, Karolinska Institutet, 171 64 Solna, Sweden., Solna, USA; 6 Department of Applied Physics, School of Engineering Sciences, KTH Royal Institute of Technology, 10691 Stockholm, Sweden, Stockholm, USA; 7 Division of Solid‐State Electronics, Department of Electrical Engineering, Uppsala University, 75121 Uppsala, Sweden, Department of Applied Physics, School of Engineering Sciences, KTH Royal Institute of Technology, 10691 Stockholm, Sweden., Stockholm, USA Introduction : Rapid, sensitive and inexpensive analysis of extracellular vesicles (EVs) with an easy‐to‐use method holds the key for the lab‐to‐clinic transition of various EV‐based diagnostic opportunities. The method should also be able to address the issues arising from EV‐heterogeneity for a robust and reproducible analysis. Here, we demonstrate the fabrication, characterization, and application of a novel silicon microchip‐based biosensor that exploits streaming current 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 size‐exclusion chromatography. The sEVs were characterized for size and amount by nanoparticle tracking analysis 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 marker was detected by means of immunocapture of sEVs on the sensor surface and measuring the changes in streaming current. Results : The electric and fluidic characterization of the biosensors showed a linear dependence on the upstream pressure (0.5‐3 bar) of the flow. The limit of detection (LoD) was estimated to be 350 sEVs/μL when targeting CD81 exhibiting a dynamic range of over 3 decades. The microchips were tested and optimized for rapid profiling (30 minutes) of CD81 and CD9 membrane proteins along with the assessment of sEV count in the sample and relative abundance of different sEV population. The results were validated using single‐EV fluorescence measurement. Summary/Conclusion : A novel microchip‐based electrical sensors was developed for rapid and sensitive analysis of sEVs. The method allows multiplexed analysis of EV‐membrane proteins along with the assessment of sEV count in the sample and relative abundance of different sEV populations. Funding : The Swedish Research Council (no. 2016–05051 and 2018–06228), the Swedish Cancer Society (no. CAN 2018/597 and CAN2021/1469), the Stockholm Cancer Society (no. 221212 and 221383) and Stockholm County Council (no. FoUI‐966345, 909121 and 750032), and The Erling Persson Foundation. Keywords : microfluidics, biosensor, EV characterization, EV detection, biomarker

Stability

Alex Chauhan 1 ; Lucas Nosal 2 ; Zakia Dahi 3 ; Enaam Merchant 3 ; Taha Haj‐Ahmad 2 ; Moemen Abdalla 4 ; Mohamed El‐mogy 3 ; Yousef Haj‐Ahmad 2 1 Norgen Biotek, Thorold, Canada; 2 Norgen Biotek Corp., Thorold, Canada; 3 Norgen Biotek Corp., Canada; 4 Department of Biochemistry (Faculty of Science), Alexandria University, Alexandria, Egypt, Egypt Introduction : Several blood collection devices are available that can stabilize cell‐free RNA (cf‐RNA) and extracellular vesicles (EVs) in plasma at room temperature, which include Streck's RNA Complete BCT tubes and Norgen's cf‐DNA/cf‐RNA preservative tubes. Amongst the cargo carried in EVs, RNA is the most instable components. In the current study, we assessed the performance of EDTA tubes, Streck's RNA Complete BCT tubes and Norgen's cf‐DNA/cf‐RNA preservative tubes to stabilize intact exosomes as well as exosomal RNA at room temperature up to 14 days. Methods : Blood from five donors was collected in EDTA, Streck, and Norgen tubes and were stored at room temperature for 0, 5, 7 and 14 days. Hemoglobin concentration was estimated using NanoDrop to determine hemolysis. Intact exosomes from all tubes were purified using Norgen's Plasma/Serum Exosome Purification Kits and were characterized and visualized on the NanoSight LM10 instrument. Furthermore, the exosomes were processed to purify exosomal RNA using Norgen's Exosomal RNA Isolation Kit, and small RNA library was constructed from all purified exosomal RNA using Norgen's Small RNA Library Prep Kit for Illumina. Libraries were quantified and sequenced using Illumina platform. Results : NanoSight data revealed that the number of intact exosomes were consistent from day 0 to day 14 for plasma collected using Norgen's tubes, while the number of exosomes increased for EDTA as well as Streck tubes. The hemoglobin concentration also showed an increase in concentration from day 0 to day 14 for Streck and EDTA tubes, however, it remained constant for Norgen tubes. The average miRNA count for Norgen and Streck tubes was consistent for 14 days and 7 days respectively, while on the other hand the miRNA count for EDTA tube showed a sharp decline on day 5 and remained low up to 14 days. Summary/Conclusion : Norgen's cf‐RNA/cf‐DNA preservative tubes are able to stabilize intact exosomes and exosomal RNA with high quality and for a relatively higher period as compared to Streck and EDTA tubes without any sign of hemolysis. Funding : Norgen Biotek Corp. funded the study. Keywords : exosomes, cf‐RNA/cf‐DNA preservative tubes, small RNA sequencing, plasma

Substrate

Sara Veiga 1 ; Rebecca Porter 2 ; Berent Aldikacti 3 ; Eric Tai 4 ; Katherine Calhoun 2 ; Vishal Thapar 4 ; David Ting 5 ; Shannon Stott 3 1 Massachusetts General Hospital Cancer Center/ Department of Medicine, Harvard Medical School, Somerville, USA; 2 Massachusetts General Hospital Cancer Center, USA; 3 Massachusetts General Hospital Cancer Center and Center for Engineering in Medicine/ Department of Surgery, Harvard Medical School, USA; 4 Massachusetts General Hospital Center for Engineering in Medicine, USA; 5 Massachusetts General Hospital Cancer Center/ Department of Medicine, Harvard Medical School, Boston, USA Introduction : Tumor microenvironment stroma is an important contributor to the physiopathology of cancer and has been shown to shape tumor stiffness. While much of our understanding about cancer comes from cell culture studies in two dimensions (2D), it is appreciated that 3D culture systems better recapitulate in vivo aspects of tumors. It is known that substrate stiffness can alter cells at multiple levels, including gene expression, yet little has been done to investigate the impact of substrate stiffness on EV biogenesis and cargo. Methods : In the present study, we wanted to explore if different cell culture conditions would affect RNA cargo of tumor derived EVs, with a specific focus on non‐coding RNA (ncRNA). Further, we explored the impact of culture conditions on EV size and number. For this, cell lines generated from ascites fluid of patients with metastatic PDAC were grown in 2D and 3D, as well as on hydrogels with defined mechanical stiffness levels (0.5 kPa, 2kPA and 8kPa). Results : Analysis of total RNA content of EVs isolated from 2D and 3D conditions showed an enrichment of more than 50% of repeat ncRNAs, specifically LINE, SINE and ERV elements, when compared with parental cells grown in the same conditions. While cells cultured in 3D produced more EVs than 2D cell culture conditions (∼ 2‐fold more), vesicle size was not impacted (around 250nm). Interestingly, PDAC cells grown on less stiff surfaces (0.5 kPa) released at least 40‐fold more EVs compared with 2D. However, substrate stiffness did not affect the levels of repeat RNAs detected in vesicle cargo, which were maintained across all conditions. Summary/Conclusion : Overall, our data shows that cells grown under non‐adherent conditions and in more elastic hydrogels present an increase in EV production, without affecting size and RNA content. Nonetheless, secreted EVs are much more enriched in repeats ncRNA when compared with their cells of origin, which can represent a novel and specific class of biomarkers in EVs from patients with PDAC.

Targeting

Hameeda Sultana University of Tennessee, at Knoxville, Knoxville, USA Introduction : Our laboratory is the first to show that arthropod exosomes from medically important vectors such as ticks and mosquitoes transmit pathogens to the vertebrate host. Our recently published studies, not only provides evidence to show that tick/mosquito‐borne flaviviruses uses arthropod‐derived exosomes for transmission from vector to the mammalian cells but also suggest that infectious exosomes from facilitates viral dissemination within the vertebrate host system. Methods : Exosomes were isolated from tick and mosquito cells, tick saliva and salivary glands by using density gradient and differential ultracentrifugation methods. In addition to exosome isolation several different functional assays were performed. Results : Overall, our studies have revealed that arthropod‐derived exosomes are important means of communication between the vector and vertebrate host. We have shown that mosquito‐borne viruses such as dengue, ZIKA, West Nile virus and tick‐borne Langat virus are transmitted from vector to the vertebrate host through arthropod exosomes. These infectious exosomes containing full length viral RNA genomes, and proteins/polyproteins were viable, secured, and highly virulent in all tested conditions such as re‐infection kinetics, trans‐migration and viral plaque formation assays. We found the first mosquito exosomal enriched marker Tsp29Fb (human CD63 ortholog), that directly interacts with dengue viral envelope protein and facilitate virus transmission. We have recently identified novel tick exosomal cargo molecules in arthropod exosomes that mediates successful blood feeding and tick‐borne virus transmission. In addition, our new findings about arthropod exosomal proteins and related mechanisms show their importance in modulation of human cytokine/chemokines at the pathogen‐vector‐host interface. Summary/Conclusion : We envision that the transmission strategies used by flaviviruses to exit arthropods via infectious exosomes and to infect human and animal host are the best approaches to develop transmission‐blocking vaccines against vector molecules or determinants that facilitate pathogen transmission. We strongly believe that this novel line of investigation is very important in understanding the potential interactions at the cross roads of virus‐vector‐host to block vector‐borne diseases from ticks and mosquitoes. Funding : This study is supported by funding from National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH) (Award number R01AI141790 and R01AI141790‐05S1 to HS). Keywords : arthropod exosomes, ticks, mosquitoes, flaviviruses, blood feeding, exosomal cargo, vector‐host interactions, vertebrate skin interface, pathogen transmission, vector‐borne diseases, transmission‐blocking vaccines

Unlocking

Edgar Gonzalez‐Kozlova 1 ; Tzu‐Yi Chen 2 ; Taliah Solemani 1 ; Ash Tewari 1 ; Carlos Cordon‐Cardo 1 ; Gustavo Stolovitzki 3 ; Navneet Dogra 1 1 Icahn School of Medicine at Mount Sinai, New York, USA; 2 Icahn School of Medicine at Mount Sinai, Department of Department of Genetics and Genomic Sciences, New York, USA; 3 IBM, USA Introduction : Extracellular vesicles (EVs) have brought great momentum to the non‐invasive liquid biopsy procedure for the detection, characterization, and monitoring of cancer. While our fundamental understanding of EVs exponentially increases, the mechanisms for cargo loading, inter‐cellular communication, organ homeostasis, and association with the disease remain unknown. Here, we explore the fundamental question of whether EV‐RNA signatures can be used to accurately report the cellular composition of the tumor microenvironment (TME). Methods : To test our hypothesis, we compared bulk EV‐RNAs from urine and serum bio‐fluids (n = 10, 40 samples), tumor tissue biopsies (n = 5, 5 samples), and adjacent normal (n = 5, 5 samples) with the composition of the TME (n = 1, 2 samples) through single‐cell RNA‐seq. We used standardized RNAseq analysis methods (Bowtie, STAR, Samtools, Cell ranger, Seurat & Monocle) combined with mixed linear effect models for differential expression analysis (R, packages Dream, MEGENA). The approach developed during this study represents a methodological and conceptual advance to overcome the technical effects of integrating two different sequencing technologies in addition to unlocking cellular to EV‐RNA comparison. While our previous results comparing EV‐RNAs with their cellular origin revealed a non‐linear correlation, common RNAs and proteins representative of lineage were identified (PMID: 35663013). Results : We report an in‐depth characterization of (1) EV‐RNA signatures enriched in urine and serum bio‐fluids, prostate cancer tumor, and adjacent normal tissue; (2) The immune landscape of the TME, including the identification of 2 tumor clusters that are potentially indicative of tumor clonal diversity. Next, we used the mRNAs identified in EVs as anchors to compare EV‐transcriptomes to single cell clusters. This allowed us to identify EV‐RNA signatures correlated (Rho > 0.7, P< 0.001) in specific cell types that are potentially informative of tumor and immune phenotypes. At the same time, stratifying EV‐RNAs by gene biotype revealed a diversity of non‐coding and coding RNAs potentially associated with the TME. Summary/Conclusion : This proof‐of‐concept study provides novel insights for using EV‐RNAs as minimally invasive tools to characterize the phenotype of the tumor microenvironment. Despite the limitations from our sample size, we lay the ground for more in depth studies unraveling EV‐RNAs as a liquid biopsy alternative to report immune and tumor phenotypes. Funding : Funding: P20CA264076 (NCI/NIH), R21AG078848 (NIA/NIH), Friedman Brain Institute at Mount Sinai. Keywords : RNAs, transcriptome, single cell sequencing, extracellular vesicles, immune cells, prostate cancer

Acidovorax

Haneen Abu‐Freih 1 ; Shani Cohen 1 ; Bibek Bhatta 2 ; Etti Azulay 3 ; Ishai Luz 4 ; Elena Voronov 1 ; Isana Veksler‐Lublinsky 1 ; Tomer Cooks 4 1 Ben‐Gurion University of the Negev, Israel; 2 Ben‐Gurion University of the Negev, Beersheva, Israel; 3 Ben‐Gurion University of the Negev, Beer Sheva, Israel; 4 Ben‐Gurion University of the Negev, beer sheva, Israel Introduction : Dysbiosis is a common feature of solid tumors, however whether this dysbiosis directly contributes to tumor development is largely unknown. We previously characterized the lung cancer microbiome where the Gram‐negative Acidovorax temperans was found to be enriched in tumors of smokers and patients harboring TP53 mutations. We showed that A. temperans exposure accelerated tumor development and burden through infiltration of proinflammatory cells in the lungs. Herein, we investigated the involvement of outer membrane vesicles (OMVs) shed by Acidovorax temperans in driving inflammatory dynamics and tumorigenesis. Methods : OMVs were extracted using standard serial centrifugations and filtrations followed by a sucrose gradient to get rid of flagella and non‐vesicular elements. The purified samples were characterized using NTA, electron microscopy and ImageStream flow cytometer microscope. RNA from the OMVs was isolated using miRNeasy kit (Qiagen) and was prepared for RNAseq using the NEBNext Multiplex Small RNA Library Prep (Illumina). Results : Our results suggest that A. temperans OMVs are taken up by A549 lung cancer cells and that A. temperans OMVs facilitate a strong pro‐inflammatory response also in A549 and THP‐1 macrophages. In addition, macrophages exposed to A. temperans OMVs overexpress SIRPα which is associated with tumor cell immune escape and tumor progression. Notably, the levels of CD47, the receptor recognizing SIRPα, were elevated in the cancer cells after the OMVs treatment. We also optimized an in vivo protocol where A. temperans OMVs were introduced into the lungs of mice by intranasal administration. Intranasal administration of A. temperans OMVs lead to an increased secretion of proinflammatory cytokines. We used OMVs RNAseq specifically focusing on short RNAs (sRNA) and identified a unique signature of RNA species including various fragmented tRNAs. Summary/Conclusion : OMVs shed by Acidovorax temperans promoted inflammatory signaling in lung carcinoma cells and elevated CD47 expression on tumor cells and SIRPα levels on macrophages. We currently investigate the capacity of these OMVs to drive lung tumorigenesis and study the molecular cargo shipped between the pathogen and cells of the host. Funding : This study is funded by the Israel Science Fund (ISF) grant #: 1178/20. Keywords : outer‐membrane‐vesicles, lung cancer, microbiome

Analytical

Mehdi (Aslan) Dehghani 1 ; Badhu Prashantika Sivasubramaniam 1 ; Jiajun Wang 1 ; Paul Keselman 2 ; Jordan Speidel 3 ; Prabuddha Mukherjee 2 ; David Pollard 1 ; Michael Olszowy 3 ; Belinda Mativenga 1 ; Ran Cheng 1 1 Sartorius Stedim North America, Boston, USA; 2 Sartorius Stedim North America, Ann Arbor, USA; 3 Sartorius Stedim North America, Arvada, USA Introduction : Extracellular vesicles (EVs) are membrane vesicles secreted by cells and distributed widely in all biofluids as well as conditioned media. Due to their role in intercellular communication, they are receiving attention for therapeutic and diagnostic applications. The first step to better understand EVs and to utilize them as therapeutic and diagnostic tools is to purify them from variety of biofluids. Robust and well‐established orthogonal techniques for quantification and characterization of individual EVs are required to utilize them as therapeutic and diagnostic tools. Methods : We have established capabilities to reliably analyze EVs samples using three different orthogonal techniques; 1) Nanoparticle tracking analysis (NTA), a scattering and fluorescence‐based technique to determine the size and concentration of nanoparticles, is the mostly widely used technique in the field. 2) Virus Counter 3100 platform (VC3100), a fluorescence‐based technique with similar principles as flow cytometry with critical enhancements to enable the effective detection of smaller particles. 3) Analytical HPLC (PATFix), a powerful technology to analyze the EV samples using three different detectors: Multi angle light scattering, UV and fluorescence. Using these three platforms, EV samples can be analyzed comprehensively, reliably, and reproducibly. Results : We have evaluated the performance of this analytical toolbox to characterize EV samples. Fundamental studies were first performed to understand the detection range as well as their limitations to understand the results more reliably. We showed that this analytical toolbox could be used to characterize EV samples from the MSC conditioned media to each step through the purification process including clarification, tangential flow filtration, and chromatography. Summary/Conclusion : This analytical toolbox will allow researchers in the field to understand the EV samples, in particular, the relevant ratio of EVs compared to other non‐EV components of the conditioned media. Additionally, this toolbox allowed us to optimize the downstream processing to achieve high yield and high purity EV samples. Funding : N.A. Keywords : analytical toolbox, reliable characterization, downstream processing

Angiocrine

Lata Adnani 1 ; Jordan Kassouf 2 ; Brian Meehan 3 ; Cristiana Spinelli 3 ; Nadim Tawil 4 ; Ichiro Nakano 5 ; Janusz Rak 6 1 Research Institute of McGill University Health Center, Montreal, QC H4A 3J1, Canada, Canada; 2 Department of Biochemistry, McGill University, Montreal, QC H4A 3J1, Canada, USA; 3 Research Institute of McGill University Health Center, Montreal, QC H4A 3J1, Canada, Montreal, USA; 4 Research Institute of the McGill University Health Center, Montreal, Canada; 5 Department of Neurosurgery, Hokuto Social Medical Corporation, Hokuto Hospital, Kisen‐7‐5 Inadacho, Obihiro, Hokkaido 080–0833, Japan, USA; 6 Research Institute of McGill University Health Center, Montreal, QC H4A 3J1, Canada. Department of Pediatrics, McGill University, Montreal, QC H4A 3J1, Canada., Montreal, USA Introduction : Glioblastoma (GBM) is an incurable form of primary astrocytic brain tumor driven by the glioma stem cell (GSC) compartment which is closely associated with the vascular niche. GSC phenotypes are heterogeneous and range from proneural to mesenchymal‐like, the latter characterized by greater invasiveness. Here, we studied the impact of cues received by GSCs via extracellular vesicles (EVs) derived from vascular endothelial cells. Methods : Cell culture studies involved EV‐depleted serum, inhibitors, Incucyte, xCelligence, bioassay (MTA, sprouting, migration assays) and FACS analysis. Molecular analysis entailed mass spectrometry, protease activity, and immunostaining assays. In vivo studies employed intracranial injections, and tissue analysis. EV analysis followed MISEV2018 guidelines. EVs were isolated by ultracentrifugation, characterized by Nanoparticle tracking analysis (NTA), and EV markers (western‐blot). EV uptake was recorded by ImageStream, confocal microscopy and Cre reporter. Results : We observed that endothelial cells and their derived EVs drive proneural‐to‐mesenchymal reprogramming of GSCs. Endothelial EVs carry matrix metalloproteinases (MMPs) which lead to inactivation of NOTCH pathway, stimulation of NFκB signaling, altered motility and responsiveness to chemotherapy, and driving infiltrative growth in vivo and in vitro in proneural GSCs. Proneural GSCs which scatter away from the tumor mass become increasingly susceptible to the angiocrine influences of EVs. Notably, endothelial EVs compete for influence on GSCs with cancer cell own EVs. Summary/Conclusion : Our work documents that endothelial secretome, including, EVs, alters GSC subtype by instigating an infiltrative growth observed in GBM thereby contributing to therapeutic failure. We postulate that a tailored modulation of endothelial cell vesiculation or EV trafficking may improve outcomes in specific subsets of GBM. Funding : Canadian Institutes for Health Research (CIHR), Foundation Charles Bruneau (FCB), Canadian Foundation for Innovation (CFI10)‐ to JR. LA received support from Whitehead‐Penny Endowment, Fonds de research du Quebec‐Sante, RI‐MUHC Desjardins Studentship in Child Health and McGill Faculty of Medicine internal postdoctoral award. Keywords : extracellular vesicles, endothelial cells, glioblastoma, angiocrine, EMT, invasion, angiogenesis, extracellular vesicle competition

Colorectal

Sarah Tassinari 1 ; Federica Collino 2 ; Benedetta Bussolati 3 ; Edoardo D'Angelo 4 ; Marco Agostini 5 ; Federico Caicci 6 ; Jacopo Burrello 7 ; Alessandro Musso 8 ; Giuseppe Giraudo 9 ; Marco Ettore Allaix 10 ; Giorgio Maria Saracco 9 ; Mario Morino 9 ; Paola Cassoni 11 1 Department of Molecular Biotechnology and Health Sciences, University of Turin, 10126, Turin, Italy, Turin, Italy; 2 Laboratory of Translational Research in Paediatric Nephro‐urology, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; Pediatric Nephrology, Dialysis and Transplant Unit, Fondazione Ca' Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy; Department of Clinical Sciences and Community Health, University of Milano, Milan, Italy, Milan, Italy; 3 2Department of Molecular Biotechnology and Health Sciences, Torino, Italy; 4 First Surgical Clinic, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padua, Italy. Institute of Pediatric Research, Fondazione Città della Speranza, Padua, Italy., Padua, Italy; 5 First Surgical Clinic, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padua, Italy. Institute of Pediatric Research, Fondazione Città della Speranza, Padua, Italy., Padova, Italy; 6 Department of Biomedical Sciences, University of Padova, Padua, Italy, Padua, Italy; 7 Department of Medical Sciences, University of Turin, 10126 Turin, Italy, USA; 8 Division of Gastroenterology, A.O.U. Città della salute e della scienza, Molinette Hospital, 10126, Turin, Italy., Turin, Italy; 9 Department of Surgical Sciences, University of Turin, Corso Dogliotti 14, 10126, Turin, Italy, Turin, Italy; 10 Department of Surgical Sciences, University of Turin, Corso Dogliotti 14, 10126, Turin, Italy, USA; 11 Department of Medical Sciences, University of Turin, 10126 Turin, Italy, Turin, Italy Introduction : Colorectal cancer (CRC) is the third most frequently diagnosed cancer in men and the second in women. In this study we aimed to characterize EVs from different sources in CRC patients. Methods : EVs have been extracted from 110 fresh biopsies or 48 decellularized tissues (tumor and coupled 10‐cm far normal tissue) and from 16 plasma samples, following the WMA Declaration of Helsinki. EVs were extracted from biopsies after enzymatic digestion and sequential centrifugations and analyzed by NTA, TEM, super‐resolution microscopy and bead‐based cytofluorimetric analysis (FACS). Results : NTA and TEM confirmed the presence of intact EVs in our preparations. The number of extracted EVs was higher in fresh biopsies compared to decellularized one. Tetraspanins expression was confirmed with super‐resolution microscopy and FACS, being CD63 the most expressed. The surface marker analysis showed different EV‐related information in the different sources analyzed. Comparing EVs extracted from fresh tumor vs normal biopsy, we observed alteration of markers related to the cellular microenvironment and tumor phenotype, such as markers of angiogenesis (CD31, CD105), platelet activation (CD41b, CD42a and CD62p), and epithelial‐to‐mesenchymal transition (SSEA‐4, CD146). Analysis of EVs extracted from decellularized tissue showed a different markers distribution related to tumor stage, and a significant increase of inflammatory/immunological molecules in the normal tissue surrounding the tumor of metastatic patients. Selected tissue markers were confirmed to be modulated in plasma of CRC patients. Summary/Conclusion : In conclusion, fresh biopsies contain EVs, exposing the heterogeneity of tumor and infiltrating cell composition (tumor cells, endothelial cells, platelets), their interactions and the resulting tumor state. At variance, EVs from decellularized tissues, entrapped in the extracellular matrix, may better embody the microenvironment alterations more prominent in the tumor surrounding tissue. Keywords : cancer, colon cancer, biomarker, biopsy, decellularized tissue, tumor microenvironment

Comparison

Jubin George 1 ; Stephanie Chidester 1 ; Joshua Welsh 2 ; Dove‐Anna Johnson 3 ; Sean Cook 1 ; Jason Savage 2 ; Jennifer Jones 4 1 NIH‐NCI, USA; 2 NIH‐NCI, Bethesda, USA; 3 Case Western Reserve University Medical School, USA; 4 NIH NCI, Rockville, USA Introduction : Extracellular Vesicles (EV) are of broad interest as carriers of molecular signatures of tumor progression and cancer treatment response. To study EVs and the nucleic acids, lipids, and proteins that comprise different types of EVs, one commonly used method to generate of EV stocks is production of conditioned cell media (CCM), followed by steps to concentrate and purify EVs. Factors that impact the yield and efficiency of EV production using CCM are not yet fully defined, so we reviewed data from a highly controlled set of 19 EV productions performed in 2022 to identify factors for future consideration or further investigation. Methods : Cells used in this comparison included 11 mouse and 8 human cell lines. Cells were cultured in serum free media to produce one liter of conditioned media for each cell line. CCM EVs were harvested and isolated by ultrafiltration (100K) followed by size exclusion chromatography. Cell density and viability were evaluated by microscopy at the time of CCM harvest. Duration of the production culture phase was documented in hours, and peak SEC fraction particle size and concentration for this analysis were documented by nanoparticle tracking analysis. Results : Pairwise comparison of average results in human and mouse samples identified factors related to human vs mouse cellular doubling times as associated with the yield and efficiency of EV production using this CCM method. Human cell lines required an average of 6 more hours than mouse cell lines to grow to a sufficient density to harvest CCM (human: 49.4 vs mouse: 43.3), and resulting CCM samples at the times of harvest produced fewer EVs on average from human cell lines than from mouse cell lines (human: 7.15e10 vs mouse: 1.42e11 particles per mL in 5mL). Summary/Conclusion : Most human tumor cell lines are known to have longer average doubling times than mouse tumor cell lines. Our results demonstrating lower EV yields and lower cellular densities despite longer average times in culture for human cells, indicate that EV production from human cell lines might be improved by extending cell culture prior to CCM harvest into a third day, rather than planning to harvest human tumor cell line CCM for EV isolation on the second day of culture. Funding : NIH, NCI, CCR Intramural Research Program. Keywords : extracellular vesicles, production, conditioned media

Convection

Steven C. George 1 ; Peter Sariano 2 ; Rachel R. Mizenko 1 ; Venktesh Shirure 2 ; Abigail Brandt 2 ; Cem Nesiri 2 ; Bhupinder Shergill 2 ; David Rocke 2 ; Randy P. Carney 1 1 University of California, Davis, Davis, USA; 2 University of California, Davis, USA Introduction : Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30‐150 nm) limits diffusion. We hypothesized that convection from interstitial flow and binding to the extracellular matrix control in vivo transport and spatial distribution Methods : We isolated EVs from the MCF10 breast cancer progression series (MCF10A, normal; MCF10DCIS, pre‐malignant; MCF10CA1, malignant), and examined their transport and binding through a laminin‐rich ECM under controlled interstitial flow using computational and experimental models of a microfluidic device. To isolate EVs, each cell line was cultured for 72 h in EV‐depleted media. Conditioned media was subjected to increasing centrifugation spins at 4°C (300xg for 10 min, 2,000xg for 15 min, 10,000xg for 30 min) and crudely purified via centrifugation through 150,000 kDA filters (4,000xg for 45min at 4°C). EVs for downstream imaging and analysis were stained with 2 mM CellTrace Far Red and incubated for 2 h at 37°C. Size exclusion chromatography (SEC) was used for final EV purification via qEVOriginal 35nm SEC columns to remove free dye, soluble proteins, and larger vesicles Results : We demonstrate an increasing presence of laminin‐binding integrins α3β1 and α6β1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs under controlled physiological interstitial flow (0.15‐0.75 μm/s) demonstrated that convection, and not diffusion, was the dominant mechanism of transport. Furthermore, binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3β1 and α6β1 Summary/Conclusion : Our studies demonstrate for the first time that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be considered in the interpretation of EVs as biomarkers and leveraged in nanotherapeutic design Funding : NSF Graduate Research Fellowship (PAS); ARCS Foundation Scholar Award (PAS); NIH (UH3 HL141800 , R01 EB030410, R21 AI161041, R01 CA241666, F31 NS120590); and the Department of Biomedical Engineering and the College of Engineering at the University of California, Davis. Keywords : biotransport, convection, diffusion, extracellular matrix binding

Diagnostic

Monica S. Y. Ng 1 ; Xiangju Wang 2 ; Andrew Kassianos 3 ; Helen Healy 1 1 Kidney Health Service, Royal Brisbane And Women's Hospital, Australia, Australia; 2 Queensland Health, Brisbane, Australia; 3 Queensland Health, Australia Introduction : Kidney fibrosis is the histological manifestation of irreversible CKD. We hypothesize protein cargo of urinary extracellular vesicles (uEVs) is differentially expressed in kidney fibrosis. Methods : Study approved by ethics committee and informed consent obtained. 29–47.5ml urine collected from 3 people without kidney fibrosis and 4 people with 40–90% kidney fibrosis. Urine spun at 650xG for 10mins and supernatants treated with protease inhibitor prior to ‐80°C storage. Urine thawed at 37°C for 15mins. Samples spun at 2000xG for 20mins and supernatant filtered through 30μm filter. Supernatant concentrated to 500μl via centrifugation at 3500xG for 40mins in 15ml centrifugal filter units with 100,000Da cut‐off regenerated cellulose membrane. Samples then washed in 50nm filtered PBS and spun at 20000xG for 30mins. uEV pellet resuspended in 500ul filtered PBS. Centrifugation steps completed at 4°C in a fixed angle rotor on maximum brake. uEVs confirmed with electron microscopy and proteomics. 50ul uEVs stained for antibodies against nephron segment markers (podocin, CD90, CD31, CD45, CD10, CD13, Tamm Horsfall Protein, CD227) and interleukin receptors (CD126, CD217, CD25, CD121a, CD121b) for 30mins. 400nm polystyrene NIST traceable size standards were added as counting beads. Samples analysed on Cytek Aurora with Enhanced Small Particle module. Results : Mean uEV concentration was 4.03 × 10^8 uEVs/ml from fibrotic kidney and 7.53 × 10^8 uEVs/ml from non‐fibrotic urine (p = 0.65). Interleukin 2 receptor, CD25 was expressed on mean 2.84 × 10^6 uEVs/ml in fibrotic kidney urine vs. 4.97 × 10^5 uEVs/ml in non‐fibrotic urine; p< 0.001. Tubular epithelial markers (CD10, CD13, CD227) were the most highly expressed nephron compartment markers. Ratio of uEVs expressing CD121a (interleukin 1 receptor type 1): CD121b (decoy interleukin 1 receptor type 2) was higher in fibrotic kidney urine (mean 1.86 vs. 0.37, p = 0.04). Summary/Conclusion : Majority of kidney uEVs are of tubular origin. There is an emerging uEV signature that can be used to distinguish kidney fibrosis.

Dissecting

Vaibhav Sharma 1 ; Saroj Kumar 2 ; Fredrik Nikolajeff 1 1 Lulea University of Technology, Lulea, Sweden; 2 Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India, New Delhi, India Introduction : Parkinson's disease (PD) is a neurodegenerative disorder that currently suffers from diagnosis at late stages. As currently there is no cure for PD, the pressing need of the hour is the discovery of molecular markers that would be potent in diagnosing the disease at its early stages. Therefore, a biomarker with interconnecting links between the proteome and miRNAs may be imperative in PD. With the proven link of exosomes in the progression of neurodegenerative diseases, these nanovesicles have extensive potential in finding novel biomarkers for PD. To date, integrated omics‐based profiling, establishing the links amongst proteomic and miRNomic of the exosomes has not been worked upon in PD. Our current work focuses on this area for opening up new avenues in the mechanistic details of this intricate neurodegenerative disease. Methods : Salivary and blood‐derived EVs from PD patients and healthy cohorts were isolated by chemical precipitation followed by antibody‐based validation through CD63, flotillin, and CD9 (universal surface marker) and confirmed neuronal origin by CD171. These nanovesicles were also characterized by an Electron microscope and via NTA. Additionally, miRNA and protein were purified from these different cohorts. Finally, miRNA analysis and proteomic analysis were performed via Illumina and Mass Spectrometry‐based platforms. Results : We have correlated the multi‐omics data from the exosomal set of PD‐diseased and healthy cohorts. We were able to find some significant differences between PD patients and healthy controls. The miRNA data shows some unique miRNAs, solely expressed in PD disease. Additionally, proteomics data also highlights some possible biomarkers for PD. The implications of these results will be discussed. Summary/Conclusion : This study highlights potential molecular biomarkers via extensive pathways analysis and interconnecting pathways between proteomes, and miRNaome respectively. These markers could later also be identified in the biofluids directly. Keywords : extracellular vesicles, parkinson's disease, biomarker, proteomics, miRNAs

Dissetting

Angela Galardi 1 ; Marta Colletti 2 ; Silvia Lampis 3 ; virginia Di Paolo 2 ; caterina ferraina 3 ; maria antonietta de ioris 3 ; franco locatelli 2 ; francesca nazio 2 ; Angela Di Giannatale 2 1 Department of Pediatric Hematology/Oncology and Cell and Gene Therapy IRCCS, Ospedale Pediatrico Bambino Gesù, Piazza di Sant' Onofrio, 4, 00165, Rome, Italy, Rome, Italy; 2 Bambino Gesù Children's Hospital IRCCS – Department of Onco‐Haematology, Gene and Cell Therapy, Rome, Italy, Rome, Italy; 3 Bambino Gesù Children's Hospital IRCCS – Department of Onco‐Haematology, Gene and Cell Therapy, Rome, Italy, Italy Introduction : Around 70% of patients with metastatic NB at diagnosis present bone marrow (BM) infiltration, this specific tropism to the BM has not been completely elucidated. Autophagy is a self‐degradative process that plays a homeostatic role in normal cells. In cancer cells, it plays a different role based on the context: suppresses and facilitates tumorigenesis. Emerging evidence suggest that, bone metastasis can be supported by MSCs through the creation of metastatic niches and that the dialogue between tumor cells and the surrounding tumor microenvironment may be mediated by exosomes. The aim of this work is to evaluate the role of autophagy in the BM metastatic niche formation through ii) the analysis of autophagy‐related proteins into NB cells‐derived exosomes; iii) the study of the autophagic pathway in MSCs cells Methods : Autophagic flux has been evaluated by western blot analysis in 8 NB cell and MSCs isolated from BM of NB patients with/without BM involvement and healthy control (HC‐MSCs). Exosomes of NB cell lines have been isolated by high‐speed ultracentrifugation. Protein content was analyzed by high‐resolution mass spectrometry for the presence of proteins implicated in autophagy. Modification in autophagy flux in MSCs has been evaluated after co‐culture with NB‐derived exosomes Results : analysis of basal levels of autophagy revealed that: i) NB cell lines have basal levels of autophagy ii) HC‐MSCs have higher basal levels of autophagy than MSCs derived from NB patients; iii) NB‐derived exosomes contain proteins implicated in autophagy pathway regulation. Summary/Conclusion : our study gives new insights into the characterization of the autophagy pathway in NB and in the BM niche. The understanding of this molecular process could help us to develop new therapeutic approaches in patients affected by metastatic NB. Funding : This work was supported by grants from the Ministero della Salute (GR‐2016‐02364088 to ADG). Keywords : exosomes, neuroblastoma, autophagy

Divergence

Janos Zempleni 1 ; Fang Zhou 2 ; Haluk Dogan 1 ; Roland Madajim 3 ; Peerzada T. Mumtaz 3 ; Juan Cui 1 1 University of Nebraska‐Lincoln, Lincoln, USA; 2 Harvard University, USA; 3 University of Nebraska‐Lincoln, USA Introduction : We reported that 50–75% of orally administered small extracellular vesicles in bovine milk (sMEVs) escape absorption and alter bacterial communities in the ceca of mice. Here, we expanded that line of investigation and tested the hypothesis that sMEVs select genetic variants in gut bacteria leading to altered metabolite production. Changes in purine metabolism were of particular interest, because of the essential roles of adenine and guanine in energy metabolism and cell signaling. Methods : Gut content of murine ceca was cultured in media containing a nutritionally relevant concentration of sMEVs (sMEV‐supplemented, mEVS) or sMEV‐free (mEVF) media under anaerobic conditions for six days. Bacterial DNA and RNA were analyzed by shotgun and RNA‐seq analysis, respectively, and genetic variants were identified using the MIDAS pipeline. Metabolites were assessed in bacteria and cell‐free media supernatant by ultra‐high‐performance liquid chromatography‐tandem mass‐spectrometry. Results : sMEVs selected approximately 55,000 genetic variants in 23 species of bacteria including a cluster of variants in purine metabolism. Genetic variants were transcribed. We identified a total of 10,508 and 12,500 mRNA‐level variants in 2,734 and 3,040 unique transcripts in 7 and 5 bacterial species in mEVS and mEVF cultures, respectively. Six hundred eighty‐nine and 226 transcripts matched the genetic variants in mEVS and mEVF cultures, respectively, including 15 out of 65 genes in purine metabolism. sMEV‐dependent selection of genetic variants was associated with changes in bacterial metabolism. For example, the concentration of purine metabolites and purine‐dependent metabolic pathways (glycolysis, tricarboxylic acid cycle energy metabolism, pentose sugar metabolism) were different in cell‐free mEVS and mEVF media. Summary/Conclusion : sMEVs participate in cross‐kingdom communication through selecting genetic variants in genes from purine metabolism in gut bacteria. Funding : Supported by NIH P20GM104320, NIFA (2016‐67001‐25301 and 2020‐67017‐30834), USDA Hatch and W‐4002, and the SynGAP Research Fund (all to J. Z.). J.Z. is a consultant for PureTech Health, Inc. Keywords : divergence, genetic variants, gut bacteria, milk extracellular vesicles

Engineered

Mahboube Shahrabi Farahani 1 ; Elham Hosseini‐Beheshti 2 ; Seyed Mohammad Moazzeni 3 ; Mehdi Forouzandeh Moghadam 4 1 Tarbiat modares university, Tehran, Iran; 2 Univers of Sydney, Sydney, Australia; 3 Tarbiat modares university, Iran; 4 Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat modares University, Tehran, Iran, Tehran, Iran Introduction : Exosomes as a type of extracellular vesicles (EVs) are natural nano‐carriers that possess the required crucial features of an ideal biomolecular delivery system. However, using unmodified exosomes may have some limitations such as low accumulation in target sites. Studies have established that most of these hurdles can be overcome by engineering exosomes against different cell surface markers. Methods : In this study, the newly designed fusion protein of ICAM‐1/LAMP2b was initially modeled, and its stability and binding affinity to interact with LFA‐1 were assessed by the MD simulation and the Z‐dock server, respectively. HEK293T cells were then stably transduced by a lentiviral vector encoding ICAM‐1/LAMP2b. The purified exosomes were characterized, and their interaction with recombinant LFA‐1 was studied by ELISA and western blot analysis. The uptake of targeted and non‐targeted exosomes was also evaluated by imaging and flow cytometry. Lastly, to assess the ability of targeted exosomes to be applied as a safe carrier, pAAVS1‐puro‐DNR plasmids were encapsulated into exosomes by electroporation and GFP expression in T cells was checked by imaging and flow cytometry. Results : The bioinformatics study indicated the acceptable binding affinity of ICAM‐1 for LFA‐1. Then, the HEKT 293 cell line was successfully modified permanently by a lentiviral vector to express ICAM‐1 on the surface of the derived exosomes. TEM imaging, DLS, and western blotting confirmed the intact nature of purified exosomes. Furthermore, the ELISA and western blot tests established the binding affinity of targeted exosomes for recombinant LFA‐1 with a significant difference from non‐targeted exosomes (1.81 OD difference). Furthermore, flow cytometry results revealed noteworthy differences in the binding of LFA‐1‐positive (54.7%), non‐targeted exosomes (18.7%), and targeted exosomes to LFA‐1‐negative cells (23.2%). Finally, flow cytometry indicated that 19.5 % of T cells were GFP positive after treating them by loaded targeted exosomes. Summary/Conclusion : Engineered exosomes expressing ICAM‐1/LAMP2b fusion protein on their surfaces were produced and isolated. The targeted exosomes were able to efficiently interact with T cells as their recipient cells. These engineered exosomes can be utilized as an ideal targeted delivery system to transfer various biomolecules to T cells, facilitating immunotherapies or other cell‐based treatments. Keywords : lentiviral vector, lymphocyte function‐associated antigen‐1, intercellular adhesion molecule‐1, targeted exosomes

Enrichment

Olesia Gololobova 1 ; Gregory E. Rice 2 ; Ramin Khanabdali 3 ; Kenneth W. Witwer 4 1 Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, USA; 2 Inoviq Limited, Notting Hill, Victoria, Australia., Notting Hill, Australia; 3 Inoviq Limited, Notting Hill, Australia., USA; 4 Johns Hopkins University, Baltimore, USA Introduction : Isolating extracellular vesicles (EVs) from human plasma can be challenging. EVs are at low concentration in plasma compared with, e.g., lipoprotein particles, which further overlap in size and density with EVs. To separate EVs, methods such as differential centrifugation, ultrafiltration, size exclusion chromatography (SEC), and combinations thereof are time‐consuming and unsuitable for clinical settings. There is an urgent need for a fast, robust and simple method for enriching vesicles in a one‐step process for downstream analysis. Here we present proteomics data of evaluation plasma‐derived EVs isolated using an affinity matrix magnetic nanoparticle technology (EXO‐NET, INOVIQ Ltd). Methods : EVs were isolated from 200 μL of healthy plasma pool using 15 μl EXO‐NET following the manufacturer's instructions. On‐bead lysis with RIPA buffer was used to recover EV‐associated proteins. Desalted peptides were analyzed by LC tandem mass spectrometry (LC‐MS/MS) on LumosETD (Thermo Fisher Scientific). MS/MS spectra were searched with Mascot 2.8.0 via PD2.4 against the UP5640_H.sapiens database. The data analysis was performed based on DAVID Knowledgebase (v2022q3). Results : 318 unique proteins were identified (protein threshold 95%), and around 200 proteins were identified as commonly EV‐associated proteins, including CD9, HSPA8, HSP90, ENO1, Annexin 2, 14‐3‐3 family members, and PLP. 200 proteins were identified as liver and lipoprotein‐specific, followed by proteins specific to blood cells, such as platelets. Interestingly, around 30 proteins are putatively CNS‐specific. The yield of CD9‐positive EVs was similar to that obtained with commercial SEC columns; however, less ApoB100 (low‐density lipoprotein marker) and ApoA1 (high‐density lipoprotein marker) were observed in the EXO‐NET output. Summary/Conclusion : The EXO‐NET platform may provide a simple and rapid enrichment EVs from human plasma, especially for clinical settings, offering minimal co‐isolation of lipoproteins and a one‐step process. Funding : The work is supported by the National Institute of Mental Health (NIMH; R21/R33MH118164). Keywords : extracellular vesicles, lipoproteins, plasma, proteomics

Evaluation

Pevindu H. Abeysinghe ; Natalie P. Turner; Murray Mitchell Queensland University of Technology, South Brisbane, Australia Introduction : Analysis of small extracellular vesicles (sEV) (30‐200nm) miRNA is critical in understanding the cascade of cellular signalling pathways in diseases. Next‐generation sequencing provides comprehensive miRNA profiles across biological fluids. However, selecting the best sEV miRNA isolation technique for next‐generation sequencing is challenging (1). Here, we compare combinations of three sEV miRNA isolation techniques for miRNA next‐generation sequencing. Methods : Blood plasma from dairy cows (n = 4) with similar genetic and physical characteristics were utilized to isolate sEV. Ultracentrifugation (UC) (100,000 x g for 2 hours at 4°C), size‐exclusion chromatography (SEC) (qEV 70nm) and ultrafiltration (UF) were used to design four methodological approaches (UC + SEC, SEC + UC, SEC + UF and UC + SEC + UF). All sEV samples were characterized using western blot markers (exosome: Flot‐1, CD81, CD9; non‐exosome: BSA), Nano‐particle tracking analysis (NTA) and transmission electron microscopy (TEM). sEV miRNAs were isolated using TRIzol and miRNeasy mini kit. Novaseq S1 platform was used for single end 100bp sequencing. Read counts were mapped using unitas and the differential expression was performed using edgeR. Results : All four methods yielded more than 1700 of miRNAs in each and differential miRNA expression demonstrated a distinct separation of miRNA profiles to the control plasma miRNA sample. The PCA determined a clear separation of expression profiles of miRNAs from all four methodological approaches to the control blood plasma circulating miRNA. However, UC+SEC yielded a higher number of sEV (characterized) which generated reliable sEV miRNA next‐generation miRNA sequence results. Summary/Conclusion : Four methodological approaches yielded a satisfactory level of sEV miRNAs, however, the selection of the best sEV miRNA isolation method depends on the downstream miRNA characterization technique. UC + SEC is the most precise and accurate bovine blood plasma sEV miRNA isolation method for next‐generation sequencing. Keywords : sEV, miRNA, ultracentrifugation, size‐exclusion chromatography, ultrafiltration

Evanalyzer

Melanie Schürz 1 ; Danmayr Joachim 2 ; Maria Jaritsch 1 ; Eva Klinglmayr 1 ; Heloisa Melo‐Benirschke 1 ; Cristian‐Tudor Matea 3 ; Patrick Zimmerebner 4 ; Jakob Rauter 5 ; Martin Wolf 6 ; Fausto Gueths Gomes 7 ; Zdenek Kratochvil 8 ; Zbynek Heger 8 ; andrew Miller 8 ; Thomas Heuser 9 ; Vesna Stanojlovic 1 ; Jana Kiefer 10 ; Tanja Plank 1 ; Litty Johnson 11 ; Martin Himly 11 ; Constantin Blöchl 12 ; Christian Huber 13 ; Martin Hintersteiner 14 ; Nicole Meisner‐Kober 3 1 Chemical Biology and Biological Therapeutics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, Austria; 2 Fernuniversität Hagen, Austria; 3 Chemical Biology and Biological Therapeutics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, Salzburg, Austria; 4 Chemical Biology and Biological Therapeutics, Department of Biosciences, University of Salzburg, Austria, USA; 5 Department of Biosciences, University of Salzburg, Austria, USA; 6 Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria, Salzburg, Austria; 7 Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS), Paracelsus Medical University (PMU), Salzburg, Austria, USA; 8 Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic, USA; 9 Vienna Biocentre Core Facilities, Vienna, Austria, Vienna, USA; 10 Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Salzburg, Austria, Austria; 11 Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Salzburg, Austria, USA; 12 Bioanalytics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, USA; 13 Bioanalytics, Department of Biosciences and Medical Biology, University of Salzburg, Austria, Salzburg, USA; 14 EvoBiotiX SA, Lugano, Switzerland, USA Introduction : Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis generally require dedicated instrumentation and proprietary software not readily accessible to non‐EV specialised labs, limiting their implementation for standard EV characterisation in the rapidly growing EV field. Methods : We developed ‘EVAnalyzer’, an ImageJ plugIn for automated, quantitative single vesicle analysis from imaging data, and established a robust protocol for capture, (immuno‐) labelling and fluorescent imaging of EVs using exclusively standard reagents and laboratory equipment. The process has been optimised and validated for a number of routine EV applications (Schürz et al, JEV 2022) and is continuously extended for new functions and applications. Results : We will present single vesicle characterization applications of EVanalyzer including the quantification of EV subpopulations based on immunostaining, validation of EV labelling reagents or optimisation of genetic EV engineering by determining population fractions and loading densities at the single vesicle level. We will further highlight the use of EVAnalyzer for automated quantification of cell uptake at the single cell ‐ single vesicle level, thereby enabling high content EV cell uptake assays and plate‐ based screens. Additionally, we will show how the program is used for the quantification of EVs from in vivo applications for quantification of EV biodistribution in biofluids and tissue. Summary/Conclusion : We propose that EVAnalyzer can be used in virtually every lab for routine as well as advanced in vitro and in vivo applications in EV and nanoparticle research. Due to its open access and open‐source character, EVAnalyzer is a versatile tool which is open to community innovation within the EV and nano‐ and advanced material research fields. Funding : This work was supported by the following grants: EV‐TT BPro (County of Salzburg, WISS2025, P1812596) and EVTT (European Union, EFRE/IWB 20102‐F1900731‐KZP) and NanoCommons (EC H2020 #731032). Keywords : cell uptake, EV immunolabelling, EV biodistributino, openinnovation, single particle imaging, single vesicle imaging

Exocas‐2

MinJu Bae 1 ; HyeonAh Seong 2 ; JunSoo Park 2 ; SeHyun Shin 3 1 Korea University, Seoul, Republic of Korea; 2 Korea University, USA; 3 Korea University (Microgentas), USA Introduction : Extracellular vesicles (EVs) are considered essential biomarkers in liquid biopsies. Despite intensive efforts aimed at employing EVs in a clinical setting, workable approaches are currently limited owing to the fact that EV‐isolation technologies are still in a nascent stage. This study introduces a magnetic bead‐based ion exchange platform for isolating EVs called ExoCAS‐2 (exosome clustering and scattering). Owing to their negative charge, exosomes can easily adhere to magnetic beads coated with a polycationic polymer. Owing to the features of magnetic beads, exosomes can be easily processed via washing and elution steps and isolated with high purity and yield within 40 min. Methods : Cationic salt is coated on the surface of magnetic beads with carboxyl group surface residues. Inject the cation coated magnetic bead (ExoCAS) into the plasma solution and incubate in 4°C for 30 minutes. By charge interaction, negative charged EVs are attached to the cationic beads. And then, pH 6 washing buffer and 1M NaCl elution buffer were used for EV isolation. Results : The present results confirmed the isolation of exosomes through analyses of size distribution, morphology, surface and internal protein markers, and exosomal RNA. Compared with the commercially available methods, the proposed method showed superior performance in terms of key aspects, including operation time, purity, and recovery rate. Summary/Conclusion : This highlights the potential of this magnetic bead‐based ion exchange platform for isolating exosomes present in blood plasma. Funding : This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government, MSIP (2016R1A5A1010148) and was funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) under the Industrial Strategic Technology Development Program (20012427). Keywords : exosome, isolation, cationic polymer, magnetic beads, ion exchange

Filtration

Jillian WP Bracht 1 ; Mandy Los 2 ; Edwin van der Pol 3 ; Rienk Nieuwland 4 1 Department of Clinical Chemistry, Amsterdam UMC location University of Amsterdam, Amsterdam, The Netherlands, Amsterdam, Netherlands; 2 Department of Clinical Chemistry, Amsterdam UMC location University of Amsterdam, Amsterdam, The Netherlands, Netherlands; 3 (2) Laboratory of Experimental Clinical Chemistry and Vesicle Observation Center, Amsterdam; The Netherlands; (8) Biomedical Engineering and Physics, Amsterdam UMC, Amsterdam, The Netherlands, Amsterdam, Netherlands; 4 (2) Laboratory of Experimental Clinical Chemistry and Vesicle Observation Center, Amsterdam; The Netherlands;, Amsterdam, Netherlands Introduction : Processing blood by double centrifugation (2 × 2500g) is common but yields plasma that still contains ∼10e5‐10e6 platelets/mL, which may affect downstream EV analyses. Remaining platelets can be removed by filtration without affecting EV concentrations. Since a filtration procedure requires additional time, we investigated whether platelet‐free plasma can also be prepared by combining single centrifugation and filtration. Methods : EDTA blood from healthy controls was centrifuged at (1) 2 × 2500g for 15 minutes (no brake; plasma collected to 10 mm above the buffy coat/cell pellet), (2) 1 × 2500g, and (3) 1 × 5000g. Plasma was then filtered using 0.8 um polycarbonate track‐edged filters to remove remaining platelets. Concentrations of platelets and EVs (>150 nm; CD61+, CD61+/CD62p+, CD45+, CD235a+) were measured in filtered and unfiltered plasma by calibrated flow cytometry (Cytek Northern Lights and Apogee A60‐Micro). Results : Plasma contained ∼10e7 platelets/mL after single centrifugation and ∼10e5 platelets/mL after double centrifugation. Filtration removed >98% of remaining platelets for all protocols. However, after single centrifugation, the removal of platelets using filtration led to a 2‐fold increase in the concentration of CD61‐EVs and an 11‐fold increase in CD61/CD62p‐EVs, the latter likely derived from activated platelets. The maximum concentration of remaining platelets that can be removed by filtration without generating CD61‐EVs was 2 × 10e6 platelets/mL. A linear regression analysis showed that on average 2.5 CD61‐EVs are generated for each removed platelet above this platelet concentration. The concentration of CD45‐EVs and CD235a‐EVs was not affected by filtration. Summary/Conclusion : The maximum concentration of platelets that can be removed by filtration without generating CD61‐EVs within the detection range of the used flow cytometers is 2 × 10e6 platelets/mL plasma, and therefore we recommend to measure the platelet concentration in plasma before filtration. Funding : This study was funded by Health Holland (AQrate project). EvdP receives funding from the Dutch Research Council (grant number VIDI 19724). Keywords : blood plasma, extracellular vesicles, filtration, platelet removal

Functional

Yashvi Sharma 1 ; Sujata Mohanty 2 1 All India Institute of Medical Sciences (AIIMS), New Delhi, India; 2 AIIMS New Delhi, Delhi, India Introduction : Mesenchymal Stem Cells (MSCs) are the key therapeutic candidate in the field of regenerative medicine and can be derived from various sourcesv(Bone Marrow (BM) and Wharton's Jelly (WJ)). Wharton's Jelly, being a naïve source and posing less ethical concerns is an interesting candidate of choice for MSCs derivation. However, in terms of their translational significance, the point of their storage and transportation poses a major complication due to eventual loss of their viability. Owing to such issues, the derivatives of these stem cells, i.e. Extracellular Vesicles (EVs), especially small EVs (size > 200nm) have secured limelight as novel therapeutic tool. These nano sized vesicles have attracted the interests of scientific and clinical community for their ease of storage, transportation and logistics, making them a strong candidate as a stem cell derived ‘off the shelf’ product. To delve further into this aspect, we have evaluated the functional and biophysical capabilities of WJ‐MSCs derived small EVs to test their durability for translational applications Methods : Wharton's jelly was collected after obtaining donor consent(IC‐SCR/120/21(R)). MSCs were cultured in serum free media for isolation of small EVs using Total Exosome Isolation Kit (Thermofischer). The small EVs were then subjected to short term storage for 1 and 3 months at different temperatures, including RT, 4°C, ‐20°C, ‐80°C to assess their shelf life. At each condition, small EVs were characterized via NTA (Size < 200nm), TEM (Cup shaped morphology), Western blotting (Surface marker‐CD63;Cytoplasmic marker‐ALIX; Negative marker‐Calnexin) for their biophysical characteristics, and for the functionality assessment, their effect on the proliferation of keratinocytes cell line (MTT), migration of keratinocytes cell line (Scratch assay), and immunosuppressive activity on PBMCs (MLR) was checked. Results : It was observed via the biophysical characterization and functional assessments including proliferation, migration, and immunosuppression studies that the small EVs are capable of maintaining their functional capabilities for upto 3 months, when stored at lower temperatures. However for upto 1 month, they are also able to maintain their functionality at a temperature of 4°C Summary/Conclusion : We can conclude from this study that WJ‐MSC‐EVs can be stored as an ‘off the shelf’ product at a common refrigerator temperature for up to 1 month, while for storage up to 3 month, lower temperatures have to be maintained

Harnessing

Jihoon Han 1 ; In‐San Kim 2 ; Gi Beom Kim 3 ; Seonghyun Kim 3 1 Korea Institute of Science and Technology, Republic of Korea; 2 Korea Institute of Science and Technology, Seoul, Republic of Korea; 3 SHIFTBIO, Republic of Korea Introduction : In order to utilize exosomes to be an ideal drug modality, it is necessary to improve the fusion rate of exosomes with target cells. In this study, we engineered oncolytic exosomes (bRSVF‐Exo) loaded with misfolded proteins (MPs) and expressing respiratory syncytial virus F protein, enabling the exosomes to fuse to cancer cells in a nucleolin‐dependent manner and directly dumping MPs into cancer cells, inducing immunogenic cell death (ICD). We evaluated the therapeutic efficacy and antitumor immunity of bRSVF‐Exo by itself and combined with PD‐1 blockade. Taken together, this study suggests an effective therapeutic strategy for using tumor‐targeting oncolytic exosomes for cancer immunotherapy. Methods : Exosomes were isolated by differential centrifugation and ultracentrifugation. Exosome markers and RSVF expression were confirmed using ProteinSimple analysis, along with particle numbers, conformation and size of exosomes. In vivo experiments were conducted with CT26.CL25 inoculated mice intratumorally injected with exosomes three times. Results : bRSVF‐Exo was engineered by isolating exosomes from RSVF‐expressing HEK293T cells treated with bafilomycin, an autophagy inhibitor which increased the expression of RSVF and MPs in exosomes. RSVF expressing exosomes spontaneously fused with cancer cells via nucleolin. Therefore, bRSVF‐Exo could preferentially fuse with cancer cells and direct dumping MPs, leading to the accumulation of aggresomes inside cancer cells, triggering ER stress and ICD. bRSVF‐Exo showed a 70% tumor regression effect in vivo. This antitumor effect was amplified when combined with PD‐1 blockade, leading to complete remission in some cases. Summary/Conclusion : Our results show that bRSVF‐Exo is a potential ICD inducer with tumor targetability that elicits anti‐tumor immunity. We present a previously unidentified strategy for effectively delivering MPs into cancer cells by tumor‐targeting oncolytic exosomes, resulting in potent ICD‐mediated immune responses against cancer. Combined therapy with bRSVF‐Exo and PD‐1 blockade augment a powerful tumor‐specific immunity.

Inhibition

Byungheon Lee ; Soyeon Jeon Kyungpook National University, Daegu, Republic of Korea Introduction : Exosomes are extracellular vesicles naturally secreted by cells and serve as carriers of RNAs and proteins for cell‐to‐cell communications. KRAS is frequently mutated and plays a vital role in many tumors. Receptor expressed in lymphoid tissue (RELT), also known as tumor necrosis factor receptor superfamily 19‐like, is upregulated in certain tumors such as lung tumor. To selectively deliver cytotoxic agents to tumor, we herein loaded mesenchymal stem cell‐derived exosomes with a siRNA against KRAS and surface‐modified them with RELTpep‐KLA comprising a RELT‐binding peptide (CRQTKN, named RELTpep) and proapoptotic peptide ((KLAKLAK)2, named KLA). Methods : Exosomes were isolated from cell culture medium using ultracentrifugation and transfected with the KRAS siRNA using ExoFect reagents. Exosomes were then surfaced‐modified with the RELTpep‐KLA using a phospholipid‐based membrane anchor, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐polyethylene glycol‐maleimide. Cytotoxicity assays were performed using CCK reagents. Antitumor therapy was performed using Balb/c nude mice bearing subcutaneous human tumor xenografts. Results : Compared with control peptide‐KLA, mesenchymal stem cell‐derived exosomes surface‐modified with RELTpep‐KLA more efficiently internalized into A549 lung tumor and Panc‐1 pancreatic tumor cells highly expressing RELT on their surface and exhibited cytotoxicity to these tumor cells. Exosomes loaded with KRAS siRNA as well as surface‐modified with RELTpep‐KLA reduced the phosphorylation of Erk and exhibited cytotoxicity in A549 and Panc‐1 cells and inhibited tumor growth in Panc‐1 tumor‐bearing mice more efficiently than those with either control siRNA or control peptide‐KLA. Summary/Conclusion : These results demonstrate that RELT‐targeted mesenchymal stem cell‐derived exosomes carrying proapoptotic peptide and KRAS siRNA hold a potential for exosome‐based targeted therapy against cancer. Keywords : exosomes, KRAS, proapoptotic peptide, RELT, siRNA

Integrated

Nagendra Verma 1 ; Adam Poe 2 ; Andrew Fealy 2 ; Mariam Ebrahami 3 ; Anokhi Patel 3 ; Xue Ying Song 4 ; Chintda Santiskulvong 5 ; Alex Rajewski 5 ; Sun Ren 6 ; Kevin Tseng 6 ; Sungyong You 7 ; Alexander V Ljubimov 8 ; Mehrnoosh Saghizadeh 9 1 Biomedical Sciences, Cedars‐Sinai Medical Center, Regenerative Medicine Institute Eye Program, Cedars‐Sinai Medical Center, Los Angels, USA; 2 Biomedical Sciences, Cedars‐Sinai Medical Center, Regenerative Medicine Institute Eye Program, USA; 3 Regenerative Medicine Institute Eye Program, Cedars‐Sinai Medical Center, University of California Los Angeles, Los Angeles, USA; 4 4Genomics Core, Cedars‐Sinai Medical Center, Los Angeles, USA; 5 Genomics Core, Cedars‐Sinai Medical Center, Los Angeles, USA; 6 5Bioinformatics Shared Resource, Cedars‐Sinai Medical Center, USA; 7 Bioinformatics Shared Resource, Cedars‐Sinai Medical Center, Los Angeles, USA; 8 Biomedical Sciences, Cedars‐Sinai Medical Center, Regenerative Medicine Institute Eye Program, David Geffen School of Medicine, University of California Los Angeles, USA; 9 Biomedical Sciences, Cedars‐Sinai Medical Center, Regenerative Medicine Institute Eye Program, 6David Geffen School of Medicine, University of California Los Angeles, Los Angeles, USA Introduction : MicroRNA‐10b (miR‐10b) is up‐regulated in the limbus vs. central human cornea and also in diabetic (DM) vs. normal (N) limbus, which may contribute to limbal cell function and diabetic corneal disease, respectively. Our purpose was to identify miR‐10b target genes/proteins using integrated genomic and proteomic analyses to elucidate its role in the normal and diabetic limbus. Methods : Human autopsy N and DM corneas were procured by the National Disease Research Interchange (NDRI) in Optisol medium. In vitro experiments were performed with stem cell‐enriched human N and DM primary limbal epithelial cells (LECs). MiRNA transfection was performed using Lipofectamine RNAiMAX. Normal LECs were transfected with miR‐10b mimic (M), or its mimic control (MC). At day 3 post‐transfection, cells were harvested for total RNA isolation using Trizol for next‐generation RNA sequencing (RNA‐seq). A matching set of transfected LECs was collected and cell lysates were prepared for proteomics analysis Results : Sets of 661 mRNAs (FC of ± 2, 305 up‐ and 356 down‐regulated) and 144 proteins (FC of ± 1.5, 72 up‐ and 72 down‐regulated) were identified as differentially expressed in miR‐10bM vs. MC with adjusted p < 0.05 using RNA‐seq and proteomics analyses, respectively. Combined proteomics and genomics analysis revealed a total of 17 overlapping genes as potential targets of miR‐10b, all considered putative or validated targets in the literature. Additionally, pathway analysis of proteomics and transcriptomics data revealed significant changes in multiple signaling pathways such as cell cycle, DNA repair and replication, Wnt, and TP53 signaling. Overexpression of miR‐10b in primary N and DM LECs significantly decreased expression levels of target proteins GCLM, CMPK1, Lig1, and TPM4 by western blot and immunostaining. In addition, down‐regulation of miR‐10b in DM organ‐cultured corneas using miR‐10b inhibitor increased expression levels of TPM4 and Lig1 involved in DNA repair and replication by immunostaining Summary/Conclusion : In conclusion, by using integrated genomic and proteomic analyses of cultured N and DM LECs, potential key target genes of miR‐10b were revealed as well as the underlying pathways affecting human LEC function. Further analysis of promising targets may improve our understanding of the molecular mechanisms underlying diabetic corneal alterations and wound healing Funding : NIH grants R01 EY025377 and EY029829 (Saghizadeh), Cedars‐Sinai Board of Governors Regenerative Medicine Institute, and Department of Biomedical Sciences. Keywords : exosome, cornea, diabetes, wound healing

Intranasal

Maheedhar Kodali 1 ; Shama Rao 2 ; Leelavathi N. Madhu 3 ; B Shuai 4 ; James Cai 2 ; Ashok K. Shetty 5 1 Texas A&M University, College Station, USA; 2 Texas A&M University, USA; 3 Institute for Regenerative Medicine, Department of Molecular and Cellular Medicine, Texas A&M University College of Medicine, College Station, Texas, USA., College Station, USA; 4 Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, USA; 5 Institute for Regenerative Medicine, Dept of Cell Biology and Genetics, Texas A&M Univ School of Medicine, College Station, USA Introduction : Introduction: Acute neuroinflammation after a traumatic brain injury (TBI) activates microglia, resulting in the perpetuation of chronic neuroinflammation linked with cognitive dysfunction. So, biologics proficient in modulating proinflammatory microglia may prevent chronic neuroinflammation after TBI. This study investigated the ability of intranasally (IN) administered extracellular vesicles (EVs), purified through chromatographic methods from cultures of human induced pluripotent stem cell (hiPSC)‐derived neural stem cells (NSCs), for modulating genes related to proinflammatory and antiinflammatory signaling cascades after a TBI. Methods : Methods: Adult mice received IN administration of hiPSC‐NSC‐EVs (25 × 109 EVs) or the vehicle 90 minutes after a unilateral moderate controlled cortical impact injury induction. Seventy‐two hours later, a cohort of mice was euthanized, microglia were isolated from the injured cerebral hemispheres, and the expression of genes linked to multiple inflammatory signaling pathways was examined through scRNA sequencing. Results : Results: Microglia from vehicle‐treated TBI mice displayed enrichment of genes related to type‐1 interferon (IFN)‐, IFN‐gamma, and interleukin 6 (IL6)‐signaling, damage‐associated microglia (DAM), and the complement system, involved in innate immune response and various inflammatory processes. Notably, the expression of many of these genes was either normalized to control levels or reduced in TBI mice receiving hiPSC‐NSC‐EVs. Moreover, the expression of multiple genes linked to antiinflammatory pathways, such as IFN‐β, IL4, and IL10 signaling, were either maintained or further enhanced with hiPSC‐NSC‐EV therapy. hiPSC‐NSC‐EV treatment also alleviated the upregulation of senescence‐related genes after TBI. Summary/Conclusion : Conclusion: IN administration of hiPSC‐NSC‐EV treatment after TBI can attenuate the expression of genes involved in type‐1 IFN, IFN‐gamma, and IL6 signaling, DAM, complement activation, and cellular senescence in microglia. Funding : Supported by a grant from the National Institute of Neurological Disorders and Stroke (1R01NS106907 to A.K.S.). Keywords : neural stem cells, extracellular vesicles, traumatic brain injury

Klebsiella

Shogo Tsubaki 1 ; Juntaro Matsuzaki 2 ; Yusuke Yoshioka 3 ; Takuma Araki 4 ; Hitoshi Tsugawa 5 1 Division of Host Defense Mechanism, Tokai Univesity school of medicine, Isehara, Japan; 2 Division of Pharmacotherapeutics, Keio University Faculty of Pharmacy, Japan; 3 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku‐Ku, Japan; 4 Support Center for Medical Research and Education, Tokai University School of Medicine, Isehara, Japan; 5 Division of Host Defense Mechanism, Tokai University Shool of Medicine, Isehara, Japan Introduction : Klebsiella pneumoniae is a commensal bacterium that colonizes human mucosal surfaces, the gastrointestinal tract, and the oropharynx. It can cause serious infections, such as pneumonia, urinary tract infections, and liver abscesses, primarily in immunocompromised patients. To date, the mechanisms behind K. pneumoniae‐host cell interactions and disease development in immunocompromised patients remain unclear. Recently, some studies indicated bacterial small RNAs may mediate host cell signaling. This study investigated how K. pneumoniae transports small RNAs into host cells. Methods : LacZ reporter AGS cells were constructed to express LacZ protein by frameshift mutation induced by Cas9‐sgRNA complexes upon internalization of specific single guide RNA (sgRNA). K. pneumoniae ATCC 43816 (WT‐Kp) was transfected via electroporation with specific sgRNA. LacZ reporter AGS cells were infected with sgRNA‐transfected K. pneumoniae (sgRNA‐Kp) using transwell insert chambers (MOI = 1:1). Bacterial extracellular vesicles (EVs) were collected using ultracentrifugation and characterized using transmission electron microscopy. Nanosight and qNano were used to measure the size and concentration of bacterial EVs. Results : Electroporation of sgRNA significantly decreased the mucoid phenotype of K. pneumoniae, and caused a loss of capsules on bacterial surfaces. Although the number of bacterial EVs produced by sgRNA‐Kp remained unchanged, the reduction of diameter in EVs isolated from sgRNA‐Kp was observed when compared to the EVs derived from WT‐Kp by a loss of capsules region. Quantitative PCR analysis detected transfected sgRNAs contained in bacterial EVs. Then, to examine whether transfected sgRNAs are transported and internalized into host cells via bacterial EVs, LacZ reporter AGS cells were infected with sgRNA‐Kp. As a result, lacZ positive cells were detected among sgRNA‐Kp‐infected LacZ reporter AGS cells. Additionally, LacZ‐positive cells were also detected by adding purified‐bacterial EVs to reporter AGS cells. These results indicate that bacterial EVs transport own sgRNAs into host cells. Our finding show that bacterial EVs derived from K. pneumoniae can deliver the small RNAs into host cells regardless of capsule structure. Summary/Conclusion : Our findings demonstrate that bacterial small RNAs transported by bacterial EVs may serve a role as the main tool in bacteria‐host cell communication.

Macrophage

Sunila Pradeep Ob‐Gyn, Medical College of Wisconsin, Milwaukee, USA Introduction : In ovarian carcinoma (OvCa), macrophages are reprogrammed toward pro‐tumorigenic phenotypes, including the release of anti‐inflammatory cytokines and expression of the immunosuppressive molecules. The expression of genes involved in the inflammatory response is tightly regulated at the transcriptional and post‐translational levels. To understand the mechanism driving pro‐tumorigenic phenotype in macrophages, we studied the role of tumor cell‐derived extracellular vesicles (EVs) in the cross‐talk with macrophages in OvCa. We found that EVs from ovarian cancer cells contain eukaryotic translation initiation factor eIF4E, an mRNA 5' cap‐binding protein. eIF4E containing EVs from ovarian cells enhanced translational activity and PD‐L1 expression in macrophages. However, a detailed mechanism explaining how eIF4E‐packaged EVs induce immunosuppressive phenotype in macrophages is necessary to develop novel immunotherapy strategies for advancing OvCa treatment. Our study aims to understand the eIF4E mediated translational mechanism driving pro‐tumorigenic phenotype in tumor‐associated macrophages. Methods : We performed EVs characterization following standard techniques and performed a co‐culture assay to evaluate the EV uptake by stromal cells. We performed SUnSET assay and polysome fractionation assay to determine whether eIF4E‐EVs affect translation in macrophages. Immunoprecipitation and proximity labeling assay were to study the encapsulation of eIF4E in EVs. We used Rab27aKO ovarian cancer in vivo model to study the effect of eIF4E packaged EVs on the immune suppressive ability of macrophages. We confirmed the expression of PD‐L1 or other immunosuppressive markers on macrophages by flow cytometry. Results : Confocal microscopy revealed that macrophages were more susceptible to EV uptake than other stromal cells. Our study shows that tumor cells employ EVs to deliver eIF4E to the tumor microenvironment, facilitating the release of cytokines such as IL‐6 and the expression of PD‐L1 on macrophages to support tumor growth. In addition, our data confirms eIF4E‐EVs enhance protein synthesis in macrophages. Altogether, our results shows that EVs mediated transfer of eIF4E to macrophages contributes to anti‐tumor immune response via PD‐L1 expression. Summary/Conclusion : In summary, we discovered that EVs enhances the immunosuppressive properties. This effect is regulated by EVs encapsulated eIF4E. This immunosuppressive phenotype of macrophages is characterized by increased de novo synthesis of PD‐L1, SIRPα and CD206. Our findings provide insight into how EVs orchestrate a pro‐metastatic phenotype by translational reprogramming in tumor associated macrophages. Funding : This work was supported in part by the OCRFA 657713, the WHRP, MCW American Cancer Society (ACS‐IRG 19‐138‐34), the DoD W81XWH‐21‐1‐0361 and NCI R01CA258433. Keywords : ovarian cancer, extracellular vesicles, protein translation, immunosuppression

Metastatic

Federico Vannuccini 1 ; Yari Ciani 1 ; Vera Mugoni 2 ; Orsetta Quaini 1 ; Caterina Nardella 1 ; Ugo De Giorgi 3 ; Gerhardt Attard 4 ; Orazio Caffo 5 ; Consuelo Buttigliero 6 ; Umberto Basso 7 ; Francesca Demichelis 1 1 Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy; 2 Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento (Italy), Italy; 3 Department of Medical Oncology, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, 47014 Meldola, Italy; 4 UCL Cancer Institute, University College London, London, Malta; 5 Department of Medical Oncology, Santa Chiara Hospital, 38122 Trento, Trento, Italy; 6 Department of Oncology, University of Turin, San Luigi Gonzaga Hospital, 10093 Torino, Italy; 7 Medical Oncology 1 Unit, Department of Oncology, Istituto Oncologico Veneto IOV IRCCS, 35128 Padova, Italy Introduction : Prostate cancer patients almost inevitably develop resistance to first‐line hormonal therapy and to second‐line castration regimens, entering a state referred to as castration‐resistant prostate cancer (CRPC). Liquid biopsies allow for the interrogation of all components shed by tumor cells into biofluids, including extracellular vesicles (EVs). In the framework of PRIME (PRostate cancer plasma Integrative Multi‐modal Evaluation), a funded program to develop multi‐modal liquid‐biopsy tailored assays for advanced prostate cancer, we profiled the EV‐associated transcriptome of 16 healthy donors (HDs) and 58 CRPC patients at multiple time points (n = 3) before and during Enzalutamide treatment. Methods : Plasma samples were collected across multiple Italian institutions with harmonized procedures and processed using ONCE (ONe Aliquot for Circulating Elements), an in‐house developed approach for the concomitant isolation of EVs and cfDNA from a single plasma aliquot. RNA was extracted from EVs with the IZON qEV RNA Extraction kit and treated with RNase‐Free DNAse I. Libraries were prepared using the SMARTer smRNA‐Seq kit for total RNA and sequenced on NovaSeq 6000 with a SR150 protocol. Results : We observed that the transcripts' integrity depends on the RNA biotype in healthy and disease conditions. Specifically, in EV‐associated RNA (EV‐RNA) from the HD and CRPC plasmas, we found intact YRNA transcripts with reads spanning the entirety of the transcripts. Also, mitochondrial transcripts show uniform coverage and longer reads than other RNA biotypes. Conversely, lncRNAs represent the RNA biotype with the shortest read length (mean < 50bp) and less uniform coverage. Furthermore, we observed that the length and coverage uniformity of protein‐coding reads depend on the putative cell of origin. Interestingly, erythrocyte‐derived, such as HBB, and whole‐blood associated EV‐RNA transcripts have high uniformity and longer reads with respect to other tissues, in line with mitochondrial transcript features, while prostate‐specific genes are highly fragmented. Stratifying our CRPC patient cohort (n = 58) by the time of response to Enzalutamide, we identified 490 genes differentially represented in plasma EVs between short‐ or long‐treatment responders (FDR< 0.05; n = 431 and n = 59 upregulated in short and in long responders, respectively). The majority of the highly represented genes in short responders (n = 313) are concordantly more expressed in CRPC tissues with respect to the whole blood, in keep with the representativeness of the plasma‐derived EVs signal of prostate cancer cell transcriptome. Summary/Conclusion : While protein‐coding and lncRNA from CRPC EV cargo are highly fragmented, their relative abundance represents an informative source for inquiring about the clinical status of cancer patients. Funding : AIRC (id 22792), CRUK (id 26822).

Microglial

Ma Hao Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, China., China (People's Republic) Introduction : Remyelination is a critical aspect after spinal cord injury, and the immune microenvironment at the injury is crucial for remyelination. The main objective of this study was to clarify that microglia cell lineage exosomes overexpressing miR‐223 can more effectively promote remyelination in mice after demyelination, and to seek the underlying mechanisms Methods : We applied RNA‐seq to analyze the levels of miRNA and mRNA in the spinal cords of healthy and demyelinated mice, and verified that the levels of miR‐223 were higher in the spinal cords of demyelinated mice than in healthy mice by RT‐qPCR. Lentiviral transfection and ultra‐high speed centrifugation were used to extract miR‐223 overexpressed and normally expressed BV2‐derived exosomes, which were identified by electron microscopy, particle size analysis, WB and qPCR. Mice were divided into four groups:miR‐223+BV2‐Exo(OE), miR‐223control‐Exo(NC), Vehicle and Sham. Transcriptomics and proteomics identified downstream signaling and binding proteins of miR‐223. Flow analysis was performed to investigate the recruitment of myeloid suppressor cells in spinal cords and peripheral blood of spleen; WB and q‐PCR were performed to detect iNOS, CD86, Arg1 and CD206 to investigate the polarization of microglia in spinal cord; EC staining was performed to observe the morphology of spinal cord injury centers, and immunofluorescence was used to statistically analyze the polarization of microglia and oligodendrocytes. Results : In demyelinated mice, miR‐223 levels were elevated. EC and immunofluorescence staining showed increased regenerative myelin and more MBP in the OE group compared to other groups. Flow analysis showed that MDSCs were 8.99% ± 1.4% higher in peripheral blood of spleen and 28.1% ± 2.3% higher in spinal cord tissue in the OE group compared with the control group, and the differences were statistically significant; WB and q‐PCR showed that M2‐type microglia were 32% ± 2.8% more and M1‐type microglia were 18% ± 2.4% less in the OE group compared with the control group, and the differences were Immunofluorescence showed that the number of 0PCs in spinal cord tissue increased by 24% ± 2.1% and the proportion of mature OLs increased by 37% ± 3.0 % in the OE group compared with the control group. Mass spectrometry analysis showed that miR‐223 activated LRP1 via the Shc1/PI3K/Akt pathway, thereby promoting recruitment differentiation of MDSCs and polarization of microglia to the M2 type. Summary/Conclusion : Our study shows that miR‐223+BV2‐EXO activates LRP1, promotes the recruitment of myeloid suppressor cells (MDSCs) and the polarization of microglia to M2 type to improve the immune microenvironment at the site of injury through the Shc1/PI3K/Akt pathway, and promotes the proliferation and differentiation of OPCs into mature oligodendrocytes (OLs) to promote remyelination. It was demonstrated that miR‐223+BV2‐EXO could be a new potential therapeutic approach.

Mitigation

Jimin Kim 1 ; Seul Ki Lee 1 ; Minyoung Jung 1 ; Joonghoon Park 2 ; Tae Min Kim 2 ; Soo Kim 1 1 Brexogen, Republic of Korea; 2 Seoul National University, Pyeongchang, Republic of Korea Introduction : Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) are now being recognized a novel therapeutics for various diseases because of their immune‐modulatory and anti‐inflammatory potential. Atopic dermatitis (AD) is chronic skin disease manifested by pruritus, impaired immunity, and skin tissue destruction. Stimulation of MSCs with inflammatory cytokines such as interferon‐γ (IFN‐γ) can improve the therapeutic potential of MSC‐derived EVs. Methods : EVs were generated from interferon‐γ‐stimulated induced mesenchymal stem cells (IFN‐γ‐iMSC‐EVs). The proteome signature of IFN‐γ‐iMSC‐EVs was analyzed. IFN‐γ‐iMSC‐EVs were subcutaneously administered into a mouse model of AD induced by 2,4‐dinitrochlorobenzene (DNCB). The repression of AD by IFN‐γ‐iMSC‐EVs was assessed by analyzing clinical features and the expression of molecular markers in AD mice. Results : IFN‐γ‐iMSC‐EVs was enriched with proteins responsible for regulating interferon activity and inflammatory signaling, as shown by proteome profiling followed by detailed bioinformatic study. The expression of major Th2 receptors (IL‐4α/13Rα1/31Rα) was diminished, the their corresponding intercellular signaling molecules also became less active. IFN‐γ‐iMSC‐EVs alleviated clinical symptoms, and blocked infiltration of inflammatory and mast cells in AD skin. In addition, IFN‐γ‐iMSC‐EVs led to a reduction in the expression of thymic stromal lymphopoietin (TSLP), NF‐κB activation, and IgE receptors. Further, disintegrated skin barrier by AD was reversed by IFN‐γ‐iMSC‐EVs, which was supported by the upregulation of key factors responsible for epidermal differentiation and lipid synthesis. Summary/Conclusion : Conclusively, we demonstrated that IFN‐γ‐iMSC‐EVs has potential to become a novel EVs based therapeutics for AD via blocking inflammation/Th2 response as well as promoting skin restoration. Keywords : extracelluar vesicle, induced mesenchymal stem cell, interferon‐gamma, atopic dermatitis

Neurogenic

Anil Tiwari 1 ; Mehak Vohra 2 ; kartik goel 3 ; Moumita Mondal 4 ; Abha Gour 5 ; Parinita Agrawal 6 ; Nisha Palanisamy Rajendran 6 ; RITU RAJ 7 ; Alka Bhat 6 ; Arun Chandru 8 ; Tuhin Bhowmick 9 ; Virender Singh Sangwan 5 1 Dr Shroff's Charity Eye Hospital and Shroff‐Pandorum Centre for Ocular Regneration, New Delhi, India, New Delhi, India; 2 Pandorum Technologies Pvt. Ltd., Bangalore, India and Shroff‐Pandorum Centre for Ocular regeneration, New Delhi, india, USA; 3 Pandorum Technologies Pvt. Ltd., Bangalore, India and Shroff‐Pandorum Centre for Ocular regeneration, New Delhi, india, India; 4 Dr Shroff's Charity Eye Hospital, India; 5 Dr Shroff's Charity Eye Hospital and Shroff‐Pandorum Centre for Ocular Regneration, New Delhi, India, India; 6 Pandorum Technologies Pvt. Ltd, Bangalore, India, India; 7 Pandorum Technologies Pvt. Ltd, Bangalore, India, Bangalore, India; 8 Pandorum Technologies Pvt. Ltd., Bangalore, India, India; 9 Pandorum Technologies, USA Introduction : Exosomes are the indicator of tissue health. Mesenchymal Stem Cell (MSCs) derived exosomes (MSC‐Exo) are one of the most popular therapeutically active extracellular vesicles. Though extensive work has been done to evaluate the immunomodulatory and anti‐fibrotic effects of MSC‐Exo, there is limited information about its neurogenic potential. Here, we showed that MSC‐Exo promotes neurite growth and multilineage differentiation of neurons. Methods : MSC cells were routinely cultured in MEM supplemented with HPL. Exosomes were prepared in MEM+ 2% HPL. Exosomes were prepared using ultra centrifugation and were characterized using a nano‐tracker for the size and zeta potential. The neurogenic potential of exosomes was evaluated on neuroblastoma cells SH‐SY5Y by evaluating i) neurite length, ii) Immunofluorescence (ß3 tubulin staining), iii) real‐time ‐PCR (RT‐PCR) and iv) immunoblot Results : Throughout the experiment, neurotrophic growth factor (NGF) is used as a positive control. We observed MSC‐Exo showed faster and better neurite growth compared to NGF and negative control (differentiation media only). We observed faster growth and migration of neuronal cells in the presence of MSC‐Exo than in NGF. Significantly increased expression of neuronal differentiation and multilineage neurogenic markers were observed in MSC‐Exo but lacks in NGF. Summary/Conclusion : NGF is an FDA‐approved drug for treating corneal diseases. Our study showed that MSC‐Exo is a better pro‐neurogenic factor than NGF. Also, MSC‐Exo were more efficient in the multilineage differentiation of neurons than NGF. In conclusion, we can say that MSC‐Exo can be an advance and next‐generation therapy not only for nerve growth but also for the pathologies associated with neuronal degeneration. Funding : None. Keywords : neurogenic, multilineage, exosomes

Optimizing

Cahyani Gita Ambarsari 1 ; Anna Maria Piccinini 2 ; Jon Jin Kim 3 ; Maarten W Taal 2 1 University of Nottingham, Nottingham, United Kingdom; 2 University of Nottingham, USA; 3 Queen's Medical Centre, USA Introduction : Urinary extracellular vesicles (uEVs) are potentially clinically valuable disease biomarkers, particularly in patients with kidney and urinary tract problems. uEVs encapsulate their cargo, including protein and nucleic acids, and stabilize it from enzymatic degradation. Polymeric Tamm Horsfall protein (THP) was reported to interfere with the number of uEVs isolated and protocol reproducibility. Methods : We optimized uEVs isolation from healthy volunteers using precipitation‐, pH and precipitation‐, and size‐exclusion chromatography (SEC)‐based method, with and without THP removal. uEVs were characterized by nanoparticle tracking analysis (NTA). Results : Among three commercial kits utilized, Total Exosome Isolation (from urine) Reagent (TEIR) resulted in the highest uEVs concentration, followed by SEC and Urine Exosome Purification Kit, in samples with and without THP removal. The implementation of the THP removal protocol reduced the uEVs isolated with TEIR and Urine Exosome Purification Kit, but not with SEC. The number of small‐size uEVs was less in THP‐removed isolates than in the samples without THP removal. A homogenous population of small uEVs was best obtained with Urine Exosome Purification Kit, followed by SEC and TEIR. Urine Exosome Purification Kit needed the smallest dilution to reach the requirement of nanoparticle tracking analysis, followed by SEC and TEIR. Summary/Conclusion : TEIR is beneficial in studies requiring large numbers of uEVs regardless of their size. Urine Exosome Purification Kit is useful in research aiming to study small uEVs. THP removal protocol causes loss of uEVs isolated with TEIR and Urine Exosome Purification Kit, in contrast, it improves uEVs isolation with SEC. Funding : The project is a part of Dr. Ambarsari's PhD research. The PhD study is funded by the Indonesian Endowment Fund for Education. Keywords : centrifugation, chromatography, exosomes, methods, nanoparticles, proteins

Orthogonal

Diana Kitka 1 ; Tatyana Vagner 2 ; Taylon Silva 3 ; Sara Veiga 4 ; Krizia Sagini 5 ; Giorgia Guerra 6 ; Javier Mariscal Avila 7 ; Valentina Minciacchi 8 ; Cecilia Lässer 9 ; Ivan Poon 10 ; Shannon Stott 11 ; Dolores Di Vizio 12 1 Department of Surgery, Cedars‐Sinai Biomedical Sciences and Translational Medicine, Los Angeles, CA US, Los Angeles, USA; 2 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, Los Angeles, USA; 3 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, USA; 4 Massachusetts General Hospital Cancer Center/ Department of Medicine, Harvard Medical School, Somerville, USA; 5 Institute for Cancer Research, OUH, Oslo, USA; 6 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, USA; 7 Department of Surgery, Cedars‐Sinai Biomedical Sciences and Translational Medicine, Los Angeles, CA US, USA; 8 Georg Speyer Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, USA; 9 Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, Gothenburg, Sweden; 10 La Trobe University, Department of Biochemistry and Genetics, Bundoora, USA; 11 Massachusetts General Hospital Cancer Center and Center for Engineering in Medicine/ Department of Surgery, Harvard Medical School, USA; 12 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles CA, USA, Los Angeles, USA Introduction : Extracellular vesicles (EVs) are membrane‐covered particles of heterogeneous sizes and cargo. Large oncosomes (LO), which are shed by cancer cells with highly metastatic properties, are atypically large (1‐10 um). Size exclusion chromatography (SEC) has been successfully used for the isolation and purification of Small‐EVs (S‐EVs) but not large EVs (L‐EVs). The aim of this study was to obtain quantitative and qualitative information about purification of L‐EVs derived from cancer cell cultures comparing SEC to density gradient centrifugation while testing microfluidics as alternative approach. Methods : Differential ultracentrifugation (UC), SEC (qEVsingle/70nm), density gradient centrifugation, tunable resistive pulse sensing (qNano), flow cytometry, western blot, EVHB‐Chip, 3D cell culture (Fiber Cell Systems). Results : We collected three crude subsets of vesicles (2.8K, 10K, 100K) and purified the vesicles with SEC. EVs were collected from the first EV fractions (EV1‐4). To exclude the possibility of apoptotic body contamination, especially in the 2.8K fraction, we induced apoptosis by TRAIL in the DU145 cell line to separate apoptotic bodies from large oncosomes. Particle concentration was determined by qNano and flow cytometry, showed that L‐ (900 ‐ 5700 nm) and S‐ (30 ‐ 250 nm) particles were most enriched in the first two EV fractions and L‐EV proteins were slightly more abundant in fraction EV2. In line with published data, the bulk of the proteins eluted in the protein fractions (P8‐11), confirmed the notion that most of the proteins present in crude low and high‐speed UC preparations are non‐EV contaminants. While both L‐ and S‐EVs eluted in the same fractions, the recovery efficiency was significantly higher for S‐EVs (63.1%) than for L‐EVs (22%). Flow cytometry analysis showed similar patterns and relatively low recovery for L‐EVs, suggesting that SEC is not appropriate for LO purification. Orthogonal experiments enabled to characterize the captured L‐ and S‐ EVs with the state‐of‐the‐art microfluidic device (EVHB‐Chip) with the ultimate goal of generating a platform that combines the use of multiple EV analytes and different populations of EVs with different biological significance for biomarker identification. Summary/Conclusion : SEC can successfully separate EVs from contaminating non‐EV proteins, but low particle yield affects its use for L‐EVs. It is essential to implement the use of the EVHB‐Chip to characterize specific subsets of EVs. Funding : R01CA218526 NIH/NCI, R01CA234557 NIH/NCI. Keywords : large oncosomes, EV purification, EV separation

Pancreatic

Baldev Singh 1 ; Pankaj Gaur 1 ; Jeyalakshmi Kandhavelu 1 ; Lu Luo 1 ; Arnav Bhatnagar 1 ; Yaoxiang Li 1 ; Shivani Bansal 1 ; Seema Gupta 1 ; Partha P. Banerjee 1 ; Vivek Verma 2 ; Amrita K. Cheema 3 1 Georgetown University, Washington, USA; 2 University of Minnesota, USA; 3 Georgetown University, USA Introduction : Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a majority of patients presenting with metastatic disease at diagnosis. Extracellular vesicles (EVs) released by cancer cells have emerged as key mediators of intracellular signaling, communication, and immune modulation within the tumor microenvironment (TME). Tumor‐associated macrophages (TAMs) are crucial components of TME that influence tumor growth and metastasis. The present study is focused on determining the role of PDAC‐derived EVs in altering macrophage phenotype, function and metabolism. Methods : EVs were isolated from the conditioned medium that was used to culture one established pancreatic cancer cell (PANC‐1), one PDX cell line (PPCL68) and a normal (non‐tumorigenic) immortalized pancreatic epithelial cell line (hTERT‐HPNE), using size exclusion chromatography. The EVs were extensively characterized using nanoparticle tracking (NTA), cryo‐electron microscopy, and immunoblot analysis as per ISEV guidelines. Macrophages were generated from the bone marrow cells of C57BL6 mice by cultured in vitro in macrophage colony stimulating factor (M‐CSF) containing medium. Macrophages were co‐cultured with isolated PDAC or normal cell derived EVs. Flow cytometry and metabolomic analyses were performed to determine the effect of EVs on macrophage phenotype, function(in vitro and in vivo) and metabolism. Results : Our results show that PDAC cell derived EVs were taken up by the macrophage in a time‐dependent manner that resulted in an immunosuppressive phenotype characterized by higher expression of CD206 and PD‐L1 and higher secretion of immunosuppressive cytokines including TGF‐beta, IL‐10 and GM‐CSF. Functionally, PDAC‐EV treated macrophages were able to suppress proliferation of CD8 T cells under in‐vitro and in‐vivo conditions. Metabolically, cancer EV‐treated macrophages showed accumulation of immunosuppressive metabolites (5‐methylthioadenosine, lactic acid, and kynurenate). Summary/Conclusion : We present novel findings suggesting that PDAC cell derived EVs are immunomodulatory as these influence macrophage function that might abet tumor progression and metastasis. Estimation of precise mechanisms of EV mediated immune modulation of macrophages is ongoing.

Presenting

Abhishek Sharma 1 ; Annika Jarvinen 2 ; Martin Albers 3 ; Sanna Auer 3 1 University of Oulu, Tampere, Finland; 2 BioNavis Oy, Finland; 3 BioNavis Oy, Tampere, Finland Introduction : Surface Plasmon Resonance (SPR) has been used already a few decades for label‐free detection and quantitation of biochemical kinetics and affinities for many different types of analytes. SPR is a well‐established technique to measure biomolecular interactions, such as drug‐protein binding kinetics and affinity, in real‐time, without labels and with high‐purity analytes. Methods : The new Multi‐Parametric Surface Plasmon Resonance (MP‐SPR) technology is a technique utilizing full SPR angular spectra in the interaction measurements, allowing for the characterization of several parameters from the SPR curve in real time. Uniquely MP‐SPR collects TIR (Total Internal Reflection) angle and SPR peak angle minima, thus monitoring changes in the bulk liquid. PureKineticsTM 1 allows real‐time bulk effect subtraction producing high‐quality results. MP‐SPR extends the applicability of the SPR technique to even more challenging interaction studies of high relevance in life sciences, quality assurance, and in vitro diagnostic applications. Results : Beyond the traditional use of MP‐SPR in biomolecular interaction studies, we present results among new application fields, such as cell‐based assays2,3, extracellular vesicle quantification3 and uptake4, and degradation of materials5 with MP‐SPR. The versatility of MP‐SPR for EV studies has been realized just lately3,4. Since the technique is powerful in detecting particles below 150 nm in their native state (e.g., without labels), it has enormous potential even for diagnostic use in this segment. We will also offer a new regenerable avidin for surface modification6,7. Regenerable avidin is an ideal reagent for reversible surface immobilization with high precision. Summary/Conclusion : In conclusion, MP‐SPR is a powerful label‐free technique for studying biomolecules such as EVs and could be used to study EV and EV corona interactions. Keywords : multi‐parametric surface plasmon resonance, biomolecular interactions, label‐free detection, small particle analyses

Production

Rachel Moen 1 ; Youn J. Jung 2 ; Hayden M. Pagendarm 3 ; John T. Wilson 2 ; Alissa M. Weaver 4 ; Ethan Lippmann 1 ; Jamey Young 1 1 Vanderbilt University, USA; 2 Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA, USA; 3 Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA, Nashville, USA; 4 Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN, USA, Nashville, USA Introduction : Mesenchymal stem cells (MSCs) naturally produce therapeutically relevant EVs that have potential to treat many immunological diseases. However, primary MSCs vary extensively between different tissues and donors, have limited replicative potential, and are typically grown in 2D monolayers. Collecting sufficient EVs for a therapeutic treatment is therefore resource‐, cost‐, and labor‐intensive. MSCs differentiated from induced pluripotent stem cells (iPSCs) can provide a reproducible non‐invasive source of MSCs that avoids obstacles due to donor‐to‐donor variability and scarcity of human tissue. This study aims to develop a scalable platform to produce EVs from tissue‐specific cell types, using iPSC‐derived MSCs as a proof of concept for other cell lines that can also be derived from iPSCs. Methods : We are optimizing EV production by encapsulating MSCs into GelMA‐Cad hydrogel microspheres. GelMA‐Cad is a gelatin covalently bonded to N‐cadherin to support cell growth by mimicking cell adhesion to the extracellular matrix. Cells are encapsulated using a custom microfluidic device, and the microspheres are cultured in suspension. EVs are collected from the culture media for characterization (NTA, western blot, and TEM) and functional assays. Results : We have shown that GelMA‐Cad supports cell growth in both 2D and 3D culture. By seeding MSCs into hydrogel microspheres, adherent cells can be grown in a pseudo‐suspension culture allowing for increased cell density and EV production per volume of culture. EV yield (per cell) and particle size were compared to traditional 2D adherent cultures. Here, we found that MSCs seeded in GelMA‐Cad produced a similar yield of small EVs but higher yield of large EVs when grown in pseudo‐suspension culture. Summary/Conclusion : Production of EVs from MSCs embedded in GelMA‐Cad leads to higher volumetric productivity than adherent 2D cultures. Future work will expand this platform to other iPSC‐derived cell types beyond MSCs. Funding : NSF MCB‐2036809. Keywords : Hydrogels, Mesenchymal Stem Cells, Induced Pluripotent Stem Cells, Microsphere

Regionally

Shuo Wang 1 ; Yosuke Tanaka 2 ; Ying Xu 2 ; Sen Takeda 1 ; Nobutaka Hirokawa 1 1 The University of Tokyo Graduate School of Medicine, Japan; 2 The University of Tokyo Graduate School of Medicine, Tokyo, Japan Introduction : Digit formation is essentially determined by a posteriorizing Sonic hedgehog (Shh) protein gradient in the mouse embryo limb buds at 10.5 days postcoitum. The most distal AER secretes FGF8 proteins to form a distal‐to‐proximal gradient of PI3K signaling. In perpendicular to this, Shh protein is propagated from the posterior organizer toward the anterior region through the mesenchyme, via EVs and/or cytonemes. However, the switching mechanism between these two propagation modalities and its relevance in development has been little understood. Methods : The polydactylous KIF3B kinesin hypomorph mouse embryos were analyzed by immunohistochemistry, whole mount in situ hybridization, and electronmicroscopy. FGF8b‐soaked bead injection to limb buds of wild type embryos, followed by whole embryo culture, was also performed. The primary cultured mesenchyme and KIF3B/talpid3‐knockdown fibroblasts were subjected to cell biological and pharmacological analyses. Talpid3 activity and its KIF3B binding capacity were investigated by biochemistry and FLIM/FRET microscopy. Results : In the limb buds of KIF3B‐hypomorph mouse embryos, the distal‐to‐proximal PI3K signaling gradient was lost by its overall activation. In addition, the posterior‐to‐anterior gradient of Shh protein was also lost by its global dispersion. Experimental elevation of PI3K signaling by implantation of FGF8b‐soaked beads in the inner layer reproduced the disorganization of Shh gradient. We detected that KIF3B essentially terminates PI3K signaling by transporting and stabilizing the PTEN‐family protein Talpid3. Interestingly, PI3K signaling generally facilitated Shh‐EV release through the general exosome releasing pathway involving nSMase2, and simultaneously suppressed Shh vesicle transport into the cytonemes. Thus, endocytosed Shh protein tends to be secreted as EVs in the outer layer, while retained into cytonemal punctata in the inner layer. This may provide differential Shh diffusion rates between these two layers, constituting a diffusion‐and‐trapping system for gradient formation. Summary/Conclusion : Here we show the experimental evidence for a regionally regulated secretion of Shh‐EVs creating a concentration gradient in developing mouse limb buds. We will also discuss on possible application of this PI3K‐mediated Shh‐EV biogenesis by presenting some preliminary data. Funding : This work was supported by JSPS KAKENHI grant numbers JP23000013, JP16H06372, and JP22K06246 to N.H; by Strategic Research Program for Brain Sciences from AMED (JP20dm0107084) to N.H. and Y.T., and by a GAP‐Fund (8th and 12th terms) from Univ Tokyo and an AMED Grant (JP22ym0126805) through the TR Center, The University of Tokyo Hospital, to Y.T. Keywords : sonic hedgehog, morphogen gradient, regulated EV biogenesis, FGFR, PI3K signaling, polydactyly

Regulation

Kristine V. Hoagstrom 1 ; Ying Huang 2 ; Heather Jensen‐Smith 2 ; Michael A. Hollingsworth 2 1 University of Nebraska Medical Center, Omaha, USA; 2 University of Nebraska Medical Center, USA Introduction : Pancreatic ductal adenocarcinoma (PDAC), a highly aggressive disease with poor prognosis, lacks early detection methods. Recent studies highlight the value of extracellular vesicles (EVs) in PDAC diagnostics and treatments. Overexpression and selective enrichment of MUC1, a type I transmembrane protein, has been found in tumor EVs, correlating with hyperplasia, migration, and invasion. Additionally, ALIX, a cytoplasmic adapter and ESCRT‐associated protein, demonstrates critical mediation of EV biogenesis. Our unpublished data reveals significant reduction in EV secretion upon knock‐out of MUC1 in multiple pancreatic cancer cell lines. This study aims to elucidate the nature of the MUC1‐ALIX interaction and its function in EV biogenesis regulation. Methods : EVs were isolated from conditioned media of pancreatic cancer cell lines, purified by centrifugation then ultracentrifugation at 110,000g for 2 hours in a Beckman SW28 Swinging Bucket rotor, and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis, and immunoblotting. MUC1‐ALIX association was analyzed by immunofluorescence microscopy (IFM), fluorescence resonance energy transfer (FRET), and co‐immunoprecipitation (CoIP). Results : IFM data illustrated colocalization between the C‐terminal subunit of MUC1 (MUC1.CT) and ALIX. FRET studies with CFP‐labeled MUC1 and YFP‐labeled ALIX showed decreased YFP intensity with photobleaching concomitant with increased CFP intensity, demonstrating a MUC1‐ALIX association. CoIP analyses using MUC1.CT and ALIX mAbs revealed an impact of knocking out MUC1 or ALIX on the protein content of EVs. Together these results implicate a role for the MUC1‐ALIX interaction in regulating EV biogenesis and packaging. Summary/Conclusion : This study demonstrates a clear MUC1‐ALIX interaction and that its disruption impacts EV biogenesis. The results lay the groundwork for ascertaining MUC1's role in modulating protein content in tumor EVs and the influence of this process on the progression of PDAC. Funding : Acquired Resistance to Therapy Network (ARTNet) 1U54CA274329‐01. Structural Biology and Molecular Biophysics (SBMB) Training program at UNMC from the Department of Education Graduate Assistance in Areas of National Need (GAANN) program fellowship.

Restrained

Gna Ahn ; Ji‐Young Ahn; Yang‐Hoon Kim Chungbuk National University, Cheongju‐si, Republic of Korea Introduction : Many studies have reported on the antimicrobial agents in bovine colostrum (BC), but that of BC‐derived exosomes (BC‐Exo) remain largely unknown. Staphylococcus aureus (S. aureus) is a typical opportunistic pathogen. To treat S. aureus infection, antibiotics‐based treatment has been used for a long time, however, the frequency of resistant strains has been on the rise; hence, alternative approaches are needed. We demonstrate the antimicrobial activity of BC‐Exo against S. aureus, and further investigate its role as a bacteriostatic agent related to the toxin‐antitoxin (TA) system. Methods : BC‐Exo isolation from BC provided by Seongan farm was basically used acetic acid treatment, and was centrifuged at 200,000 × g for 1 h at 4°C (beckman coulter, USA). Acquired BC‐Exo was confirmed to morphology observation through TEM and cryo‐EM. Size and concentration were also measured (iZon, Australia). TSG101 and CD81 were used to identify exosome protein. Purified BC‐Exo was used for physiological properties such as growth, hemolysis, bio‐film formation, surface morphology through SEM, and ATP quantification. Last, after gDNA extraction from S. aureus, TA protein expression and purification was used to E. coli BL21(DE3). Purified TA protein was treated to S. aureus and BC‐Exo, the result was confirmed to SDS‐PAGE or native page. Results : The antimicrobial properties of BC‐Exo were identified by the growth inhibition, especially showed bacteriostatic effect by using live/dead assay. Furthermore, hemolysis inhibitory effects and interrupted biofilm formation were also confirmed. Though SEM data, we observed that BC‐Exo induced winkled cell surface. Also, BC‐Exo reduced ATP production and affected overall phenomenological events in S. aureus. We also demonstrated that TA system was partially related to S. aureus and BC‐Exo. Summary/Conclusion : We demonstrated that BC‐Exo can exhibit clear antimicrobial activity against S. aureus. These results suggest that BC‐Exo has potential in antibiotic research as a natural substance that can replace chemical antibiotics in the future. Keywords : bovine colostrum exosome, staphylococcus aureus, antimicrobial effect, bacteriostatic

Retroviral

John JS Cadwell 1 ; Kenneth W. Witwer 2 ; Zhaohao Liao 3 1 FiberCell Systems Inc., New Market, USA; 2 Johns Hopkins University, Baltimore, USA; 3 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, USA Introduction : Various methods have been applied to the transfer of genes into MSC. The generation of stable MSC transfectants is hampered by the limited number of passages MSC can undergo before they start to differentiate and difficulty in performing at scale. Current data suggests that mesenchymal stem cells, when seeded into a 3‐D hollow fiber bioreactor show little proliferation and may be maintained in culture and produce EVs continuously for extended periods of time. Transient transfection under these conditions could result in a usefully stable MSC transfectant. To this end a retroviral transfection of bone marrow MSC using a hollow fiber bioreactor was performed. Methods : Bone marrow MSC from ATCC were expanded to 5 × 10e7 cells using DMEM/10% FBS and 6 T300 flasks. Retrovirus encoding for green fluorescent protein was produced in culture. A FiberCell C2025D 20 kd MWCO polysufone cartridge with 450cm2 of area and a 2.8 ml volume was seeded as follows. 5 × 10e7 cells in a volume of 5 mls was attached to one side‐port and 4.5 × 10e9 retrovirus in a volume of 4 mls was attached to the opposite side port. Equal volume of cell/virus mixture was flushed into each syringe and then injected into the ECS of the cartridge with the excess volume flowing through the fibers into the medium reservoir, concentrating the cells and the virus together. After 24 hours the ECS was drained and the medium replaced with basal DMEM (no serum). Harvests were performed at 24 hour intervals. Results : 1) Isolated EVs from the first 16 days of harvest collection show strong Nluc signal and approximately 5–12% of all detected particles in those EV samples were GFP positive, indicating release of the GFP‐Nanoluc fusion reporter proteins via EVs by MSC transfectants. 2) The particle count of EVs produced by palmGRET MSCs declined from 1.4E+10 particles/mL in the first harvest to 3.9E+06 particles/mL measured in the last harvest (day 27). These data strongly suggest that EV production was drastically reduced after 2 weeks of MSC transduction in HFBR 3) It was not possible to directly determine transduction efficiency under these conditions. Summary/Conclusion : A hollow fiber bioreactor can reduce the volume required to perform transductions by 100X, and utilizes a closed, cGMP compatible format. Overall, these promising preliminary data warrant further optimization and refinement of the transduction protocol, particularly by modifying the viral titer, selection strategy, and length of the experiment. Cell viability assays will also be performed to determine whether EV concentration declined as a result of decreased cell viability or if other factors are at play. Increasing scale by a factor of 100x or more is possible using existing systems. Funding : na. Keywords : retroviral transduction, palm‐GRET, hollow fiber

Separation

Soha Alkhaldi ; Ching‐An Peng University of Idaho, USA Introduction : Extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) are membrane‐bound vesicles that mediated the intercellular communication between cells through transporting active biological cargoes, such as proteins, as lipids, and RNAs. However, the most commonly used techniques for EV isolation (e.g., differential ultracentrifugation and hydrophilic polymer precipitation) have disadvantages regarding feasibility and purity. Low purity and retention of polymer can interfere with further downstream analysis. Here, we developed a novel method for the separation of MSC‐derived EVs using chitin magnetic particles bound with chitin‐binding domain (CBD) fusion with self‐cleaving intein tag and lactadherin C1C2 which has high affinity to phosphatidylserine (PS) exposed on EV membranes. Methods : The gene of human lactadherin C1C2 was amplified by polymerase chain reaction and cloned into the N‐terminal of intein and CBD from pTXB1vector, then expressed in E. coli. Prior to thiol‐induced cleavage of thioester bond between the intein tag and lactadherin C1C2, cell conditioned medium (CCM) containing EVs was mixed with chitin magnetic beads for 1hr at 4oC to allow the binding between lactadherin C1C2 and PS on EVs membranes. Then, lactadherin C1C2‐EVs were isolated from the beads by the cleavage buffer containing 50 mM dithiothreitol. Results : Our approach yielded high purity and high specificity of EVs better than those obtained using the classical methods. Relevant EV biomarkers (i.e., CD63, CD9, CD81, Tsg 101, and Alix) were detectable by the Western blot. The size and charge of EVs were determined respectively to be 111.21 ± 11 nm and −14.47 ± 2.2 mV, which are within the size ranges of exosomes and microvesicles. Summary/Conclusion : The specific binding affinity between lactadherin C1C2 and PS‐expressing EVs was harnessed to effectively capture EVs from MSC CCM using chitin magnetic beads tethered with CBD‐intein‐lactadherin C1C2 fusion protein. Funding : N/A. Keywords : Mesenchymal stem cell, chitin magnetic bead, chitin binding domain, intein, lactadherin C1C2, phosphatidylserine

Simplified

Kathryn Neville 1 ; Sarah Brabant 2 ; Jessica Ritz 2 ; Joshua Miller 2 ; Samuel Walker 3 ; Jonathan Flax 4 ; James McGrath 3 ; James Roussie 2 1 SiMPore inc., West Henrietta, USA; 2 SiMPore Inc., West Henrietta, USA; 3 University of Rochester, Rochester, USA; 4 University of Rochester Medical Center, Rochester, USA Introduction : Developing and characterizing methods for separation and concentration of extracellular vesicles (EVs) remain an area of significant interest. EV applications increasingly require isolated EVs of high purity and yield to perform basic biological, diagnostic, and especially therapeutic analyses. An ability to characterize EVs by simple and rapid means before, during, and after isolation steps would benefit understanding of both currently used methods and development of new methods. However, assessing EV preparations for EV size, concentration, and presence/absence of molecular cargo currently requires multiple dedicated instruments and practiced knowledge of these systems’ procedures and pitfalls. We aim to bridge this gap by developing a user‐friendly EV visualization procedure for determining concentration and detection of molecular markers within EV preparations. Methods : Here, we report on our assessment of a microfluidic device capable of rapid visualization of EVs using simple pipet‐driven loading and on‐membrane EV capture and imaging. This device, named the μSiM‐EV, was assessed for its ability to catch and visualize EVs by a number of microscopy techniques. On‐membrane immunofluorescence was used to detect EV surface proteins, while comparisons to nanoparticle tracking analysis was used for assessing EV concentration. Results : We assess nanoporous silicon nitride membranes, which are incorporated into the μSiM‐EV device, for their ability to capture small‐ and medium‐sized EVs. We showcase the ability to visualize EVs when captured on‐membrane by optical and electron microscopy. We further compare nanoparticle tracking analysis to the μSiM‐EV for particle concentration determination. Summary/Conclusion : The μSiM‐EV offers ease of use for rapid EV visualization during separation and concentration procedures. Future work will focus on expanding the variety and number of EV preparations from different sources that are analyzed via the μSiM‐EV procedure. Funding : NIH Grant No. NIGMS 2‐R44‐ GM137651 ‐02 to SiMPore Inc. Keywords : EV characterization, single EV analysis, microfluidics, microscopy

Successful

Stephen Lenzini 1 ; Jae Jung 1 ; Grishma Patel 2 ; Sanket Jadhav 2 ; Priyanka Gupta 2 ; Rukmini Ladi 2 ; Michael Boychyn 1 ; Elie Zakhem 1 ; Jon Rowley 1 1 RoosterBio, Inc., Frederick, USA; 2 Sartorius Stedim North America Inc, Bohemia, USA Introduction : Extracellular Vesicles (EVs) are produced by many cell types, particularly from mesenchymal stromal cells (MSCs). The number of clinical trials investigating EVs from MSCs as a therapeutic agent has been increasing. The goal of this study was to identify and optimize scalable parameters for EV production in a 3D stirred‐tank bioreactor. Methods : Human bone marrow MSCs were expanded in xeno‐free RoosterNourish™ medium followed by EV collection in RoosterCollect™‐EV medium (RoosterBio) in Ambr®250 bioreactor at 250mL scale. Different microcarriers were screened for optimal MSC attachment and growth. Microcarrier concentrations and cell seeding densities were evaluated to maximize cell yield. Agitation ramping, culture duration and feed strategies were evaluated. The robustness and reproducibility of the process were verified across multiple donors. Optimal parameters were verified at a large scale (15L) in Biostat STR® bioreactor (Sartorius). Critical quality attributes (CQAs) of EVs were evaluated to confirm identity and potency. Results : Synthemax II was selected as the optimal microcarrier with over 90% of cell attachment and maximal cell yield after 5 days of growth in the bioreactor. Optimized cell seeding density and microcarrier concentration generated over 1 × 1010 particles/mL by day 5 of collection. Agitation ramping maintained homogenous suspension during the culture. Over 90% of the generated particles stained positive with lipid bound membrane dye. Samples also showed positivity for the canonical EV identity tetraspanin markers CD81, CD9 and CD63 in western blot. Potency assays demonstrated the biofunctionality of the generated EVs. The developed process was successfully scaled to 15L to generate a total of over 10 × 1014 particles while maintaining CQAs. Summary/Conclusion : This study developed an optimized, reproducible, and scalable xeno‐free microcarrier‐based 3D bioreactor process for MSC‐EV production. Keywords : scale‐up, bioreactor, MSCs

Uncovering

Ella Fouhy 1 ; Neil O'Gorman 2 ; Luisa Weiss 3 ; Kieran Wynne 4 ; Fionnuala Ní Áinle 5 ; Mary Higgins 6 ; Jennifer Donnelly 7 ; Patricia Maguire 3 1 School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland, Ireland; 2 Department of Obstetrics and Maternal Fetal Medicine, Coombe Women & Infants University Hospital, Dublin Ireland., Ireland; 3 School of Biomolecular and Biomedical Science, University College Dublin, Ireland, Dublin, Ireland; 4 Systems Biology Ireland, University College Dublin, Ireland, Dublin 4, Ireland; 5 Mater Misericordiae University Hospital, Rotunda Hospital and School of Medicine UCD, Dublin, Ireland., Ireland; 6 UCD Perinatal Research Centre, School of Medicine, University College Dublin, Ireland; 7 Department of Obstetrics and Gynaecology, Rotunda Hospital and Mater Misericordiae University Hospital, Dublin, Ireland., Ireland Introduction : Pre‐eclampsia (PE) is a serious complication affecting 1 in 12 pregnancies, defined by new‐onset hypertension and proteinuria after 20 weeks of gestation. Annually, PE claims the lives of 76,000 mothers and 500,000 babies, making it the world's deadliest pregnancy complication. Early detection of PE is key for appropriate clinical decision‐making throughout pregnancy to reduce life threatening complications. Extracellular vesicles (EVs) are important circulating messengers regulating a myriad of biological and pathological processes and may be highly relevant to the pathophysiology of PE, with levels shown to increase as gestation progresses. We hypothesise that EVs circulating in early pregnancy contain markers that may potentially predict occurrence of PE. Methods : Ethical approval was gained to access the Biological Resource Bank in the Coombe Women and Infants University Hospital Dublin. Early pregnancy (12‐15 weeks) plasma samples from healthy women (n = 67) and women who subsequently went on to develop a confirmed diagnosis of PE during their pregnancy (n = 27) were randomly selected from the biobank. Circulating EV particle concentration and size was determined using Nanoparticle Tracking Analysis and flow cytometry. For proteomic analysis, EVs were isolated using IZON qEV single columns and subjected to label‐free quantitative proteomics analysis. Results : We observed a trend towards an increase in particles in the size range of 50–200 nm in the plasma of women who went on to develop PE relative to control patients. Comparative proteomic analysis of EVs revealed that proteins originating from platelets were among the proteins significantly altered between the groups, which we confirmed using ELISA. Summary/Conclusion : We have found differential levels of circulating vesicles in plasma of expectant mothers who went on to develop PE. Such anomalies identified at this crucial early stage of pregnancy when the placenta becomes definitive and takes over from the yolk sac, may underpin aberrant placental implantation and future pregnancy complications. Our results may provide clinicians with the much‐needed tools to identify women at future risk of PE. Funding : Science Foundation Ireland. Keywords : early pregnancy, preeclampsia, platelets, placenta, proteomics, NTA, flow cytometry, plasma

Antioxidant

Shynggys Sergazy 1 ; Sanzhar Zhetkenev 2 ; Laura Chulenbayeva 3 ; Mohamad Aljofan 4 ; Alexandr Gulyayev 5 1 National Laboratory Astana, Nazarbayev University, Nur‐Sultan, Kazakhstan; 2 National Laboratory Astana, Astana, Kazakhstan; 3 Nazarbayev University, Nur‐Sultan, Kazakhstan; 4 Nazarbayev University, NUSOM, Nur‐Sultan, Kazakhstan; 5 National Laboratory Astana, Nazarbayev University, Kazakhstan Introduction : Mare's milk is the national drink of the indigenous population of Kazakhstan. Similar to other milk types, mare's milk is a rich source of EVs, which can be loaded with flavonoids such as quercetin that may have some valuable therapeutic properties. In this study, we examine cytoprotective properties of quercetin‐loaded exosomes (ExoQuer) on doxorubicin induced cellular toxicity as well as antioxidant and radical scavenging activity. Methods : EVs obtained 3 different methods: total exosome isolation kit (Invitrogen™), isoelectric precipitation and size exclusion chromatography (Izon™). Radical scavenging activity was measured using the DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) test. ABTS assay kit was used to measure antioxidant activity (Sigma‐Aldrich). Cell viability was determined by MTT assay. HDFn cells were seeded in a 96‐well plate at a density of 105 cells/well and incubated at 37°C for 24 h. Toxicity was induced by doxorubicin and left for 24 h. The cells were then treated with ExoQuer, free quercetin for 24 h. Subsequently, 0.5 mg/mL MTT reagent dissolved in DMEM was added to each well, and the plate was further incubated at 37°C for 3 h. Each well was measured at 570 nm. Results : Antioxidant activity of free quercetin and ExoQuer did not differ significantly, 2.4 and 2.2 TEAC respectively. The DPPH radical scavenging activity (ES50) of free quercetin was 5.5 μM and for ExoQuer it was 5.9 μM. Viability of cells treated with ExoQuer was 71% while cells treated with free quercetin was 64%, compared to control. Summary/Conclusion : Whereas ExoQuer and free quercetin have the same level of antioxidant and radical scavenging activity, ExoQuer has higher cytoprotective activity on the model of doxorubicin‐induced cellular toxicity. Funding : This research was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP13067844) and Nazarbayev University Collaborative Research Program 2021–2023 (Award no. OPCRP2021006).

Application

Lauren A. Newman ; Zivile Useckaite; Andrew Rowland Flinders University, Bedford Park, Australia Introduction : Hepatic metabolism by cytochrome P450 (CYP) 3A4 is a primary determinant of exposure for >50% of drugs. Drug metabolising enzymes, including CYP3A4, have been detected in extracellular vesicles (EVs) isolated from blood. Metabolic associated fatty liver disease (MAFLD) causes steatosis (SS) and inflammatory injury (NASH), and alters CYP expression. Given the major role of CYP3A4 in drug clearance, reduced expression places MAFLD patients at higher risk of toxicity. We aimed to characterise CYP3A4 expression in circulating liver derived EVs to track changes in drug clearance in MAFLD patients. Methods : EVs were isolated from plasma of subjects with SS (n = 10) or NASH (n = 5) and from healthy controls (n = 13) by a two‐step protocol using size exclusion chromatography followed by anti‐ASGR1 immunoprecipitation (IP). EVs morphology and abundance was characterised by TEM and NTA. The abundance of global EV, liver specific EV and co‐isolation markers was quantified using targeted LCMS proteomics. Differences in CYP3A4 expression in ASGR1+ EVs isolated from control, SS and NASH subjects was used to predict changes in drug exposure in patients with MAFLD. Results : Anti‐ASGR1 IP successfully captured plasma EVs of hepatic origin. A significant decreasing trend in CYP3A4 expression was observed in ASGR1+ EVs with increasing MAFLD severity (p = 0.049) and predicted changes in exposure to clinically relevant drugs. Summary/Conclusion : Liver derived EVs accurately reflect the impact of MAFLD on hepatic CYP3A4 expression and represent a valuable liquid biopsy platform to understand clinically relevant changes in drug exposure. Keywords : drug exposure, tissue‐specific isolation

Association

Chih‐Chi Li 1 ; Andrew Man‐Chung Wo 2 ; Yufeng Jane Tseng 1 1 Biomedical Electronics and Bioinformatics. National Taiwan University, Taiwan (Republic of China); 2 Institute of Applied Mechanics, National Taiwan University; Reliance Biosciences, Taiwan (Republic of China) Introduction : Bacterial extracellular vesicles (BEVs) play a crucial role in neurodegenerative diseases, suggesting they may be involved in the gut‐brain axis. However, little research has examined the relationship between gut microbiota, stool‐derived BEVs (stBEVs), and plasma‐derived BEVs (plBEVs). In this study, we aim to isolate and analyze stBEVs from healthy individuals and explore the connection to their gut microbiota and plBEVs. Methods : Four models were employed to optimize the purification process for isolating stBEVs: culture medium from gram‐positive bacteria, gram‐negative bacteria, eukaryotic cells, and human stool specimens. The composite protocol, which included differential centrifugation, filtration, and density gradient centrifugation, yielded high amounts of stBEVs. The isolated stBEVs were characterized using TEM, NTA, BCA assay, WB, and NanoFCM. Functional tests were also performed on gram‐positive and gram‐negative bacteria using endotoxin, PBMC stimulation, and LTR2 receptor assay to ensure successful isolation of stBEVs. DNA was extracted from stBEVs and plBEVs, and PCR was used to target the v3‐v4 and full‐length regions of the 16s rRNA gene for sequencing. Electrophoresis confirmed the success of the PCR amplification. PacBio sequencer or NGS platform Illumina MiSeq was used to sequence DNA from stools, stBEVs, and plBEVs. Results : Our analysis revealed that stBEVs in density gradient fractions F5&6 contain exosomal markers (CD9, syntenin‐1), while F8&9 contain bacterial markers (OmpA, LTA). Notably, Flotillin and TGS101 were present not only in eukaryotic cell‐derived EVs, but also in bacterial EVs. PCR results indicated that plBEVs were able to amplify the v3‐v4 region, but not the full‐length region, suggesting that the plBEVs may no longer have an intact 16s rRNA sequence. Metagenomic analysis results showed a correlation between the relative species abundance of stBEVs and gut microbiota at the phylum level. Summary/Conclusion : Eukaryotic‐EVs and Bacteria‐EVs from stool were distinctly separated. A comparison of stBEV DNA and plBEV DNA revealed that plBEV DNA had become fragmented. Sequencing results suggest that BEVs may act as a means of transporting information from the microbiota. Funding : This work was supported in part by funding from National Taiwan University's Advanced Education Deep Cultivation Plan ‐ Core Research Group Plan (grant agreement: ntu‐cc‐111L890203).

Biochemical

Alice Gualerzi 1 ; Silvia Picciolini 1 ; Valentina Mangolini 2 ; Francesca Rodà 3 ; Angela Del Prete 4 ; Luana Forleo 4 ; Marzia Bedoni 1 1 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milan, Italy; 2 (1) IRCCS Fondazione Don Carlo Gnocchi ONLUS, (2) Università degli Studi di Brescia, Milan, Italy; 3 (1) IRCCS Fondazione Don Carlo Gnocchi ONLUS, (2) University of Modena and Reggio Emilia, Milan, Italy; 4 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Italy Introduction : Saliva is an interesting complex and easily available liquid biopsy and its use in diagnostics is fast increasing as well as the identification of specific salivary biomarkers for several disorders, spanning from neurodegenerative to cancer diseases. Extracellular Vesicles (EVs) are known to be present in saliva, although their low concentration has limited their use in clinics despite the remarkable potentialities. In the present study, we compare the biochemical profile of salivary and blood‐derived vesicles to investigate the use of saliva as a valuable source of EVs that could be studied as biomarkers in an easily accessible biofluid. Methods : EVs were isolated from serum and saliva of 5 healthy volunteers with the same combined protocol that includes 2 steps: size exclusion chromatography and ultracentrifugation. Nanoparticle tracking analysis, western blot and CONAN test were performed for the physico chemical characterization of the obtained EV preparation. Later on, samples were used for the biochemical profiling using a Raman microspectrometer equipped with 532 nm laser. Results : Using a comparable protocol for the isolation of EVs from both liquid biopsies, salivary EVs showed greater purity in terms of co‐isolates (evaluated by nanoparticle tracking analysis and CONAN test). Besides, the use of Raman spectroscopy was not hampered by the limited yield of EVs isolated from saliva, on the contrary we were able to provide a comprehensive characterization of EVs, in a high throughput and repeatable manner. The obtained molecular fingerprint together with the calculated protein‐to‐lipid and nucleic acid‐to‐protein ratios allowed us to hypothesize differences in the content of vesicles. Summary/Conclusion : Raman spectroscopy can represent a turning point in the application of salivary EVs in clinics, taking advantage of the simple method of collection of the liquid biopsy and of the quick, sensitive and label‐free biophotonics‐based approach. Funding : This research was funded by the Italian Ministry of Health, Ricerca Corrente 2020–2022. Keywords : raman spectroscopy, saliva, biophotonics, molecular characterization, biomarkers, liquid biopsies

Bottom‐Up

Meline Macher 1 ; Rahul Rimal 2 ; Smriti Singh 3 ; Rinho Kim 4 ; Assa Yeroslaviz 4 ; Nils Chapin 5 ; Hannah Kempf 5 ; Eva Bertosin 6 ; Alessandro Strada 7 ; Ilia Platzman 8 ; Joachim Pius Spatz 5 1 Max Planck Institute for Medical Research (MPI MR) Heidelberg, Heidelberg, Germany; 2 MPI MR Heidelberg, Germany; 3 MPI MR Heidelberg / DWI Aachen, USA; 4 MPI BC Munich, USA; 5 MPI MR Heidelberg, USA; 6 Biomolecular Nanotechnology TU Munich, USA; 7 Istituto di Scienze e Tecnologie Chimiche Milan, USA; 8 MPI MR Heidelberg, Heidelberg, USA Introduction : The pro‐regenerative and immunomodulatory effects of human mesenchymal stem cell (hMSC) EVs are promising for applications such as therapy of atopic dermatitis (AD), an inflammatory skin disease. However, despite large potential, their conventional enrichment from culture medium hampers translation, as EV biogenesis is difficult to control, leading to high EV heterogeneity and challenges in production and mechanistic understanding. Thus, we apply bottom‐up synthetic biology to produce fully synthetic EVs (synEVs) mimicking hMSC EVs as potential therapeutic for atopic dermatitis. Methods : We have isolated EVs from adipose hMSCs by differential ultracentrifugation and characterized them by NTA, dynamic light scattering, cryo‐TEM, MACSPlex, Western blot and miRNA sequencing. Based on this and a previously published lipid composition, we have assembled synEVs. We assessed their functions in human organotypic AD models in vitro. These are very physiologically relevant full‐thickness skin models at an air‐liquid‐interface. Results : We show we can build synEVs mimicking MSC EV diameter, surface charge, lipid composition and certain protein and miRNA contents, while allowing more efficient production. The tested synEVs show great effects in AD models, restoring the physiological skin architecture after only 6 treatment days. This was achieved by direct effects on skin cells, without immune cell contribution. To clarify the mechanisms, we are now performing transcriptomics and expect to identify key pathways whose expression is changing between the healthy, atopic dermatitis and synEV‐treated condition until the conference. Summary/Conclusion : In this work we have developed synEVs with therapeutic functions based on hASC EV analysis to help overcome challenges of clinical translation. Moreover, synEVs allow probing the function of individual EV components. Regarding atopic dermatitis, we may have identified a potential new treatment strategy, which could complement the currently limited options. Funding : Federal Ministry of Education and Research of Germany, Grant Agreement no. 13XP5073A, PolyAntiBak, MaxSynBio Consortium, which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society, Volkswagen Stiftung (priority call ‘Life?’), German Science Foundation SFB 1129, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy via the Excellence Cluster 3D Matter Made to Order (EXC‐2082/1 – 390761711), Gottfried Wilhelm Leibniz Award (funding for J.P.S.), Max Planck School Matter to Life. Keywords : hASC EVs, mesenchymal stem cells, synthetic EVs, EV engineering, atopic dermatitis, skin disease, synthetic biology, biomimetic vesicles

Cd39‐Rich

Tomasz Brzoska 1 ; Elizaveta V. Menchikova 2 ; Tomasz W. Kaminski 2 ; Omika Katoch 2 ; Rikesh K. Dubey 2 ; Stevan P. Tofovic 2 ; Edwin K. Jackson 2 ; Mark T. Gladwin 3 ; Prithu Sundd 2 1 University of Pittsburgh, Pittsburgh, USA; 2 University of Pittsburgh, USA; 3 University of Maryland, USA Introduction : Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. CD39 maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP‐dependent platelet activation, its role in the pathophysiology of SCD is still unidentified. Methods : To evaluate SCD platelet response to ADP in vivo we used a state‐of‐the‐art intravital lung microscopy and a novel in vivo model of ADP‐triggered thrombocytopenia in transgenic humanized SCD mice. Additionally, both mouse and human SCD platelet ADP‐dependent aggregation was examined using in vitro turbidimetric aggregation assay. Hemin, a major host‐derived damage associated molecular pattern associated with SCD, was incubated with Human Lung Micro‐Vascular Endothelial Cells (HMVEC‐L). Extracellular vesicles (EVs) were obtained from mouse and human plasma samples, and cell culture medium samples using size exclusion chromatography. Obtained EVs were subjected to nanoparticle tracking analysis. EVs CD39 levels and activity were determined using ELISA, western blot and malachite green phosphate assays, respectively. Results : Intravital lung microscopy and in vivo thrombocytopenia studies revealed that intravascular administration of ADP triggered acute pulmonary thrombosis in control but not in SCD mice. In vitro aggregation study confirmed our in vivo findings and demonstrated impaired SCD mouse and human platelet response to ADP, which was, further, significantly augmented by a specific CD39 inhibitor. Hemin triggered shedding of CD39‐bearing EVs by HMVEC‐L. Indeed, we discovered that isolated from mouse and human plasma endothelial cell‐derived SCD EVs expressed higher CD39 levels and activity in comparison to control EVs. Summary/Conclusion : Our findings suggest that CD39‐bearing EVs prevent ADP‐mediated platelet aggregation and pulmonary thrombosis in SCD. Current study explains why P2Y12 blockers are not effective in SCD therapy. Funding : NIH‐NHLBI 1R01HL128297‐01, American Heart Association 18TPA34170588, Vascular Medicine Institute startup funds, American Society for Hematology Scholar Award, American Society for Hematology Research Restart Award, Pittsburgh Heart, Lung and Blood Vascular Medicine Institute Pilot Project Program in Hemostasis and Vascular Biology (P3HVB). Keywords : sickle cell disease, extracellular vesicles, platelets, purinergic signaling, CD39

Circulating

Kentaro Jingushi 1 ; Atsunari Kawashima 2 ; Takuro Saito 3 ; Akinaru Yamamoto 2 ; Toshihiro Uemura 2 ; Yu Ishizuya 4 ; Yoshiyuki Yamamoto 4 ; Sassi Nesrine 4 ; Norio Nonomura 2 ; Kazutake Tsujikawa 1 1 Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan; 2 Department of Urology, Graduate School of Medicine, Osaka University, Suita, Japan; 3 Department of Surgery, Graduate School of Medicine, Osaka University, Suita, Japan; 4 Department of Urology, Graduate School of Medicine, Osaka University, Japan Introduction : Reportedly, bacterial information (16S rRNA gene) exists in the blood; however, the mode and source of this bacterial information remains unclear. Therefore, in this study, we aimed to determine the source of bacteria information present in the blood by analyzing human cell‐derived EVs (hEVs) recovered from the serum. Methods : Clinical specimens: Clinical specimens were collected from healthy individuals at Osaka University Hospital. Written informed consent was obtained from each patient, and the study was approved by the Ethics Review Board of the Osaka University Hospital. Serum EVs isolation: Serum EVs were isolated using different methods following the manufacturer's instructions: ultracentrifugation, qEV columns, Exosome Isolation Kit, and MagCapture Exosome Isolation Kit. Subsequently, EVs were treated with DNase I. Fecal samples: The fecal swab‐immersed medium was centrifuged at 2,000 × g, for 30 min, and the supernatants were filtered through a 0.2 μm syringe filter and subjected to ultracentrifugation for recovery of fecal EVs. hEVs isolation from serum: Biotinylated mAb (anti‐CD9 mAb, anti‐CD81 mAb) were incubated with streptavidin magnetic beads. Subsequently, serum filtered with 0.2 μm syringe filter was co‐incubated with mAb‐coated magnetic beads. The bead‐EV complexes were collected using a magnet and subjected to DNA isolation, western blot, and TEM analysis. 16S metagenomic sequencing: V1‐V2 region was amplified by PCR and 16S rRNA metagenomic sequencing was performed on a MiSeq platform. Results : The 16S rRNA gene was detected in serum EVs by various recovery methods. A comparison of the bacterial composition in serum EVs, feces, and fecal EVs revealed that the 16s rRNA gene in serum EVs was similar to that in feces but not to that in fecal EVs. hEVs recovered from the serum revealed the presence of 16S rRNA gene; Firmicutes, Actinobacteria, and Proteobacteria predominantly contributed to the bacterial genomic DNA population in hEVs. Furthermore, bacterial genomic DNA was found in phagocytosed macrophage‐released EVs, suggesting these EVs to be a bacterial source in the blood. Summary/Conclusion : Circulating hEVs harbor bacterial genomic DNA. Keywords : circulating EVs, blood, bacterial genomic DNA, 16S rRNA gene

Comparative

Meenakshi Mendiratta 1 ; Sujata Mohanty 2 1 All India Institute of Medical Sciences, Delhi, India; 2 AIIMS New Delhi, Delhi, India Introduction : Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have gained popularity in regenerative medicine due to their ability to differentiate, immunomodulatory capabilities, and secretion of paracrine mediators such as small extracellular Vesicles (sEVs). These sEVs carry miRNA and mRNA, proteins, and other cargo that help heal damaged or diseased tissues and organs. Several studies have shown the potential role of small EVs cargo, especially the role of miRNAs in hepatoprotection and hepatocyte regeneration. Naive sEVs face some of the biggest problems, such as repeatability, scalability, limited cargo encapsulation, and target specificity. Surface modification techniques can be employed to enhance the homing of these sEVs which improves the target specificity of these small extracellular vesicles. The current prospective study aims to develop an EV‐based treatment method by developing Bioengineered small Extracellular Vesicles (BioEn‐EVs) and assessing their potential applicability in liver disease with a specific focus on hepatic cells. Methods : Human MSCs were isolated from Wharton's Jelly with due consent forms and characterized according to the International Society of Cellular Therapy (ISCT) guidelines. MSCs were cultured in serum‐free media for the isolation of small Extracellular Vesicles (sEVs). sEVs were characterized for size (NTA), morphology (TEM), and surface and cytoplasmic marker profiling (Western blotting). Bioengineering of the surface of sEVs with hepato‐specific ligands as well as cargo of sEVs with selective miRNA exhibiting hepatoprotective activity was done. BioEn‐EVs were characterized via NTA, TEM, Western blotting, zeta potential, and their cargo content was validated by qRT‐PCR. The hepatoprotective and antioxidant effect of naïve and BioEn‐EVs was assessed in vitro. Results : The aggregation of BioEn‐EVs was observed with a TEM as compared to naïve sEVs. The diameter and zeta potential of BioEn‐EVs were increased and became positive as compared to naïve sEVs. The cellular uptake efficiency of BioEn‐EVs were significantly greater extent than naïve sEVs. It was observed that there was a significant ROS scavenging potential and hepato‐regenerative potential of BioEn‐EVs. Summary/Conclusion : It is concluded that the Bioengineering of MSC‐derived sEVs enhanced their ability to efficiently deliver its content into hepatic cells under stress/injured hepatic cells. Thus, BioEn‐EVs are cell‐free, promising, and customizable translational approaches to improve the outcomes of patients with liver disease.

Comparisons

Derya Sağraç 1 ; Oğuz K. Kırbaş 2 ; Pakize N. Taşlı 2 ; Bahar S. Özdemir 3 ; Fikrettin Şahin 2 1 Yeditepe University, Atasehir, Turkey; 2 Yeditepe University, Istanbul, Turkey; 3 Yeditepe University, Turkey Introduction : Cells secrete differential‐sized vesicles called “Extracellular vesicles ” to communicate with neighbor cells and the environment. It has been extensively revealed that EVs especially small EVs exhibit cellular identity in protein, lipid, and nucleic acid manner. Extracellular vesicles derived from various plant sources (PDEVs) has been started in the late 60s with carrot culture. Although extracellular vesicles derived from mammalian cells (MDEVs) have a higher rate of EV studies than PDEVs, there is increasing interest in PDEVs. Nowadays the isolation of PDEV from a wide variety of plant sources has been studied. Isolation of PDEVs from a plant cell suspension system instead market‐derived products provides more accurate results in both characterization and functionality experiments. In this comparison study, PDEVs were isolated from Vitis vinifera suspension medium (VVSM) and MDEVs from Neural Progenitor Cells (NPCs). Methods : MDEVs were isolated from the collected, serum‐free medium of NPCs by mixing ultracentrifugation and ultrafiltration protocols. On the other hand, PDEVs were isolated from VVSM by using ultrafiltration. Nanoparticle Tracking Analysis (NTA) was used to quantify EVs and determine their size distribution. The atomic force microscopy technique was preferred to exhibit EVs in nanoscale imaging. LC/MS proteomic analysis was used to identify EV cargo proteins. Fatty acid profiles (FAME) of PDEVs and MDEVs were investigated by GC‐MS. The peaks were identified by the Sherlock software. Differences between MDEVs and PDEVs were analyzed by the Student t‐test and P< 0,05 was considered statistically significant. Results : The size of PDEVs was close to 150 nm while the size of MDEVs was 100 nm. AFM images verified NTA results. AFM Images of MDEVs showed that the mean size of MDEVs was 180 ± 45 nm. Moreover, PDEVs showed a similar size to NTA which is 150 ± 20 nm. LC/MS proteomic analysis showed that origin cell features are mostly passed to the EVs. Furthermore, FAME profiling determined that PDEVs had more saturated fatty acids (45%) than MDEVs (15% and 39%). The mono‐unsaturated fatty acid content of MDEVs (59% and 27%) was higher than PDEVs (13,3%). Summary/Conclusion : In conclusion, both mammalian and plant suspension cell‐derived EVs had similar EV sizes and quantities. However, their membrane‐bound fatty acid pattern was different. This difference gives EVs stability and membrane rigidity and will open a new scientific window that intensively needs to be investigated. Funding : This study is supported by TUBITAK (2211/C) and Yeditepe University. Keywords : FAME, PDEVs, MDEVs

Controlling

Hans Radhoe employee, Delft, Netherlands Introduction : The characterization of liposomes/polymersomes typically involves a combinatorial approach. Resolving the details with nanometer resolution usually requires one of three TEM techniques: staining, freeze−fracture or cryoTEM. Staining is a fast process but it can alter the sample structure and composition. The freeze fracture processing preserves the sample and allows for the analysis of fine structural details, but is time‐intensive and can result in morphological changes and sample fusion. Cryo‐TEM is a direct imaging and enables high resolution but the liposomes are removed from the liquid environment making a dynamic and functional analysis of the structures impossible. As a consequence not only the systematic errors are introduced but the process prevents the possibility of enabling high‐throughput studies. Methods : We introduce our technology for in‐situ Liquid Phase Transmission Electron Microscope (TEM) studies which is an add on and a plug‐and‐play system. The latter relies on a Microfluidic Lab on a Chip device as a smart sample carrier the Nano Cell which allows to flow the biological sample in its native liquid environment inside the TEM. The Microfluidic Lab on a Chip device contains an integrated set of biasing electrodes or an integrated microheater i.e. to perform liquid heating experiments. Furthermore the Nano Cell contains an integrated microheater and/or electrodes. This enables comprehensive in‐situ studies on liposomes/polymersomes and pharmaceutical research as a function of temperature/biasing/pressure/flow rate. Results : As a result the user can study the structure/size/morphology/composition and visualize real‐time dynamics with high resolution. As a reference we have reported single particle analysis of proteins with 4.5 angstrom resolution while being in liquid The different stimuli e.g. temperature/pressure/flow/pH and the mass transport are controlled simultaneously enabling the user to optimize the biochemistry on the fly. We have shown that these capabilities allow to visualize in‐situ the synthesis of liposomes/polymersomes. Furthermore the user can visualize the exact moment when a cargo molecule gets encapsulated by the liposome providing a unique opportunity to optimize the drug delivery vehicle before using it for e.g. cancer immunotherapy purposes. Moreover the system enables HT studies. Summary/Conclusion : Our development provides the unique possibility to visualize biological processes in real time with high resolution, while the bio sample is in its native environment (as a function of different stimuli). This opens up several possibilities for pharmaceutical applications of liposomes/polymersomes and other EVs e.g. disease biomarkers drug delivery vehicles or vaccines and fundamental studies in biophysics and biochemistry. We believe that this technique will become a very powerful complementary technique to cryo EM and other techniques conventionally used within EVs research.

Deformation

Premanshu K. Singh 1 ; Patricia Sarchet 2 ; Raphael E. Pollock 2 ; Shaurya Prakash 1 1 Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, Ohio, USA, Columbus, USA; 2 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA, Columbus, USA Introduction : Biomechanical analysis of single extracellular vesicles (EVs) can provide label‐free insights to morphological structure, biomarkers, and potential targets for therapeutics. However, reliable and quantitative measurements on the mechanical properties for single EVs are scarce as these are compliant, nanoscale particles with limited tools for mechanical property measurements. Conventional methods use nano‐indentation with instruments such as an atomic force microscopy, which lack high‐throughput and are limited to a small number of EVs. EV isolation and separation methods use mechanical forces on the entire EV in centrifugation and filtration. Here, we report on a flow‐based method complemented by transmission electron microscopy (TEM) imaging to quantify the change in the shape of EVs due to the mechanical forces acting on the entire EV. We show that the well‐established thin shell theory can be used to quantify the EV deformation and provide an estimate of the elastic modulus. Methods : Cells from Human liposarcoma (LPS) cell line Lipo246 were cultured in serum‐free media for 48 h for EV production and LPS cell‐conditioned media (LCCM) collection. LCCM was centrifuged at 2000g for 20 mins to remove cellular debris and the supernatant was microfiltered with a PVDF membrane filter (pore size 220 nm). Nanotracking analysis and TEM characterized EVs in pre‐ and post‐filtration LCCM. TEM image analysis with ImageJ quantified EV shape and size for calculating EV deformation due to microfiltration. Thin shell theory was used to calculate elastic modulus (E). Results : TEM images (N∼ 400) of microfiltered LCCM confirmed particles in 10–953 nm range as observed by NTA.The E of EVs yielded E = 0.89 ± 0.09 MPa (mean ± SE) for small EVs (sEVs; 30–150 nm) and E = 0.95 ± 0.13 MPa (mean ± SE) for large EVs (lEVs; >150 nm). E for individual EVs shows that the sEVs E ranges from 0.1‐10 MPa while lEVs range from 0.5‐2 MPa. Summary/Conclusion : This is the first report on the mechanical property estimation of LPS‐derived EVs. This study uses a flow‐based methodology to provide a high throughput, whole EV deformation analysis method for estimating statistically significant mechanical properties as function of EV size. Funding : This work was supported by the National Institutes of Health under grant number DOD CDMRP Grant CA210874. Keywords : liposarcoma, extracellular vesicles, elasticity modulus, TEM

Development

leila darzi 1 ; Mehdi Forouzandeh Moghadam 1 ; Mehdi shamsara 2 1 Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat modares University, Tehran, Iran, Tehran, Iran; 2 National Research Center for Transgenic Mouse, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran, Iran Introduction : Finding a safe gene delivery system is crucial for gene therapy in a broad range of diseases. Exosomes as natural nanocarrier are considered as ideal delivery platforms for gene therapy that can efficiently and selectively deliver their cargo to the target cells. The significant potential of exosomes for being investigated and applied for gene therapy purposes is mainly derived from their high level of biocompatibility and low level of immunogenicity. The potential of using exosome for delivery of functional DNA to target cell should be further investigated. Methods : HEK293T was engineered to express DARPin against HER2 and targeted exosomes were isolated from modified HEK293T using Exo‐spin kit. Purified exosomes were characterized by TEM, zeta sizer and exosomal CD markers. Purified pEGFP‐C1 plasmid was introduced to DARPin/Exos using electroporation and termed to pEGFP‐C1/Exos. PKH26‐labeled pEGFP‐C1/Exos were exposed to HER2‐positive SKBR3 cell line and exosome uptake was validated by florescent microscopy and GFP expression was analyzed using real‐time PCR, florescent microscopy and flow cytometry. Effect of increase in exosome quantity and GFP expression was measured. Results : In this study, we demonstrated delivery of GFP‐encoding plasmid to target cells by exosomes the quantity of pEGFP‐C1 plasmid in pEGFP‐C1/Exos was measured through Real‐time PCR. Transferring of pEGFP‐C1/Exos to the target cells and GPF expression in mRNA and protein level proved the sufficient delivery of the plasmids. PKH26 labeled‐pEGFP‐C1/Exos were uptaken by SKBR3 and expressed GFP. Increase in pEGFP‐C1/Exos quantity caused more GFP expression in target cells. Summary/Conclusion : Exosome as natural, nontoxic, and non‐immunogenic carriers have potential for using in gene delivery system in vitro. In this study, through using proper electroporation protocol the 4 kbp GFP‐encoding plasmid, pEGFP‐C1were loaded into HER2‐targeted exosomes successfully and delivered them to the target cells functionally. Based on these results, targeted exosomes may be promising vehicle for cancer therapeutics in the future. Broader assessments of this method using various types of cargoes and target cells can highlight the findings and guarantee their in‐clinic applicability for gene delivery and gene therapy purposes. Keywords : HER2, exosome, gene delivery, gene therapy, breast cancer

Elucidating

Javier Ramirez Ricardo 1 ; Ariana von Lersner 2 ; Bong Hwan Sung 2 ; Dolores Di Vizio 3 ; Andries Zijlstra 4 ; Alissa M. Weaver 2 ; Heather H. Pua 5 1 Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville TN, USA, Nashille, USA; 2 Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN, USA, Nashville, USA; 3 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles CA, USA, Los Angeles, USA; 4 Department of Research Pathology, Genentech, South San Francisco CA, USA, USA; 5 Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville TN, USA, Nashville, USA Introduction : Activated Leukocyte Cell Adhesion Molecule (ALCAM) is a widely expressed transmembrane adhesion protein that regulates cell motility and metastasis, especially in bladder cancer. Given the important roles of extracellular vesicles (EVs) in tumorigenesis, we asked whether ALCAM might also impact EV biogenesis or function. Methods : To study the effects of ALCAM on cancer cells, we generated ALCAM‐deficient HT1080 fibrosarcoma and UMUC3 bladder tumor cell lines using CRISPR‐Cas9. EVs were isolated from control and ALCAM knockout cell culture supernatants by serial ultracentrifugation to generate 10,000g and 100,000g pellets. Nanoparticle tracking (ZetaView), western blots, and single EV flow cytometry with dimensional reduction analysis after staining with di‐8‐ANEPPS were performed. Results : Although no differences were observed in EV counts between HT1080 WT and ALCAM deficient cells, we observed an increase in EV counts by nanoparticle tracking in both the 10,000g and 100,000g pellets of UMUC3 ALCAM‐deficient bladder tumor cells compared with controls. By western blot, we observed reduced amounts of CD9 and Syntenin, with relatively preserved levels of Alix and TSG101 in the lysates of 100,000g pellets of UMUC3 ALCAM‐deficient cells. Flow cytometry identified differences in EV subpopulations between control and ALCAM‐deficient cell lines. Summary/Conclusion : This work identifies changes in EV number and protein cargos in a bladder tumor cell line in the absence of ALCAM expression. This finding suggests that ALCAM may regulate EV biogenesis and/or cargo loading in cancer cells. Funding : NIH R01CA249424 (HHP).

Endocytosis

Thach Tuan Pham 1 ; Anh Le 2 ; Cong Dang 2 ; Vinh Do 2 ; Boya Peng 2 ; Hong Boon Ong 3 ; Chen Huan 2 ; Han Wei Hou 4 ; Minh T.N Le 1 1 National University of Singapore, Singapore, Singapore; 2 National University of Singapore, Singapore; 3 School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, Singapore, Singapore; 4 School of Mechanical and Aerospace Engineering, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore, Singapore Introduction : Red blood cell‐derived extracellular vesicles (RBCEVs) display excellent characteristics for an EV‐based drug delivery platform. Many studies have shown efficient delivery of therapeutic cargo using RBCEVs to treat cancer and other diseases in mouse models, paving a way for the application of RBCEVs in clinics. However, the biological functions of RBCEV's endogenous components are little known. In this study, we investigate the biodistribution of RBCEVs, study how RBCEVs are taken up, and track the fate of haemoglobin(the major RBCEV cargo) and their potential therapeutic effects. Methods : Red blood cells were induced with calcium ionophore to release RBCEVs. After removal of RBCs and debris, RBCEVs were obtained from supernatant by ultracentrifugation (50 000 xg for 70 min at 4oC) and 60% sucrose cushion (50 000 xg for 16 h at 4oC). The concentrations were measured using a haemoglobin quantification kit. Size and number of RBCEVs were measure by nanoparticle tracking analysis. RBCEVs markers were checked using CD235a, BAND3 and ALIX. Results : We dissected the biodistribution of RBCEVs at the cellular level and found F4/80+ macrophages took up the majority of RBCEVs in livers, while in the spleen, RBCEVs ended up mainly in CD169+ macrophages at the marginal zone. The uptake was strongly mediated by the phosphatidylserine on the RBCEVs. Upon endocytosis, RBCEVs were trafficked to late endosomal and lysosomal compartments, and the proteins were broken down rapidly within 2 hours. Incubating RBCEVs with human peripheral blood mononuclear cell‐derived macrophages led to the differentiation of macrophages into a Mheme‐like phenotype. The macrophages showed reduced CD86 expression, increased the expression of CD163, a marker of Mheme and M2. They also exhibited anti‐inflammatory effects indicated by reduced secretion of the TNF‐alpha cytokine after lipopolysaccharide (LPS) stimulation. In addition, RBCEV‐induced macrophages upregulated heme oxigenase 1 (HO‐1) and a cholesterol efflux protein, ABCG1. Mechanistically, the heme transporter HRG‐1 mediated the release of heme into the cytoplasm, which eventually upregulated HO‐1 and induced the Mheme‐like phenotype in macrophages. Finally, we found that RBCEV treatment prevented the accumulation of oxidized low density lipoprotein in macrophages in vitro and on the artery‐wall‐on‐a‐chip model, suggesting an anti‐atherosclerosis effects. Summary/Conclusion : RBCEVs robustly taken up by macrophages in vivo and in vitro. Incubating RBCEVs with macrophages induced them into Mheme‐like phenotype which has anti‐inflammatory and anti‐atherosclerosis effect. Funding : This project is supported by the Singapore Ministry of Education (MOE‐T2EP30121‐0016), Ministry of Health (MOH‐000643), and the National University of Singapore (NUHSRO/2019/076/STARTUP/02).

Endothelial

Bin Gong Pathology Department, UTMB, Galveston, USA Introduction : Rickettsioses are devastating human infections3 that are caused by obligately intracellular bacteria of the genus Rickettsia (R). R are transmitted to human hosts by the bite of an infected tick. Microvascular endothelial cells (MECs) are the primary targets of infection, and edema resulting from EC barrier dysfunction occurs in the brain and lungs in most lethal cases in humans, but the underlying mechanisms remain unclear. The aim of the study is to explore the potential role of Rickettsia (R)‐infected, EC‐derived exosomes (Exos) during infection. Methods : Using size‐exclusion chromatography (SEC), we purified Exos from conditioned, filtered, bacteria‐free media collected from R‐infected human dermal MEC (DMECs) (R‐ECExos) and plasma of R‐infected mice (R‐plsExos). Results : We found that both R‐ECExos and R‐plsExos induced disruption of both tight junction TJs and adherens junctions AJs, two elemental components of the EC paracellular barrier, and barrier dysfunction in normal recipient brain microvascular ECs (BMECs), depending on exosomal RNA cargos1,2. Deep‐seq and stem‐loopPCR showed microvasculopathy‐related (MVP) microRNAs are selectively enriched in human R‐ECExos. In separate studies using a traditional in vitro model and a novel single living‐cell biomechanical assay, we demonstrated that ECExos have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase‐abundant environment; miR23a anti‐sense oligonucleotide‐enriched ECExos ameliorate R‐ECExo‐provoked recipient BMEC dysfunction in association with stabilization of ZO‐1 in a dose‐dependent manner. Summary/Conclusion : These results suggest that Exo‐based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation. Funding : This work was supported by NIH grant R01AI121012 (BG), R21AI137785 (BG), R21AI154211(BG), R03AI142406 (BG), R21AI144328 (BG) and John Sealy Distinguished Chair in Alzheimer's diseases (XF). Keywords : exosome, extracellular vesicle, endothelial cell, endothelial barrier function, spotted fever group rickettsial infection, intracellular bacterium, fluidic AFM, single living cell study

Engineering

Prerak Gupta 1 ; Koushik Debnath 1 ; Gina Chung 2 ; Jae‐Won Shin 1 1 Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL 60612, USA, Chicago, USA; 2 Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL 60612, USA, USA Introduction : Extracellular vesicles (EVs) are lipid membrane‐bound nanoscale mediators known to present various surface ligands and activate cell membrane receptors over distance. Despite a number of existing strategies, engineering EVs to present full‐length surface proteins has not been straightforward. Here, we developed a facile platform to rapidly functionalize surface proteins with correct orientation. We demonstrate the efficacy of this approach by activating the Notch pathway to restore endothelial barrier function upon acute lung injury. Methods : EVs were obtained from mouse D1 MSCs following EV‐TRACK ID: EV150007 . We tethered lipid conjugated Fc binding peptides into the membrane of EVs or synthetic liposomes. We used Fc‐FITC protein (∼50 kDa) to confirm Fc‐fusion protein functionalization. We introduced the recombinant Notch ligand Jagged1 (JAG1)‐Fc (∼150 kDa) to EVs or liposomes, and titrated the number of JAG1 molecules per EV by ELISA. We tested the efficacy of JAG1 functionalized EVs or liposomes by delivering them into mice 4 h after injection of lipopolysaccharide (LPS), followed by evaluating edema (wet/dry ratio) and vascular permeability (Evans blue albumin) after 24 h of treatment. Results : The results show a dose‐dependent membrane tethering of Fc‐FITC proteins into EVs (EC50 ∼ 1 mM). Strikingly, introducing ∼6 JAG1‐Fc molecules per EV is sufficient for EVs (1.5 × 108 per 20g) to treat edema and vascular hyperpermeability after LPS injury in mice, while unmodified EVs do not show the therapeutic effect at the same dose. In contrast, JAG1‐liposomes were not able to restore edema or vascular permeability even at a higher dose (1.5 × 109 per 20g). Summary/Conclusion : Our results show that EV‐mediated, but not liposome‐mediated presentation of JAG1 can treat LPS‐induced lung injury. Future studies will test the notion that EVs are biophysically optimized for surface ligand presentation to activate the Notch pathway. This platform is general and can be broadly used to activate membrane receptors over distance. Funding : This work was supported by National Institutes of Health Grant R01‐ HL141255 (to J.‐W.S.). Keywords : extracellular vesicles, ligand presentation, notch, vascular permeability

Enhancement

Alexander Rojas 1 ; Rabindra Tirouvanziam 2 ; Brian S. Dobosh 3 1 Emory University, Hampton Bays, USA; 2 Emory Unviersity, Atlanta, USA; 3 Emory University, Atlanta, USA Introduction : One of the challenges of actualizing the use of RNA therapeutics is the lack of versatile delivery vehicles that are also non‐immunogenic. Engineered EVs could have lower immunogenicity since they are generated by host cells. Unfortunately, methods to load an RNA of interest (ROI) into EVs are not as well explored and a “good‐enough” approach is taken wherein an ROI is overexpressed whilst alternative approaches are not tested. This leaves a hole in the therapeutic EV landscape where better cargo‐loading methods remain uninvestigated. Sometimes, EV “zipcodes” or motifs recognized by RNA‐binding proteins (RBPs) fused to EV‐localized proteins are included. Currently, the best method to load mRNA into EVs is to fuse the archaea RBP, L7Ae, to CD63 and have the cognate C/Dbox motif contained in the 3’ UTR or end of the ROI. Methods : Here, we use the designer EV toolkit (DEVKit), a modular cloning toolkit developed in our lab, to test and combinatorically clone endogenous RNA zipcodes and benchmark them against CD63‐L7Ae + the ROI with the 3’ C/Dbox. Stable H441 epithelial cell lines, and HEK293T cells for orthogonal validation, were created and EVs were purified by differential ultracentrifugation. In concordance with MISEV2018, structure, size, concentration, contents and contamination were evaluated by transmission electron microscopy, nanoparticle tracking analysis, western blotting, and qRT‐PCR. Results : We consistently found four motifs that promote at least an 8‐fold increase in the loading of EGFP mRNA into EVs compared to the no‐motif control. Importantly, this is a one‐plasmid system. CD63‐L7Ae is a two plasmid or two‐component system which can be severely hampered when using difficult‐to‐engineer cells. Summary/Conclusion : This is an endogenous RNA delivery system that can be customized to deliver any mRNA with broad applicability to a variety of diseases. EV‐based RNA platforms can be further enhanced by combining other EV‐engineering methods such as modified targeting and lysosomal escape. Funding : Emory I3 Program. Keywords : RNA therapeutics, cargo loading, designer EVs

Exploration

Ruben R. Lopez 1 ; Prisca Bustamante 1 ; Vahe Nerguizian 2 ; Julia Burnier 3 1 McGill University, Verdun, Canada; 2 École de technologie supérieure ÉTS, Montreal, Canada; 3 Gerald Bronfman Department of Oncology, and Department of Pathology Faculty of Medicine and Health Sciences, McGill University, Montreal, Canada Introduction : Tumour‐derived extracellular vesicles (TEVs) play an important role in cell‐to‐cell communication; they transfer biomolecules that contribute to malignant transformation, preparation of the pre‐metastatic niche and are essential in TEV organotropism. Transmembrane proteins such as integrins ITGB5 and ITGA6 play a role in cell adhesion, signaling and are particularly implicated in organotropism to the liver and lung. Other transmembrane proteins such as prominin‐1 are used as markers of cancer stem cells (CSC) and are known to be found in colorectal cancer cells. The aim of our study was to determine whether there was EV enrichment for transmembrane proteins implicated in metastasis using two cancer models with organotropism to the liver. Methods : Colorectal (CRC) (HT29) and uveal melanoma (UM) cell lines (MP41, MP46, and OMM 2.5) were cultured until 80% confluency was reached. Then, the cell culture medium was replaced by an EV‐depleted supplemented medium. After 24h, the medium was collected and spun to remove cell debris and apoptotic bodies. Then, the medium was concentrated using 100 kDa Amicon ultrafiltration units. 50 μL of diluted medium per cell line was added on top of a microarray chip coated with capture antibodies (CD9, CD63, and CD81). Fluorescent antibodies CD133 (APC), ITGA6 (Alexa‐488), and ITGB5 (PE) were added according to the manufacturer's protocol and measured in the Exoview 200. Results : EVs derived from UM liver metastasis cell line OMM 2.5 and liver metastatic CRC HT29 cells had significantly higher expression of prominin‐1 (CD133) across the three different capture probes (CD9, CD63, CD81) with 35% and 36% of the total averaged counts compared to MP41 and MP46 cells (6% and 3%). All studied cell lines EVs showed a low % count for ITGB5., while ITGA6 was found in all cell lines EVs (59%‐93% total averaged counts). Summary/Conclusion : Transmembrane proteins that have been implicated in site‐specific metastasis were found in EVs from liver metastatic cells. Interestingly, stem cell marker CD133 was enriched in liver metastatic cell line‐derived EVs. This result has potential implications for the understanding the communication between cancer cells and their microenvironment during organotropic metastasis. Funding : New Frontiers Research Fund. Keywords : transmembrane proteins, liver metastatic, colorectal cancer, uveal melanoma, cancer stem cells, tumour‐derived extracellular vesicles

Fabrication

Sung‐Wook Nam School of Medicine, Kyungpook National University, Daegu, Republic of Korea Introduction : Nanopillar structures have been utilized as a separating or sorting structure for biomolecules. For sorting of EV with the size of 50 to 200 nm, it is needed, a stable fabrication of the nanometer‐sized structures. Methods : We employed semiconductor‐based fabrication methods like electron‐beam lithography (EBL), photolithography, and reactive ion etching (RIE). For the fabrication of nanopillar array, we started with the thin film stack of 100 nm silicon‐oxide (SiO2) hard mask layer and 400 nm low‐pressure chemical vapor deposition (LPCVD) amorphous silicon (a‐Si) layer upon 300 nm thermal oxide layer on 6 inch silicon wafer. By EBL, the array of nanohole patterns were generated, which was transferred to SiO2 hard mask by RIE. For the patterning of microstructure, large scale micropatterns were generated by photolithography, which was transferred to the SiO2 hard mask by RIE. Accordingly, the SiO2 hard mask layer has both EBL and photolithography patterns together, which was transferred to a‐Si layer by the second RIE. On top of the patterned a‐Si layer, tetraethyl orthosilicate (TEOS) oxide and plasma‐enhanced chemical vapor deposition (PECVD) oxide were back‐filled followed by being planarized by chemical‐mechanical polishing (CMP). Vent‐hole structures were generated in the oxide layer. By using the a‐Si layer as sacrificial materials, we released the a‐Si by XeF2 gas phase etching to convert the a‐Si nanochannels to nanofluidic channels. To seal the vent holes, 2 micrometer PECVD oxide was deposited. Results : By designing an optimized jig structure, we introduced EV sample into the nanopillar‐based sorting device. The device has one inlet and five outlets. From HEK293 cell‐lines, EV was isolated by ultracentrifuge process. Nano tracking analysis (NTS) shows 100–200 nm size particles. The HEK293‐driven EV was sorted into five different ports. RNA and protein analysis were under investigation. Summary/Conclusion : We present a method to fabricate nanopillar‐based EV sorting device. HEK293‐drivien EV was sorted into five different sub‐groups. Our method provide an EV sorting process based on physical interactions between EV and nanopillar array under continuous flow. Keywords : EV sorting, semiconductor, HEK293, nanopillar, sacrificial process

Genetically

Hongbo Chen 1 ; Peilin Shi 2 ; cheng fang 3 1 School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat‐sen University, Shenzhen 518107, China, Shenzhen, USA; 2 Sun Yat‐sen University, USA; 3 School of Pharmaceutical Sciences (Shenzhen), Sun Yat‐sen University, USA Introduction : Heart transplantation is the ultimate and effective treatment for end‐stage heart failure or severe coronary heart disease. However, ischemia‐reperfusion injury will inevitably occur in donor hearts during transplantation, which seriously affects the postoperative survival rate of heart transplantation.Plant derived vesicle‐like nanoparticles from most edible plants have been shown to have no detectable toxicity or immunogenicity. In recent years, some studies have proved that PDVLNs participate in the pathophysiological processes of plant cells themselves, and can resist infection by pathogenic microorganisms, and have the function of protecting plant cell tissues. Methods : Western Blotting Cells were lysed with radioimmunoprecipitation assay buffer (RIPA) lysis buffer (Thermo Scientific). Cell lysates and purified membrane vesicles were loaded for 10% SDS‐PAGE. Primary antibodies for GFP, β‐actin,were detected by horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse secondary antibodies with an ECL kit (Protein Tech, China). Quantitative Real‐Time PCR qPCR was performed to determine the expression of genes. Results : In this study, through small RNA sequencing, we found that different sizes vesicles derived from pueraria may have different effects. Secondly, we found that pueraria‐derived vesicles(PNV)have antioxidant stress and anti‐inflammatory effects, which can affect macrophage polarization. To be able to enhance its targeting ability and influence macrophage polarization, we designed a genetically engineered macrophage that overexpresses CSF1(CSF1‐RAW NV). We fused pueraria derived vesicles‐like nanoparicles with genetically engineered macrophage‐derived nanovesicles (Fusion EV) to enhance its ability to target the heart. In vivo, fusion vesicles can reduce I/R injury of transplanted hearts, reduce the expression of related apoptotic proteins, have anti‐inflammatory effects, and inhibit the occurrence of acute early immune rejection. Summary/Conclusion : We screened out one plant‐derived vesicle, using the pueraria derived vesicles‐like nanoparicles itself rich in active ingredients and MiRNA, etc. To fuse it with genetically engineered macrophage nanovesicle (Fusion EV) to produce a targeted effect, affect macrophage polarization and anti‐inflammatory effects.It can successfully reduce the oxidative stress response of cardiomyocytes, inhibit apoptosis of cardiomyocytes, and reduce the expression of Caspase3 and Bax, and reduce reperfusion injury in ischemic transplantation, and the occurrence of early acute immune rejection.

Identifying

Sheela Abraham 1 ; Tristan Robertson 2 ; Christopher Wells 1 ; Kathrin Tyryshkin 1 ; Jelle Penders 3 ; Molly M. Stevens 4 ; John Rudan 5 ; Eric Bonneil 6 ; Pierre Thibault 6 ; Xiaoyan Jiang 7 ; Mark D. Minden 8 ; Isabelle Grenier‐Pleau 1 1 Queen's University, Kingston, ON, Canada, USA; 2 Queen's University ‐ Canada, Mississauga, Canada; 3 SPARTA Biodiscoveries Limited, UK, USA; 4 Department of Materials, Department of Bioengineering & Institute of Biomedical Engineering, Imperial College London, London, UK., London, United Kingdom; 5 Queen's University Department of Surgery, Kingston, ON, Canada, USA; 6 Institute for Research in Immunology and Cancer (IRIC), Montreal, QC, Canada, Montreal, USA; 7 British Columbia Cancer Research Institute, Vancouver, BC, Canada;, USA; 8 University Health Network (UHN), Toronto, ON, Canada, USA Introduction : Intercellular communication events mediated by extracellular vesicles (EVs) are becoming increasingly relevant in cancer research. Although EVs are known to influence haematopoietic stem cell (HSC) proliferation and differentiation, very little is known about how malignant EVs affect both healthy and leukaemic stem cells. This study aims to elucidate the composition and functionality of leukaemic EVs, to understand their role in disease. Methods : EVs were enriched using a combination of iodixanol density cushion (IDC) and size exclusion chromatography (SEC) from the blood plasma of healthy, chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) subjects. Single particle raman spectroscopy and mass spectrometry were performed on leukaemic and healthy EVs to identify novel spectral and proteomic biomarkers for CML and AML. Colony forming cell (CFC) assays were used to assess functional effect of leukaemic EVs on the proliferation and differentiation HSCs. Results : Results revealed significantly more EVs in the blood plasma of patients with CML and AML compared to healthy controls. Raman spectroscopy identified unique EV profiles in samples from healthy controls, CML and AML patients, suggesting different lipid, nucleic acid and protein content. Machine‐learning techniques were used to identify and rank EV proteins quantified by mass spectrometry, identifying potential EV protein biomarkers for leukaemia. Finally, CFC assays displayed an antiproliferative effect of leukaemic EVs on healthy HSCs. Summary/Conclusion : Our group has identified unique Raman spectral and protein EV signatures that distinguish normal healthy individuals from leukaemia patients. Functional studies using human primary samples have also demonstrated a biological role of leukaemic EVs on HSC function, supporting the possibility of EVs as a therapeutic target in disease. Funding : Canadian Institutes of Health Research, Canadian Foundation for Innovation, Bickell Foundation. Keywords : leukaemia, CML, AML

Integrating

Nadim Tawil 1 ; Laura Montermini 2 ; Hélène Page‐Veillette 1 ; Dongsic Choi 3 ; Brian Meehan 4 ; Cristiana Spinelli 4 ; Lata Adnani 5 ; Ekaterina Yurchenko 1 ; Anthoula Lazaris 1 ; Peter Metrakos 1 ; Janusz Rak 6 1 Research Institute of the McGill University Health Center, Montreal, Canada; 2 Research Institute of the McGill UHC, Montreal, Canada; 3 Department of Biochemistry, Soonchunhyang University, College of Medicine, Cheonan, Republic of Korea; 4 Research Institute of McGill University Health Center, Montreal, QC H4A 3J1, Canada, Montreal, USA; 5 McGill University, Montreal, USA; 6 Research Institute of McGill University Health Center, Montreal, QC H4A 3J1, Canada. Department of Pediatrics, McGill University, Montreal, QC H4A 3J1, Canada., Montreal, USA Introduction : Research on extracellular vesicles (EVs) and particles (EPs) continues to expand, constantly pushing against analytical barriers. Heterogeneity of EVs/EPs and their inner complexity render traditional approaches insufficient to fully map, characterize and understand their landscapes across biological systems. While multiple technologies have evolved to address this gap, integrated broad‐spectrum assemblies of required instrumentation remain rare. Here we describe an institutional initiative, the Center for Applied Nanomedicine (CAN), as a comprehensive instrumental pipeline for EV/EP studies at the Research Institute of the McGill University Health Centre (RI‐MUHC) in Montreal, Canada. Methods : The instrumentation forming the CAN platform includes nanoparticle tracking analysis (NTA), micro‐fluidic resistive pulse sensing (MRPS), nano‐flow cytometry (nFC), imaging flow cytometry (iFC), chip‐based fluorescent imaging (ChipFI), fluorescence‐activated vesicle sorting (FAVS) and super‐resolution microscopy (single‐molecule localization microscopy; SMLM). Funding was provided by Canada Foundation for Innovation (CFI) and instruments were integrated with RIMUHC Technology Platforms. Results : The CAN analytical pipeline includes all steps of EV/EP preparation, analysis and downstream applications. A preparation suite is equipped with isolation and purification equipment (Ultracentrifugation, SEC columns), and a profiling suite features an array of orthogonal NTA and MRPS profilers (NS300, PMX120, nCS1) allowing for enumeration, sizing and zetapotential measurement. Molecular maps of individual EV/EPs can be subsequently built using nFC (CytoFLEX), iFC (ImageStream) and ChipFI (ExoView R200) instruments, with further validation through super‐resolution microscopy (Nanoimager), electron microscopy and other technologies. Finally, EV/EP sorting capacity is being actively developed at CAN (CytoFLEX SRT). This would allow to further focus on EV/EP subsets of interest, characterize them, and better elucidate their functions and involvement in various physiological/pathological contexts. Summary/Conclusion : Integrated instrumental platforms, like CAN, incorporating versatile analytical workflows may offer unique insights into complexity of EV/EP landscapes in health and disease. Funding : Canada Foundation for Innovation (CFI). Keywords : extracellular vesicles, extracellular particles, EVs, EPs, nanoFlow, FAVS, NTA, MRPS, Super‐resolution microscopy, Single‐molecule localization microscopy, SMLM, Nanoimager, EV/EP sorting, NS300, PMX120, nCS1, ExoView, CytoFLEX, CytoFLEX SRT, ImageStream

Integrative

Tatyana Vagner 1 ; Taylon Silva 2 ; Elizabeth Hutchins 3 ; Wenyan Zhao 4 ; Yari Ciani 5 ; Minhyung Kim 1 ; Sungyong You 6 ; Krizia Sagini 7 ; Wei Yang 1 ; Michael Freeman 1 ; Edwin Posadas 8 ; Jlenia Guarnerio 9 ; John P. Nolan 10 ; Andries Zijlstra 11 ; Clotilde Thery 12 ; Francesca Demichelis 5 ; Paul Boutros 13 ; Kendall Van Keuren‐Jensen 14 ; Dolores Di Vizio 15 1 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, Los Angeles, USA; 2 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, USA; 3 2Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, U, Phoenix, USA; 4 Department of Human Genetics, Institute for Precision Health, UCLA, Los Angeles, CA, USA, USA; 5 Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy; 6 Bioinformatics Shared Resource, Cedars‐Sinai Medical Center, Los Angeles, USA; 7 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles, US, Oslo, USA; 8 Division of Cancer Biology and Therapeutics, Departments of Surgery, Biomedical Sciences and Pathology and Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars‐Sinai Medical Center, Los Angeles, USA; 9 Samuel Oschin Comprehensive Cancer Institute, Cedars‐Sinai Medical Center, Los Angeles, los angeles, USA; 10 Scintillon Institute, San Diego, USA; 11 Department of Research Pathology, Genentech, South San Francisco CA, USA, USA; 12 INSERMU932, Institut Curie, Paris, France; 13 Department of Human Genetics, Institute for Precision Health, UCLA, Los Angeles, CA, USA, Los Angeles, USA; 14 Neurogenomics Division, Translational Genomics Research Institute, Phoenix, USA; 15 Department of Surgery, Cedars‐Sinai Medical Center, Los Angeles CA, USA, Los Angeles, USA Introduction : Extracellular Vesicles (EVs) are membrane covered particles of heterogeneous size and cargo. While small EVs (S‐EVs) have been widely investigated, little is known about Large EVs (L‐EVs). The paucity of studies comparing protein cargo of L‐ and S‐EVs, the absence of studies focused on protein coding RNA and integrative analyses to compare the protein and transcript expression in different EV fractions, prompted us to perform mass spectrometry to profile EVs from different cancer cell models. The prostate cancer model was selected for additional complementary RNA‐Seq to enable an integrative, multi‐analyte approach of three EV populations in this model. Methods : LC‐MS/MS, SWATH‐MS,, RNA‐seq, vFC, TRPS, TEM, immunoblotting, single‐EV RNA‐seq, UC, and Iodixanol gradient. Results : We identified novel marker candidates for L‐ and S‐EVs, some of which were present both at the protein and transcript level. The correlation between protein and protein‐coding mRNA was higher in L‐ than S‐EVs. Additionally, the protein cargo differed among L‐ and S‐EVs more than the transcript, suggesting that the identity of the EV populations is defined by their proteins more than by their mRNA cargo. Pathway analysis demonstrated that L‐EVs are enriched in mRNAs and proteins involved in essential metabolic activities including mitochondrial functions. GSEA of the most abundant transcripts confirmed enrichment of mitochondrial function in L‐EVs versus S‐EVs, and this finding was corroborated in single EVs. The proteins/RNAs enriched in prostate cancer cell‐derived L‐EVs were also identified in L‐EVs from the plasma of patients with metastatic prostate cancer by a SWATH proteomic assay. L‐EV signature, but not the S‐EV signature correlated with progression to metastasis. Summary/Conclusion : The integrated L‐EV proteomic and transcriptomic signature enabled distinction between benign and localized prostate cancer, as well as between localized cancer and metastatic castration‐resistant cancer. Funding : R01CA218526 NIH/NCI and R01CA234557 NIH/NCI.

Intravenous

Yeongju Byun 1 ; Jihwa Chung 2 ; Kyoung Hwa Kim 2 ; Tae Hee kim 2 ; MyeongKwan Choi 2 ; Shung Hyun An 2 ; DaeHo Bae 2 ; Kihwan Kwon 3 1 Exollence Co., Ltd., Seoul 07985, Korea and Medical Research Institute, School of Medicine, Ewha Womans University, Seoul 07985, Korea, Republic of Korea; 2 Exollence Co., Ltd., Seoul 07985, Korea, Republic of Korea; 3 Department of Internal Medicine, Cardiology Division, School of Medicine, Ewha Womans University, Seoul 07985, Korea and Exollence Co., Ltd., Seoul 07985, Korea, Seoul, Republic of Korea Introduction : Exosomes (EXOs), one of extracellular vesicles derived from cells, have been focused on as a promising drug delivery system. However, a systemic delivery technique for efficiently delivering EXOs to a specific tissue or cell has not yet been developed. We previously identified a dysfunctional endothelium‐specific peptide (DSP) that specifically binds to the surface of dysfunctional vascular endothelial cells (ECs) caused by low oscillatory shear stress (OSS), and1 we developed a method for successfully loading diverse therapeutic materials into EXOs using extracorporeal shock waves (ESW). In this study, we investigated the targeted delivery of short interfering (siRNA) by systemic administration to disturbed flow‐induced atherosclerotic dysfunctional ECs using DSP‐coated EXO as the delivery vehicles. Methods : Using click chemistry, DSP was conjugated to the surface of EXOs derived from cow milk (DSP‐EXO). ICAM‐1‐specific siRNA (siICAM‐1) was encapsulated into the DSP‐conjugated exosomes (DSP‐EXO‐siICAM‐1) by ESW. EXO and DSP‐EXO are treated to ECs cultured under pro‐atherogenic oscillatory shear stress (OSS, ± 5 dyne/cm 2 ) and anti‐atherogenic laminar shear stress (LSS, 20 dyne/cm 2 ), respectively. A mouse model of flow‐induced atherosclerosis was produced by partial ligation of the left carotid artery (LCA). Results : DSP‐EXO, unlike EXO, preferentially attached to ECs cultivated under OSS rather than LSS. Following injection into the tail vein, DSP‐EXO attached preferentially to the OSS area of the LCA in the mouse model, whereas EXO did not. Furthermore, DSP‐EXO‐siICAM‐1 significantly reduced endothelial ICAM‐1 expression in ECs under OSS conditions and in the LCA region in the mouse model. Summary/Conclusion : The results of this study demonstrate that surface‐engineered EXOs can be delivered selectively to specific tissues or cells by systemic delivery, such as intravenous injection. This is anticipated to be a promising strategy for future development of EXO‐based therapies. Keywords : exosome, target delivery, surface engineering, dysfunctional endothelial cell‐specific peptide (DSP), atherosclerosis

Liposarcoma

Premanshu K. Singh 1 ; Patricia Sarchet 2 ; Raphael E. Pollock 2 ; Shaurya Prakash 1 1 Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, Ohio, USA, Columbus, USA; 2 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA, Columbus, USA Introduction : Dedifferentiated liposarcoma (DDLPS) is among the most common sarcoma subtypes. In addition to growing to massive size before detection, DDLPS can also present or recur as synchronous multifocal lesions and are characterized by ultimately lethal multicentric recurrence in approximately 60% of patients, resulting in an overall survival rate of 10% at 10 years. At the molecular level, practically all DDLPS are characterized by MDM2 gene amplification. Recent studies have shown a possible role for extracellular vesicle (EV) cargoes in cancer initiation, progression and metastasis. Major cargoes in LPS EVs include MDM2 DNA which has been described to play an important role inducing tumour progression and metastasis. Our past work has shown upregulation of MDM2 DNA within EVs from DDLPS patient serum as well as from DDLPS cell lines. Given the distinct pathways for the biogenesis and secretion of EVs of varied size, it is hypothesized that MDM2 DNA expression is EV size dependent. Methods : Cells from Human liposarcoma (LPS) cell line Lipo246 were cultured in a serum‐free medium for 48 h for EVs production and LPS cell‐conditioned media (LCCM) collection. LCCM was centrifuged at 2000g for 20 mins to remove cellular debris and the supernatant was filtered first with 200 nm (pore size) PVDF membrane filter followed by 100 nm (pore size) PDVF filter. The quality of isolated particles at each filtration step was assessed through TEM and Nanotracking analysis. EV MDM2 DNA was analyzed using real‐time PCR and results were normalized to GAPDH. Results : The pre‐filtered LCCM showed the highest upregulation in MDM2 DNA by 3.4x over the control case. 2.4x MDM2 DNA upregulation was observed in LCCM with EVs< 200 nm, while the LCCM with EVs< 100 nm gave an upregulation of 1.9x. Summary/Conclusion : Our study represents the first investigation of EV size dependence on MDM2 gene expression and the initial findings suggest that the EV size is related to the MDM2 DNA upregulation in LPS‐derived EVs. Future experiments will quantify the number of MDM2 molecules as a function of EV size. Funding : This work was supported by the National Institutes of Health under grant number DOD CDMRP Grant CA210874. Keywords : liposarcoma, MDM2 DNA, biomarker, extracellular vesicles

Macrophages

Alon Nudelman 1 ; Anjana Shenoy‐Katapadi 1 ; Dror Chorev 1 ; Irit Rosenhek‐Goldian 2 ; Paula Abou Karam 2 ; Inna Solomonov 1 ; Neta Regev‐Rudzki 2 ; Irit Sagi 1 1 Weizmann Institute of Science, Rehovot, Israel, Israel; 2 Weizmann Institute of Science, Rehovot, Israel, Rehovot, Israel Introduction : Proteolysis is an integral process of the immune response to infection. While the research focus is on secreted soluble proteases, recent studies have also identified proteases borne by extracellular vesicles (EVs). However, the role of EVs as protease‐bearing particles, especially in the context of bacterial infections, is poorly understood. Here we shed light on the composition and function of proteolytically active EVs secreted from macrophages infected with Salmonella enterica serovar typhimurium (S. typhimurium). Methods : EV samples were isolated by differential centrifugation and characterized using atomic force microscopy (AFM), nanoparticle tracking analysis (NTA), sucrose gradient pelleting, and protein characterization. EV quantification and standardization was performed using NTA and total protein quantification. Results : We discovered that EVs derived from S. typhimurium‐infected macrophages were enriched in proteolytically active Matrix Metalloproteinase 9 (MMP‐9). Interestingly, the signaling pathway responsible for MMP‐9 upregulation in response to S. typhimurium exposure is selective to immune cells and regulated by Toll‐like Receptor 4 (TLR‐4) recognition of bacterial lipopolysaccharide (LPS). Co‐immunoprecipitation of MMP‐9 with numerous membrane proteins indicates co‐localization of MMP‐9 to the EV membrane. We further demonstrated that specific inhibition of EV‐associated MMP‐9 significantly decreased macrophage ability to invade through basement membrane in vitro, suggesting that proteolytic EVs may play a role in macrophage response to bacterial infection. Summary/Conclusion : Our findings reveal that upon exposure to S. typhimurium, macrophages secrete EVs enriched in membrane‐associated proteolytically active MMP‐9, crucial for macrophage invasion through the extracellular matrix and basement membrane. Future work includes investigating the role of proteolytic EVs on bacterial infectivity. Keywords : proteolysis, matrix metalloproteinases, immune response

Mesenchymal

Ji Yong Choi 1 ; Da Eun Jeong 2 ; Hong Seok Kang 2 ; Ji Soo Park 2 ; Ji Hye Lee 2 ; Joo Youn Lee 2 1 Xcell Therapeutics, Seoul, Republic of Korea; 2 Xcell Therapeutics, Republic of Korea Introduction : In recent decades, extracellular vesicles (EV) have been studied in various fields and have been evaluated for their wide utilization and high value. These studies found that EVs have cell‐specific cargo proteins, lipids, and are substances that have functions that reflect the characteristics of cells. In particular, EV derived from stem cells have been reported to have stem cell characteristics, and they function similar to stem cells as cell free materials. Therefore, EVs secreted according to the culture environment of stem cells produce different results. Methods : Mesenchymal stem cells were cultured using different culture media and then EVs were separated by tangential flow filtration (TFF) system from conditioned media. Size distribution and particle number of EVs were measured by using Zetaview and confirmed by expression of EV markers (CD9, CD63, and CD81) by western blot. Isolated EVs were treated on human umbilical vein endothelial cells (HUVECs), HaCaT cell (human epithelial keratinocyte), dermal fibroblast and epidermal keratinocyte to evaluate vascular and skin regeneration. Results : We confirmed that EVs secreted from cells cultured with serum free serum free chemically defined medium (CDM) have remarkable skin regeneration ability and vascular regeneration ability. Through the experimental results, compared to MSC‐derived EV secreted from the medium containing FBS, we found that HUVEC cells and HaCaT cells of the MSC‐derived EV treatment group from CDM showed higher angiogenesis index and wound area recovery rates, respectively. In addition, normal human skin fibroblasts (NHDF) and epidermal keratinocytes were treated with each EV to confirm that genes (Involucrin, Loricrin) and proteins (Hyaluronic acid, pro‐collagen) related to skin barrier, moisturizing and anti‐aging were highly expressed MSC‐derived EV isolated from CDM. Summary/Conclusion : The application of serum free chemically defined medium is a method of obtaining MSC‐derived EVs with the ability to regenerate stem cells while maintaining the characteristics of stem cells in cell culture. By cultivating cells in animal‐derived components free, it is possible to improve the quality of the stem cell culture environment and obtain ‘human stem cell‐derived EV’ that completely exclude animal‐derived EV. Ultimately, this study shows that CDM lead to the only stem cell‐derived EVs you want to get. Keywords : CellCor EXO CD, serum free chemically defined media, Stem cell

Metabolites

Syeda Tayyiba Rahat 1 ; Mira Mäkelä 2 ; Maryam Nasserinejad 3 ; Tiina Ikäheimo 4 ; Anni Nieminen 5 ; Sylvain Sebert 6 ; Nsrein Ali 7 ; Seppo Vainio 8 1 Faculty of Biochemistry and Molecular Medicine, Laboratory of Developmental Biology,University of Oulu., Oulu, Finland; 2 Faculty of Biochemistry and Molecular Medicine, Laboratory of Developmental Biology, University of Oulu, Oulu, Finland; 3 Center for Life Course Heath Research, Faculty of Medicine, University of Oulu., Finland; 4 Research Unit of Population Health Research, Faculty of Medicine, University of Oulu., Finland; 5 FIMM Metabolomics Unit, Institute for Molecular Medicine Finland, University of Helsinki., Finland; 6 ) Center for Life Course Heath Research, Faculty of Medicine, University of Oulu., Finland; 7 Faculty of Biochemistry and Molecular Medicine, Laboratory of Developmental Biology, Flagship GeneCellNano, University of Oulu, 90220, Oulu, Finland, Finland; 8 Faculty of Biochemistry and Molecular Medicine, Laboratory of Developmental Biology,Flagship GeneCellNano, University of Oulu, 90220, Oulu, Finland, Oulu, Finland Introduction : Human body has its own ways of communication which are still to be fully discovered, and one of those is the sweat. Lately sweat has been a subject of interest as a non‐invasive tool to monitor physiological changes in healthy and disease conditions, because of its components i.e., analytes, ions, and extracellular vesicles (EVs), and easy accessibility. Methods : A pilot study* with 11 healthy individuals was conducted, where sweat was induced, at rest, by heat exposure and collected using clinical grade absorbent patches along with monitoring of blood glucose levels. By our novel method, sweat EVs were isolated from patches and enriched by ultracentrifugation and analyzed by LC‐MS. *Ethical License EETMK:199/2016 *Clinical trials Registration: NCT02855905 Results : A targeted metabolomics analysis was performed using the isolated sweat EVs, in which 41 metabolites were examined. Based on samples quality control (QC) and Blank, 24 metabolites were produced at an acceptable range for detection by LC‐MS. Upon testing the association between the metabolite concentration and blood glucose by applying Spearman's rank association, a significance was revealed. Summary/Conclusion : Our study provides first evidence of the metabolic composition of sweat EVs and proposes the use of metabolites as a novel approach to study the physiological responses of the human body to the health‐related problems, hence offering a novel strategy for monitoring health and well‐being. Keywords : diabetes, diseases, biomarker

Microbially

Ranya M. Elsayed 2 ; Mahmoud Elashiry 1 ; Christopher Cutler 3 1 Periodontics Department, Dental College of Georgia, Augusta University, Augusta, USA; 2 Periodontics Department, Dental College of georgia, Augusta university, Augusta, USA; 3 Periodontics Department, Dental College Of GEorgia, Augusta university, Augusta, USA Introduction : Exosomes (exo) can cross the blood brain barrier (BBB) and have recently been linked to neuroinflammation and Alzheimer's disease (AD) pathogenesis. Periodontitis (PD) is a chronic oral degenerative disease that affects 70.1% of Americans 65 years and older and has been linked to age‐related disease such as AD. P.gingivalis (Pg) is considered a “keystone” pathogen in PD due to its outsized influence on the local oral microflora. Our recent work has shown that exo derived from Pg‐infected dendritic cells (DC) transmit immune senescence to bystander cells and induce inflammatory alveolar bone loss in‐vivo. The aim of the current study was to test the hypothesis that microbially induced exo in the oral cavity cross the BBB and contribute to neuroinflammation and pathogenesis of AD. Methods : Isolation of exo from Pg‐infected DCs (PgDCexo) was performed using ultracentrifugation and ultra‐filtration methods. Quantification and characterization of PgDCexo were performed using NTA analysis, western blot and miRNA array. DiI labeled PgDCexo or exo from immature non‐infected DCs (control) were injected intragingivally in wild type (WT) mice or APPNL‐G‐F knock in mouse model of AD (n = 6 per group). Detection of exo in the brains was performed using the in‐vivo live imaging system (IVIS) and confocal microscopy. Brains were dissected and analyzed for signs of AD pathology and neuroinflammation by WB and qPCR. Maxillae were analyzed for bone loss by micro‐CT. Results : In‐vitro analysis revealed a∼2‐fold increase in secreted exosomes from DCs infected with Pg.These exo were enriched in age‐related, anti‐apoptosis/anti‐autophagy miRNAs, Pg fimbrial adhesin protein mfa1, and pro‐inflammatory cytokines IL6/TNFa/IL1b. Orally injected PgDCexo were detected in the brains of APPNL‐G‐F knock‐in and WT mice using IVIS and colocalized with Iba1 positive microglial cells in the hippocampus using immunofluorescence. Hallmarks of AD including, neuroinflammation, hyperphosphorylation of Tau (Ptau), and beta amyloid deposition were increased in brains of APPNL‐G‐F knock‐in injected with PgDCexo compared to control. This was associated with inflammatory alveolar bone loss as evident by micro CT 3D analysis. Summary/Conclusion : Oral microbial infection induces senescence and exo release from DC. These pathologic exo promote paracrine senescence of normal bystander cells, induce inflammatory alveolar bone loss and can cross BBB, contributing to AD pathogenesis. Keywords : periodontitis, exosome, immune senescence, BBB, alzheimer's disease

Modulations

Sujata Mohanty 1 ; Yashvi Sharma 2 ; Suchi Gupta 3 1 AIIMS New Delhi, Delhi, India; 2 All India Institute of Medical Sciences (AIIMS), New Delhi, India; 3 AIIMS, New Delhi, USA Introduction : Mesenchymal Stem Cells (MSCs) have been recognised widely for their regenerative & immunomodulatory potential. They can be isolated from various sources (Bone Marrow (BM) & Wharton's Jelly (WJ)). Studies have reported that hypoxia exposure to MSCs enhances their therapeutic function, especially via augmenting the release & content of their EVs. Currently the focus of regenerative medicine has shifted to cell free therapy, and EVs serve as the perfect candidate for that. This study explores the comparative immunomodulatory functionality of tissue specific MSCs derived small EVs (less than 200nm), cultured in Normoxia and Hypoxia. Moreover, it also gives a cue into the mechanism of these small EVs to further their applications in immune‐implicated disorders. Methods : BM & WJ MSCs were isolated after obtaining donor consents (IC‐SCR/120/21(R)). MSCs were cultured in serum free media & exposed to 1% hypoxia for 24 hours in cGMP compliance. Small EVs were isolated from conditioned media using ultracentrifugation. They were characterized via NTA (Size < 200nm), TEM (Cup shaped morphology) and Western blotting (Surface marker CD63, Cytoplasmic marker ALIX and Negative marker Calnexin). These EVs were tested for their immunomodulatory effect via MLR and macrophage polarization assay. Microarray based mechanistic analysis was performed for identification of miR 125b‐5p & its upregulation was done via mimics transfection for further pathway analysis. Furthermore, excisional wound model was created in Wistar rats (226/IAEC‐1/2019) and EVs treatment was given for validation. Results : It was observed that there was a significant immunosuppressive effect of both BM & WJ‐MSCs derived exosomes. This activity was augmented significantly when the exosomes were derived forth hypoxic priming of MSCs. Moreover, there was also a significant difference in the immunosuppressive abilities of BM & WJ as a source, where WJ‐MSCs derived exosomes were faring better. Furthermore, it was observed that the wound healing and inflammatory scenario was improved upon exosomes treatment in rats. This was evidenced by the increase in miR125b‐5p in hypoxia derived EVs of WJ‐MSCs. Summary/Conclusion : This study hereby concludes that hypoxia priming mediated increase in miRNA 125b‐5p can be considered as a minimum manipulation strategy to enhance the regenerative capabilities of MSCs & their EVs to further their implication in immune disorders, and while both the sources respond well to this priming strategy, WJ‐MSCs exhibit augmented immunomodulation as compared to BM‐MSCs. These results could be accredited mechanistically in terms of their enhanced miRNA content and this gives us a hope for developing future therapeutic strategies taking miRNA as a target molecule.

Multiplexed

Mi Ho Jeong 1 ; Yoseob Han 2 ; Taehwang Son 3 ; Hyungsoon Im 1 1 Massachusetts General Hospital, Boston, USA; 2 Department of IT Convergence, Soongsil University, USA; 3 Massachusetts General Hospital, Chalestown, USA Introduction : Idiopathic pulmonary fibrosis (IPF) is a lung disorder in which various cell types are intertwined. Extracellular vesicles (EVs) in bronchoalveolar lavage fluid (BALF) could play important roles as cellular surrogates for molecular IPF diagnosis. However, the current bulk EV analysis prevents the accurate evaluation of EV biomarkers derived from various cell types. Here, we developed a single EV sensing platform based on plasmon‐enhanced multi‐color fluorescence detection to evaluate IPF markers on cell‐specific EVs. Methods : We developed in vitro IPF models using lung epithelial cells (Beas‐2B), macrophages (THP‐1), and fibroblasts (MRC5) that represent epithelial‐mesenchymal transition, M1 or M2 transition, and fibroblast‐myofibroblast transition, respectively. We isolated small EVs from cell line culture media using size‐exclusion chromatography and characterized the size and concentration using nanoparticle tracking analysis. We used nanoplasmonic EV sensing (nPLEX) chips for single EV analysis. We used the Cy7 channel for all EV detection, the AF555 channel for cell‐specific EVs, and the AF647 channel for IPF markers. We then analyzed cell‐specific EV counts and marker intensities in single EVs. Results : For EVs from Beas‐2B epithelial cells, the rate of EpCAM+/Fibronectin+ EVs was significantly higher in the IPF model than in control. For EVs from MRC5 fibroblast cells, CD44+/Fibronectin+ and CD44+/Col1A1+ EVs were increased in the IPF model. For THP‐1 EVs, CD45+/CD80+ was elevated in EVs derived from M1 macrophages, while CD45+/Arg1+ was increased in M2 macrophages’ EVs. Summary/Conclusion : This study evaluated IPF marker levels in the cell‐specific EVs using the 3‐channel single EV analysis. We will apply the assay to human BALF samples and establish a correlation of IPF marker levels between cell‐specific EVs and cells in lung tissues. Funding : This study is supported by Basic Science Research Program (NRF‐2021R1A6A3A14039686) of the National Research Foundation of Korea (NRF).

Osteoclasts

Takaaki Tamura 1 ; Akiko Kogure 2 ; Yusuke Yoshioka 3 ; Shinichi Sakamoto 4 ; Tomohiko Ichikawa 5 ; Takahiro Ochiya 6 1 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku, Japan; 2 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku‐ku,, USA; 3 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku‐Ku, Japan; 4 Associate Professor, Chiba, Japan; 5 Graduate School of Medicine and School of Medicine, Chiba University, Japan; 6 Department of Molecular and Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shimjuku‐ku, Japan Introduction : Prostate to bone cancer metastases induce mixed lesions containing areas of bone destruction and formation that are directed by osteoclasts (OCs) and osteoblasts (OBs), respectively. OCs play an essential role in the tumor invasion areas even in osteogenic bone metastases of prostate cancer (PCa). OC‐derived extracellular vesicles (EVs) are reported to regulate OB activity in normal bone homeostasis; however, there is no study investigating the role of EVs from OC educated by PCa cells in the tumor bone microenvironment. Methods : We prepared four types of OCs and EVs from these cells; OCs differentiated from RAW264.7 cells (OC cells), OC cells co‐cultured with normal prostate epithelial cells (OCN cells), osteolytic PCa cells (OCP cells), and osteoblastic PCa cells (OCC cells). OC differentiation was induced in the presence of RANKL. EVs were purified from culture supernatant using ultracentrifugation.◻To observe the changes in PCa‐educated OCs, we investigated the response to denosumab (anti‐RANKL antibody) and signaling pathways in OCP and OCC cells. Further, to reveal the function of EVs from PCa‐educated OCs, we added EVs to mineralizing OBs (MC3T3‐E1). The expression levels of OB marker genes such as ALP and BGLAP, which are stably expressed in MC3T3‐E1, were compared by qPCR and ALP staining was conducted. Next‐generation sequencing (NGS) was performed to identify EV‐delivered miRNAs regulating OB activity. Results : OCP and OCC cells showed denosumab resistance. Some signaling pathways, which are reported to promote OC differentiation independent of the RANKL pathway, were increased. Moreover, OCP and OCC cell‐derived EVs significantly inhibited OB activity; these EVs down‐regulated OB marker genes. NGS identified some candidate miRNAs as EV components regulating OB activity. Summary/Conclusion : We report the role of EVs derived from OCs educated by cancer cells for the first time. OCs educated in the tumor invasion areas may release EVs, inhibiting OB activity and leading to further bone destruction. Funding : This work was supported by Project for Cancer Research and Therapeutic Evolution (P‐CREATE) grant number: JP20cm0106402 (to T.O.) from the Japan Agency for Medical Research and Development (AMED).

Preclinical

Kevin C. Hicok 1 ; Aline Betancourt 2 ; Timothy Moseley 1 1 Direct Biologics LLC, La Jolla, USA; 2 Direct Biologics LLC, USA Introduction : Many studies have now documented the safety and molecular profile of bone marrow‐mesenchymal stem cells and their secretome. Still, safety and molecular content of each therapeutic is unique and must be characterized to support their clinical use. Herein are the results of murine preclinical safety, proteomic and extracellular vesicle (EV) identity studies of a BM‐MSC EV enriched biologic currently being evaluated for its clinical safety and efficacy. Methods : Safety was evaluated in murine models to assess acute tolerability, toxicity, and long term tumorigenicity. IgE levels were measured to assess acute allergic response after either subcutaneous or IV injection. Renal and liver histopathology was performed to assess acute toxicity (7 days). Long term (3 Month) tumorigenicity was determined in NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mice using up to 20x human dose equivalent concentration. Animal studies were performed in ALAAS approved facilities. In molecular characterization studies, the EV content was evaluated using electron microscopy, STED Microscopy, SP‐IRIS, and NTA. Proteomic analysis was performed using standard mass spectrophotometric (MS) protein sequencing. Results : Safety: The therapeutic was deemed safe. No evidence of acute immune response, toxicity, or of tumor induction was observed within the tested time and dose ranges. Blood analyses were within normal ranges after 7 days and after 90 days. Identity: EVs were enriched for CD63+ EVs versus CD9+ or CD81+ EVs. At least 1173 different proteins were identified by MS sequencing of 5 different lots. Protein content was consistent with previously published data sets for MSC EVs. Summary/Conclusion : These data demonstrate a superior safety profile relative to many traditional small molecule and single protein biologics, consistent with prior BM‐MSC therapeutics studies. The complex protein composition indicates a potential multi‐molecular, multi‐modal mechanism of action for efficacy of this BM‐MSC therapeutic. Funding : Studies were funded by industry company employing authors of this abstract. Keywords : GLP preclinical safety, In vivo, proteomics, EV therapeutic identity

Recombinant

Bruna Andrade Aguiar Koga 1 ; Alice Marcela Sampaio Del Colletto 1 ; Mari Cleide Sogayar 2 ; Ana Claudia Oliveira Carreira 3 1 Surgery Department, Faculty of Veterinary and Animal Science, University of São Paulo; NUCEL, School of Medicine, University of São Paulo, São Paulo, Brazil; 2 NUCEL, School of Medicine, University of São Paulo; Biochemistry Department, Chemistry Institute, University of São Paulo, Brazil; 3 Center of Natural and Human Sciences, Federal University of ABC; Surgery Department, Faculty of Veterinary and Animal Science, University of São Paulo; NUCEL, School of Medicine, University of São Paulo, Santo André, Brazil Introduction : Regenerative Medicine aims to achieve treatments that accelerate the different stages of the healing process in both humans and animals, and the manipulation of the growth factors (GF) composition can improve or modify the repair and remodelling process of injured tissues, as well as providing more aesthetic and functional scars. The present study aimed to evaluate the effect of recombinant human platelet‐derived growth factor (PDGF‐BB) and/or combined with endothelial vascular growth factor (VEGF165) on adipose canine mesenchymal/stromal stem cells (AD‐MSC) and their secretome. Methods : AD‐MSCs were isolated and characterized, cultured and the conditioned media were collected under conditions without SFB for isolation of exosomes by ultrafiltration and ultracentrifugation. Samples were quantified resulting in higher yield in exosomes of cells treated for 24 hours using 10ng/mL of each GF or their combination. GF were biologically tested in Rowett nude rats, where 3μg/mL of GF were applied alone or combined in wounds on the animals' backs, and after 7 days the healing evolution was evaluated macroscopically and histopathologically. Results : GFs were obtained from purified media by heparin affinity chromatography and characterized by ELISA and Western blot, and their biological activity were tested in vitro and in vivo in a rat model. Mesenchymal stem cells were obtained, characterized, cultured and treated with GF and exosomes were isolated from conditioned media. The present study demonstrated for the first time the potential of rhPDGF‐BB and/or rhVEGF165 supplementation in the healing of skin wounds in seven days, stimulating angiogenesis, fibroblast reaction and re‐epithelialization, but a long‐term evaluation is necessary. The treatment with both factors combined improved the therapeutic profile of adipose mesenchymal stem cells by stimulating cell migration and exosome secretion. Summary/Conclusion : Together, the data support future investigations into the effect of GF supplementation in vitro, on cellular and molecular content of the mesenchymal stem cell secretome and in vivo, in a safe and reproducible model, opening a new perspective for a cell‐free therapeutic approach for wound healing. Funding : Coordination for Improvement of Higher Education Personnel (CAPES), National Council for Scientific and Technological Development (CNPq), The State of São Paulo Research Foundation (FAPESP No. 2016/05311‐2). Keywords : wound healing, PDGF‐BB, VEGF, mesenchymal stem cells, Exosomes

Rivaroxaban

Luisa Weiss 1 ; Paulina Szklanna 1 ; Sarah Kelliher 2 ; Kieran Wynne 3 ; Alfonso Blanco 4 ; Aideen O'Doherty 5 ; Barry Kevane 2 ; Jan Zivny 6 ; Séan Murphy 7 ; Martin O'Donnell 5 ; Fionnuala Ní Áinle 8 ; Patricia Maguire 1 1 School of Biomolecular and Biomedical Science, University College Dublin, Ireland, Dublin, Ireland; 2 Department for Haematology, Mater Misericordiae University Hospital, Ireland, Dublin, Ireland; 3 Systems Biology Ireland, University College Dublin, Ireland, Dublin 4, Ireland; 4 Conway Institute Flow Cytometry Core Facility, University College Dublin, Ireland, Dublin, Ireland; 5 National University of Ireland Galway, Ireland, Ireland; 6 Institute of Pathological Physiology, Charles University, Czech Republic, Czech Republic; 7 Department for Stroke Medicine, Mater Misericordiae University Hospital, Ireland, Dublin, USA; 8 Department for Haematology, Mater Misericordiae University Hospital and Rotunda Hospital, Ireland, Dublin, USA Introduction : Despite secondary prevention with aspirin (ASA), patients with stable cardiovascular disease (CVD) remain at heightened long‐term risk of major cardiovascular events (MACE). The Cardiovascular Outcomes for People Using Anticoagulation Strategies (COMPASS) double‐blind randomized clinical trial, including nearly 28,000 CVD patients, demonstrated that aspirin combined with low‐dose Rivaroxaban (R) significantly decreased the incidence of MACE by 24% and cardiovascular mortality by 18% compared with ASA alone. However the underlying molecular mechanisms remain elusive. Extracellular vesicles (EVs) are crucial messengers regulating a myriad of biological/pathological processes and are highly implicated in CVD. We hypothesised that circulating EV profiles reflect the cardioprotective properties of adding R to ASA. Methods : A cohort of stable CVD patients (n = 40) who participated in the COMPASS trial ( NCT01776424 ) and were previously randomised to receive ASA, were prospectively recruited following informed written consent according to the declaration of Helsinki and assigned a revised regimen of open label ASA+R. Blood samples were obtained at baseline (ASA only) and 6‐months follow‐up (ASA+R). Plasma EV concentration, size and origin were analysed by NTA and flow cytometry. EVs were enriched by ultracentrifugation for proteomic analysis. Results : ASA+R fundamentally altered small (< 200nm) and large (200‐1000nm) EV concentration and size, compared to ASA alone. Crucially, levels of platelet‐derived, tissue‐factor positive and myeloperoxidase‐positive EVs significantly decreased at follow‐up, potentially contributing to the reduced incidence of thrombotic events associated with adding R to ASA. Comparative proteomic characterisation further revealed a significant decrease in highly pro‐inflammatory protein expression at follow‐up. Summary/Conclusion : The observed changes in EV subpopulations, together with the differential protein expression profiles, suggest amelioration of an underlying pro‐inflammatory and pro‐thrombotic state when low‐dose R is added to ASA in patients with CVD, which may be of clinical relevance towards understanding the fundamental mechanism underlying the previously reported superior cardiovascular outcomes associated with this antithrombotic regimen. Keywords : cardiovascular disease, anti‐thrombotic therapy, rivaroxaban, aspirin

Scalability

John JS Cadwell 1 ; Laura Perin 2 ; Paolo Neviani 3 ; Sargis Sedrakyan 4 1 FiberCell Systems Inc., New Market, USA; 2 Children's Hospital Los Angeles‐University of Southern California, Los Angeles, USA; 3 Children's Hospital Los Angeles, Los Angeles, USA; 4 Children's Hospital Los Angeles ‐ University of Southern California, Los Angeles, USA Introduction : Introduction: EV clinical translation is inhibited by limitations in the scale‐up of EVs production. Hollow fiber bioreactors (HFBR) support culture of large numbers of cells, at high densities, with concentrated EVs. Culture conditions may affect EV composition and potency. Here we compare the production, potency, identity, and therapeutic potential of EVs collected from cells grown in culture dish (2D) vs a small and medium‐sized HFBR (3D). Methods : Methods: 1 × 10e6 human clonal stem cells from amniotic fluid (hAFSC from consented donors) were seeded in 2D (145cm2), 1.6 × 10e7 hAFSC were seeded on a small cartridge (FiberCell C2025D; 450cm2), and 1.8 × 10e8 hAFSC on medium cartridge (FiberCell C2011; 4,000cm2) with fibronectin coating. All cultures used Chang medium with 20% of ES‐FBS, starved for 24hr and then EVs collected. The effect of harvest frequency was tested (8hr, 24hr, 72hr). 2D‐EVs and 3D‐EVs were compared by Nanosight, potency assay (by WB), identity (by Exoview), and therapeutic effect (in vivo injections in an animal model of chronic kidney disease, Alport Syndrome). Results : Results: 2D production was ∼5.5 × 10e9EV/ml/24hrs while 3D was ∼2.8 × 10e10EV/ml (first four 24hrs) and ∼4.4 × 10e10EV/ml (two days of hourly harvests). The medium cartridge produced similar concentrations of EVs but at 10X harvest volume indicating linear scalability. Very little difference in EV concentration was observed during harvest intervals, with a very similar size distribution. This could indicate either significant EV re‐uptake or inhibition of EV secretion dependent upon free EV in the supernatant. Both 3D‐EVs trapped VEGF in vitro (an in vitro established potency assay) and expressed CD9, CD81, CD63, CD80, CD86, and VEGFR1 as 2D‐EVs. 3D‐EVs ameliorated proteinuria and histology when injected into Alport mice and also trapped VEGF in vivo as 2D‐EVs. Summary/Conclusion : Summary/Conclusion: 3D‐EVs had comparable properties and bio‐activity to 2D‐EVs, but the HFBR produced 10x to 100X more EVs. Cell culture conditions for hAFSC still need optimization in the HFBR, however, a currently available 1.2m2 cartridge provides a 50X scale‐up potential. The HFBR is a closed system that can be cGMP compliant. In conclusion, the HFBR can produce a sufficient number of EVs to support pre‐clinical and clinical applications of EVs with at least similar properties to EVs produced in 2D methods. Funding : na. Keywords : 3D, hollow fiber bioreactor, human amniotic fluid stem cells

Specificity

Olesia Gololobova 1 ; Gabriela Lima 2 ; Shivni Patel 3 ; Tanina Arab 2 ; Julian Cornejo 3 ; Antonio Giuseppucci 3 ; Erin Shirk 3 ; Linglei Jiang 2 ; Kenneth W. Witwer 4 1 Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, USA; 2 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, USA; 3 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, USA; 4 Johns Hopkins University, Baltimore, USA Introduction : Establishing the specificity of EV surface proteins opens new opportunities for studies of otherwise inaccessible tissues, such as the central nervous system (CNS). Capturing and detecting brain‐origin EVs that leak or are transported into peripheral samples is a type of ‘liquid biopsy.’ Most EVs in peripheral biofluids are irrelevant to brain disease, but one might boost signal above noise by enriching cell/organ‐specific EVs with immunocapture methods. Although numerous studies have attempted to harvest EVs of neuronal origin from peripheral samples, EVs from other CNS cells have been relatively under‐studied. Here, single‐particle interferometric imaging sensing (SP‐IRIS) and other platforms were used to capture and phenotype EVs from several CNS cell types. Methods : EVs were separated from cell cultures representing oligodendrocytes (HOG), astrocytes (U‐87 MG), microglia (SV40), and neurons (SH‐SY5Y), as well as differentiated iPSCs using ultracentrifugation followed by size exclusion chromatography and ultrafiltration. Characterization was done with Western blot, nanoflow cytometry, and transmission electron microscopy. SP‐IRIS was performed by capturing with CD9, CD63, and CD81 antibodies and visualized by fluorescent imaging with tetraspanins (all cell lines); myelin oligodendrocyte glycoprotein (MOG); neural cell adhesion molecule (CD56); transmembrane protein 119 (TMEM119); and disialoganglioside (GD2). Super‐resolution microscopy was done with tetraspanin labeling. CNS‐specific antibody‐coated magnetic beads were used to compare the presence and abundance of surface markers via flow cytometry. Results : Approximately half of EVs derived from microglia, astrocytes, and oligodendrocytes were triple‐positive for CD9, CD81, and CD63, while only 9% of neuronal EVs expressed all three tetraspanins. Additionally, 84% of microglial and 94% of neuronal EVs were positive for CD81, while astrocytes and oligodendrocytes had higher percentages of CD63+ EVs, at 91% and 82%, respectively. However, putative cell‐specific markers were displayed at a much lower abundance than tetraspanins. Some markers, such as oligodendrocyte proteins MOG, were not detected on EVs. Summary/Conclusion : Biological barriers and low expression of cell‐specific markers are challenges for diagnostic utility of CNS EVs that may need to be overcome by implementing multi‐target capture strategies. Funding : The work is supported by the National Institute of Mental Health (NIMH; R21/R33MH118164). Keywords : extracellular vesicles, SP‐IRIS, super‐resolution microscopy, iPSCs, central nervous system

Tetraspanin

Luca Musante 1 ; Adam Himebauch 2 ; Andrew Hoffman 3 1 University of Pennsylvania, Philadelphia, USA; 2 The Children's Hospital of Philadelphia, USA; 3 University of Philadelphia, USA Introduction : Single particle interferometric reflectance imaging sensing (SP‐IRIS) based ExoViewR100 is a platform capable of detecting EV markers without extensive sample preparation and with a minimal sample volume. However, the kit assay protocol recommends purifying the starting sample by size exclusion chromatography (SEC) to eliminate plasma components that interfere with the analysis. The objective of this study was to compare the detection of tetraspanin of pEVs using 1 microL of platelet poor plasma (PPP), 50 microL of PPP for dialysis (PPPdial) as alternative to SEC to remove interference, and finally 150 microL of PPP purified using SEC (PPPsec) Methods : Whole human blood samples collected in EDTA and centrifuged at 1,500 g for 10' at room temperature (RT) within 30 minutes of collection. Two centrifugations were subsequently carried out at 2,500 g for 10' at RT to obtain PPP. The PPP was dialyzed using a microdialyzer system 500 and a dialysis membrane with a MWCO of 1,000 kDa. The dialysis buffer (120 mL PBS‐EDTA) was changed 3 times within 24 hours. A single qEV 35 nm column was used for SEC. Samples were diluted 60 times PPP, 60 times PPPdial and 4 times PPPsec in incubation buffer II respectively. Samples and antibodies were incubated in accordance with the ExoView kit assay protocol. Results : EVs could be detected using PPP, PPPdial, and SEC fractions. In comparison to PPPsec, background noise detected on the mouse isotype control for PPP was elevated, whereas it was reduced for CD63 (AF647) and CD9 (AF488), but not completely for CD81(AF555) for PPPdial. A close distribution of tetraspanins was seen except for CD81 on both CD41a and CD81 capture spots for the PPPsec. Dialysis followed by SEC did not result in the detection of any tetraspanin on CD41a capture, suggesting that the SEC column may remove preferentially platelet‐derived particles. As a result of SEC, the background noise in the isotype control can be reduced. By contrast, when we calculated EV counts based on the dilution factor, we observed a significant decrease in the counts in the PPPsec, particularly for platelets derived particles. As a result of dialysis, the noise on the isotype control was substantially reduced while maintaining a distribution of tetraspanin close to that of PPP. Summary/Conclusion : In conclusion, dialysis could be an alternative method to SEC to remove plasma component that interfere and it may provide a more native vision of the tetraspanin distribution in pEVs using less sample.

Therapeutic

Jinhee Park; Kyeongeun Park; Sung‐Soo Park; Seung Wook Oh MDimune Inc., Seoul, Republic of Korea Introduction : Cell‐derived vesicles (CDVs) produced by serial extrusion of diverse human cells are emerging as novel delivery vehicles due to their superior biocompatibility and excellent cellular uptake and tissue penetration capabilities. Here, we present an alternative approach for mRNA delivery using CDVs to overcome the immunogenicity and toxicity concerns of synthetic vehicles used in mRNA therapeutics. In addition, the therapeutic potential of mRNA‐carrying CDVs was evaluated by examining key characteristics in vitro and in vivo. Methods : mRNAs were complexed with CDVs via positively‐charged lipids. Fluorescent‐labeled mRNAs or reporter mRNAs such as luciferase and EGFP were used to determine the potential of CDVs for mRNA delivery. Flow cytometry and luciferase reporter assay estimated mRNA uptake and protein expression level. The in vivo delivery and biodistribution of mRNA‐loaded CDVs were assessed by IVIS imaging. Furthermore, acute toxicity and inflammatory response were evaluated by assessing a hematology profile and multiplex cytokine/chemokine panel. All the animal experiments were conducted in comparison with lipid nanoparticles (LNP). Results : In this study, we first established a method of loading mRNAs to CDVs using cationic lipids. The mRNA‐loaded CDVs showed a uniform size distribution with higher than 90 % loading efficiency. We observed that CDVs facilitated the delivery of mRNAs to target cells, resulting in robust protein expression. Additionally, we assessed the biodistribution and toxicity of mRNA‐loaded CDVs in mice. The hematology and cytokine analyses did not detect noticeable alterations in CDVs, whereas multiple immunogenic responses were evident in LNP. Summary/Conclusion : This study demonstrated the potential of CDVs as an mRNA delivery carrier with high loading efficiency and in vivo safety. This finding will facilitate the development of more efficient mRNA‐based therapeutics for cancer and other debilitating diseases.

Unravelling

Daniele D'Arrigo 1 ; Caterina Severi 2 ; Cataldo Schietroma 2 ; Stephanie Mangenot 3 ; Quentin Lubart 2 1 Université Paris Cité, CNRS, Laboratoire MSC, UMR7057, 45 Rue des Saint Pères, 75006 Paris, France; Abbelight, 191 Avenue Aristide Briand, 94230 Cachan, France, Paris, France; 2 Abbelight, 191 Avenue Aristide Briand, 94230 Cachan, France, Cachan, France; 3 Université Paris Cité, CNRS, Laboratoire MSC, UMR7057, 45 Rue des Saint Pères, 75006 Paris, France, Paris, France Introduction : Single‐molecule localization microscopy (SMLM) is gaining interest in the extracellular vesicle (EV) field thanks to high resolution. In SMLM, diffraction‐limited images are used to localize single fluorophores with nanometric precision, generating a super‐resolution picture. Even if used mostly for surface markers, SMLM allows evaluation of several EV features, such as 3D morphology and vesicle cargo. However, SMLM techniques offer lower axial (Z) resolution (∼50nm vs ∼15nm in XY), an issue for precise 3D reconstruction of small EVs. Further optimization is thus required. We used large unilamellar vesicles (LUVs) with typical diameter of 100 nm. These synthetic lipid vesicles with controlled size distribution, are used as model system to mimic in a first approach the EVs, to optimize the EV reconstruction and to improve EV characterization with SMLM. Methods : The LUVs were produced with different lipids composition (DOPC, cholesterol, DHPE‐Cy5). To perform SMLM imaging, the LUVs were captured and immobilized on an optimized surface and the effect of the lipid composition on the imaging was also evaluated. In addition, to determine with higher accuracy and to correct the axial reconstruction, we imaged other reference structures with a precise and known size, such as DNA origami. Finally, we applied this procedure to visualize and assess the morphology and the size of EVs. Results : This approach allowed the accurate size determination of mono‐ and polydisperse LUV preparations, data confirmed by other established techniques, including NTA. The lipid composition seemed to affect the LUV interaction with the capture surface, representing a potential crucial point in the imaging of EVs with SMLM. The optimized 3D reconstruction allowed an improved evaluation of the size and the 3D morphology of both the LUVs and the EVs. Summary/Conclusion : In this work we optimized the 3D resolution of our SMLM microscope in imaging LUVs and EVs, allowing a better size and 3D morphology evaluation. The more precise 3D reconstruction will improve the future characterization of EVs, the cargo assessment and the identification of different EV subpopulations with SMLM. Funding : Research supported by the company Abbelight and the CNRS through the “Plan France Relance.” Keywords : super‐resolution microscopy, single‐molecule localization microscopy, single‐EV imaging, standardization

Upregulated

Judy WP YAM 5 ; Yue Yao 1 ; Yi Xu 1 ; Liang Yu 2 ; Zhi‐Jie Xiao 3 ; Pui‐Chi Tin 2 ; Hiu Ling Fung 2 ; Hoi Tang Ma 2 ; Jing Ping Yun 4 1 The University of Hong Kong, USA; 2 The University of Hong Kong, Hong Kong; 3 Sun Yat‐sen University, China (People's Republic); 4 Sun Yat‐sen University Cancer Center, China (People's Republic); 5 The University of Hong Kong, Hong Kong, Hong Kong Introduction : The successful entry of small extracellular vesicle (sEV) into cells is a prerequisite for the influence of sEVs on the recipient cells. sEV derived from hepatocellular carcinoma (HCC) promote cancer progression and metastasis via multifarious pathways. However, how sEV enters HCC cells remains obscure. This study aims to elucidate the mechanistic basis underlying the internalization of sEV by HCC cells. Methods : The uptake of sEV by cells was examined by fluorescent microscopy. The properties of cells with sEV uptake were evaluated by in vitro and in vivo functional assays. The clinical significance of sEV uptake regulator was studied by multiplex fluorescent immunohistochemistry of HCC patients tissue microarray. Results : Compared to normal liver and non‐metastatic HCC cells, metastatic HCC cells showed the highest capacity to take up TMR‐dextran, an indicator of macropinosome, and PKH67‐labeled sEV. The uptake of TMR‐dextran and PKH67‐sEV by cells was compromised by 5‐(N‐Ethyl‐N‐isopropyl)amiloride (EIPA) which is an inhibitor of macropinocytosis. EIPA reduced HCC cell‐derived sEV in promoting cell growth, motility and tumor formation in mice. EIPA is a Na+/H+ exchanger (NHE) inhibitor that targets solute carrier family 9 family. Among all family members, NHE7 expression was well correlated with the sEV uptake ability of HCC cells. Knockdown of NHE7 in metastatic HCC cells reduced the ability of cells to internalize TMR‐dextran and PKH67‐sEV resulted in diminished HCC growth, motility and tumorigenesis. Conversely, HCC with overexpressing NHE7 displayed enhanced ability to internalize sEV and increased cancer properties. NHE7 localized at late endosomes revealed by its colocalization with Rab21. NHE7 induced cytosolic alkalinity and regulated expression of Rab21. The increased alkalinity promoted maturation of macropinosome thus facilitated sEV uptake and cancer properties of cells. NHE7 was frequently upregulated in HCC, associated with poorer survival and well correlated with Rab21 expression. Lastly, inducible‐knockout of NHE7 in tumors developed in mice remarkedly suppressed liver tumor formation and distant metastasis. Summary/Conclusion : This study reveals the sEV uptake by HCC cells via NHE7‐driven macropinocytosis and provides therapeutic insights into targeting dysregulated NHE7. Funding : Research Grants Council General Research Fund (Reference number: 17105322). Keywords : sEV uptake, hepatocellular carcinoma, macropinocytosis, pH regulator

Vaccination

Hanne C. Winther‐Larsen University of Oslo, Oslo, Norway Introduction : Secretion of extracellular vesicles from the bacterial body (BEV) is associated with a range of phenotypes including cell‐cell communication, host‐pathogen interactions, antimicrobial resistance development and microbial biofilm formation. BEV may also elicit a humoral and antibody mediated immune response after immunization of a host making them an interesting vaccine candidate against an infectious agent. We have investigated the use of BEV as a potential vaccine against several intracellular bacterial pathogens creating problems in aquacultured fish, including Francisella noatunensis, Piscirickettsia salmonis and Yersinia ruckeri. Methods : BEVs were isolated from the bacterial cultures by either ultra centrifugation or tangential flow, and resuspended in PBS. The BEV were further characterized by electron microcopy, mass‐spectrometry and differential light scattering. The zebrafish were immunized with 20 ug BEV‐PBS solution before the fish were challenged with the specific pathogen. Tilapia, Atlantic cod and Atlantic salmon were immunized with 10 ug BEV resuspended either in PBS or formulated with a mineral oil adjuvant before challenged with the same bacterial species from where the BEV was derived. The fish immune response from the BEV immunization and bacterial challenge was determined together with the cumulative survival. Results : Immunization with BEV revealed protection against the diseases in a zebrafish challenge model. However, less protection was detected in the natural hosts; tilapia, Atlantic cod and Atlantic salmon, respectively. While the challenge experiments performed in the natural host was performed in BEV immunized fish together with an oil adjuvant, the zebrafish was immunized with naked BEV. Summary/Conclusion : We hypothesis that the immune protective potential of BEV is host dependent and that the oil adjuvant may have a destructive effect on the immune‐protective potential for BEV. Funding : The Research Council of Norway and The University of Oslo. Keywords : bacterial extracellular vesicles, BEV, aquaculture, immunization, therapeutics, vaccine formulation

Amelioration

Limei Xu 1 ; Yujie Liang 2 ; Jiang Xia 3 ; Li Duan 1 1 Department of Orthopedics, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, China (People's Republic); 2 Department of Child and Adolescent Psychiatry, Shenzhen Kangning Hospital, Shenzhen Mental Health Center, China (People's Republic); 3 Department of Chemistry, and Center for Cell & Developmental Biology, School of Life Sciences, the Chinese University of Hong Kong, Shatin, USA Introduction : The upregulation of miR‐214 in osteoclasts is considered one of the pathogenic factors of osteoporosis (OP), a disease that currently cannot be cured. Suppressing the level of miR‐214 by anti‐miR‐214 in osteoclastsreverses bone absorption and provides a possible remedy. Delivery of anti‐miR‐214 requires a reliable, efficient, and safe vehicle that can specifically target osteoclasts. Red blood cell (RBC) extracellular vesicles (RBCEVs) are a natural‐born, nano‐sized carrier featuring a low risk of gene transfer (RBCEVs do not contain genomic or mitochondrial DNA) and high availability (RBCs are renewable and can be obtained in large quantities), but lack osteoclast‐binding specificity. Methods : A drug delivery system targeting osteoclasts was constructed by modifying RBCEVs with TRAP affinity peptide and loading miR‐214. The effect of miR‐214 targeted delivery by RBCEVs on osteoclast‐related mRNA and protein was detected using qRT‐PCR and western blotting. The targeted effect in vivo was observed using small animal imaging. The therapeutic effect of targeted delivery of anti‐miR‐214 on osteoporosis in mice was tested by immunohistochemistry and micro‐CT. HE staining was used to evaluate the biological safety of the targeted delivery of miR‐214 by RBCEVs. Results : Peptide functionalization displays the osteoclast‐binding motif on the surface of RBCEVs. It endows RBCEVs osteoclast‐targeting capability to deliver anti‐miR‐214 to osteoclasts and pre‐osteoclasts both in vitro and in vivo. Osteoclast‐targeting RBCEVs (OT‐RBCEVs) administered through intravenous injection concentrated in the bone skeleton of mice, inhibited the osteoclast activity, promoted the osteoblast activity, and improved the bone density of the osteoporotic mice. Summary/Conclusion : Altogether, functionalization by the bi‐functional fusion peptide proves to be a facile and modular method to modify the surface of RBCEVs and construct cell‐targeting delivery vehicles. This work also shows that targeted delivery of anti‐miR‐214 by OT‐RBCEVs is a viable method for OP treatment. Funding : National Natural Science Foundation of China (81972116), Shenzhen Science and Technology Projects (SGDX20201103095800003, GJHZ20200731095606019, and JCYJ20170817172023838); Guangdong International Cooperation Project (2021A0505030011). Keywords : osteoporosis, red blood cells, extracellular vesicles, targeted delivery, anti‐miR‐214

Differential

Cherie Saffold 1 ; Carleigh Gray 2 ; Heather H. Pua 3 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA, Nashville, USA; 2 Vanderbilt University Medical Center, USA; 3 Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville TN, USA, Nashville, USA Introduction : YRNAs and YRNA fragments (YsRNAs) are small, noncoding RNAs that cells secrete as ribonucleoprotein complexes (RNPs) or in extracellular vesicles (EVs). Cells can increase or decrease EV‐bound YRNA/YsRNA secretion levels in response to inflammatory stimuli. However, little is known about how YRNAs are secreted during inflammation in vivo. Previous work from our lab shows that select EV‐bound miRNA levels increase in murine bronchoalveolar lavage fluid (BALF) during experimentally induced allergic airway inflammation. In this study, we tested if murine YRNAs (RNY1 and RNY3) and their YsRNAs are differentially secreted in EVs during allergic airway inflammation. Methods : Whole BALF from mice was treated with RNase, proteinase K, and detergent. qPCR was used to quantify YRNA in the steady state lung. To induce allergic airway inflammation, mice were sensitized and challenged with ovalbumin (ova). EVs were purified by sucrose density‐gradient ultracentrifugation from ova‐challenged and vehicle‐challenged mouse BALF. qPCR was used to quantify YRNA levels in each fraction. Results : We detected RNY1, RNY3, and their respective fragments in whole BALF and all three sucrose‐gradient fractions. Whole BALF YRNAs were RNAse‐sensitive in the presence of detergent, but not proteinase K. Additionally, a population of RNY1 fragment was only RNAse sensitive in the presence of proteinase K and detergent. In whole BALF and density gradient purified BALF, RNY1 fragment levels increased in mice with allergic lung inflammation compared to controls. Summary/Conclusion : Murine BALF contains YRNA, and YRNA is present in BALF EVs. A portion of extracellular RNY1 fragment may be bound to protein in EVs. RNY1 fragment levels increase in BALF EVs during allergic airway inflammation. These data contribute to the growing evidence that YRNAs are selectively secreted and may contribute to cell‐cell communication in inflammatory states. Funding : NIH DP2‐ HL152426 (HHP). Keywords : YRNA, extracellular RNA, allergy, inflammation, lung

Establishing

Boyang Su 1 ; Xiaojing Yang 2 ; Jina Nanayakkara 2 ; Jenie Marian Cruz Burgos 3 ; Hon S. Leong 1 ; Neil Renwick 2 1 Department of Medical Biophysics, University of Toronto, Biological Sciences Platform, Sunnybrook Research Institute, Toronto, Canada; 2 Laboratory of Translational RNA Biology, Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada; 3 Instituto Nacional de Medicina Genómica, Cuautitlán Izcalli, Mexico Introduction : Neuroendocrine neoplasms (NENs) are a constellation of rare cancers arising from neuroendocrine cells of many different anatomical sites (lungs, pancreas, etc.). miR‐375 has been reported to be overexpressed in NEN tissues compared to non‐NEN tissues, suggesting its strong promise as a universal biomarker for NENs. Peripheral expression of miR‐375 has not been demonstrated before and for the first time we show its expression in extracellular vesicles (EVs) isolated from NEN patient plasma samples. Methods : EVs were isolated using aqueous two‐phase separation (ATPS) method from plasma samples of NEN patients and healthy volunteers. Classic EV biomarkers (CD9, CD63, CD81) were measured with nanoscale flow cytometry (nFC). Multiple RNA isolation methods (Trizol, RNeasy, exoRNeasy) were tested to determine RNA content of isolated EVs. RNA length was analyzed using Bioanalyzer and miR‐375 expression was measured with RT‐qPCR. Results : Classic EV biomarkers were detected on plasma EVs isolated using ATPS. exoRNeasy kit was found to be the most effective method for RNA isolation from plasma EVs. Length of plasma EV RNA was determined to be between 25 – 200 nt. miR‐375 expression was enriched in NEN patient samples compared to healthy control and statistically significant. Summary/Conclusion : Given that miR‐375 is known to be associated with NEN tissues, we found that it is also a plasma biomarker supporting its use as a promising liquid biopsy target for NENs.

Glioblastoma

Salomé Araujo‐ Abad 1 ; Enrique Rodríguez‐Cañas 1 ; María Fuentes‐Baile 2 ; Pilar García‐Morales 1 ; Ricardo Mallavia 1 ; Miguel Saceda 3 ; José Luis Neira 4 ; Camino de Juan Romero 5 1 Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández, Avda, Universidad s/n, Ed. Torregaitán, Elche, 03202 Alicante, Spain., Spain; 2 Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Camí de l'Almazara 11, Elche, 03203 Alicante, Spain., Spain; 3 Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Camí de l'Almazara 11, Elche, 03203 Alicante, Spain, Spain; 4 Universidad Miguel Hernández, Spain; 5 Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Camí de l'Almazara 11, Elche, 03203 Alicante, Spain, Elche, Spain Introduction : Glioblastoma (GBM), characterized by a fast growth and invasion into adjacent tissue, is the most aggressive cancer of brain origin. Current protocols that include cytotoxic chemotherapeutic agents effectively treat localized disease, however, these aggressive therapies present side effects due to the high doses being administrated. In order to reduce the therapeutic exposure of the patients, we look for more efficient ways of drug delivery. Methods : We have isolated and fully characterized small EVs from seven patient derived GBM cell cultures. Besides, we loaded them with two chemotherapeuric drugs (Temozolomide and EPZ015666) by incubation methods and, the amount of drug loaded was measured by high‐performance liquid chromatography (HPLC) method. Finally, we test their antiproliferative effect on different cancer cell lines. Results : Our study indicates that the direct incubation method was the most efficient loading protocol and that a minimum total amount of drug triggers an effect on tumor cells. Moreover, we have seen that GBM derived small EVs, although with lower target specificity, can also induce an effect on cancer cells of different nature. Summary/Conclusion : GBM derived small EVs represents a promising drug delivery tool that can be further investigated in preclinical studies and be potentially developed in clinical trials for the treatment of GBM. Funding : This research was funded by the Miguel Servet Program to C.d.J.R. from Instituto de Salud Carlos III (CP19/00095) and GVA (CIAICO/2021/135). M.S was funded by FISABIO intramural grants UGP‐20‐135 and UGP‐20‐254.C.d.J.R. and M.S were funded with PI22/00824 by ISCIII.S.A.A was recipient of “Carolina foundation predoctoral fellow‐ship” 2020. The FESEM equipment used in this work was funded by Generalitat Valenciana (Spain) and co‐financed with ERDF funds (OP ERDF of Comunitat Valenciana, Spain) GVA‐IDIFEDER_2018/020, “Una forma de hacer Europa.” Keywords : glioblastoma, small EVs, nanocarriers, chemotherapy, FESEM

Label‐Free

Marina Marjanovic 1 ; Jaena Park 1 ; Janet E. Sorrells 2 ; Eric Chaney 3 ; Edita Aksamitiene 1 ; Amro Abdelrahman 4 ; Jennifer Leiting 4 ; Jennifer Yonkus 4 ; Peter Groves 3 ; Jonathan Harrington 5 ; Isaac Lynch 4 ; Colleen Bushell 3 ; Heidi Nelson 4 ; Mark Truty 4 ; Stephen A. Boppart 1 1 University of Illinois at Urbana‐Champaign, Urbana, USA; 2 University of Illinois at Urbana‐Champaign, Champaign, USA; 3 University of Illinois at Urbana‐Champaign, USA; 4 Department of Surgery, Mayo Clinic, USA; 5 Center for Individualized Medicine, Mayo Clinic, USA Introduction : Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at late stages when surgical and pharmacological treatments become ineffective. Cancer‐associated extracellular vesicles (EVs) can potentially serve as a diagnostic and prognostic biomarker to determine effective personalized treatment regimens and improve patient outcome. In this study, the in vivo chemotherapy treatment response of pancreatic tumor patient‐derived xenografts (PDX) in mice was evaluated using simultaneous label‐free autofluorescence multi‐harmonic (SLAM) microscopy. Methods : Specimens of human pancreatic tumor tissue were surgically placed subcutaneously in NOD SCID mice. A total of 24 mice were used in this 5‐week study, divided between treatment groups (responsive or resistant) that received one chemotherapy regimen and control groups injected only with saline. Chemotherapy regimens included either a combination of 5‐fluorouracil, irinotecan, and oxaliplatin, or gemcitabine and nab‐paclitaxel. Tumors were imaged both intravitally and ex vivo after the animals were euthanized. Label‐free multimodal multiphoton imaging was used to provide simultaneous, co‐registered structural and functional images of tumors and EVs in untreated samples. Heterogeneous populations of EVs could be identified by their unique optical signatures. Results : Elevated EV density was observed in both chemotherapy responsive and resistant tumors during the early response (weeks 1 and 3) and remained elevated (thru week 5) in the resistant tumors. The optical redox ratio of EVs decreased with treatment (weeks 1 and 3), but by week 5, returned to baseline in the resistant tumors, and remained low for the responsive tumors. Summary/Conclusion : These results suggest the potential for using this label‐free optical technology and methodology to detect and characterize EVs by their optical signatures, which can be utilized as possible biomarkers for determining chemotherapy responsive vs. resistant tumors. Funding : Funding was provided by a Seed Grant from the Mayo Clinic & Illinois Alliance for Technology‐Based Healthcare, and grants from the National Institutes of Health (R01CA213149, P41EB031772, S.A.B.). Keywords : multiphoton microscopy, pancreatic cancer, chemotherapy, drug resistance

Metabolomics

Elaine M. Richards 1 ; Stephanie Merfeld‐Clauss 1 ; Dmitry Traktuev 2 ; Keith L. March 1 1 University of Florida, Gainesville, USA; 2 University of Florida, USA Introduction : Secretome, and the extracellular vesicles (EVs) it contains, has been progressively recognized as mediating most physiologic and therapeutic effects of mesenchymal stem/stromal cells. Many studies have characterized various proteomic and miRNA components of MSC secretome and its EVs but relatively little is known about its metabolome, and its role in the activity of secretome and its EVs. Methods : We compared the metabolome of human adipose‐derived stem/stromal cell (ASC) secretome derived from expanded ASC of two healthy donors and control media (n = 4 per donor), using global metabolomics profiling with a Thermo Q‐Exactive Orbitrap mass spectrometer with Dionex UHPLC and autosampler. Samples were analyzed in positive (POS) and negative (NEG) heated electrospray ionization with mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18‐pfp 100 × 2.1 mm, 2 μm column with mobile phases A 0.1% formic acid in water and B acetonitrile with flow rate 350 μL/min and column temperature 25°C. MZmine (freeware) was used to identify features, deisotope, align features and fill gaps. Adducts and complexes were removed. Results : PCA plots of both modes of all metabolites revealed distinct separation of the secretomes from control media (74 and 68% of total variance explained by PC1+PC2 in POS and NEG mode respectively); the secretome groups were substantially overlaid in the POS mode but separated in PC2 in NEG mode. PLS‐DA plots showed separated plots in both modes in PCA2 (observed statistic 0.99 in both modes). Thus, these two ASC secretomes share many common metabolites, and also display inter‐donor differences. Summary/Conclusion : These data provide a foundation for mechanistic insight into activities of MSC‐S and MSC‐EV that influence metabolic processes, and also may assist in defining donor variability and batch‐to‐batch consistency in the production of MSC‐S and MSC‐EV for clinical therapeutics.

Multi‐Omic

Yue Zhang 1 ; Tao‐Tao Tang 2 ; Lin‐Li Lv 2 ; Bi‐Cheng Liu 3 1 Southeast University, China (People's Republic); 2 medical school of southeast university, Nanjing, China (People's Republic); 3 Zhong Da Hospital, Southeast University, Nanjing, China (People's Republic) Introduction : Mesenchymal stem cell‐derived extracellular vesicle (MSC‐EVs) are a bright star in regenerative medicine. A growing body of evidence has demonstrated the effectiveness of MSC‐EVs in animal models of renal disease and cardiovascular disease by virtue of their cargo. However, before clinical translation, there is a need to optimize MSC culture condition without any substrate of animal origin for EV harvest. In the present study, we aimed to identify the optimal xeno‐free culture medium for the clinical‐grade production of MSC‐EVs and employed multi‐omic analysis for the molecular and functional profiling of these vesicles. Methods : MSCs were isolated from human umbilical cords and cultured with xeno‐free media until passages 6 (P6) to evaluate differentiation potential, cell doubling time, metabolic activity, apoptosis and cellular senescence. EVs were prepared from the culture supernatants by differential centrifugation (2000g, 4°C, 30min; 13500g, 4°C, 30min; 100000g, 4°C, 2h) and were further purified via size exclusion chromatography. The purified EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, NanoFCM and western blotting analysis of exosomal markers. MSC‐EVs from four donors were subjected to multiomics analysis, including transcriptomics, proteomics, metabolomics and lipidomics. Results : First, we analyzed the influence of different xeno‐free media on biological properties of MSCs and found that human platelet lysate (HPL)‐supplemented media support the isolation and long‐term proliferation of MSCs. Compared to FBS‐based media, HPL augmented proliferation and metabolic activity of MSCs while improving apoptosis and senescence, suggesting that HPL is an efficient alternative to FBS for MSC culture. Next, we purified the EVs from FBS or HPL‐cultured MSCs and verified them according to the MISEV2018 guideline. Nonsignificant differences in protein markers, size, and morphology were found between these two vesicles. Interestingly, multiomics analysis clarified that differences in the composition of MSC‐EVs under FBS and HPL culture conditions were mainly focused on proteins and miRNAs but not mRNAs, lncRNAs, cirRNAs, hydrophilic metabolites and lipids. However, more than 80% of differential miRNAs and 90% of differential proteins were not highly expressed in HPL‐produced MSC‐EVs, indicating that the therapeutic potential of MSC‐EVs may not be affected by HPL. GO enrichment analysis of the highly expressed miRNAs showed they are associated with regeneration and development, immune regulation, and extracellular matrix composition. Moreover, principal component analysis revealed a similar molecular content of MSC‐EVs from different donors. Summary/Conclusion : HPL‐based culture condition well‐maintained the molecular composition and therapeutic potential of MSC‐EVs and could serve as an efficient xeno‐free alternative for the production of clinical‐grade MSC‐EVs.

Neurotrophic

Jonathon Sewell ; Ashley Mason; Austin Keeler; Bettina Winckler; Christopher Deppmann University of Virginia, USA Introduction : In the developing peripheral nervous system, sympathetic circuits are tuned by limiting amounts of nerve growth factor (NGF) that is secreted by target organs (e.g. salivary gland or eye). NGF binds its receptor, TrkA, at the distal axon of innervating sympathetic neurons and internalizes onto retrogradely trafficked signaling endosomes. Insufficient NGF uptake leads to cell death. These neurons are innervated by autonomic motor neurons from the spinal cord, the survival of which is also NGF‐dependent, despite lacking TrkA or access to soluble NGF. How these motor neurons survive without access to NGF is unknown. We hypothesize that TrkA positive extracellular vesicles (EVs) secreted from sympathetic neurons provide this neurotrophic support. Methods : Using cultured sympathetic neurons from mouse superior cervical ganglia, we isolated EVs from serum‐free, conditioned media by differential centrifugation and characterized them using nanoparticle tracking analysis, immunoblot assays, and cryo‐electron microscopy. To assess neurotrophic capacity, mouse spinal cord neurons were cultured with sympathetic EVs and cell death in ChAT+ motor neurons was assessed by Annexin V staining. Results : The sizing distribution of isolated EVs show a predominant peak at 45–75nm with a shoulder of larger sizes, while protein analysis shows an enrichment of EV markers (Alix, CD63, CD81) and absence of intracellular markers (Cytochrome C, calreticulin). We identify TrkA as an EV cargo, which can be present in its active, phosphorylated state. Using a compartmentalized culture system, we show that TrkA originating from distal axons can be packaged in EVs secreted from the somatodendritic domain. In addition, sympathetic EVs promote motor neuron survival, suggesting they provide neurotrophic support. Summary/Conclusion : We have rigorously characterized EVs from sympathetic cultures and shown that retrogradely trafficked TrkA can be a cargo. These EVs are capable of supporting autonomic motor neuron survival, suggesting a novel mechanism in sympathetic circuit tuning.

Optimisation

Nihar Godbole 1 ; Akhilandeshwari Ravichandran 2 ; Andrew Lai 3 ; Dominic Guanzon 4 ; Falvio Carrion 5 ; Priyakshi Kalita ‐ de Croft 6 ; Laura Bray 2 ; Carlos Salomon 7 1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, UQ Centre for Clinical Research (UQCCR), Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, QLD, Australia., BRISBANE, Australia; 2 Institute of Health and Biomedical Innovation (IHBI), Faculty of Engineering, School of Mech., Medical & Process Engineering, Queensland University of Technology, QLD, Australia, USA; 3 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia, Brisbane, Australia; 4 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, QLD 4029, Brisbane, Australia; 5 Departamento de Investigación, Postgrado y Educación Continua (DIPEC), Facultad de Ciencias de la Salud, Universidad del Alba, Santiago, 8320000, Chile., USA; 6 Exosome Biology Laboratory, Centre for Clinical Diagnostics, UQ Centre for Clinical Research (UQCCR), Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, QLD, Australia, Brisbane, USA; 7 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia., Brisbane, Australia Introduction : Ovarian cancer (OC) is the most common gynaecological malignancy and globally it is the eighth commonly diagnosed cancer in females. Studies have implicated liquid biopsies like Extracellular Vesicles (EVs) could be used as a potential biomarker for multiple diseases including OC. Studies indicate culturing cells in 3D system can better mimic the cellular microenvironment than the 2D monolayers. Therefore, this study aimed to optimise and establish a standard technique for developing 3D models, as well as to isolate and characterise the EVs and cells from these models. Methods : OC cell lines used in this study ‐ SKOV‐3 and OVCAR‐3 were cultured as a 2D monolayers and were also encapsulated using Gelatin methacryloyl hydrogels at the seeding density of 50,000 cells per 20ul gel. Cultured for nine days, multiple assays were performed on 3D cells to observe the spheroid formation inside the hydrogel, which were later characterised histologically. The EVs were isolated from the cell‐conditioned media using differential centrifugation and characterised using Nanoparticle Tracking Analysis, Transmission Electron Microscopy, and ExoView®. qPCR was performed to analyse the expression of the different miRNA associated with chemoresistance in ovarian cancer isolated from EVs and spheroids. Results : Based on the initial experiments performed in this project, on cell‐laden hydrogels, we observed the ability of OC cells to grow, proliferate, and aggregate inside the hydrogel, thus characterising this as an ideal 3D model. The structures of these spheroids were further validated histologically. Furthermore, we were able to successfully isolate and characterise EVs from these 3D models based on the size, morphology, and surface markers. Lastly using qPCR, we were able to observe miRNAs differentially expressed within spheroids and EVs. Summary/Conclusion : Results indicated that the encapsulated cells were able to grow and proliferate into spheroids and were also used for harvesting the different populations of EVs. Therefore, characterising this as an optimised 3D model that can be used for extracellular vesicles studies. Furthermore, this in vitro model could be used for understanding and developing therapeutics for ovarian cancer.

Optimization

Chiranth K. Nagaraj 1 ; Truc K. Nguyen 2 ; Xilal Y. Rima 3 ; Minh‐Dao Duong‐Thi 2 ; Jacob Doon‐Ralls 4 ; KARUNYA ALBERT 2 ; Eduardo Reátegui 5 1 The Ohio State University, columbus, USA; 2 The Ohio State University, Columbus, USA; 3 The Ohio State University, USA; 4 The ohio state university, USA; 5 Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, USA Introduction : The advances in droplet microfluidics in the last decade show immense potential to produce lipid nanoparticles (LNPs). The enhanced mixing capabilities of microfluidics allow lipid fragments to recombine as novel therapeutic carriers. Lipid membranes (LM) are considered waste in various protein isolation processes. In contrast, these LM on EVs contain sensitive information on physiological and pathological disorders in humans. The production of recombinant extracellular vesicles (rEVs) using conventional sonication methods suffer from a wide range of size distribution, low reproducibility, and reduced stability of encapsulated molecular cargo. Therefore, we developed a microfluidic approach to control the size of rEVs and the contents of encapsulated molecular payload. Methods : The lipid components are fed as a dispersed phase on an array of flow‐focusing and herringbone microfluidic devices. The continuous high throughput process is optimized by considering hydrodynamic forces, capillary number, geometry, and flow rate ratios ranging from 1:4 ‐ 1:20 μL/min to control size. The device can produce highly homogeneous nanodroplets of sizes including 80, 100, 120, 150, and 200nm. Characterization methodologies include Tunable Resistive Pulse Sensing (TRPS) for size, Total Internal Reflection Fluorescence Microscopy (TIRFM) for analyzing the internal contents of rEVs with molecular beacons. Results : The microfluidic devices were effectively compared and optimized to produce a monodispersed hydrodynamic diameter of ∼ 120nm. Large‐scale production is underway with the herringbone device for volumes required to perform clinical trials. rEVs with sizes over 200nm can also be produced with low polydispersity. Summary/Conclusion : The novel rEVs carrying advanced therapeutics can escape immunogenic responses and cross the blood‐brain barrier to accurately target and cure pathological diseases. Keywords : recombinant extracellular vesicles, personalized therapeutics, droplet microfluidics

Purification

Paschalia Pantazi 1 ; Vladimir Bokun 1 ; Blair Strang 2 ; Yan Liu 3 ; Beth Holder 1 1 Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, United Kingdom; 2 Infection and Immunity Research Institute, St George's, University of London, London, USA; 3 Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, USA Introduction : Investigation of extracellular vesicles (EVs) produced by virus‐infected cells is challenging due to the variety of particle types that are released into the cell culture supernatants and their similar physical and biochemical features. In this work, we developed a simple centrifugation‐based approach for isolating EVs in human cytomegalovirus (HCMV) ‐infected cell conditioned medium free from HCMV virions and dense bodies, and validated our results using a state‐of‐the‐art flow nanoanalysis (FNA) method. Methods : We utilized uninfected or HCMV‐infected human foreskin fibroblast (HFF) cells to generate conditioned culture media, which was clarified by low‐speed centrifugation and 0.45‐μm filtration. Cell‐free extracellular particle suspensions were fractionated by centrifugation at 25,000xG for 2.5hr over 16% iodixanol cushions. EVs were then purified from resulting supernatants by ultrafiltration and size exclusion chromatography, and fractions analyzed by nanoparticle tracking analysis (NTA), ELISA for EV markers, electron microscopy (EM), and FNA for sizing and nucleic acid content. Results : Nucleic acid labelling combined with FNA enabled visual separation of virions and EVs. As evidenced by FNA and EM, a near‐complete fractionation of the particles following the centrifugation step was achieved. Due to higher particle density, HCMV virions and dense bodies penetrated through 16% cushions and formed pellets, while less dense EVs were remained in the supernatant, thereby affording a successful separation of viral particles and EVs. EVs purified from the post‐centrifugation supernatants were positive for beta‐actin and CD63 by ELISA. Summary/Conclusion : Our method for purification of EVs from HCMV‐infected cells is simple, gentle, does not require specialized equipment, and could be adapted to other viruses. These methods will enable future wide‐ranging functional and biochemical investigation of EVs produced by infected cells. Funding : This project was supported by Imperial College President's PhD scholarship awarded to Vladimir Bokun and a Genesis Research Trust grant to Vladimir Bokun, Yan Liu and Beth Holder. Acquisition of nanoFCM instrument was supported by a Wellcome Trust multi‐user equipment grant awarded to Dr Beth Holder and Dr Richard Kelwick. Keywords : human cytomegalovirus, extracellular vesicle isolation, viral infection

Quantitative

Xiaoyu Ren ; Changsun Kang; Dongin Kim University of Oklahoma Health Sciences Center, USA Introduction : Exosomes, one of the subtypes of extracellular vesicles, range from 50 to 200 nm in diameter and regulate cell‐to‐cell communication in the biological and pathological processes. Although exosomes derived from tumors have various functions in cancer progression, resistance, and metastasis through cancer exosome‐derived tropism, there is no quantitative information on cancer exosome‐derived tropism that will be beneficial to guide cancer therapy by inhibiting exosome release or uptake. Methods : Using two ovarian cancer cell lines (OVCA4 and OVCA8) that were transfected with the fluorescent protein (mkate2), tropism of cancer cell exosomes was quantified by measuring the release of exosome number from “parent” ovarian cancer cells and determining the uptake of these exosomes amounts into “parent” ovarian cancer cells, 3‐D spheroids, and tumors from tumor‐bearing mice models. Results : An OVCA4 cell releases about 50 to 200 exosomes, and single OVCA8 cell secretes about 300 to 560 exosomes. Multifold exosome uptake from ovarian cancer cells into the “parent” ovarian cancer was observed compared to non‐cancer cells. In vivo tumor‐bearing mice models, most exosomes (200 to 600 million) are home to the “parent” cell tumors. Summary/Conclusion : Quantification of the release of cancer‐derived exosomes and the uptake of the exosomes into their “parent” cancer cells, displayed the target tropism of cancer‐derived exosomes. These results will be beneficial for future diagnosis and therapeutic applications. Funding : This publication's research was partly supported by NIH/NIGMS COBRE grant (P20GM103639).

Reproducible

Andrew Lai 1 ; Dominic Guanzon 2 ; Carlos Palma 2 ; Flavio Carrion 3 ; Ryan Cohen 4 ; Sunil Lakhani 5 ; Andreas Obermair 6 ; Andreas Moller 7 ; Carlos Salomon 8 1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia, Brisbane, Australia; 2 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia, Australia; 3 Departamento de Investigación, Postgrado y Educación Continua (DIPEC), Facultad de Ciencias de la Salud, Universidad del Alba, Santiago, Chile, Santiago, Chile; 4 School of Biomedical Sciences, The University of Western Australia, and Colorectal Research Unit, St John of God Healthcare, Perth, Western Australia, Australia., Australia; 5 UQ Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, QLD, Australia. Pathology Queensland, The Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia, Brisbane, USA; 6 Queensland Centre for Gynaecological Cancer Research, The University of Queensland Centre for Clinical Research, Herston, Queensland, Australia, Brisbane, USA; 7 Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia., Brisbane, Australia; 8 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia., Brisbane, Australia Introduction : The current method of isolating extracellular vesicles, such as ultracentrifugation, can be time‐consuming, requiring specialised equipment, labour intensive, and quality control can be challenging. Here we describe the development of a reproducible, automated method to isolate extracellular vesicles using high‐performance liquid chromatography (HPLC) that can be applied to routine pathology laboratories. Methods : A modular HPLC system with a fraction collector coupled with either a multimodal chromatography or size‐exclusion column was used for this study. To optimise the HPLC method, replicates of fetal bovine serum or human serum were processed (200 μL), with multiple elution fractions collected from each starting sample. EVs containing fractions were selected based on Nanoparticle Tracking Analysis (NTA), Western blots, and electron microscopy. Reproducibility was calculated based on NTA and protein quantification data. To further demonstrate this isolation platform, the isolated EVs from human plasma were subjected to next‐generation sequencing (NGS) and mass‐spectrometry proteomic analysis. Finally, to show the real‐world clinical utility of this automated isolation method, we isolated EVs and investigated the proteomic and miRNA profile of EVs from patients with ovarian cancer (n = 36), colorectal cancer (n = 26), endometrial cancer (n = 20), lung cancer (n = 30), brain metastasis (n = 36), and controls. control. A pooled plasma sample was also isolated intersperse throughout as a quality control. Results : A highly reproducible method of isolating extracellular vesicles from bovine and human plasma was achieved. Fractions containing EVs were confirmed. The average inter CV for among the technical replicate was below 10% for the particle mean (3.75 %), mode (7.68 %) and protein concentration (8.45 %). This suggests that this method is reproducible with low technical variations. In addition, using the optimised automated method from one biological sample, most of the total vesicles can be purified into only one tube. Furthermore, the purity of the isolated vesicles was comparable to current isolation techniques and can be used for downstream analyses such as NGS and proteomics. Using PCA analysis on the proteomic and miRNA data, we demonstrated that we could identify cancer‐type‐specific plasma EVs using our HPLC system. Summary/Conclusion : Using automated systems to isolate extracellular vesicles could have important implications in diagnostics, where high throughput and reproducibility is essential. Keywords : isolation, size exclusion chromatography

Scaling‐Up

Ana Meliciano 1 ; Ana D. Louro 2 ; Pedro Vicente 3 ; Cláudia Diniz 3 ; Margarida Serra 4 1 iBET ‐ Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal, Oeiras, Portugal; 2 iBET ‐ Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal, Lisboa, Portugal; 3 iBET ‐ Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal, Portugal; 4 iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal, Lisboa, Portugal Introduction : Human‐induced pluripotent stem cells (hiPSC) are a promising source of native EV for cardiac regeneration, providing advantages over stem‐cell based therapies for repairing adult cardiomyocytes with limited self‐renewal after injury. However, large‐scale manufacture of clinical grade hiPSC‐EV remains a bottleneck for clinical translation. In this study, we developed a scalable bioprocess for hiPSC‐EV mass production in stirred‐tank bioreactors (STB). Methods : Briefly, hiPSC were expanded as monolayers in static adherent cultures or as 3D aggregates in STB (200 mL working volume), operated in perfusion (D = 1.3 day‐1) with low levels of dissolved O2 (4% O2). EV were isolated from conditioned culture medium by density gradient ultracentrifugation. We investigated the size, morphology, and protein content of EV using electron microscopy, nanoparticle tracking analysis, and western blotting. EV bioactivity was assessed in endothelial cells and hiPSC‐derived cardiac fibroblasts. The EV's functional cargo (miRNAs) was assessed using RT‐qPCR. Results : Our results demonstrated a 2.9‐fold increase in cell concentration in STB compared to the static culture, which resulted in a 3.1 increase in total particles isolated. hiPSC‐EV produced in the STB presented a cup‐shaped morphology and were positive for EV markers. Tube formation assays showed increased pro‐angiogenic activity for hiPSC‐EV produced in STB versus static 2D monolayer culture, and a different miRNA content was found between these EV. Summary/Conclusion : Overall, our study validated the STB system for large‐scale EV production and showed the impact of cell culture on EV composition. Ongoing work aims at scaling the current process for 2 L bioreactors to accelerate clinical development even more. Funding : FCT PhD fellowships (SFRH/BD/145767/2019; UI/BD/151255/2021); CARDIOPATCH Interreg SUDOE (SOE4/P1/E1063); BRAV3 (H2020‐SC1‐BHC‐874827); iNOVA4Health (UIDB/04462/2020; UIDP/04462/2020); LS4FUTURE (LA/P/0087/2020).

Simultaneous

Lien Lippens 1 ; Niké Guilbert 2 ; Sofie Van Dorpe 2 ; Sarah Deville 2 ; Robin Boiy 2 ; Quentin Roux 2 ; Nicolaas Lumen 3 ; Ilkka Miinalainen 4 ; Pekka Rappu 5 ; Katrien Vandecasteele 6 ; Hannelore Denys 7 ; Bruno De Geest 8 ; Olivier De Wever 2 ; An Hendrix 1 1 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium, Ghent, Belgium; 2 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium, Gent, Belgium; 3 Department of Urology, Ghent University Hospital, Ghent, Belgium, Belgium; 4 Biocenter Oulu, University of Oulu, Oulu, Finland, Oulu, Finland; 5 Department of Life Technologies, University of Turku, Turku, Finland, Turku, Finland; 6 Radiotherapy, Department of Human Structure and Repair, Ghent University, Ghent, Belgium, Belgium; 7 Medical Oncology, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent, Belgium, Ghent, Belgium; 8 Department of Pharmaceutics, Ghent University, Ghent, Belgium, Ghent, Belgium Introduction : Extracellular vesicles (EVs) are increasingly studied as diagnostic and prognostic biomarkers. Typically, the analysis of EV‐associated biomarkers requires their separation from biofluids, which warrants larger input volumes, leads to longer turn‐over times, and increases technical variability. To overcome these issues we aimed to develop a one‐step workflow for simultaneous size‐based fractionation and biomarker detection. Methods : We coupled a multi‐angle light scattering detector (MALS) and a fluorescent light detector (FLD) in‐line with the asymmetrical flow field‐flow fractionation (AF4) equipment. We first optimized the AF4‐MALS‐FLD parameters using recombinant EVs (rEVs) including spacer (350 μm), membrane (10 kDa regenerated cellulose), running buffer (PBS + 0.02% NaN3), detector flow, and cross flow profile. Next, we evaluated the performance of the AF4‐MALS‐FLD set‐up using diverse biofluids including cell culture medium, urine and blood plasma. Fractions were collected and quality was controlled using complementary characterization methods in compliance with MISEV2018 guidelines. Relevant experimental parameters were submitted to the EV‐TRACK knowledgebase (EV‐TRACK ID: EV210212 ). Results : We were able to analyze EV surface markers CD9, CD63 and CD81, and cancer biomarkers HER2, EpCAM and PSMA in pre‐purified EV preparations from different breast and prostate cancer cell lines. Analysis of increasing numbers of EVs revealed a linear correlation with the number of particles as measured by NTA (4E+9 – 2E+10 particles). Furthermore, the AF4‐MALS‐FLD workflow allowed to detect EVs in samples with increasing complexity as cell culture supernatant, urine and blood plasma. Finally, we validated the one‐step workflow by confirming the presence of PSMA‐positive EVs in urine from prostate cancer patients (n = 25), and by demonstrating the presence of EpCAM‐ or HER2‐positive EVs in blood plasma enabling discrimination of breast cancer patients (n = 15) from healthy donors (n = 15). Summary/Conclusion : We have successfully optimized and validated the AF4‐MALS‐FLD workflow, that allows simultaneous size‐based fractionation and surface marker characterization in a fast, reproducible and sensitive manner. Funding : This work was supported by UGent, FWO, CRIG, and KOTK (the Flemish cancer society).

Transcardiac

Mohsin Khan 1 ; Jennifer Pennise 1 ; Antonia Yuko 2 ; Daria Harlamova 2 ; Sadia Mohsin 2 ; Fabio Recchia 2 1 Lewis Katz School of Medicine, Temple University, Philadelphia, USA; 2 Lewis Katz School of Medicine, Temple University, USA Introduction : The heart is often considered as a recipient of signals from the periphery to fine tune blood pumping in response to physiological and pathological changes. Cardiac secretory function consisting of proteins and peptides termed as cardiomyokines has founded the concept of heart mediated organ crosstalk. Studies have shown extracellular vesicles (EVs) are major players regulating organ crosstalk between peripheral organs and the heart. Nevertheless, whether cardiac tissue can release EVs with unique composition and content to deliver messages to peripheral organs and the heart itself remains unknown. Methods : Paired serum samples isolated from coronary sinus (CS) and aorta (AO) blood of dogs under normal and tachypacing induced heart failure were used for extracellular vesicle (EVs) isolation, characterization and miRNA array analysis. In vitro functional assays tested the effect of CS and AO EVs under normal and heart failure conditions on cardiomyocytes and adipocytes. Transplantation of EV from CS and AO samples under normal and heart failure conditions in wild type mice was done to determine changes in whole body metabolism by CLAMs, modulation of adipose tissue metabolism, changes in the heart and immune response. Results : Results showed heart failure promotes increased secretion of EVs in the CS compared to AO blood suggesting heart produces more EVs under diseased conditions. miRNAs array analysis identified top elevated miRNAs as miRNAs 16, 92 and 137 significantly enriches in CS EVs under heart failure conditions. Measurement of miRNAs 16, 92, 137 is different tissues showed significantly increased expression in brown adipose tissue (BAT) compared to all other tissue assessed. Next, 3TC‐LI adipocytes treated with CS EVs from heart failure dogs showed decreased OXPHOS measured by Seahorse, reduced expression of mitochondrial markers and low BODIPY labelling together with increase in whole body oxygen consumption rates, food intake and energy expenditure as measured by CLAMS. Mechanistically, EV derived miRNAs 16, 92, 137 significant transcriptomic changes BAT assessed by bulk RNA‐sequencing. Top altered genes identified in BAT were UQCR10, CPT1a and PGC1a after administration of CS EVs from heart failure animals and are binding targets for miRNAs 16, 92 and 137 respectively. Summary/Conclusion : In conclusion, cardiac tissue communicates to peripheral organs including adipose tissue by releasing EVs derived miRNAs enriched differently in response to heart failure or under normal conditions. Top enriched EV derived miRNAs released by the heart regulate metabolic changes in adipose tissue suggesting heart‐adipose crosstalk.

Blood‐Based

Sonal Sukreet 1 ; Elise Kim 2 ; Melissa Petersen 3 ; Fan Zhang 3 ; Sid O'Bryant 3 ; Michael Rafii 4 ; Robert A. Rissman 5 1 University of California‐San Diego, San Diego, USA; 2 UCSD, USA; 3 UNTHSC, USA; 4 Keck School of Medicine and Medical Director of the Alzheimer's Therapeutic Research Institute (ATRI), USA; 5 UC San Diego, USA Introduction : Individuals with Down Syndrome (DS) show AD neuropathology and cognitive impairment at an accelerated rate as they age. There are limited resources for treating AD in DS patients (DSAD). Using biobanked plasma samples from a previously completed AD/DS clinical trial of anti‐inflammatory treatment, we aimed to characterize AD neuropathology in blood‐based small extracellular vesicles (EVs) over time and as a function of treatment to develop a precision medicine approach for use in DS trials. We hypothesized that changes in amyloid β (Aβ) peptides, total tau (taut), Nf‐L, and GFAP in neuronal and astrocyte EVs (NDEVs/ADEVs) might reflect treatment response in DSAD individuals. Methods : We used archived plasma samples (n = 138; with 106 non‐demented and 32 demented subjects) from the previously completed Phase 3 clinical trial, Vitamin E in Aged Persons with Down Syndrome ( NCT00056329 ). Small EVs were isolated from baseline and 36‐month plasma of DSAD individuals treated with Vitamin E or placebo. NDEVs and ADEVs were enriched with magnetic immunocapture using neuronal and astrocyte‐specific proteins CD171 and GLAST, respectively and fluorescence‐activated cell sorting. NDEVs and ADEVs were characterized for size, integrity and homogeneity as per the guidelines of the International Society of Extracellular Vesicles using nanoFCM, electron microscopy, immunoblots, and ELISA. AD marker proteins Aβ40–42, taut, Nf‐L and GFAP were quantified by SIMOA assays in native plasma, NDEVs and ADEVs of DSAD individuals in treatment vs. control group. Results : Blood‐based NDEVs and ADEVs demonstrated the expected size, shape, and distribution of small EVs i.e. exosomes. Immunoblots and ELISA showed the presence of small EVs marker proteins. Various AD biomarkers were significantly altered in NDEs and ADEs of the vitamin E‐treated DSAD group compared to the control. Summary/Conclusion : Changes in AD protein markers can serve as a biomarker of treatment response in DSAD individuals. Funding : INCLUDE‐NIH R01AG073979.

Cerebrospinal

Daniel Offen 1 ; Shay Herman 2 ; Brit mollenhauer 3 ; Ruth djaldetti 2 1 Tel Aviv University, Tel Aviv, Israel; 2 Tel Aviv University, Israel; 3 University Medical Center Göttingen, Germany Introduction : Parkinson's disease (PD) is characterized by the gradual appearance of intraneuronal, Lewy aggregates (Lewy bodies), which are primarily composed of misfolded α‐ synuclein (α‐syn), resulting in cytotoxicity and neural death. Methods : death. To examine whether aggregated α‐syn can actively spread from the nasal cavity to the brain via exosomes and initiate pathological aggregate in the brain, we isolated exosomes from patients CSF‐ PD patients or non‐PD patients. Results : We found that intra nasal delivery of CSF‐derived exosomes from PD patients induce impairments in motor behavior, hyposmia, elevated anxiety and gained less weight. Summary/Conclusion : These data suggest that exosomes are involved in the propagating α‐syn aggregation and in the initiating of PD‐like symptoms. Funding : NA. Keywords : parkinson's disease, CSF

Complementing

Subhadip Ghatak 1 ; Anu Sharma 2 ; Xuyao Zeng 3 ; Adam Anthony 4 1 Indiana University, Indianapolis, USA; 2 Indiana University, Apt 42, USA; 3 Indiana University, Bloomington, USA; 4 Indiana University, USA Introduction : Paracrine effects via exosome emerged as the primary mechanisms of cell‐cell crosstalk at the wound site. Resolution of wound inflammation relies on successful crosstalk between keratinocytes that must re‐epithelialize, and the blood‐borne wound‐site macrophage, which must mount and timely resolve inflammation. We tested the hypothesis that keratinocyte‐originated exosomes (EXO‐k) are specifically directed to inflammatory macrophages for the resolution of wound inflammation. Methods : Murine EXO‐k were genetically labeled with GFP reporter using tissue nanotransfection and isolated using differential ultracentrifugation followed by immunomagnetic separation. Isolated wound‐edge (WE) EXO‐k was characterized per MISEV 2018 guidelines and reported in EV‐track (EV 220292; EV‐metric score 100%). Protein cargo on EXO‐k surface was detected, validated, and quantified using LC‐MS/MS, dSTORM imaging, and flow cytometry. The cellular source of Complement Factor I (CFI) was determined following transfection with either KRT14 or Lyz2 promoter‐driven CFI plasmids with an “in frame” reporter. N‐glycans compositions from EXO‐k were analyzed by capillary electrophoresis‐mass spectrometry (CE‐MS). The Glycoproteome of EXO‐k was generated using LC‐MS/MS. Results : In non‐diabetic WE, EXO‐k were selectively taken up by inflammatory macrophages. CE‐MS identified 51 unique N‐glycan compositions of which 7 glycans including mannose were exclusively present in WE‐ EXO‐k. CFI is known to be released by macrophages for opsonization, and also by keratinocytes for complement activation. dSTORM imaging identified that 58% and 7% of CFI on EXO‐k were of keratinocytes and macrophage origin respectively. Uptake assay showed that unlike EXO‐k isolated from m+/db WE, uptake of diabetic EXO‐k was significantly compromised by inflammatory macrophages. Glycoproteomics analysis of EXO‐k isolated from m+/db and diabetic db/db WE tissue identified a total of 584 glycopeptides. A comparative analysis revealed that glycoprotein CFI was present in significantly lower abundance in diabetic EXO‐k. Glycosylation mapping of the 3 glycosylation sites of CFI showed significant fucosylation in diabetic EXO‐k. A sialylation profile analysis of CF1 revealed that the percentage of triply sialylated glycoforms is significantly lower in EXO‐k under diabetic conditions (* p< 0.05). Summary/Conclusion : This work provides a novel insight into the pathophysiological mechanism of diabetic EXO‐k that results in compromised crosstalk between WE keratinocytes and macrophages. Hyperglycemia dysregulates exosome surface glyco modification of EXO‐k. These perturbations compromise crosstalk between keratinocytes and wound macrophages complicating the resolution of wound inflammation. Funding : NIH R56DK129592 to SG. Keywords : keratinocyte‐originated exosomes, resolution of inflammation, complement factor I, macrophages

Encapsulating

yaoyao Lu 1 ; Cedric Happi‐mbakam 1 ; Jacques‐p Tremblay 2 1 CHU de Quebec research centre, Laval university, Canada; 2 CHU de Quebec research centre, Laval University, Canada Introduction : The CRISPR‐Cas9 system is a promising technology that revolutionized genome editing applications for the potential treatment of a variety of genetic diseases. However, efficiently delivering the CRISPR‐Cas9 components to the target organ or cell remains a significant challenge. Our study aimed to encapsulate CRISPR/Cas9 protein into extracellular vesicles (EVs) by protein S‐palmitoylation, which is the reversible addition of fatty acids to the cysteine residues of proteins. It is an important post‐translational modification that regulates multiple aspects of protein function, including the localization to membranes, intracellular trafficking, protein interactions, protein stability, and protein conformation. In this study, we demonstrate that S‐palmitoylation proteins were preferentially encapsulated into EVs. Methods : We obtained the peptide sequence from the N‐terminus of a few membrane‐related proteins, which is the ideal substrate for S‐acyltransferase, the enzyme that catalyzes S‐palmitoylation. We fused those peptides onto the N‐terminus of both eGFP and SpCas9, which promoted the palmitoylation and encapsulation of those proteins into EVs. The encapsulation efficiency of palmitoylated eGFP was verified by flow cytometry and western blotting. Meanwhile, the packaging efficiency of SpCas9 was verified in reporter cells. Results : The palmitoylation modification permitted 87.2% of eGFP entry into EVs. The N‐terminus palmitoylation of SpCas9 did not affect its activity and was successfully encapsulated into EVs. EVs coated with VSV‐G encapsulating palmitoylated SpCas9/sgRNA complex restored 12.3% mCherry expression from the Ai9 reporter cells. Summary/Conclusion : Our study provides a novel approach to encapsulating CRISPR/Cas9‐sgRNA complex into EVs. This may open an effective avenue for using EVs as vehicles to deliver CRISPR/Cas9 for genome editing.

Establishment

Ayaulym Nurgozhina 1 ; Shynggys Sergazy 2 ; Laura Chulenbayeva 3 ; Sanzhar Zhetkenev 1 ; Mohamad Aljofan 4 1 National Laboratory Astana, Astana, Kazakhstan; 2 National Laboratory Astana, Nazarbayev University, Nur‐Sultan, Kazakhstan; 3 Nazarbayev University, Nur‐Sultan, Kazakhstan; 4 Nazarbayev University, NUSOM, Nur‐Sultan, Kazakhstan Introduction : Mare's milk has a very good hygienic and sanitary status, differs from the milk of other farm animals in that it has the lowest somatic cell content and a very low total number of microorganisms. The main hypothesis was that exosomes obtained from mare's milk can be a beneficious drug transport system compared to well‐studied exosomes from bovine milk. Objective of the project was to establish extraction method of exosomes obtained from a mare's milk, for future use as drug‐delivery system. Methods : To obtain pure exosomes of optimal size, three methods were investigated ‐ isoelectric precipitation, size‐exclusion chromatography, total exosome isolation. Each approach differs in price, equipment and materials, duration and complexity of implementation as well as mechanism of action. Results : Exosomes obtained from all methods have smooth membrane and isolated without surface rupture and damage, which indicates their possible further loading of drugs and use for therapeutic purposes in experiments according to transmission electron microscopy (TEM) data. Exosomes were purified well by total exosome isolation (TEI) and size‐exclusion chromatography (SEC) methods from dead cells, cell debris, and fat globules, according to Zetasizer data, whereas immunoprecipitation purification was unsatisfactory. Also, exosomes obtained from all three methods were of optimal size. TEI and SEC showed good clearance of casein, the main protein contaminant in milk. Summary/Conclusion : In our study, we consider that TEI method is the best choice for both laboratory research and large‐scale production. This approach requires less time, is less methodology sensitive, is more consistent with small volumes of biological samples, and does not demand any special equipment in comparison with other methods. Funding : Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No.AP13067844), Nazarbayev University Collaborative Research Program 2021–2023 (Award no.OPCRP2021006).

Ev‐Mediated

Dong‐Gyu Jo 1 ; Jihoon Han 2 ; Jae Hyung Park 3 ; Yong Woo Cho 4 1 School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea; 2 School of Pharmacy, Republic of Korea; 3 School of Chemical Engineering, Suwon‐si, Republic of Korea; 4 Department of Materials Science & Chemical Engineering, Hanyang University ERICA Campus, Ansan, Republic of Korea Introduction : Effective intracellular delivery of therapeutic proteins can provide treatments for a wide array of diseases. Exosomes, nanosized vesicles naturally secreted by various types of cells, are emerging as promising nanocarriers for therapeutic biomolecules. However, efficient delivery of functional proteins across the cell membrane remains a challenge. Methods : In this study, we introduce a new exosome‐based intracellular cargo protein delivery system, named ‘mMaple3‐mediated protein loading into and release from the exosomes’ (MAPLEXs). By fusing a photocleavable protein, mMaple3, between a cargo protein and an exosomal marker, we are able to load the cargo proteins into the exosomes and release the cargo proteins from the exosomal markers via 405‐nm light‐mediated photocleavage of mMaple3. Results : Using this novel cargo protein release system, we were able to successfully deliver soluble proteins across the target cell membrane both in vitro and in vivo. First, we showed that transcription factors can be delivered to the target cells via MAPLEX and regulate their target genes. Secondly, we achieved in vivo gene editing in Cre reporter mice. As reported previously, without any targeting moiety, most of intravenously injected MAPLEXs accumulated in the liver. Lastly, we showed that in vivo epigenetic editing can be induced in the brain of AD mouse model via MAPLEX. Summary/Conclusion : Our results suggest MAPLEXs as an efficient protein delivery system, which can be utilized for delivering diverse therapeutic proteins for various types of diseases.

Extracellular

Vidyanand (Anand) Sasidharan 1 ; Viraj Doddihal 2 ; Fengyan Deng 3 ; Sean Mckinney 3 ; Kexi yi 3 ; Eric Ross 3 ; Selene swanson 3 ; Chongbei Zhao 2 ; Alejandro Alvarado Sanchez 2 1 Stowers Institute, Kansascity, USA; 2 Stowers, USA; 3 stowers, USA Introduction : Upon amputation, planarian stem cells (neoblasts) first exhibit body‐wide proliferation, followed by mobilization and localized expansion of a subset of cells to the vicinity of the wound. These coordinated steps are critical to blastema formation and suggest an essential role for intercellular communication during wound healing and tissue regeneration. Yet, little is known about the factors affecting the observed cellular biology. Recently, Extracellular Vesicles (EVs) have been shown to play a role in cell‐cell communication in multiple systems. Methods : EVs were isolated from 2 DPA animals by combining Ultracentrifigation and Ultrafiltration methods. NTA was performed on isolated EVs to quantify the particles, and TEM was performed for qualitative analysis. MassSpectrometry was performed to study the EV proteomics profile and the cargo packaged inside them. RNAi was performed to validate the significance of the EV cargo‐related genes during planarian regeneration and Homeostasis. Small RNA sequencing was performed to understand the diversity of small RNA species packaged inside the EVs. EVs derived from RNAi animals were transplanted back into healthy animals to understand the role of EVs in planarian RNAi transport. Results : Proteomics and small RNA sequencing revealed the molecules packaged inside the EVs during planarian regeneration. RNAi on EV biogenesis pathway genes leads to lethality in regenerating and intact animals, suggesting that their pathway is conserved evolutionarily. Perturbing the EV cargo genes by RNAi leads to multiple defects in regenerating planarians, such as lethality, stem cell maintenance, etc. Interestingly, we could also prove that Evs are used as a mode of transport to carry out RNAi‐mediated gene knockdowns in planarians. Small RNA profiling of EVs from OVO and UNC RNAi animals showed an array of 20–25 nucleotide gene‐specific fragments packaged inside the EVs. Transplanting EVs derived from RNAi animals showed gene‐specific RNAi phenotypes in healthy animals. Summary/Conclusion : Overall, these results reveal EVs essential role in planarian regeneration and Homeostasis. We were also able to find out a novel role of EVs as RNAi transporter in planarians. The latter finding brings out an opportunity for performing transgenics in planarians. Funding : Stowers Institute and HHMI. Keywords : stem cells, regeneration, RNAi

Heterogeneity

Chul Won Seo ; Chaeyeong Jung; Dongsic Choi Department of Biochemistry, Soonchunhyang University, college of Medicine, Chenonan, Chungcheongnam,31151, Repubilic of Korea, Cheonan‐si, Republic of Korea Introduction : Extracellular vesicles (EVs) are nano‐sized vehicles equipping with diverse molecular components for the intercellular communication. In particular, it has been reported that the release of various EV subtypes are dynamically affected by oncogenic transformation, resulting in different functionality and uptake efficiency. In this study, we applied the nano‐flow cytometry, quantitative proteomics, and high‐throughput uptake screening of EV subtypes affected by mutant KRAS and BRAF. Methods : EVs were isolated from wildtype (Caco2), mutant BRAF (HT29, WiDr), and mutant KRAS (HCT116, LoVo) colorectal cancer cells by size exclusion chromatograph. EVs were labeled with fluorescent labeled antibody (CD147, CD44, CD63, CD9, CD81) and their subtypes were analyzed by nano‐flow cytometry. Proteomes of EVs were analyzed by Orbitrap Eclipse Tribrid Mass Spectrometer. EV uptake and their subcellular trafficking (cytosol, nucleus, lysosome) in recipient cells was measured by Lionheart FX automated microscope. Results : We found increased CD147‐ and CD63‐positive EV subtypes in mutant BRAF HT29 and WiDr cells. However, exosomal CD63‐positive EVs were downregulated in mutant KRAS HCT116 and LoVo cells. CD9‐ and CD81‐positive EVs are upregulated in both mutant RAS/RAF cells than wildtype cells. Moreover, mutant RAS/RAF cell‐derived EV showed differential uptake and subcellular trafficking efficiency in the cytosol, lysosome, and nucleus. Quantitative EV surface proteomes showed the overexpression of specific integrins (ITGB4) and heparan sulfate proteoglycans (GPC1) in mutant RAS/RAF EVs relating in their increased EV uptake. Summary/Conclusion : In this study, we discovered the heterogeneity of EVs and their differential EV uptake efficiency according to the oncogenic transformation. Our study shed light the new landscape of EV subtype‐depend EV uptake and thus this knowledge can be used as a novel drug delivery platform in efficient transfer of therapeutic cargo from lysosome degradation.

Igf2Bp3‐Evs

Katia Scotlandi; Caterina Mancarella; Alessandra De Feo IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy Introduction : Ewing sarcoma (EWS), a pediatric bone and soft‐tissue cancer, is characterized by high high level of intra‐tumor heterogeneity and aggressiveness. EWS is defined by the fusion of EWSR1 to the FLI1 gene. Several studies supported the role of EWSR1::FLI in governing EWS heterogeneity but other molecules were also reported to be involved. Here we focused on IGF2BP3, an RNA‐binding protein with an oncogenetic role in cancer. Cellular IGF2BP3 was described to be associated with EWS progression when highly expressed. In this study, we investigated whether and how IGF2BP3 is released by EWS cells and whether extracellular IGF2BP3 confers functional variations in recipient EWS cells. Methods : 11 patient‐derived EWS cell lines and experimental models modified for IGF2BP3 expression were used. Elisa assay detected expression of IGF2BP3 in cell supernatants. EVs were extracted from cell culture medium using the ExoQuick or the Ultracentrifugation methods.EVs were characterized using a NanoSight NS300 system. Labelling of EVs was made using the fluorescent dye PKH67. Cells were exposed to 15 ug of EVs derived from cells deprived or not of IGF2BP3 and tested for cell proliferation or migration. Western blotting was used to evaluate EVs markers or signaling mediators. RNAseq and bioinformatic analyses identified the miRNA cargo of IGF2BP3‐pos or ‐neg EVs and associated targets. Results : IGF2BP3 is released from EWS cells. IGF2BP3 is present in the EVs but not in the vesicles‐depleted supernatants. When A673 or TC‐71 EWS cells received EVs from the IGF2BP3 knockdown cell lines their migratory capability was significantly decreased compared with when they received IGF2BP3‐positive EVs. No difference were observed with respect to proliferation. The uptake of IGF2BP3‐positive EVs determined an increase of IGF2BP3 in recipient cells in a dose‐dependent manner, indicating that EVs may transfer IGF2BP3 in surrounding cells and modify their behavior. Analysis of miRNA cargo identified a signature of differentially expressed miRNAs. capable to cluster tissue tumor samples with different expression of IGF2BP3. From a mechanistic point of view, the analysis of 11 miRNAs that were found to be in common between two experimental models indicated alterations in the IGF1R_PI3K_Akt pathway in recipient cells. Summary/Conclusion : We identified in IGF2BP3 a player of Intra‐tumour phenotypic heterogeneity, which has been defined as a major determinat of EWS malignancy. IGF2BP3 is secreted via EVs and affects PI3K/Akt signaling in recipient cells, ultimately altering their migratory abilities. This indicates that cells with high expression of this RBP can increase the malignancy of neighbouring cells. Funding : The research leading to these results has received funding from AIRC under IG 2019—ID. 22805—P.I. Scotlandi Katia.

Investigating

Susannah J. Entwistle 1 ; Deborah Briggs 2 ; Ian Kerr 1 ; Beth Coyle 3 ; Alistair Hume 1 1 University of Nottingham, Nottingham, United Kingdom; 2 University of Nottingham, United Kingdom; 3 University of Nottingham, Nottingham`, United Kingdom Introduction : Medulloblastoma is the most common malignant paediatric brain cancer with poorer prognosis related to the onset of metastasis. It has four molecular subgroups; Wingless (WNT), Sonic Hedgehog (SHH), group 3 and group 4, of which group 3 is the most likely to be metastatic and is therefore associated with the poorest prognosis. Increased exosome release is connected with disease progression and metastasis in multiple cancers. Rabs are a family of small GTPases (70 in humans) which regulate vesicle trafficking. Several Rabs are known to regulate exosome biogenesis and secretion, including Rab11A, and may thereby contribute to cancer progression. The role of Rabs in metastatic medulloblastoma is unclear. We aim to explore whether Rabs contribute to the progression of metastatic medulloblastoma through the exosome biogenesis and secretion pathways. Methods : An analysis of the literature, databases such as ExoCarta.org and the R2: Genomics analysis and visualisation platform was completed to highlight Rab GTPase targets of interest. RT‐qPCR was used to assess gene expression of target Rabs across SHH, group 3 and group 4 cell lines. CRISPR‐Cas9 technology was used to generate knock out (KO) group 3 medulloblastoma cell lines of key Rab targets. KO cells were imaged using a ZOE fluorescent cell imager. Extracellular vesicles (EVs) were isolated using ultrafiltration followed by size exclusion chromatography using qEV original 70nm Gen 2 columns. Size and concentration of EVs were characterised using the ZetaView and the EV fraction imaged by transition electron microscopy. Results : Literature and database analysis identified Rab11A as a candidate which may contribute to disease progression in group 3 medulloblastoma. RT‐qPCR showed increased gene expression of Rab11A in group 3 and group 4 medulloblastoma patient‐derived cell lines. Imaging of Rab11A KO cells showed altered morphology compared to a control cell line. Summary/Conclusion : Initial findings suggest that Rab11A KO is affecting cellular phenotype. Current and future work aims to continue characterisation of this phenotype to determine the potential role of Rab11A in medulloblastoma pathogenesis through vesicular trafficking and secretion pathways. Funding : Funded by the BBSRC DTP programme. Keywords : rab, cancer, medulloblastoma

Investigation

Daiki Masuda 1 ; Yuta Shimizu 1 ; Tatsutoshi Inuzuka 1 ; Hiroyuki Awano 2 ; Tatsushi Toda 3 ; Mariko Taniguchi‐Ikeda 4 1 Business Development Office, H.U. Group Reseach Institute G.K., Akiruno, Japan; 2 Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan; 3 Department of Neurology, Graduate School of Medicine, The University of Tokyo, Bunkyo‐ku, Tokyo, Bunkyo‐ku, Japan; 4 Department of Clinical Genetics, Fujita Health University Hospital / Department of Pediatrics, Kobe University Graduate School of Medicine, Toyoake, Japan Introduction : Fukuyama congenital muscular dystrophy (FCMD) is a congenital muscular dystrophy characterized by central nervous system abnormalities and ocular involvement, and is an autosomal recessive genetic disorder. In recent years, myomiR, a skeletal muscle‐specific microRNA (miRNA) referred to as a biomarker for muscular dystrophies such as Duchenne‐type, has been reported in the blood. However, there have been few reports on biomarkers, including miRNAs, that are specific to FCMD. In this study, we aimed to identify FCMD‐specific miRNA markers by collecting extracellular vesicles (EVs) from serum samples of FCMD patients and healthy controls and performing miRNA analysis on these samples. Methods : Serum samples were collected from 12 FCMD patients and 8 healthy controls with informed consent. EVs were collected from 1 ml of serum using magnetic particles conjugated with anti‐CD9 and anti‐CD63 antibodies. miRNA was extracted from the recovered EVs using the mirVana miRNA Isolation Kit. Small RNA‐seq was performed on samples from 4 FCMD patients and 2 healthy controls. The remaining samples were subjected to qPCR targeting a total of 19 genes, including miRNAs that were highly expressed by small RNA‐seq and the myomiRs miR‐206, miR‐1, and miR‐133a, for validation. Results : As a result of small RNA‐seq, 153 miRNAs whose expression was found to be upregulated in FCMD patients were extracted. These included the myomiRs miR‐206 and miR‐133a. As a result of qPCR validation for 19 genes, the expression of level miR‐26 was upregulated in FCMD patients compared to healthy controls (2.28‐fold, 2‐ddCt method), and the difference was statistically significant. (p < 0.001, U test). Summary/Conclusion : Our study demonstrates that miRNA analysis on EVs collected by immunoprecipitation can identify miRNAs whose expression levels are specifically increased in FCMD patients. This result suggests that the immunoprecipitation method is effective for miRNA analysis and may pave the way for the development of a novel FCMD diagnosis. Funding : This study was funded by H.U. Group Reseach Institute G.K. Keywords : miRNA, immunoprecipitation, muscular dystrophy, small RNA‐seq, myomiR

Large‐Scale

Yijun Zhou 1 ; Runjie Yuan 2 ; Allaura Cone 2 ; Kyle Shifflett 3 ; Gabriel Arias 4 ; Alice Peng 2 ; Meredith Chambers 5 ; Ryan McNamara 6 ; Smaranda Willcox 5 ; Justin Landis 5 ; Yue Pan 7 ; Jack Griffith 8 ; Dirk Dittmer 9 1 University of North Carolina at Chapel Hill, Chapel Hill, USA; 2 University of North Carolina at Chapel Hill, USA; 3 UNC‐Chapel Hill Department of Microbiology and Immunology, USA; 4 UNC‐Chapel Hill Department of Biochemistry, USA; 5 UNC‐Chapel Hill Lineberger Cancer Center, USA; 6 Ragon Institute of MGH, USA; 7 UNC‐Chapel Hill Department of Biostatistics, USA; 8 UNC‐Chapel Hill Lineberger Cancer Center, Chapel Hill, USA; 9 [email protected], Chapel Hill, USA Introduction : Most conventional extracellular vesicle (EV) purification methods are limited to a smaller scale. We use a tangential‐flow filtration and Capto Core 700 (TFF‐CaptoCore700) based method for large‐scale EV purification and intentionally avoid strong physical force to preserve EV integrity. Separating EV into subclasses that carry unique cargos and markers is essential. This is the prerequisite for any accurate phenotypic tests and even more importantly for EV manufacturing. Methods : EV purified by TFF‐CaptoCore700 was further separated into sub‐populations by heparin chromatography. The biophysical properties of EV sub‐populations were analyzed using nanoparticle tracking analysis, transmission electron microscope, and super‐resolution microscopy. Protein contents were analyzed using mass spectrometry. Cell intake was analyzed by immunofluorescent assays. ERK phosphorylation was investigated by western blot upon EV treatment. Results : Two main EV sub‐populations were obtained from heparin chromatography. The first does not bind to the heparin column, namely a non‐heparin‐binding (NHB) fraction. The second binds to the heparin column and can be eluted by higher salt, thus is a heparin‐binding (HB) fraction. NHB carries most conventional EV protein markers while HB is enriched in extracellular matrix binding protein and histones. Both NHB and HB can be taken‐in by human endothelial cells but only HB induce ERK phosphorylation. Summary/Conclusion : Heparin chromatography would be an effective novel step to isolate EV subclasses at larger scale and can separate pyrogenic EV from those prepared for clinical applications. Funding : This work was founded by the NIH under Grant 5R01DE018304 to DPD, RO1ES031635 to JDG, P01CA019014, and R01CA228172 to DPD and JDG. The UNC Proteomics Core Facility is supported in part by NCI Grant (2P30CA016086‐45) to the UNC Lineberger Comprehensive Cancer Center. Keywords : extracellular vesicles, heparin, large‐scale purification, tangential flow filtration, Capto Core 700

Non‐Contact

Mi Ho Jeong 1 ; Hyungsoon Im 2 ; Joanna B. Dahl 3 1 Massachusetts General Hospital, USA; 2 Massachusetts General Hospital, Boston, USA; 3 University of Massachusetts Boston, Dorchester, USA Introduction : Most fundamental extracellular vesicle (EV) research has characterized nanoscale EVs and focused on EV biochemical content. There is much less understanding of large microscale EVs and EV mechanical properties. Here we test our new microfluidic technique to distinguish between large EV populations (L‐EVs, >1 μm diameter) from glioblastoma cell lines with mutations in isocitrate dehydrogenase 1 (IDH1) that is known to disrupt lipid metabolism and after temozolomide (TMZ) treatment. Methods : L‐EVs were purified via centrifugation from cell culture supernatants of GBM cell lines. L‐EVs were characterized per MISEV18 using dynamic light scattering, Western Blot, and optical microscopy (L‐EV morphology, size, and concentration). Drug‐treated T98G cells were exposed to 90 μM TMZ for 3 days. Lipidomics assessed L‐EV lipid content. Non‐contact stiffness measurements of single L‐EVs were done using microfluidics in which L‐EVs were gently stretched by the suspending fluid. Results : L‐EVs from IDH1‐mutated Gli36 and MGG cells were stiffer than wild‐type (∼20% higher median, small overlap of median 95% confidence intervals, classical and bootstrap hypothesis tests p < 0.05). Lipidomics analysis indicated a reduction of unsaturated lipids and relatively more saturated lipids in L‐EVs from IDH1‐mutated cell lines. L‐EVs from TMZ‐treated T98G cells may be softer than the control, though the statistical evidence was less strong. Summary/Conclusion : The changes in lipid composition (more saturated lipids) with the IDH1 mutation is consistent with L‐EV stiffening; tighter‐packing saturated lipids increase biomembrane rigidity. Microfluidics could be a complementary technique with atomic force microscopy to measure EV stiffness, specifically for microscale L‐EVs, with the advantages of higher throughputs and a non‐contact mode that eliminates substrate adhesion effects. Funding : NIH (R21CA217662, R01GM138778 to H.I.), Nat. Res. Fdn. of Korea (NRF‐2021R1A6A3A14039686 to M.H.J.). Keywords : large extracellular vesicles, microfluidics, stiffness, glioblastoma, IDH1 mutation

Repeatability

Edita Aksamitiene ; Marina Marjanovic; Darold R. Spillman; Stephen A. Boppart University of Illinois at Urbana‐Champaign, Urbana, USA Introduction : Reliability of quantitative urinary extracellular vesicle (uEV) assessment methods should be evaluated using healthy subject samples before test results can be applied to screen for pathological conditions, including cancer. Our goal was to estimate the short‐term precision that a lab may expect to obtain when analyzing naïve chemically untreated subpopulations of uEVs isolated by classical differential velocity centrifugation (DC) or novel silicon carbide precipitation (SiC‐P) techniques. Methods : Varying size uEVs were isolated in parallel from 6 technical replicates of 34 ml raw or pre‐processed (0.45 μm vacuum‐filtered) healthy consented adult human morning urine samples by DC at 800 × g (10 min), 2K × g (0.5 h), 12K × g (1 h) and 100K × g (1 h) at 4°C, followed by 12K (uMV) or 100K (uEXO) pellet resuspension in 55 μL or 100 μL PBS‐25 mM Trehalose buffer, and an optional 0.45 μm spin‐filtration (SF) step of PBS‐diluted final analyte solutions. uEXO2 nanoparticle fraction was captured from 800 × g (10 min) pre‐cleared pH 8.6‐adjusted urine on 3 ml 1000 grit size SiC slurry for 0.5 h and eluted in 3 ml PBS (pH 6.0) for 15 min followed by 1 μm gravity filtration and 0.45 μm SF steps. We fitted a random‐effects nested ANOVA model to determine the extent and relative contribution of procedural (sample processing, (sub)sampling) & instrumental (between‐run, within‐run) errors to the total technical variability of uEV concentration and size measurement process by nanoparticle tracking analysis. Closeness of agreement between measured variables was expressed as coefficient of variation (CV%). Results : There was a strong linear relationship between raw urine volume input and uEV yield. Sample pre‐processing caused a 30% to 40% loss of uEVs. Intra‐individual bimonthly variations (CVI) of uMV counts were ∼1.6‐fold higher than CVI of uEXO (41.1% vs 26.3%). CVI of uMV and uEXO size was comparable (7.6% vs. 7.8%). CV of uEV sedimentation and collection process was 7.3% & 2.4% for concentration and size variables, respectively. The corresponding estimates for samples generated by the SiC‐P method were 17.5% & 2.2%. 6 min long run‐to‐run variability of DC‐derived uEXO and uMV sample concentration reads was 2.35% & 2.9%, and only 0.75% & 1.9% for size reads. Runs analyzing uEXO2 counts were less precise (4.6%). Due to minor drift, within‐run repeatability was 6.2%, 7% or 6% for uMV, uEXO or uEXO2 concentration and 1.8%, 2% or 1.5% for their size. Summary/Conclusion : With the estimated < 10.5% combined analytical CVA for uEV concentration and < 3.5% CVA for size measurements, only DC met the desired allowable limit of analytical performance (CVA< 0.5 × CVI) goals, favoring it over the novel more rapid SiC‐P uEV separation method. Keywords : repeatability, precision, ultracentrifugation, precipitation, rigor, analytical variability, performance, NTA

Reprogramming

Priyanka Ghosh ; Kyo Sasaki; Isabel Aranzazu Pulido‐Ruiz; Kayla E. King; Steven A. Weinman; Ann L. Wozniak University of Kansas Medical Center, USA Introduction : Macrophage (MΦ)‐derived extracellular vesicles (EVs) play a key role in intercellular communication. In the liver, EV signaling is linked to both the progression and resolution of inflammatory diseases including nonalcoholic steatohepatitis (NASH). We previously showed that caspase‐1‐mediated cleavage of the trafficking adaptor protein RILP changes EV content generating pro‐inflammatory EVs. Preventing RILP cleavage with a dominant negative, non‐cleavable analog of RILP (ncRILP) reversed this effect. The aim of this study was to determine if manipulation of the RILP‐cleavage state in MΦs could prevent the transmission of an injury signal to hepatocytes. Methods : Cell crosstalk was assessed in transwell co‐cultures with LPS/ATP‐treated MΦs in the upper chamber and hepatocytes in the lower chamber. MΦs and monocytes were transfected using lentiviral vectors. Effects on target hepatocytes were assessed by RT‐PCR and ELISA. EVs were isolated by differential ultracentrifugation and characterized by western blot. Human peripheral blood monocytes were prepared from patients with inflammatory (NASH) and non‐inflammatory (NAFLD) fatty liver disease. Results : LPS‐ATP treatment of MΦs caused injury to hepatocytes in the transwell system. Expression of ncRILP in MΦs had no effect on cytokine production but it blocked the transmission of an injury signal to hepatocytes. Similarly, purified EV preparations from ncRILP‐expressing MΦs directly suppressed inflammatory effects in hepatocytes. To assess the disease relevance of this finding we examined the RILP cleavage state in circulating monocytes from patients. Monocytes from NASH patients had increased RILP cleavage compared to those from NAFLD patients. Treatment of NASH monocytes with ncRILP‐derived EVs abrogated the inflammatory phenotype. Summary/Conclusion : Macrophages transfer an inflammatory injury signal to hepatocytes via EVs. Prevention of macrophage RILP cleavage reprograms this EV signal and suppresses injury transmission. Funding : R01AI147276.

Selenoprotein

Victor Bodart‐Santos 1 ; Bridgette Melvin 2 ; Ikshu Pandey 3 ; Zhi Ruan 4 ; Seiko Ikezu 1 ; Tsuneya Ikezu 1 1 Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL 32224, USA, Jacksonville, USA; 2 Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL 32224, USA, USA; 3 Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA, USA; 4 Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL 32224, USA, Boston, USA Introduction : Microglia are primary innate immune cells in the central nervous system and display a neurodegenerative phenotype (MGnD) in response to amyloid plaque deposition in human and mouse models of Alzheimer's disease (AD). We previously reported that MGnD hyper‐secrete extracellular vesicles (EVs) compared to homeostatic microglia in APPN‐L‐GF knock‐in mouse model of AD. We recently identified selenoprotein P (Sepp1), a secreted heparin‐binding glycoprotein, as a potential regulator of EV and IL‐1B secretion from microglia under a pro‐inflammatory stimulus. We hypothesize that silencing of Sepp1 suppresses EV secretion in plaque associated MGnD microglia in vivo. Methods : In this study, we employed the small interfering RNA (siRNA) to determine the effects of silencing Sepp1 on EV secretion. We monitored EV secretion from BV‐2 cells, a murine microglial cell line, constitutively expressing tdTomato‐CD63 EV reporter molecule with or without downregulation of Sepp1 by Nanoimager, which can detect the tdTomato signal at a single molecule level. To track EVs secreted by microglia in vivo, a microglia‐specific lentivirus expressing mEmerald‐CD9 (mEm‐CD9) reporter molecule was co‐injected into the hippocampus of aged APPN‐L‐GF mice with lentivirus expressing Sepp1 or scramble shRNA and mCherry. Mice were euthanized at 2 weeks post injection and immunostained for galectin‐3 (Mac2, MGnD marker), RFP (mCherry), GFP (mEm‐CD9) and fluorostyrylbenzene (FSB, amyloid plaque). The images of mEm‐CD9+ voxels (EV particles) in the proximity of Mac2+/RFP+/GFP+ microglia were captured by Lightning super‐resolution confocal microscopy and the number of EV particles were quantified after 3D surface rendering of EV particles using IMARIS software. Results : Under pro‐inflammatory stimulation of BV‐2 cells with lipopolysaccharide followed by ATP for rapid EV secretion, we observed a reduction in the loading of tdTomato‐CD63+ molecules to EVs secreted in three independent Sepp1‐shRNA clones. At seven months of age, we mostly detected mEm‐CD9+ microglia as Mac2+ MGnD surrounding amyloid plaques in APPN‐L‐GF mice. Lentiviral shRNA‐induced Sepp1 silencing reduces the EV secretion (mEm‐CD9+ voxels) from mEm‐CD9+/Mac2+/RFP+ microglia compared to the scramble shRNA‐transduced mEm‐CD9+/Mac2+/RFP+ microglia in the APPN‐L‐GF mouse brain. Summary/Conclusion : These data demonstrate that in vivo silencing of Sepp1 suppresses EV secretion from MGnD microglia. Since activated microglia secrete neurotoxic and pathogenic molecules, our data suggest Sepp1 as a potential target for ameliorating microglia‐mediated disease progression in neurodegenerative conditions including AD. Keywords : microglia, alzheimer's disease, selenoprotein P

Visualization

Steven Ting‐Yu Chuo 1 ; Yen‐Ju Chen 2 ; Wendy Wan‐Ting Wong 2 ; Yi‐Wen Chang 3 ; Ju‐Chen Chuang 1 ; Hsueh‐Fen Juan 3 ; Charles Pin‐Kuang Lai 4 1 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan, Taipei, Taiwan (Republic of China); 2 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan, Taiwan (Republic of China); 3 Department of Life Science, National Taiwan University, Taipei, Taiwan, Taipei, Taiwan (Republic of China); 4 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan (Republic of China) Introduction : Extracellular vesicles (EVs) play an important role in cell‐to‐cell communication under (patho)physiological conditions. However, the fate of delivered EVs, as well as interacting proteins which mediate EV intracellular transport remained largely unexplored. We developed a multi‐functional EV imaging and proximity labeling system, PalmGREX, to enable visualization and interactome analysis of EVs with spatiotemporal accuracy. Methods : Bioluminescence resonance energy transfer (BRET)‐based PalmGRET EV reporter (Charles Lai lab) comprising eGFP‐NanoLuc (Antonio Amelio lab) and inner membrane labeling palmitoylation moiety of growth cone associated protein 43 was molecularly fused with APEX2 ascorbate peroxidase (Alice Ting lab) to create PalmGREX. Human embryonic kidney 293T cells (293T) were stably transduced to express PalmGREX followed by isolation of small EVs (sEVs) via differential centrifugation. Labeled sEVs were administered to 293T, and the sEVs were tracked subcellularly at super‐resolution, followed by proximity labeling and proteomic analysis of EV‐interacting proteins during intracellular trafficking. Trypin was applied to EV‐recipient cells to mitigate non‐internalized sEVs. Results : PalmGREX enables live‐cell tracking of sEVs under super‐resolution microscopy, where PalmGREX‐sEV signals significantly overlap with the endoplasmic reticulum (ER) and are in close proximity to the mitochondria at 3‐hour post‐sEV treatment. In addition, PalmGREX‐sEVs were semi‐quantitated and confirmed to be internalized by the recipient cells via bioluminescent and BRET‐fluorescent signals in a time‐dependent manner. Concurrently, PalmGREX enables proximal protein labeling of internalized sEV during subcellular trafficking. Downstream proteomics and functional enrichment analyses based on GeneOntology reveal that PalmGREX‐sEV proximity‐labeled proteins are enriched with the mitochondrial and endoplasmic reticulum (ER) annotated proteins. Summary/Conclusion : Using PalmGREX, we identified that internalized sEVs are trafficked to the ER and mitochondrial periphery, which is corroborated by enriched ER and mitochondrial proteins as identified by sEV proximity labeling. Efforts are currently underway in identifying sEV‐interacting proteins involved in subcellular trafficking and the fate of internalized sEVs following their uptake. Funding : National Science and Technology Council (NSTC) grants NSTC 111‐2628‐B‐001‐004 (C.P.L.), Academia Sinica Innovative Materials and Analysis Technology Exploration (i‐MATE) Program AS‐iMATE‐107‐33 (C.P.L.), Institute of Atomic and Molecular Sciences grants IAMS 30‐08 (C.P.L.), and Academia Sinica Career Development Award 109‐M04 (C.P.L.). Keywords : EV uptake, small EV, subcellular trafficking, proximity labeling

“Lumin‐Ev

Olga Volpert 1 ; Erez Eitan 2 ; Emma Vande Brake 3 1 NeuroDex, Natick, USA; 2 NeuroDex, Arlington, USA; 3 NeuroDex, USA Introduction : We developed an immunoassay based on Luminex principle, in order to evaluate in a multiplexed format, surface protein composition of intact EVs in unprocessed plasma. Methods : plasma samples. EVs bound to capture beads are detected with biotinylated antibodies against distinct EV surface proteins followed by streptavidin‐PE. Luminex reader separates capture beads by index colors and measures PE levels associated with each color range. We developed assays that capture tetraspanins, synaptic proteins, or cell‐specific markers, which are subsequently detected with one more tetraspanin antibody. Each assay also contains an internal negative control (IgG). Results : Assay specificity for EVs was shown by abolishing signal by EV depletion via size exclusion or detergent treatment. Capture specificity is indicated by detection of neuronal markers GAP43 and L1CAM in EV preparations from neuronal but not HEK293 cell culture. GAP43‐positive neuronal EVs are detected with antibodies for tetraspanin or another neuronal (L1CAM) but not erythrocyte marker (CD235). Our assay for synaptic EV proteins GluR2, Syntaxin1, NRGN, and GAP43, with CD63 and CD9 for normalization and isotype control to exclude non‐specific binding generates signals for all analytes, significantly above non‐specific background and shows dilution‐dependent linearity. Importantly, the results were not affected by multiplexing, as similar values were obtained in a multiplexed and single‐plexed formats. When applied to plasma samples from patients with Parkinson's disease, Alzheimer's disease (AD) and age‐matched controls (N = 20 each) GluR2, Synataxin1, and GAP43 showed significant reduction in AD. Summary/Conclusion : The intact EV LuminEV assay has high potential as a user‐friendly medium‐throughput method for surface protein detection on plasma EV, to be used in fundamental studies and biomarker analysis. Keywords : intacg = t EV assay, luminex

Administration

Lina Antounians 1 ; Rebeca Lopes Figueira 1 ; Bharti Kukreja 2 ; Cadia Chan 3 ; Michael Wilson 2 ; Brian Kalish 2 ; Augusto Zani 1 1 The Hospital for Sick Children, Toronto, Canada; 2 The Hospital for Sick Children, USA; 3 The Hospital for Sick Children, Toronto, USA Introduction : Incomplete lung development, also known as pulmonary hypoplasia (PH), is a recognized cause of neonatal death. In search for a regenerative therapy, we reported that administration of rat amniotic fluid stem cell extracellular vesicles (AFSC‐EVs) restored features of impaired lung branching morphogenesis in experimental PH. Herein, we aimed to 1) identify if in vivo AFSC‐EV administration promotes lung maturation; 2) determine AFSC‐EV cargo miRNAs that induce lung cell‐specific responses. Methods : AFSC‐EVs were isolated from rat AFSC conditioned medium by differential ultracentrifugation (100,000g) and characterized by size (NTA), morphology (TEM), and expression of canonical markers (CD63, TSG101, Flot‐1; Western blot, WB). We induced fetal PH by nitrofen gavage to dams at embryonic day (E)9.5. At E18.5, fetuses received an intra‐amniotic injection of either saline (control+PBS, n = 8; PH+PBS, n = 8) or AFSC‐EVs (PH+AFSC‐EV, n = 9). At E21.5, lungs were harvested and assessed for branching morphogenesis (mean linear intercept, radial alveolar count) and expression of maturation markers (Pdpn, Sftpc; qPCR/WB). Lung nuclei were subjected to single nucleus RNA‐seq (snRNA‐seq; 10X Seurat/R). Cargo‐seq of AFSC‐EVs was performed with SeraMir/NextSeq. AFSC‐EV cargo miRNA levels were correlated with differentially expressed mRNA in PH+AFSC‐EV lungs (MultiMiR). Results : Lung branching morphogenesis was impaired in PH+PBS vs. control+PBS fetuses and restored to control levels by AFSC‐EVs. Similarly, Pdpn, and Sftpc expression was reduced in PH+PBS vs. control+PBS group and restored to control levels in PH+AFSC‐EV group. snRNA‐seq analysis identified macrophages and inflamed epithelial and mesenchymal cells unique to PH+PBS lungs. A network analysis of AFSC‐EV miRNAs and downregulated mRNAs in PH+AFSC‐EV lungs revealed 820 miRNA‐mRNA targets (32 validated) that were involved in inflammatory responses. Summary/Conclusion : In vivo AFSC‐EV administration in experimental PH restores features of impaired lung development and can regulate key genes relevant to pathological processes. AFSC‐EV‐based therapy could be a promising avenue to treat fetal PH. Funding : SickKids Foundation, Canadian Institutes of Health Research.

Characterizing

Sarah Catherine B. Baker 1 ; Ursula S. Sandau 2 ; Trevor J. McFarland 2 ; Julie A. Saugstad 3 1 Oregon Health and Science University, Portland, USA; 2 Oregon Health & Science University, Portland, USA; 3 Department of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, PORTLAND, USA Introduction : Niemann Pick Disease Type C (NPC), also known as Childhood Alzheimer's, is a rare neurodegenerative disease. NPC is caused by endolysosomal disruptions driven by abnormal cholesterol storage. Extracellular vesicles (EVs) can be formed through the endolysosomal pathway, and have been implicated in neurodegeneration. However, whether EVs are affected by NPC is unknown. Here, we isolated and characterized EVs from both cerebrospinal fluid (CSF) and fibroblasts of NPC patients. Methods : NPC and control CSF (n = 12) and dermal fibroblasts (n = 8) were obtained from Oregon Health & Science University, Rush University, and Coriell Institute. CSF was ultrafiltered and probed for EV markers with immunoblots. EVs were enriched from CSF and cell conditioned media using ultrafiltration (Millipore 100kD) and size exclusion chromatography (Izon 35nm). Fractionated EVs were validated using immunoblots, fluorescent nanoparticle tracking analysis (fNTA), and transmission electron microscopy. fNTA, vesicle flow cytometry (VFC), and multiplexed bead‐based flow cytometry (MBFC, Miltenyi Biotec) were then used to characterize EV populations. Groups were compared using Student's t‐test or Mann‐Whitney. Results : Immunoblots show NPC CSF contains significantly more Flotillin 1 and CD81 protein compared to controls (p = 0.041, p = 0.015). Similarly, fNTA and VFC show NPC fibroblasts release more 100nm‐sized EVs than controls (p = 0.004). MBFC shows NPC EV surface proteins differ from controls in both CSF and cell culture media, including enrichment of proteins implicated in NPC pathology. Summary/Conclusion : Our results indicate that human NPC CSF and cell culture media contain more EVs compared to controls, and that proteins on NPC EVs are distinct from those on control EVs. Current work is investigating whether these increases result from altered EV biogenesis or uptake, and further identifying alterations to NPC EV cargo. Ultimately, this work demonstrates how EVs are altered in NPC, a devastating neurodegenerative disease that has no cure. Keywords : niemann pick disease Type C, extracellular vesicles, neurodegeneration

Considerations

Amy C. Kauffman 1 ; Ana Maria Pardo 2 ; Audrey Bergeron 2 ; Hannah Gitschier 2 1 Corning Life Sciences, Kennebunk, USA; 2 Corning Life Sciences, USA Introduction : Naïve extracellular vesicles (EVs) have the potential to revolutionize therapeutic treatments for regenerative medicine on a broad scale. The paracrine factors retained in EVs from parent stem cells have the ability to orchestrate complex physiological changes. The lack of standardization in culture of adherent mesenchymal stem cells has resulted in a diversity of methods for expansion and maintenance. Further, it is unclear how cell culture conditions influence cell expansion, behavior, and EV production. In this work, we analyzed human bone marrow‐derived MSCs (hMSCs) and subsequent EV production in various cell culture devices to understand the environmental effects that drive changes in MSC physiology and EV functional performance. Methods : hMSCs were cultured in xeno‐free medium to understand the importance of polymer surfaces, gas‐permeable film, surface chemistry, and shear stimuli on hMSC properties and derived EVs. EVs were isolated via tangential flow filtration. EV concentration and size was assessed via Nanoparticle Tracking Analysis, tetraspanin expression was profiled via single particle interferometric reflectance imaging sensor, and biological functionality was assessed via wound healing assay. hMSCs biomarker expression was analyzed via flow cytometry. Results : Cell culture conditions influenced the expansion and characteristics of hMSCs, which can impact the quantity and quality of EVs produced, supporting the need for process standardization. While material choice and surface treatment are important factors to consider when expanding MSCs, the addition of mild shear stress can enhance EV production with no biological consequences. Summary/Conclusion : This data suggests that material choice, surface treatment, and environmental stimuli are important factors to consider when developing in vitro cell culture processes, and highlights the impact they can have on the biology and functional performance of naïve EVs for regenerative medicine.

Extravesicular

Laura Patras 1 ; Kayleen Bailey 2 ; Miho Hatano 2 ; Angela Di Giannatale 2 ; Ayuko Hoshino 3 ; Daniela Freitas 4 ; Henrik Molina 5 ; Celso Reis 4 ; Jack Bui 6 ; Doru Paul 7 ; Irina Matei 8 ; David C. Lyden 9 1 Department of Pediatrics, Drukier Institute for Children's Health, and the Meyer Cancer Center, Weill Cornell Medical College, New York, USA and Department of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes‐Bolyai” University, Cluj‐Napoca, Romania, New York, USA; 2 Department of Pediatrics, Drukier Institute for Children's Health, and the Meyer Cancer Center, Weill Cornell Medical College, New York, USA, USA; 3 School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan; Japan Science and Technology Agency, PRESTO, Tokyo, Japan, USA; 4 i3S‐Institute for Research and Innovation in Health, University of Porto, Rua Alfredo Allen 208, Porto, Portugal, USA; 5 Proteomics Resource Center, The Rockefeller University, New York, NY, USA, USA; 6 University of California, San Diego, La Jolla, CA 92093, USA, USA; 7 Department of Medicine, Weill Cornell Medicine, New York, USA, USA; 8 Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, New York, USA; 9 Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA, New York, USA Introduction : Unveiling mechanistic insights into the immunoregulatory role of tumor‐derived extracellular vesicles (TEV) could improve the outcome of immunotherapies. To study TEV‐mediated crosstalk between cancer and adaptive immunity, we used a murine fibrosarcoma model of progressing and regressing tumors. We hypothesized that regressor and progressor TEV cargo induces functional changes in adaptive immune cells, regulating antitumor immune responses. Methods : Small EVs were isolated by sequential ultracentrifugation and characterized by nanoparticle tracking analysis, transmission electron microscopy, western blot, and mass spectrometry. CRISPR/Cas9 was used to generate CD147 knockout (KO) progressor cells. TEVs were injected into the footpad for biodistribution and chronic education experiments (10 μg of EV protein every 3 days). Immunofluorescence and flow cytometry were used to assess intratumor immune cell infiltration. Intratumor and lymph node immune cell populations as well as TEV‐induced changes in these cells were assessed by scRNAseq. Results : Our study shows that chronic cross‐education of naïve mice with progressor and regressor TEV prior to tumor inoculation could skew tumor growth in vivo towards a regressor or progressor phenotype, respectively, through a direct effect of EVs on T cells. Proteomic analysis of EVs identified CD147, a pan‐cancer EV biomarker and immunoglobulin superfamily member with pleiotropic functions as a mediator of progressor phenotype. CD147 ablation in progressor cells was sufficient to induce complete tumor regression in vivo. Chronic education with regressor or CD147 KO TEV inhibited progressor tumor growth and promoted CD4+ and CD8+ T cell tumor infiltration. Importantly, regressor intra‐tumor T cells displayed a higher activation state and antitumor functions, whereas T cells isolated from progressor tumors displayed an exhausted phenotype. Summary/Conclusion : This study reveals a novel role for extravesicular CD147 in modulating antitumor immune responses, identifying this protein as a promising therapeutic target for cancer immunotherapy. Funding : STOP Cancer Foundation, Hartwell Foundation, Children's Cancer and Blood Foundation. Keywords : extracellular vesicles, adaptive immunity, tumor regression

Identification

Yerim Kwon 1 ; Jihye Lee 2 ; Yoonjeong Kim 2 ; Jik‐Han Jung 1 ; Seongmin Na 2 ; Seung Wook Oh 1 ; Sung‐Soo Park 1 1 MDimune Inc., Seoul, Republic of Korea; 2 MDimune Inc., Republic of Korea Introduction : BioDroneTM, developed at MDimune, is a state‐of‐the‐art drug delivery system based on cell‐derived vesicles (CDVs) produced by serial extrusion from diverse cell sources. CDVs exhibit physicochemical similarity to extracellular vesicles (EVs) but a tremendous advantage in production scalability over EVs. Furthermore, with genetic engineering of mother cells, CDVs can potentially acquire additional biological features such as targeting and cargo loading capabilities. Methods : In this study, we identified stable anchor proteins exclusively for HEK‐CDV. First, we analyzed the HEK‐CDV proteome and selected a set of CDV‐specific membrane proteins highly abundant in the CDVs over cells or EVs. Then, these anchor candidates were overexpressed with a tag and GFP in the HEK293 cell. Antibiotic selection and cell sorting established a pool of stable HEK293 cells. After CDV production, the anchor fusion proteins were quantified via GFP ELISA, and their distribution in a single‐particle resolution was examined by nanoflow cytometry. Results : Four HEK‐CDV anchors among candidates showed a stable presence in the CDVs, with 30 to 150 molecules per CDV particle and 42 to 66 % of the GFP (+) population among total particles, depending on the anchor proteins. Moreover, their proper topology was confirmed via protease cleavage assay, while their abundance in the CDVs was also confirmed from a large‐scale extrusion. Summary/Conclusion : Together, we present a set of HEK‐CDV anchors for the versatile engineering of the BioDroneTM platform. Currently, we are developing various BioDroneTM therapeutics utilizing these anchors. Keywords : cell‐derived vesicle(CDV), BioDrone platform, anchor proteins

Ipsc‐Derived

Qing Li 1 ; Xinyu Niu 2 ; Yuguo Xia 3 ; Yang Wang 1 ; Zhifeng Deng 4 1 Institute of Microsurgery on Extremities, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China (People's Republic); 2 Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, China (People's Republic); 3 Department of Neurosurgery; National Clinical Research Center for Geriatric Disorders Xiangya Hospital, Central South University, Shanghai, China (People's Republic); 4 Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, USA Introduction : Blood−brain barrier (BBB) breakdown after ischemic stroke exacerbates brain injury and BBB senescence can cause severe neurological deficits in the aged ischemic stroke population. Recent evidence reveals that inducible pluripotent stem cell‐derived small extracellular vesicles (iPSC‐sEVs) possess the phenomenal antisenescence capability. However, whether iPSC‐sEVs can rejuvenate BBB senescence to improve stroke outcomes in aged mice remains unknown. Methods : Aged mice were treated with iPSC‐sEVs for 2 months, and transient middle cerebral artery occlusion (MCAO) was conducted. BBB senescence, BBB leakage, infarct volume, immune cell infiltration, neuroinflammation, neural death, and sensorimotor functions were detected. Next, D‐gal was utilized to induce BBB senescence, and oxygen and glucose deprivation (OGD) was performed. BBB senescence and BBB leakage were further evaluated in vitro. Mechanistically, proteomics analysis of iPSC‐sEVs was performed to explore the bioactive factors. eNOS inhibitor, AKT1 and CALM inhibitors were used to verify the mechanism. Results : In aged mice long‐term treatment with iPSC‐sEVs alleviated aging‐induced BBB senescence. In aged stroke mice, iPSC‐sEVs significantly mitigated BBB integrity damage, reduced the following infiltration of peripheral leukocytes, and decreased the release of pro‐inflammatory factors from the leukocytes, which ultimately inhibited neuronal death and improved neurofunctional recovery. Mechanism studies showed that iPSC‐sEVs could activate the endothelial nitric oxide synthase (eNOS) and up‐regulate sirtuin 1 (Sirt1) in senescent endothelial cells. Blocking the activation of eNOS abolished iPSC‐sEV‐ mediated rejuvenation of BBB senescence and the protection of BBB integrity. Proteomics results demonstrated that iPSC‐ sEVs were enriched with bioactive factors including AKT serine/threonine kinase 1 (AKT1) and calmodulin (CALM) to activate the eNOS−Sirt1 axis. Further investigation showed that AKT1 and CALM inhibitors blocked iPSC‐sEV‐afforded activation of the eNOS−Sirt1 axis in senescent endothelial cells. Summary/Conclusion : iPSC‐sEVs can protect against ischemic stroke in aged mice by rejuvenating BBB senescence, partially, through delivering AKT1 and CALM to activate the eNOS−Sirt1 axis. Funding : This study was funded by the National Natural Science Foundation of China under Grant Nos. 82071371, 82201543, and 81871833, National Postdoctoral Program for Innovative Talent under Grant No. BX20220356, China Postdoctoral Science Foundation under Grant No. 2022M723562, Natural Science Foundation of Hunan Province under Grant No. 2022JJ40828, Natural Science Foundation of Changsha city under Grant No. kq2202377, Young Foundation of Xiangya Hospital under Grant No. 2021Q01. Keywords : induced pluripotent stem cell‐derived small extracellular vesicles (iPSC‐sEVs), blood−brain barrier (BBB), cell senescence, ischemic stroke, aging

Matrix‐Bound

Camilo Mora 1 ; Sahimy Ayus 2 ; Lyana Rivera 3 1 University of Puerto Rico, MAYAGUEZ, USA; 2 Unversity of Puerto Rico, USA; 3 University of Puerto Rico, USA Introduction : Heart disease remains the leading cause of death in Puerto Rico and the US. There are limited options for monitoring cardiovascular diseases, and current monitoring methods involve high radiation exposure, which has a grade of invasiveness and other side effects. Endothelial extracellular vesicles (end‐EVs, CD144+ ‐endothelial cell marker‐) are new biomarkers associated with the prognostic and diagnostic of cardiovascular diseases. We need an end‐EV model to develop a selective biosensor; however, end‐EVs are found in body fluids in combination with other EVs and biomolecules as a heterogenous population hampering its use as an EV model. Matrix‐bound vesicles (MBVs), EVs embedded within the decellularized extracellular matrix (dECM), are a new class of tissue‐specific EVs. We hypothesize that MBVs isolated from endothelial tissues (end‐MBVs) will be CD144+, playing a pivotal role as an EV model in developing end‐EV biosensors. We seek to build an EV biosensor for the selective detection of end‐EVs CD144+. Methods : To accomplish this, we first must i) derive end‐dECM from porcine endothelium, ii) isolate end‐MBVs, and iii) characterize them as CD144+ to use them as an EV model for biosensor construction. We have developed a method based on sodium deoxycholate to decellularize endothelium–ECM. The MBVs were isolated using dECM solubilization via KCl solution. Then, the MBVs were quantified and characterized using methods validated for exosomes (i.e., nanotracking analysis ‐NTA‐, Exocet, microRNA staining). Finally, an immunoblotting technique (dot blot) was used to detect CD144 in the isolated end‐MBVs. Results : The decellularization protocol developed yielded 20–30 mg of end‐dECM. Using a 48 hours long‐lasting solubilization, the KCl solution could extract the MBVs embedded within the end‐dECM, which were further purified using a qEV column. The MBVs particle size was about 120 nm, characterized using NTA. The MBV concentration was quantified as 10^8 EVs/ uL. The immunoblotting test identified the endothelial protein marker CD144 in the MBVs Summary/Conclusion : This project provides evidence of the isolation of CD144+ MBVs that will be used as a target model for engineering the endothelial‐EV biosensor. Funding : We acknowledge the startup funding support provided to Dr. Mora‐Navarro by the CAWT under NSF grant OIA‐1849243. Keywords : MBVs, dECM

Milk‐Derived

Jueqin Lu ; Sanoji Wijenayake University of Winnipeg, Winnipeg, Canada Introduction : To reduce maternal obesity (MO)‐related health problems in children, milk‐derived extracellular vesicle (MEVs) supplementation is proposed as a solution. MEVs convey pro‐survival effects to neonates, but its association with neuroinflammation is unknown. MO leads to pro‐inflammatory responses including NF‐κB pathway activation and NLRP3 inflammasome formation. My objective is to investigate the pro‐survival properties of MEVs in response to MO‐induced pro‐inflammatory pathways. Methods : Immortalized human microglia (HMC3, ATCC #CRL‐3304) were activated by 10 or 50 ng/μL interferon‐γ (IFN‐ γ, Millipores‐Sigma #01‐172), to mimic a MO‐induced pro‐inflammatory (M1) phenotype. Activation was confirmed by measuring abundance of M1 markers (IL‐1β, SAA), anti‐inflammatory (IL‐10, CD200R1) and general activation markers (CD68, IBA‐1). Post M1 transition, HMC3s were supplemented with 200ug MEVs and harvested 0/6/12/24 for downstream analysis (n = 6/treatment). MEVs were isolated via serial centrifugation, ultra filtration, combined with differential ultracentrifugation (Beckman Coulter XL‐100). MEV concentration, size, and particle distribution were measured via tunable resistive pulse sensing (TRPS) technology (IZON: The Exoid). MEVs were visualized via transmission electron microscopy (TEM, Thermo Fisher: F200X S/TEM). Western immunoblotting (WB) was used to characterize exosome markers (CD63, CD9, CD81) and negative control (Calnexin). NF‐κB and NLRP3 inflammasome markers were quantified via RT‐qPCR and WB. Results : Post IFN‐γ treatment, abundance of M1 markers increased, indicating a M1 transition. With MEVs supplementation, NF‐κB (TLR4, TRAF6, IκBα, NF‐κB p65) and NLRP3 inflammasome (pro‐caspase‐1, IL‐1β, and IL‐18) decreased in abundance. Summary/Conclusion : MEV supplementation may reduce the activation of NF‐κB and NLRP3 inflammasome formation and promote pro‐survival effects in the microglia. MEVs could potentially combat negative health effects caused by MO. Funding : This work was funded by Natural Sciences and Engineering Council (NSERC) Discovery Grant (Fund#:03805) and Manitoba Medical Service Foundation New Investigator Research Grant (Fund #:2021‐18 awarded to Dr. Wijenayake. Keywords : milk‐derived extracellular vesicle, neuroinflammation, human microglia, NF‐κB pathway, NLRP3 inflammasome

Mir‐150‐5P

Jenie Marian Cruz Burgos 1 ; Boyang Su 2 ; Eduardo Martínez Martínez 3 ; Sergio Alberto Cortés Ramírez 4 ; Hon S. Leong 2 ; Mauricio Rodríguez Dorantes 4 1 Instituto Nacional de Medicina Genómica, Cuautitlán Izcalli, Mexico; 2 Department of Medical Biophysics, University of Toronto, Biological Sciences Platform, Sunnybrook Research Institute, Toronto, Canada; 3 Instituto Nacional de Medicina Genómica, USA; 4 Instituto Nacional de Medicina Genómica, Mexico Introduction : Prostate cancer is the fifth most common cause of death in men. PSA levels in the blood are the gold standard for diagnosing prostate cancer (PCa), but they are highly unspecific. Liquid biopsies allow us to detect miRNAs within extracellular vesicles (EVs) in biological fluids such as blood or urine. Differential expression of miRNAs has been linked to cancer development. The purpose of this study is to determine the presence of miRNAs plasma Evs from PCa patients. We isolated EVs using aqueous two‐phase separation (ATPS) and identified EV markers using nano‐scale cytometry. We discovered that the expression of miR‐150‐5p in plasma EVs differed according to Gleason score. Methods : To extract EVs from PCa cell lines (LNCaP, PC3, DU145, BPH‐1, NHPrE and BHPrE), two rounds of ultracentrifugation were performed. We used electron microscopy to examine the size and shape of EVs, as well as the EV markers TSG101, CD63, and CD9 by western blot. Based on Gleason score, human plasma samples were split into three categories. Every group had 7–8 samples and 12 healthy donor samples. All samples were obtained with proper informed consent and the approval of the Sunnybrook Research Institute's ethics committee. The ATPS and Qiagen's Exo RNeasy Midi kit were used to isolate EVs from 500uL of plasma. Nano‐scale cytometry was used to detecting canonical EV biomarkers. RT‐qPCR was used to detect the presence of miRNAs using Taqman MicroRNA Reverse Transcription kits and Taqman Small RNA probes for each miRNA. RNU6B acted as a normalizer. Results : We confirm the presence of CD9, CD63, and TSG101 in LNCaP and PC3 EVs from PCa cell lines. TEM images show the presence of EVs with traditional shapes and sizes in LNCaP, PC3, and DU145 samples. The presence of CD63, CD81, and CD9 in all EV samples from PCa cell lines and EVs from plasma samples was revealed by nanoscale cytometry, with CD9 being the most prevalent in plasma EVs. RT‐qPCR revealed that MiR‐150‐5p was considerably higher in EVs from LNCaP and BPH‐1. The miR150‐5p expression was found to be inversely proportional to Gleason score in PCa samples with a high Gleason score >4+3. Summary/Conclusion : In most of the cell lines EVs we found overexpression of the miRNAs. This study demonstrates the detection of EVs and miRNAs in 500uL of plasma using ATPS and the presence of tetraspanins using nano‐scale cytometry. The miR‐150‐5p levels in plasma vesicles are inversely proportional to Gleason score, suggesting possible use as an indicator of more advanced PCa. Keywords : biomarker, EVs, prostate cancer, PCa, miRNAs, ATPS, nano‐scale flow cytometry

Non‐Invasive

Yohan Kim 1 ; Edlira Horjeti 2 ; R Jeffrey Karnes 1 ; Eugene Kwon 1 ; Fabrice Lucien 1 1 Mayo Clinic, Rochester, USA; 2 Mayo Clinic, USA Introduction : Tumor‐derived extracellular vesicles (tdEVs) are promising biomarkers for cancer liquid biopsy. In metastatic prostate cancer, we found that high blood levels of prostate cancer derived EVs (ProstEVs) are associated with reduced levels of tumor‐reactive CD8 T cells and rapid disease progression. Our clinical findings support an immunosuppressive function of tdEVs. Thus, there is a need to characterize the molecular cargo of ProstEVs to develop novel therapies that can restore antitumor immunity and improve patient outcome. To that end, we developed a highly specific and efficient method to capture ProstEVs directly from patient blood leading to the first characterization of the ProstEV molecular cargo in prostate cancer. Methods : Blood was drawn from healthy men and prostate cancer patients following IRB approval. ProstEV‐negative platelet‐free plasma samples were spiked with 5.0 × 107 EVs/mL of cell line‐derived PSMA+‐ProstEVs for optimization. ProstEV concentrations were measured using microfluidic resistive pulse sensing and nanoscale flow cytometry. ProstEVs were captured by magnetic bead‐based immunoaffinity. Capture efficiency and specificity was analyzed with PSMA antibody and isotype control. Protein content of captured ProstEVs was analyzed by western blotting and mass spectrometry. Results : PSMA Ab‐Mg beads successfully captured 91, 99, 99% of PSMA+ EVs from spiked‐plasma samples (N = 3). PSMA‐Mg beads also captured PSMA+ EVs (4.3 × 108, 7.7 × 107, 8.3 × 107 EVs/mL) from high‐risk prostate cancer patient plasma samples (N = 3). EVs captured by PSMA‐Mg beads showed the presence of prostate cancer‐associated proteins (PSMA and AMACR), but not from isotype‐Mg beads. Proteomic profiling is ongoing and original data will be presented at the annual meeting. Summary/Conclusion : In conclusion we developed a simple and efficient method to capture tdEVs in prostate cancer blood with high specificity. This work will serve as methodological framework for a new wave of liquid biomarker studies in prostate cancer.

Pan‐Specific

Marina Cretich 1 ; Roberto Frigerio 2 ; Paola Gagni 3 ; Stefano Panella 4 ; Riccardo Vago 5 ; Lucio Barile 6 ; Alessandro Gori 2 1 Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Milano, Italy; 2 Istituto di Scienze e Tecnologie Chimiche, Consiglio Nazionale delle Ricerche, Milano, Italy; 3 Istituto di Scienze e Tecnologie Chimiche, Consiglio Nazionale delle Ricerche, Italy; 4 Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland, Switzerland; 5 IRCCS San Raffaele Scientific Institute, Milano, Italy, Milan, Italy; 6 Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland, Lugano, Switzerland Introduction : Affinity based systems for isolation of Extracellular Vesicles (EVs) from complex biosamples are commonly plagued by poor recovery and often requires a pre‐concentration step in the analytical workflow. Here we present a pan‐specific isolation method of small EVs (sEVs) from minimally pretreated biological fluids (serum, plasma and urine) based on membrane sensing peptides (MSP) as general EV binders, providing efficient EV recovery and minimum co‐isolation of contaminants. Methods : Magnetic agarose beads (40 micrometer diameter) are modified by MSPs using conventional His‐tag chemistry. 100 microliters of bead suspension are incubated for 1 hour in 1 mL urine (following 2 clearing steps of 15 min at 3000g) or 50 microliters of serum or platelet free plasma (EDTA, Heparin, Citrate) diluted 1:10 in PBS. Intact EVs are released by a mild treatment with a saline buffer for subsequent characterization by microscopy (AFM, TEM), Nanoparticle Tracking Analysis and Western Blotting (WB) of EV external and luminal markers. Common contaminants like uromodulin for urine and albumin and lipoproteins (ApoA, ApoB, ApoE) were also checked by WB. Following isolation, EVs are subjected to immunophenotyping by ExoView and nanoFCM platforms and RNA extraction and RT‐PCR analysis. Results : Highly efficient isolation of sEVs was observed from both serum/plasma and from urine, with minimal recovery of lipoproteins and undetectable albumin contamination for blood derivatives and uromodulin for urine. Electron microscopy confirmed the recovery of EVs with an average diameter in the 80–110 nm range. EVs were captured propaedeutic to the RT‐PCR analysis of specific miRNA and lncRNA and immunophenotyping by single‐vesicle techniques. The protocols were compared to reported standard procedures using antibody modified beads, ultracentrifugation (UC), ultrafiltration (UF), size‐exclusion chromatography (SEC). Summary/Conclusion : We developed highly efficient, easy, timesaving and robust protocols for capture‐and‐release of sEVs from biological fluids. We envision that the integration of MSP as pan‐selective molecular tools on different platforms (beads, resins, microplates) may find broad application in blood and urine analytical workflows. Funding : EIC funded project MARVEL. Grant agreement ID: 951768. Keywords : affinity isolation, blood workflow, urinary EVs

Quantification

Daniela Boselli 1 ; Norma Maugeri 2 ; Chiara Buracchi 3 ; Simona Di Terlizzi 1 ; Monica Romanò 1 ; Emanuele Canonico 1 ; Giuseppe Gaipa 3 ; Angelo A. Manfredi 2 ; Chiara Villa 1 ; Achille Anselmo 1 1 San Raffaele Scientific Institute, Experimental Imaging Center, FRACTAL, Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory, Italy; 2 Autoimmunity and Vascular Inflammation Unit San Raffaele Scientific Institute & Vita‐Salute San Raffaele University, Italy; 3 Tettamanti Research Center, Pediatric Clinic University of Milano Bicocca, Monza (MB), Italy, Italy Introduction : Extracellular vesicles (EVs) have excellent potential to become biomarkers for diagnosis, prognosis, or monitoring of disease. They can be found in cell culture supernatants as well as in bio fluids such as blood, urine, tears and mother milk. They carry proteins, nucleic acids, and other cellular components and play an important role in the communication between cells. Flow cytometry is one of the most used techniques for EVs analysis and quantification, however, since the EV size is close to the electronic noise of common conventional flow cytometers, classical procedures which are usually applied for cells, cannot be applied for EVs. Therefore, the analysis of such small particles is still challenging. Methods : Moving in this technological field, we have developed a twenty‐two color multiparametric panel for in‐depth immunophenotyping and quantification of circulating EVs from human plasma, using both lineage and activation / exhaustion markers, through the new generation spectral flow cytometry. Of note, sample collection, processing and analysis have been performed following MISEV (Minimal Information for Studies of Extracellular Vesicles) recommendations. Results : We were able to identify EVs ranging between 100 nm and 900 nm by exploiting the detection of the Side Scatter (SSC) on the violet laser, instead of the conventional blue one. The spectral approach minimized spreading error and spectral overlay thus allowing the simultaneous identification of several EV subpopulations. In addition to the most common EV subsets, such as erythrocyte‐derived EVs, platelet‐derived EVs, leukocyte‐derived EVs and endothelial‐derived EVs, we observed EV subsets carrying only activation and exhaustion markers. Moreover, we observed that most of the extracellular vesicles exhibited only one antigen on their surface probably due to their small size. Indeed, the unsupervised analysis performed on platelet‐, neutrophil‐ and endothelial‐derived EVs, showed only up to 5% of the EVs carrying two, three or four antigens together on their surface. Summary/Conclusion : Overall, these data highlight the importance of a spectral flow cytometry approach for deciphering the high heterogeneity of the circulating plasma derived EV thus paving the way for a routine use of high dimensional fluorescent antibody panels to study circulating EVs under different pathophysiological conditions. Keywords : spectral flow cytometry

Brain‐Derived

Tanina Arab 1 ; Yiyao Huang 2 ; Rajini Nagaraj 3 ; Evan Gizzie 4 ; Javier Redding‐Ochoa 5 ; Juan C C. Troncoso 5 ; Olga Pletnikova 6 ; Tatiana Boronina 7 ; Robert N Cole 7 ; Vasiliki Machairaki 5 ; David A A. Routenberg 4 ; Kenneth W. Witwer 8 1 The Johns Hopkins University School of Medicine, Baltimore, USA; 2 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, BALTIMORE, USA; 3 Meso Scale Diagnostics, USA; 4 Meso Scale Diagnostics, Rockville, USA; 5 Johns Hopkins University School of Medicine, Baltimore, USA; 6 Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Baltimore, USA; 7 Johns Hopkins University School of Medicine, USA; 8 Johns Hopkins University, Baltimore, USA Introduction : Parkinson's disease (PD) is a progressive neurodegenerative movement disorder. Currently, PD diagnosis relies on late clinical manifestations that overlap with those of other pathologies. Extracellular vesicles (EVs) have emerged as important participants in PD pathophysiology. Interestingly, brain tissue‐derived EVs (bdEVs) participate in cell‐to‐cell communication in the brain and can leave the tissue of origin to more accessible peripheral biofluids such as blood plasma. Methods : bdEVs were separated per an established protocol (Huang et al., JEV, 2020) from brain tissue (Johns Hopkins Brain Resource Center) of PD (n = 24), progressive supranuclear palsy (n = 25), and control (n = 24). Basic characterization was conducted per MISEV2018 guidelines. bdEV surface proteins were profiled using multiplexed ELISA. A subset of the samples was profiled by mass spectrometry for quantitative protein analyses. Results : Interestingly, several markers showed differences between pathological groups and control. Microglial markers TMEM119 and CX3CR1 and neuronal markers CD90, NCAM, and NRCAM were significantly (p‐value < 0.05) more abundant in PD and PSP. Quantitative proteomics revealed the presence of 26 significantly differentially abundant proteins in PD vs. control and/or PSP (Log 2‐fold‐change = 0.32 and p‐value < 0.05). Biologically, some of these proteins are implicated in solute carrier transport, e.g., SLC8A2 differed between PD vs. PSP and PD vs. Control. In contrast, SLC1A3 and SLC2A1 differed only between PSP and control. Several EV markers were also statistically different, e.g., clathrin protein implicated in endocytosis pathway was significantly less abundant in PD vs. PSP. Summary/Conclusion : In this study, we identified several proteins that are differentially abundant between PD and control and that may serve as biomarkers and therapeutic targets. We will test abundant cell‐specific EV surface proteins for utility in immunoprecipitation‐based separation. Funding : This work was supported by the Michael J. Fox Foundation for Parkinson's Research [00900821]. The Witwer lab is also supported in part by NIH grants AI144997, MH118164 , and DA047807 . Keywords : Parkinson's disease, microglia, astrocytes, neurons, extracellular vesicles, ectosomes, exosomes, biomarkers, proteomics, progressive supranuclear palsy

Brain‐Seeking

Sara Busatto 1 ; Marsha Moses 1 ; Golnaz Morad 2 1 1Vascular Biology Program, Boston Children's Hospital, Boston, MA, United States 2Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA, United States, Boston, USA; 2 1Vascular Biology Program, Boston Children's Hospital, Boston, MA, United States 2Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA, United States, USA Introduction : Breast‐to‐brain metastasis (BrM) affects ∼10‐16% of breast cancer patients, predicts a dismal patient prognosis and lacks effective therapeutic and diagnostic options. We demonstrated, for the first time, that EVs derived from brain‐seeking breast cancer cells (BrEVs) breach the intact blood‐brain barrier (BBB) via transcytosis and cause a significantly increased incidence of BrM. Within this context, we are now studying the interactions between BrEVs and BBB endothelial cells (ECs). Methods : BrEVs were isolated by ultracentrifugation, characterized following MISEV guidelines and tested in vitro using primary human brain ECs and in vivo using our mouse model of BrM. ECs were treated with BrEVs and tested using transwell assays, impedance measurements and genetic engineering. Our in vivo models of BrM were administered intravenously with BrEVs and mouse brain microvessels were isolated and analyzed for protein expression. The results were analyzed using machine learning approaches. Results : Our mechanistic studies show that (1) BrEVs target BBB ECs both in vitro and in vivo causing significant variations of multiple proteins involved in intracytoplasmic vesicles' long‐recycling loop (2) BrEVs are responsible for specific EC barrier function and morphological changes (3) BrEVs mediate, at least in part, their effect through the delivery of specific micro RNAs. All molecules cannot be disclosed due to a pending patent. Summary/Conclusion : BrEVs can alter BBB basal metabolism and help to prepare the pre‐metastatic niche, a microenvironment that promotes and sustains BrM in an otherwise hostile microenvironment. Our mechanistic studies increase our understanding of the early events that facilitate BrM and have the potential to identify key regulators in BrM formation and contribute to the development of EV‐based therapeutic and diagnostic strategies for BrM. Funding : This work was supported by NIH R21 CA253051‐01, the Breast Cancer Research Foundation, NIH T32 5T32HL007917‐22 and the Nile Albright Research Foundation. Keywords : breast cancer, breast‐to‐brain metastasis, extracellular vesicles

Cell‐Secreted

Koushik Debnath 1 ; Ik Sung Cho 2 ; Saiumamaheswari Saichellappa 2 ; Peter Toth1 3 ; Dolly Mehta 4 ; Jae‐Won Shin 1 1 Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL 60612, USA, Chicago, USA; 2 Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL, USA; 3 Fluorescence Imaging Core, Research Resources Center, University of Illinois at Chicago College of Medicine, Chicago, IL, USA; 4 Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL, Chicago, USA Introduction : Nanoscale mediators secreted from mesenchymal stromal cells (MSCs) were shown to be efficacious in animal models of tissue injury that disrupts vascular function. However, recent studies highlight the heterogeneity of nanoscale mediators, including extracellular vesicles (EVs) and non‐vesicular extracellular particles (NVEPs). Restoration of endothelial barrier function requires the activation of extracellular matrix (ECM) signaling. Thus, we tested the hypothesis that an ECM‐containing nanoscale fraction is essential for the restoration of vascular permeability upon tissue injury. Methods : The crude fraction was isolated by following EV‐TRACK ID EV150007 from mouse MSCs. We used immunoaffinity‐based approaches along with Triton‐X sensitivity assay and nanoparticle tracking analysis to quantify the subpopulations that contain fibronectin (FN). FN+ fractions were pulled down via FN antibody‐functionalized magnetic nanoparticles (∼8 nm) for characterization by transmission electron microscopy (TEM). The fractionated nanoscale subpopulations were delivered to mice 4 h after treatment with lipopolysaccharide (LPS), and the mice were analyzed for edema and vascular permeability in the lungs. Results : Our results show that MSC‐secreted nanoscale mediators consist of Triton‐X sensitive CD63+FN‐ (40%) and Triton‐X resistant CD63‐FN+ (33%) subpopulations, while the CD63+FN+ population is less than 5%. TEM analysis confirms that the FN+ fraction is non‐vesicular with a single particle size of ∼30 nm and is more irregular in shape than EVs. We show that FN+ NVEPs are essential for the restoration of endothelial barrier function after LPS‐induced lung injury. Summary/Conclusion : This study reveals the importance of FN+ NVEPs as a novel nanoscale mediator to restore vascular integrity in response to tissue injury. Future studies will investigate the biogenesis mechanisms of ECM‐presenting NVEPs and mechanisms of action by which FN+ NVEPs restore endothelial barrier function. Funding : This work is supported by NIH Grant No. R01‐ HL141255 (to J.‐W.S.). Keywords : non‐vesicular extracellular particles, extracellular matrix, fibronectin

Functionalizing

Daniel Humphrys 1 ; Neil King 1 ; Wes Sundquist 2 ; Ben Schmitz 2 1 University of Washington, USA; 2 University of Utah, USA Introduction : Extracellular vesicles (EV) offer a promising alternative to current methods of drug packaging and delivery, but engineering EV is challenging. To enable more precise engineering of EV, we developed Enveloped Protein Nanocages (EPN): EV generated by designed self‐assembling proteins which induce their own release from cells inside cell‐derived membrane envelopes. EPN proteins provide a genetically addressable and modular platform to produce EV. Here we describe methods that enable the functionalization of EPN membranes through the incorporation of natural and designed Transmembrane Proteins (TMP). In contrast to conventional EV‐based technologies which rely on passive pseudotyping, we leverage covalent conjugation of the TMP with the EPN protein to create Transmembrane Protein‐Conjugated Enveloped Protein Nanocages (TMP‐C‐EPN). These TMP‐C‐EPN ensure efficient incorporation and display of the TMP on the EPN membrane, and can result in the formation of monocage EPN consisting of a single protein nanocage per membrane envelope. Different TMP have been incorporated to functionalize the EPN, allowing cell‐specific targeting and vaccine development. EPN are a promising new class of genetically encoded biomaterials, and generally highlight the utility of designed protein scaffolds that induce EV release. Methods : EPN were produced and purified from adherent HEK 293T and suspension expi293F cells. Use of the SpyCatcher‐SpyTag system allowed for conjugation of the intravesicular EPN nanocage with the intravesicular tail of the designed TMPs, ensuring presentation of a particular TMP on the surface of released EPN. Western blots were used to probe for the presence of designed TMP, to check for membrane integrity, and to evaluate conjugation efficiency of the TMP‐cage interaction. CryoEM was used to image particles and evaluate EPN morphology. Flow cytometry analysis was done to evaluate cell‐specific EPN targeting efficiency. Results : EPN cages were successfully conjugated via SpyCatcher‐SpyTag covalent interaction to a designed minimal TMP. A small population of TMP‐C‐EPN were seen as monocage EPN. Design of an EGFR‐targeting TMP allowed for targeting of the EPN towards only EGFR‐expressing cells. Inclusion of the SARS‐CoV‐2 spike protein resulted in CoV‐2 decorated EPN, and delivery of an mRNA encoding for the EPN nanocage and designed CoV‐2 TMP resulted in an immune response in mice. Summary/Conclusion : EPN are a versatile, modular, and tractable platform to induce release of TMP‐decorated‐EV from producer cells. Here we functionalized EPN by directing them towards target cells in a mixed cell population, and tested their use as a vaccine platform with an mRNA‐based vaccine against SARS‐CoV‐2 to mice. The EPN technology shows promise for efficiently engineering EV as therapeutic and basic science tools. Keywords : therapeutics delivery vaccine

Multiparametric

Zach A. Troyer 1 ; Olesia Gololobova 2 ; Zhaohao Liao 1 ; Aakash Koppula 3 ; Mona Batish 3 ; Kenneth W. Witwer 4 1 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, USA; 2 Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, USA; 3 Department of Medical and Molecular Sciences, University of Delaware, USA; 4 Johns Hopkins University, Baltimore, USA Introduction : Enveloped viruses, with a lipid bilayer and host components, can be seen as a type of EV hijacked by the viral life cycle to further viral replication. We and others have shown that HIV displays EV surface markers, including tetraspanins CD63 and CD81. Individual HIV virions could be distinguished from the EV background, however, using the size and surface marker analysis afforded by the SP‐IRIS technique in conjunction with fluorescent anti‐HIV gp120 antibody. Here, we report an improvement on SP‐IRIS analysis of HIV, using smFISH to fluorescently label HIV genomic RNA (gRNA) and allow discrimination between infectious (containing gRNA) and non‐infectious (lacking gRNA) virions. Methods : Virions and/or host EVs were concentrated from the conditioned medium of chronically‐infected (HIV‐1, BaL strain) and uninfected PM1 cells using differential ultracentrifugation. Samples were UV‐inactivated before incubation on SP‐IRIS chips. An AF488‐conjugated anti‐gp120 antibody was used as a fluorescent probe. smFISH‐AF647 probes were designed against the HIV‐1 BaL gRNA. SP‐IRIS was done using ExoView R100 and tetraspanin capture chips from Unchained Labs (former: NanoView). Results : SP‐IRIS detected an smFISH‐AF647+ population of gp120/CD81+ particles around 100–120 nm diameter, likely infectious HIV‐1 virions. This smFISH‐AF647+ population was not seen in control EVs isolated from uninfected cells. Use of non‐HIV‐specific smFISH‐AF647 probes also did not result in smFISH‐AF647+ signal. Summary/Conclusion : The detected particles are likely to be mature, infectious HIV virions carrying HIV gRNA. In ongoing studies, we will phenotype these particles for additional surface proteins and determine if different smFISH probe sets might be used to detect diverse HIV strains. The technique could also be applied to image highly abundant RNAs in host EVs. Funding : This work was funded by the ION‐ARPA initiative. Keywords : SP‐IRIS, smFISH, HIV, virion, enveloped virus, rna detection, extracellular vesicles

Neuroprotective

Leila Noori 1 ; Francesco Cappello 2 ; Somayeh Arabzadeh 3 ; Yousef Mohamadi 4 ; Sina Mojaverrostami 5 ; Mohammad Akbari 5 ; Gholamreza Hassanzadeh 5 1 Department of Biomedicine, Neurosciences and Advanced diagnosis (BIND), University of Palermo, Palermo, Italy, Palermo, Italy; 2 Department of Biomedicine, Neurosciences and Advanced diagnosis (BIND), University of Palermo, Palermo, Italy, Italy; 3 Department of Biology, School of Basic Sciences, Ale Taha Institute of Higher Education, Tehran, Iran, Iran; 4 Department of Anatomy, School of Medicine, Ilam University of Medical Sciences, Ilam, Iran, Iran; 5 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran, Iran Introduction : In the early stages of CNS disorders including spinal cord injury (SCI), neuroinflammation causes to activation of the astrocytes, overexpression of glial fibrillary acidic protein (GFAP) and lead to formation of glial scar which are considered as one of the main obstacles in the regeneration of spinal cord tissue (1). Further, neural progenitor cells (NPCs), located in ependymal layer around the central canal of spinal cord, effectively contributed to tissue repairment after injury and trophic factors increase the Nestin, NPC marker, expression (2). Death of neurons is also a destructive outcome of CNS injuries which leads to functional impairment (3). Extracellular vesicles (EVs), heterogeneous bilayer nanovesicles containing growth factors, lipids, nucleic acids and proteins, derived from mesenchymal stem cells (MSCs‐EVs) showed neuroprotective properties via crossing BBB and providing growth factors, support neuronal survival and conserving from apoptosis during neurodegeneration (4). Intrathecal delivery of EVs ensures their distribution around the injured site (5). The damaged BBB is permeable within 1 h till 5 days after SCI with a peak in 24 h post‐injury (6). We investigated potential neuroprotective and neuro‐regenerative effects of human Wharton's jelly mesenchymal stem cells derived extracellular vesicles (hWJ‐MSC‐EVs) on injured spinal cord tissue one week after SCI in rats. Methods : We applied three intrathecal different doses (1, 2 and 3 μg) of human Wharton's jelly mesenchymal stem cells derived extracellular vesicles (hWJ‐MSC‐EVs) 24 h after injury in a compressive SCI rat model. Immunohistochemistry (IHC) was done to measure the GFAP and Nestin expressions. Cell death was studied by TUNEL assay. Results : Our finding showed that intrathecally administrated hWJ‐MSC‐EVs attenuated GFAP expression in all treatment groups both in gray and white matter of epicenter, although this reduction was significantly valuable in mid and high doses groups. Nestin expression remarkably improved with mid and high doses of WJ‐MSCs‐EVs. Moreover, it reduced neuronal death more significantly in mid and high doses and saved higher number of typical neurons in ventral horn of spinal cord tissue in those groups. Summary/Conclusion : We came to the conclusion that hWJ‐MSC‐EVs are promising neuroprotective tools to modulate the astrocytes activity and neuronal cell death as well as potential neuro‐regenerative agents to stimulate NPCs after SCI in rats. Funding : This work was funded by Tehran university of medical sciences. Keywords : neurodegeneration, neuro‐regeneration, extracellular vesicles, mesenchymal stem cells

Plant‐Derived

zhu zhao Univ of Arizona, Phoenix, USA Introduction : We focus on the exploration of plant kingdom in order to identify both new sources and delivery systems of therapeutics. For instance, plant produce plant‐derived extracellular vesicles (pEVs) that can be found in their paramural space. Similar to animal‐derived exosomes, these pEVs exhibit low immunological risk and good bioavailability but also offer many more resources of raw materials, cost efficient production and do not harbor zoonotic or human pathogens. Interestingly, these pEVs display two features of relevance for drug discovery and development: (1) they carry pre‐encapsulated natural cargos that could be readily available targets for the screening of new therapeutic compounds and (2) they can be loaded with synthetic cargo to overcome current limitations of animal‐derived nanoparticles drug delivery systems. So in overall pEVs can not only act as a certain type of drug, but also work as a cargo vesicle to deliver drug. Our lab targeted on exploring pEVs which potentially have high contents of anti‐oxidants by sorting of many types of plant. Some of them potentially show radio protective effects on human cells and some of them show radio sensitivity effects. And the meantime, we explored new methodologies to encapsulate drug into selected pEVs. Both radio protector drug and anti‐cancer drug are tested to be encapsulated. In this case, pEVs potentially generate benefits if a combination of chemotherapy and radiation therapy induced for cancer treatment, by enhancing chemotherapy working as a anti‐cancer drug delivery cargo and at the mean time, reducing the side effects of radiation therapy since pEVs's radiation protective characteristic. Methods : Isolation: step centrifugation; sucrose gradient; Size‐exclusion Chromatography (SEC) Characteristics: NTA/SEM/TEM/Fluorescence Microscopy. Biology tests: MTT test/ clonegenic test. Results : Spinach, tumeric, clove, duckweeds, grape, green olive, black olive etc… Many plants were tested. Olive potentially generate radio sensitivity on human cancer cells while no significant effects on normal human cells. Grapes generate radio protectivity on human normal cells, while no significant effects on human cancer cells. Summary/Conclusion : Because of the minimal cytotoxicity of each plant EVs, also variation of radiation effects on cancer and normal human cells in different types of pEVs, pEVs have strong potential benefits with loading certain types of drug to meet some special treatment requirement, such as if a combination of radiation therapy and chemotherapy is required.

Proinflammatory

Jerry Xu ; Arianna Harris Kawano; Emily K. Sims Pediatric Endocrinology, Wells Center for Pediatric Research, School of Medicine, Indiana University, Indianapolis, USA Introduction : β cell EVs may act as paracrine effectors in islet health. However, mechanisms linking β cell stress to changes in EVs and whether activation of these pathways can impact diabetes remain unexplored. In other cells, neutral sphingomyelinase 2 (nSMase2) induces ceramide‐dependent EV formation and release. We hypothesized that β cell inflammatory stress engages ceramide‐dependent EV formation pathways, and that increased ceramide‐enriched EVs could then impact surrounding β cells. Methods : We treated INS‐1 β cells with 24 hrs of 5 ng/mL IL1β. nSMase 2 and ceramide expression were quantified with immunoblot and/or flow cytometry and EVs were isolated using ultracentrifugation or tetraspanin (CD9, CD63, CD81) bead‐based pulldown. Results : IL1β increased β cell nSMase2 and ceramide expression in concert. Direct nSMase2 activation with 24 hrs of Caffeic acid phenethyl ester also increased ceramide staining. Both treatments also increased β cell EV ceramide staining. In contrast, nSMase2 knockdown reduced IL1β induction of cellular and EV ceramide staining. Human islets exhibited similar increases in cellular nSMase2 and ceramide staining after 24‐hr cytokine mix treatment (IL1β, 10ng/mL TNFα, and 100ng/mL IFNγ). To test potential for reversal, cells were treated with IL1β +/‐ the GLP‐1 agonist Exendin‐4. Exendin‐4 also reduced IL1β induction of cellular nSMase2 and cellular and EV ceramide staining. To test for β cell EVs transfer to surrounding cells, we generated INS‐1 cells with GFP tagged CD9. Flow cytometry staining in mCherry+ INS‐1 cells after 24‐hr coculture with CD9‐GFP cells showed that cocultured cells exhibited increased double‐positive fluorescence, suggesting CD9‐GFP+ EV transfer. This was enhanced by 24‐hr IL1β treatment. Summary/Conclusion : Our findings suggest that nSMase2 activity regulates β cell EV subpopulations under conditions of inflammatory stress. Future work will interrogate impacts on other EV cargo and physiologic impacts of EV transfer. Keywords : EV, diabetes, beta cell, ceramide, lipid, inflammation

Reproducibility

Andrew Lai 1 ; Dominic Guanzon 2 ; Carlos Palma 3 ; Ramin Khanabdali 4 ; Lewis Perrin 5 ; John Hooper 5 ; Jim Coward 6 ; Gregory E. Rice 7 ; Carlos Salomon 8 1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia, Brisbane, Australia; 2 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, QLD 4029, Brisbane, Australia; 3 Inoviq Limited, Notting Hill, Australia., Brisbane, USA; 4 Inoviq Limited, Notting Hill, Australia., USA; 5 Mater Research Institute‐University of Queensland, Translational Research Institute, Woolloongabba, Australia., Brisbane, USA; 6 Mater Research Institute‐University of Queensland, Translational Research Institute, Woolloongabba, Australia., USA; 7 Inoviq Limited, Notting Hill, Victoria, Australia., Notting Hill, Australia; 8 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia., Brisbane, Australia Introduction : Despite advances in treatments over the last few decades, epidemiological studies indicate only a marginal impact on the course of ovarian cancer (OVCA) due to the lack of reliable early detection methods. Current Extracellular vesicles (EV) isolation methods are time consuming, and quality control can be challenging to achieve. Here, we evaluate the performance and reproducibility of an EV capture technology (EXO‐NETÒ) to characterize a suite of EV biomarkers associated with ovarian cancer and the classification accuracy of multivariate models based on EXO‐NET ‐isolated EV biomarkers. Methods : Plasma samples were obtained from healthy women (controls, n = 20), benign (n = 20), and high‐grade serous ovarian cancer (n = 40). EVs were isolated using EXO‐NETÒ (INOVIQ LTD, Australia) and characterised by Nanoparticle Tracking Analysis (NTA), protein abundance (CD63, CD9, Alix, TSG101 and CD81), and morphology using NanoSight, Western blot, and electron microscopy (EM). Optimisation experiments were performed to determine the optimal ratio of plasma/ EXO‐NETÒ. The number of EV‐CD9+ve were quantified using an ELISA kit. RT‐PCR and targeted proteomic analysis were performed against 7 EV biomarkers (patent under review), and spike‐in control miRNAs and proteins were used. Results : EVs isolated using EXO‐NETÒ were positive for CD63, CD9, Alix, TSG101, and CD81, confirming the presence of EV‐associated proteins. NTA showed a 20% (equivalent 2.5 × 109) decrease in particles between 50 to 200 nm after EV isolation compared with total plasma. Similar results were observed with EM (i.e., plasma before and after incubation with EXO‐NETÒ). Dose‐response analysis based on CD9+ve EVs showed an optimal concentration using 200μl of plasma and 30μl of EXO‐NETÒ. At optimal ratio, a total of 6.3 ± 2.5 mg protein equivalent to 4.3 ± 1.5 mg of peptides, and 7.3 ± 2.5 ng of small RNA was obtained. Treatment with Proteinase K and RNase A did not significantly (p>0.05) change the concentration of peptides and small RNA. The coefficient of variation based on the quantification of CD9+ve EVs was 8.2 ± 5.3 %. The classification accuracy rates (i.e., the number of correct predictions) of a select group of EV biomarkers used for the detection of ovarian cancer were 100%, 95%, 100%, 80%, and 100% using LogitBoost, J48, Random Tree, Decision Table, and Random Forest algorithms, respectively. Interestingly, leave‐one‐out cross validation showed minimal loss of performance. Summary/Conclusion : We have optimised a simple, rapid, and scalable capture technology (EXO‐NETÒ) to isolate EVs with biomarker potential in the context of ovarian cancer. This will allow for the translation of EV‐based research into clinical applications and platforms that are available in pathology laboratories. Funding : MRF1199984), NHMRC 1195451, and INOVIQ LTDA.

Transcriptomics

Trevor J. McFarland 1 ; Ursula S. Sandau 1 ; Tzu‐Yi Chen 2 ; Taliah Solemani 3 ; Edgar Gonzalez‐Kozlova 3 ; Navneet Dogra 3 ; Julie A. Saugstad 4 1 Oregon Health & Science University, Portland, USA; 2 Icahn School of Medicine at Mount Sinai, Department of Department of Genetics and Genomic Sciences, New York, USA; 3 Icahn School of Medicine at Mount Sinai, New York, USA; 4 Department of Anesthesiology and Perioperative Medicine, Oregon Health & Science University, PORTLAND, USA Introduction : RNA sequencing of EV RNAs isolated from small CSF volumes is challenging and the number of publications on this topic are limited. Traditionally, RNA‐seq of CSF has been performed either on total CSF or from EVs enriched from larger volumes, typically milliliters. Due to the small volumes available from human biorepositories, methods that allow complete sequencing of EV RNA are especially needed. Methods : The Institutional Review Board of the Oregon Health & Science University approved all human participant procedures. CSF from six healthy participants was collected via lumbar puncture under fasting conditions. A volume of 300 μL from each donor was pooled, mixed and aliquoted into three 500 μL samples. Each replicate sample was concentrated using a 30 kD cut‐off ultra‐filtration column to a final volume of 150 μL. Each concentrated sample underwent size exclusion chromatography (SEC) using 35 nm resin. The EV enriched fractions were pooled from each replicate at a final volume of 800 μL. RNA was isolated using the Urine miRNA purification kit (Norgen) and eluted with 30 μL of elution solution. RNA QC, library prep, RNA‐seq and data analysis were performed at the Icahn School of Medicine using their liquid biopsy analysis pipeline. Results : Ultra‐filtration and EV enrichment by SEC followed by RNA isolation yielded 2–7 ng of RNA and passed input RNA Bioanalyzer QC metrics. Following successful cDNA library construction and sequencing, the data analysis revealed a diversity of coding and non‐coding RNAs. The majority of sequences were mapped to known genes (∼75%), while the remaining RNA species consisted of lncRNA, rRNA, and pseudogenes. As observed with other studies, miRNA represented a very small percentage of the mapped sequences. Pathway analysis of the top 10% most expressed genes revealed that these EV RNAs are associated with regulation of neuron differentiation, projection development, transmembrane transport and glutamate signaling. Summary/Conclusion : Here we report that EV RNA from 0.5 mL of CSF is suitable for RNA‐seq. Future aims include performing RNA‐seq on CSF EVs from larger cohorts of disease and control donors. Funding : NIH/NIA, R21AG07848. Keywords : cerebrospinal fluid, RNA, transcriptomics

Tumor‐Derived

Jiae Lee; Hyungjoon Park ; Young V. Kwon University of Washington, Seattle, USA Introduction : Tumor‐derived extracellular vesicles (EVs) play important roles in communication between tumor cells and immune cells, which elicits anti‐ and pro‐tumor immune responses depending on the context. Considering the powerful genetic tools available in Drosophila and the transparency of the larvae, Drosophila could be a desirable in vivo model for studying the mechanisms underlying the production of EVs from tumor cells and the processing of EVs in immune cells, as well as the physiological roles of the EV‐mediated communications. However, it is unknown whether Drosophila tumors also produce EVs that play a role in communication with immune cells. Methods : We employed a well‐characterized Drosophila epithelial tumor model to address whether Drosophila tumors produce EVs. Live imaging techniques allowed us to trace tumor‐derived large EVs in live animals and observe the production of large EVs from tumors. Moreover, we could biochemically enrich tumor‐derived large EVs and transplant them into either wild‐type or genetically modified larvae to investigate their physiological roles (Track ID: EV140287 ). Results : We found that the production of large EVs (larger than 1 micrometer) from tumors is a conserved process in Drosophila. Interestingly, the injection of large EV fractions was sufficient to induce a systemic immune response in a manner dependent on Drosophila immune cells hemocytes. cGAS‐STING signaling was elevated in tumors, and STING knockdown in tumors suppressed the production of large EVs. Interestingly, injection of large EV fractions also increased cGAS‐STING signaling in hemocytes, and STING knockdown in hemocytes suppressed the large EV‐induced systemic immune response. Summary/Conclusion : Our study visualizes how cGAS‐STING signaling propagates via the generation of tumor‐derived large EVs to induce a systemic immune response and establishes Drosophila as an animal model for studying the biology of tumor‐derived large EVs. Furthermore, we elucidate the conserved role of cGAS‐STING signaling in controlling the production of large EVs from tumor cells. Keywords : tumor, large extracellular vesicle, drosophila and systemic immune response

Water‐Soluble

Sarah J. Cox‐Vazquez Acoerela Pte Ltd, Singapore Introduction : We developed a series of transmembrane conjugated oligoelectrolytes (COEs) with tunable optical emissions from the UV to the near IR to address the false‐positive problem when detecting nanometer‐sized extracellular vesicles (EVs) by flow cytometry. The amphiphilic molecular framework of COEs is defined by a linear conjugated structure and cationic charged groups at each terminal site. Consequently, COEs have excellent water solubility and the absence of nanoaggregates at concentrations up to 50 μM, and unbound COE dyes can be readily removed through ultrafiltration. These properties enable unambiguous and simple detection of COE‐labeled small EVs using flow cytometry with negligible background signals. We also demonstrated the time‐lapsed tracking of small EV uptake into mammalian cells and the endogenous small EV labeling using COEs. Briefly, COEs provide a class of membrane‐targeting dyes that behave as biomimetics of the lipid bilayer and a general and practical labeling strategy for nanosized EVs. Methods : N/A Results : N/A Summary/Conclusion : N/A

Antigen‐Loaded

Hayden M. Pagendarm 1 ; Youn J. Jung 2 ; Alissa M. Weaver 3 ; John T. Wilson 2 1 Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA, Nashville, USA; 2 Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA, USA; 3 Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN, USA, Nashville, USA Introduction : Autoimmune diseases are driven by adaptive immune responses that are inappropriately directed towards an autoantigen. Tolerogenic vaccines capable of sequestering immune responses to autoantigens pose a potential treatment without the drawbacks of broad immunosuppression. However, development of such therapies has been limited by the inability to deliver and present adequate antigen without co‐stimulation. As many extracellular vesicles (EVs) are immunologically inert and can load a variety of cargos, we developed an EV platform for the co‐delivery of antigens and immunoregulatory cues. Methods : EVs were isolated from HEK293SF‐3F6 cells using an iodixanol density gradient and characterized in accordance with MISEV 2018 guidelines. Azide‐functionalized EVs were generated by adding Ac4ManNAz to the culture media. HEK cells expressing BASP1‐OVA were generated using a PEI MAX transfection reagent. Ovalbumin (OVA) was functionalized with AF488 and DBCO via NHS/TFP ester chemistry. Tolerance induction was assessed using in vitro and in vivo models involving bone marrow dendritic cells (BMDCs) or DC2.4s and OT‐I and OT‐II T cells. Results : We first demonstrated that HEK EVs can be loaded with the model antigen OVA via both an exogenous metabolic glycoengineering approach and an endogenous producer cell engineering approach. Furthermore, we demonstrated that OVA‐loaded HEK‐EVs induce antigen presentation without activating the innate immune system of DCs. Treating mice with engineered EVs after adoptive transfer of OT‐I and OT‐II cells, followed by OVA + LPS challenge, indicated that EV treatment suppressed T cell proliferation and shifted OT‐II cells towards a regulatory phenotype. Summary/Conclusion : These results indicate that EVs may be capable of serving as a tolerance‐inducing vaccine platform. Future work aims to load multiple immunosuppressive cues into EVs and demonstrate that treatment with engineered HEK EVs can downregulate the adaptive immune response in an experimental autoimmune encephalitis model. Funding : NSF FMSG 2036809 NIH ITED T32 DK101003 NSF GRFP 2022305770. Keywords : autoimmunity, immune tolerance, metabolic glycoengineering, immune regulation

Biomanufacturing

Theresa D'Souza 1 ; Jae Jung 2 ; Robert Kirian 3 ; Jian Ling 4 ; Nicholas Mcmahon 4 ; Kreg Zimmern 4 ; Jon Rowley 2 1 RoosterBio Inc, Frederick, USA; 2 RoosterBio, Inc., Frederick, USA; 3 RoosterBio, Frederick, USA; 4 Southwest Research Institute (SwRI®), San Antonio, USA Introduction : As the interest and need of extracellular vesicles (EVs) as therapeutic product or as carrier for bioactive cargo rises, so does the necessity of biomanufacturing platform that is cost effective and can generate sufficient material for clinical translation. In this study, we evaluated a novel 3D‐printed, scalable, and perfusion‐based bioreactor developed by SwRI, along with PBS Biotech Vertical‐Wheel® microcarrier based bioreactor for EV production. The objective of the study was to identify and optimize parameters to increase the EV productivity from human bone marrow mesenchymal stem/stromal cells (hBM‐MSCs) after growth expansion, with prototypes of different scales of perfusion‐based bioreactor as well as the microcarrier‐based bioreactor platform. Methods : The xeno‐free RoosterVial‐hBM (RoosterBio, MD) were grown in planar as seed train prior to seeding on to the 3D‐printed ‐perfusion bioreactors or microcarriers for suspension culture in PBS Biotech bioreactors. The cells were expanded in xeno‐free RoosterNourish‐MSC growth medium before EV collection in RoosterCollect‐EV (RoosterBio). The process was scaled up from surface area of 250 to2500 cm2 (46 – 460 mL) for the perfusion bioreactors and 100 to 3000 mL for PBS bioreactors. Key process parameters evaluated were seeding density, microcarriers, growth and EV collection duration, and number of media exchanges and monitored in‐process by key metabolites with Nova Flex (Nova Biomedical, MA), pH, and particle counts. The harvested conditioned media were analyzed for metabolites and EV productivity with NanoSight through Nanoparticle Tracking Analysis software (Malvern Panalytical, MA). Results : For the microcarrier‐based bioreactor scales of 500 mL and 3000 mL, the optimal microcarrier seeding density and cell growth were achieved when the cell density reached to 2E05 cells/mL and 4E05 cells/mL by the end of growth phase, respectively. The highest EV productivity of 1.2E10 particles/mL achieved at the 3000mL scale. For the 2500cm2 perfusion‐based bioreactor, the cell number was estimated at approximately 180E6 at the end of growth phase, with the EV productivity of 3.2E9 particles/mL, which was comparable to similar surface area of suspension culture 500mL scale with EV productivity of 3.0E9 particles/mL. Summary/Conclusion : The 3D‐printed perfusion‐based bioreactor can harvest EVs multiple days or even continuously. The highest particle size frequencies were observed 100–200 nm in diameter across all processes. The novel 3D‐printed perfusion‐based bioreactor, as an alternative, scalable EV biomanufacturing platform, will serve the biotech industry as a cost‐effective path in achieving the demand of translational EVs. Funding : MTEC‐19‐01‐Biomfg‐0004 (ESG# MT19007.04).

Characterization

Karina S. Zitta 1 ; Lars Hummitzsch 2 ; Rene Rusch 2 ; Katharina Heß 2 ; Markus Steinfath 2 ; Joachim Cremer 2 ; Frank Lichte 3 ; Fred Fändrich 2 ; Rouven Berndt 2 ; Kerstin Parczany 2 ; Christopher Schnoor 2 ; Martin Albrecht 1 1 UKSH Kiel, Kiel, Germany; 2 UKSH Kiel, Germany; 3 CAU Kiel, Germany Introduction : We have recently shown that human monocyte derived anti‐inflammatory macrophages (regulatory macrophages, Mreg) bear pro‐angiogenic potential in‐vitro (Hummitzsch, L. et al. Stem Cells Int (2019)). Mreg also release large extracellular vesicles (L‐EV), which may be involved in the transport of active molecules that participate in cell communication (Paolicelli, R. C. et al., Neuroscience (2018)). Methods : Mreg were differentiated for 7 days using blood monocytes from healthy donors (N = 9). L‐EV populations were enriched from Mreg culture supernatants by differential centrifugation. Characterization of L‐EV was performed by cell/particle analysis, brightfield/transmission electron microscopy (TEM) and flow cytometry. Culture media metabolites were analyzed using a blood gas analyzer. Results : L‐EV can be reproducibly isolated from Mreg cultures at day 7 by using 2 standard centrifugation steps. Mreg release about 1.5 L‐EV/Mreg into the culture medium. Release of L‐EV is negatively correlated with a lactate concentration (6‐15mmol/l) and positively correlated with pH (6.8‐7.3). The average diameter of Mreg is 13.77 ± 0.87μm (volume: 1.38 ± 0.25pl) whereas L‐EV reveal a size of 7.45 ± 0.28μm (volume: 0.22 ± 0.03pl). While Mreg size increased during the differentiation process (t0: 9.95 ± 3.73μm, t7: 13.77 ± 0.87 μm; P0.05). Several morphological similarities between Mreg and L‐EV were evidenced by TEM, such as numerous intracellular membrane vesicles and pseudopodia‐like extensions. A lipid‐bilayer structure was demonstrated by the presence of transmembrane anchored proteins specific for EV (CD81:62.3%; CD9:47.5%; CD63:78.7% and LAMP1:54.9%) (Witwer, K. W. et al. J. Extracell. Ves.(2021)). Summary/Conclusion : L‐EV can be reproducibly isolated from Mreg cultures. Their morphological appearance and membrane characteristics suggest that they are derived from Mreg and may possess Mreg‐like functions. Funding : TRIZELL GmBH. Keywords : large extracellular vesicles (LEV), regulatory macrophages (Mreg)

Drug‐Resistant

Min Woo Kim 1 ; Sol Moon 2 ; Suji Lee 2 ; Young Kim 2 ; Hyojung Lee 2 ; Joon Ye Kim 2 ; Jee Ye Kim 2 ; Seung Il Kim 2 1 Department of Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea; 2 Department of Surgery, Yonsei Unversity College of Medicine, Seoul, Republic of Korea Introduction : Predicting tumor response after neoadjuvant chemotherapy (NAC) is critical for predicting prognosis and deciding the treatment strategy in patients with breast cancer (BC); however, there are no reliable blood biomarkers that can assess tumor responses effectively. Therefore, we aimed to validate the clinical feasibility of miRNA and protein markers in extracellular vesicles (EV) collected for predicting tumor response during NAC. Methods : Drug‐resistant clones were generated from three triple‐negative breast cancer (TNBC) cell lines. Profiling of drug‐resistant TNBC identified potential drug resistance‐related biomarkers. We isolated tumor‐derived EVs and validated that drug‐resistant biomarkers were also significant in EVs released from drug‐resistant tumor cells. The putative drug‐resistant EV markers were validated in plasma samples from 72 BC patients, including 42 individuals showing no tumor response and 30 individuals showing a complete response. Results : Compared with wild‐type EVs and drug‐resistant EVs, 5 EV miRNAs (miR‐125b, miR‐146a, miR‐484, miR‐1246, and miR‐1260b) and 3 EV membrane proteins (MDR1, MRP1, and BCRP) were confirmed as biomarkers contributing to the acquisition of drug resistance. The optimal combination of drug‐resistant EV markers represented the best performance to differentiate tumor response. We also analyzed The GEO datasets to identify target genes of EV mRNAs related to drug resistance. The miRNA‐target gene networks correlated highly with cell mitosis, metabolism, drug transport, and immune response. Summary/Conclusion : Our study suggests that drug‐resistant EV markers effectively predict tumor response, which can be clinically applicable. Moreover, drug‐resistant EV markers seem to increase with repeated drug treatment in the tumor EV population. This approach allows real‐time monitoring of drug‐resistant EV marker alterations potentially sensitive to targeted therapy or associated with treatment resistance in patients with BC during NAC. Funding : This study was supported by a Severance Hospital Research fund for Clinical Excellence (C‐2022‐0018) and the National Research Foundation of Korea Grants (2021R1I1A1A01051594 and 2022R1F1A1074605). Keywords : TNBC, neoadjuvant chemotherapy, liquid biopsy, predictive biomarker, extracellular vesicles, microRNA, EV protein

Iedda‐Mediated

Migara Kavishka Jayasinghe 1 ; Yock Sin Lay 2 ; Dawn Xiao Tian Liu 2 ; Chang Yu Lee 2 ; Dong Van Hoang 1 ; Rebecca Carissa Prajogo 2 ; Minh T.N Le 1 1 National University of Singapore, Singapore, Singapore; 2 National University of Singapore, Singapore Introduction : Extracellular vesicles serve as excellent drug carriers, enhancing biodistribution and cellular uptake of drugs. Current immunotherapeutic approaches for cancer therapy are limited by a balance between therapeutic efficacy and off‐target side‐effects and can benefit from conjugation onto EVs. In this study, we developed a novel IEDDA‐mediated surface functionalization approach to engineer red blood cell‐derived EVs post‐isolation with immunomodulatory ligands at high efficiency. We hypothesised that the resulting EVs would mimic ligand multimerization, facilitating efficient receptor crosslinking and formation of immune synapses, thereby enhancing the effect of the EV‐associated immunomodulatory ligands as compared to free ligands. Methods : Extracellular vesicles were isolated from human red blood cells and subsequently purified via differential centrifugation, density centrifugation and size exclusion chromatography. The resulting EVs were surface functionalized with a combination of immunomodulatory ligands using bioorthogonal IEDDA click chemistry. These surface functionalized EVs were assessed on their ability to activate immune cells and suppress tumor progression in vivo as compared to treatment with an equivalent dose of free ligands. Results : IEDDA‐mediated conjugation resulted in efficient EV surface functionalization, capable of achieving over 50,000 copies per EV. Conjugation of immunomodulatory ligands such as agonistic CD137 antibodies and IL‐2 on the EV surface significantly enhanced their therapeutic efficacy over free ligands via the formation of immune synapses and improved receptor clustering. EVs conjugated with a complementary combination of immune stimulatory ligands and immune checkpoint inhibitors were able to significantly shift the tumor immune milieu towards an anti‐tumorigenic phenotype in a lung metastatic B16 F10 allograft and suppress tumor progression to a greater extent than a equivalent dose of free ligands. Conjugation of ligands onto EVs also limited their biodistribution to the tumor microenvironment, resulting in lower off‐target toxicity than the free ligand treatment. Summary/Conclusion : Conjugation of immunomodulatory ligands onto EVs via IEDDA reactions presents a potent and biocompatible approach that can enhance the efficacy of existing immunotherapeutics while addressing much of the drawbacks faced by current immunotherapeutic approaches for cancer treatment. Funding : Ministry of Health (grant MOH‐000643). Keywords : extracellular vesicles, cancer, immunotherapy, EV engineering, EV surface functionalization, IEDDA

Outer‐Membrane

Qianbei Li 1 ; Zihao Ou 2 ; Situ Bo 1 ; Lei Zheng 3 1 Nanfang Hospital, Southern Medical University, China (People's Republic); 2 Nanfang Hospitol, Guangzhou, China (People's Republic); 3 Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China (People's Republic) Introduction : Preeclampsia (PE) is a multisystem disorder with high maternal morbidity and mortality rates. No practical therapeutic approach is available, except for early delivery. Gut dysbiosis is also associated with PE development. However, the host response to microbiome‐based therapy for PE remains unknown. Methods : Previous data showed that the abundance of Akkermansia muciniphila (Am) was lower in patients with PE than in normotensive pregnant women, which was also found using a mouse model of PE. Results : TPre‐supplementation with Am significantly mitigated pre‐eclamptic symptoms in the murine model. Am‐derived outer‐membrane vesicles (AmOVs) entered the placenta and improved the placental pathology in mice with PE. These beneficial effects of AmOVs were mediated by enhanced trophoblast invasion of the spiral artery (SpA) and SpA remodeling through activation of the EGFR–PI3K–AKT signaling pathway. Summary/Conclusion : Together, our findings indicate the potential benefit of using AmOVs for PE treatment and shed insight into host–microbiota interactions. Funding : This work was funded by the National Natural Science Foundation of China (82071669); the Natural Science Foundation of Guangdong Province (2022A1515011730); the Natural Science Foundation of Guangdong Province (2019A1515010637); the National Science Fund for Distinguished Young Scholars (82025024); College Students' Innovative Entrepreneurial Training Plan Program (202112121021). Keywords : outer‐membrane vesicles, akkermansia muciniphila, preeclampsia, EGFR–PI3K–AKT pathway

Self‐Assembled

Rachel R. Mizenko 1 ; Bryan Nguyen 2 ; Dylan Lanser 2 ; Steven C. George 1 ; Angella Gelli 2 ; Randy P. Carney 1 1 University of California, Davis, Davis, USA; 2 University of California, Davis, USA Introduction : While biodistribution studies have long suggested that EVs cross the BBB, little is known about how or which EVs cross this barrier. To this end, both a representative model including multiple cell types to best recapitulate the in vivo BBB and a comparison of EV types will be necessary to better understand this mechanism. Here we apply BBB spheroids, wherein brain microvascular endothelial cells, pericytes, and astrocytes spontaneously form an intricate BBB‐like structure, as a high‐throughput model to explore the extent to which brain metastasis associated EVs cross the BBB. Methods : Spheroids were prepared by combining brain endothelial cells (hCMEC/D3) with primary human pericytes and astrocytes (1:1:1) in 96 well spheroid microplates and allowing to assemble for at least 1 week. EVs were collected from brain‐metastasizing breast cancer cells (231‐Br), breast cancer cells (MDA‐MB‐231), and control HEK293T cells via ultracentrifugation, labeled with carboxy‐fluorescein succinimidyl ester (CFSE) and separated from dye via size‐exclusion chromatography. After incubation with EVs, spheroids were fixed and labeled for imaging via confocal microscopy. Results : Spheroids assembled into appropriate layers with endothelial cells forming an external layer, having limited permeability to fluorescent albumin and expressing tight junction proteins, and pericytes and astrocytes forming the core. EVs could be identified in spheroids at 6 and 24 h with increasing uptake. Similarly, EV puncta were visible after incubation at ≥ 4*10^10 p/mL with more puncta for high concentrations. EVs penetrated the endothelial layer to different extent across EV types. Summary/Conclusion : Here we introduce a new high‐throughput BBB spheroid model for screening of BBB crossing of various EV types, showing that both increased concentration and exposure time increase internalization. While all EV types showed uptake, we were able to detect differences that may be important to discerning a mechanism of transcytosis. Funding : The research was supported by the NIH by Award Number F31NS120590 (RRM). Keywords : blood‐brain barrier, spheroid

Size‐Exclusion

Evelyn Lattmann 1 ; Luca Räss 2 ; Marco Tognetti 2 ; Julia M. Martínez Gómez 3 ; Valérie Lapaire 3 ; Roland Bruderer 4 ; Lukas Reiter 5 ; Yuehan Feng 2 ; Lars M. Steinmetz 6 ; Mitchell P. Levesque 3 1 University of Zurich, Schlieren, Switzerland; 2 Biognosys AG, Schlieren, Switzerland, Switzerland; 3 Department of Dermatology, University of Zurich, University Hospital Zurich, Schlieren, Switzerland, Switzerland; 4 Biognosys AG, Schlieren, Swaziland; 5 Biognosys AG, Schlieren, Switzerland; 6 Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA, USA Introduction : Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood‐derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Methods : Here, we provide a streamlined and reproducible experimental workflow to identify plasma‐ and serum‐ derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC‐DIA‐MS couples size‐exclusion chromatography to EV concentration and deep‐proteomic profiling using data‐independent acquisition. Results : From as little as 200 μl of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2’311 EV‐associated proteins, achieving a 2.8‐fold increase in depth compared to previously published melanoma studies. To compare the EV‐proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native plasma and serum. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers such as MCAM, TNC, and TGFBI were upregulated in melanoma plasma‐derived EV but not in depleted melanoma plasma samples, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T cell biology. Summary/Conclusion : Taken together, we demonstrated the increased sensitivity of an EV‐based proteomic workflow for biomarker discovery in plasma and serum. The ease of automating and scaling up such an approach enables generalized application to larger cohorts from melanoma and other indications. Keywords : extracellular vesicle, EV, exosome, melanoma, proteomics, mass spectrometry, liquid biopsies, plasma, serum, size‐exclusion chromatography, SEC, biomarker

Functionalization

Ainara González‐Moro 1 ; Elena Cercas 2 ; Arturo González‐Camuñas 3 ; Miriam Morales Rodríguez De Lope 4 ; Jordi Royes 3 ; Luis A Campos 3 ; Milagros Castellanos 3 ; Carlos Félix Sanchez‐Ferrer 2 ; Concepcion Peiro 5 ; Álvaro Somoza 3 ; Fernando de la Cuesta 6 1 Universidad Autónoma de Madrid, Algete, Spain; 2 Universidad Autónoma de Madrid, Spain; 3 IMDEA, Spain; 4 Universidad Autónoma De Madrid (Spain), Madrid, Spain; 5 Department of Pharmacology, Medicine School, Autonomous University of Madrid, Madrid, Spain, Spain; 6 Universidad Autonoma de Madrid, Spain Introduction : Cellular senescence has been associated with the progression of atherosclerosis. Senolytic drugs are compounds that induce apoptosis of such senescent cells and two of the most used are quercetin (Q) and dasatinib (D). Several studies have demonstrated that mesenchymal stem cell‐derived EVs (MSC‐EVs) exert beneficial effects. Thus, the objective of our study is to develop a novel therapy using MSC‐EVs as a vehicle for Q and D delivery in a context of atherosclerosis. Methods : EVs were isolated from immortalized adipose tissue MSCs by ultrafiltration and size exclusion chromatography (SEC), and characterized by NTA, western blot and TEM. NTA and light dispersion analyses were used to determine the optimal conditions for Q and D internalisation. Functionalization of the EVs with both senolytics was evaluated by HPLC and MRM mass spectrometry. EV's uptake by HUVECs was observed with CFSE staining. β‐galactosidase assay was used for analysing a potential senolytic effect. Besides, aptamers conjugated or not with cholesterol were synthesized in order to compare their incorporation into EVs, with the final goal of specifically delivering the treatment to the vascular tissue. Results : Optimal concentration of the different agents for EVs functionalization were: DMSO 3%, Q 20 μM and D 4 μM. Efficient functionalization of the senolytics was evidenced. CFSE staining showed that MSC‐EVs are internalized by HUVECs and in vitro experiments suggest a potential senolytic effect of EVs‐QC. Finally, oligonucleotide's modification with cholesterol displayed a better incorporation into EVs. Summary/Conclusion : We have been able to encapsulate quercetin and dasatinib in MSC‐EVs and the first in vitro experiments showed a senolytic effect of the treatment in senescent human endothelial cells. Cholesterol modified aptamers were efficiently incorporated into MSC‐EVs. Funding : Talent Program,Comunidad Autónoma de Madrid (2019‐T1/IND‐13794); Ministry of Science (PID2021‐126274OB‐I00). Keywords : atherosclerosis, senescence, extracellular vesicles, senolytics

High‐Throughput

Ramin Khanabdali 1 ; Carlos Palma 2 ; Sara Nikseresht 3 ; Khairul Ansari 4 ; Susan Susan Belzer 4 ; Laura Dagly 5 ; Laura J. Vella 6 ; Gregory E. Rice 7 1 Inoviq Limited, Notting Hill, Australia., USA; 2 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia, Australia; 3 Inoviq Limited, Notting Hill, Victoria, Australia., Melbourne, Australia; 4 Inoviq Limited, Notting Hill, Victoria, Australia., USA; 5 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, Australia; 6 The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia; Melbourne Dementia Research Centre, Parkville, VIC, Australia; The Department of Surgery, The University of Melbourne, Parkville, VIC, Australia., Melbourne, Australia; 7 Inoviq Limited, Notting Hill, Victoria, Australia., Notting Hill, Australia Introduction : Extracellular vesicles (EVs) including exosomes have great potential for both diagnostic and therapeutic applications. The lack of standardized methods for efficient and high‐throughput isolation and analysis of EVs, however, has limited their use and application for the clinical practice. Here we present a novel bead‐based immunoaffinity system (EXO‐NET®) that captures a highly enriched subpopulation of EV. EXO‐NET is 3‐D monoclonal antibody matrix constructed on magnetic bead‐based immunoaffinity for isolation and enrichment of EVs. Methods : EVs were isolated from human pooled plasma (500 μl) using EXO‐NET (300 μl) and 3 others commercial EV isolation kits according to manufacturers’ instructions. Nanoparticle tracking analysis (NTA, ZetaView) was used to analyze the particle size and number. The protein content of captured EVs was characterized by mass spectrometry and Western blotting. RT‐qPCR was used to evaluate and quantify mRNA (GAPDH and OAZI) and microRNA cargo (miR‐16, let‐7a, miR‐21) of isolated EVs from EXO‐NET and other commercial kits. Results : NTA established that EXO‐NET captured a greater proportion (>75%) of EVs (50‐130 nm size) from plasma when compared to other comparable commercial kits (< 65%). Western blotting analysis demonstrated that EXO‐NET not only outperformed other kits in term of enrichment of EVs marker (flotillin) but also reduced commonly co‐isolated contaminant protein (ApoB). Mass spectrometry analysis indicated that contaminant albumin peptide intensity was 3‐fold higher in other isolation kits compared to EXO‐NET. In addition, the total peptide intensity of known EV associated markers was 6 to10‐fold higher in EXO‐NET than other kits. At the transcriptomic level, qPCR analysis showed that EXO‐NET had higher yield and recovery (4‐8‐fold) of both mRNAs and microRNAs compared to other commercial kits. Summary/Conclusion : The EXO‐NET is a novel method for the rapid, efficient, and scalable enrichment and purification of EVs that reduces contaminants which may confound downstream analysis. Keywords : EXO‐NET, extracellular vesicles, EV isolation, EV enrichment

“Immunoaffinity

Erez Eitan 1 ; Sara Rau 2 ; Emma Vande Brake 2 ; kashfia Haque 2 ; Olga Volpert 3 1 NeuroDex, Arlington, USA; 2 NeuroDex, USA; 3 NeuroDex, Natick, USA Introduction : Blood extracellular vesicles contain lipids, proteins, and nucleic acids that image, to a certain extent, their cell of origin. Immunoaffinity Isolation of EV subpopulations based on cell‐specific surface proteins can provide a liquid biopsy platform with increased specificity. Methods : An optimized immunoaffinity isolation procedure called ExoSORT was used to screen over 70 antibodies against neuron‐specific surface markers. We identified that combining antibodies against GAP43 and NLGN3 provides increased specificity toward neuronal‐derived EVs (NDEs). Antibodies against oligodendrocyte and liver‐specific EV markers were also identified. Results : ExoSORT ability to isolate EVs was demonstrated according to MISEV—including electron‐microscopy, nanoparticle‐tracking‐analysis, western‐blot, ELISA, FACS analysis for EV markers (CD63, CD9, CD81, and FLOT1), and negative markers (Calnexin, Albumin, and ApoA), and by unbiased proteomic analysis. ExoSORT specificity toward NDEs was demonstrated in comparison to isotype control antibody (IgG) and EV‐depleted plasma by measuring neuronal‐specific proteins (SYP, NFL, ENO2, proBDNF, and RGMa) and mRNA (NRGN, ENO2, NEFL, and HCRT). Enrichment was also demonstrated by unbiased proteomic analysis and RNAseq. ExoSORT efficiency was shown by spike‐in recovery experiments with EVs isolated from IPS‐derived neurons and HEK293 cells genetically engineered to express the neuronal markers. ExoSORT precision (CV< 16%) was demonstrated by repeating the isolation over nine independent experiments. ExoSORT diagnostic potential is demonstrated by measuring alpha‐synuclein aSYN and TDP43 in plasma samples from 30 Parkinson's, 25 Lewy‐Body Dementia, and 30 Multiple‐System Atrophy patients than in 45 healthy controls. Significant differences in aSYN could identify synucleinopathies with over 80% specificity and sensitivity. In addition, NDE‐associated TDP43 was significantly elevated in plasma samples from ALS patients. Summary/Conclusion : ExoSORT is an optimized immunoaffinity EV isolation method that works well for NDEs and potentially for EVs from additional cell types. It has the potential to serve as a novel biomarker platform with increased cell specificity. Keywords : biomarkers, immunoaffinity, cell‐specific

Charge‐Modulated

Hyun‐Kyung Woo 1 ; Young Kwan Cho 2 ; Chang Yeol Lee 2 ; Haeun Lee 2 ; Cesar Castro 2 ; Hakho Lee 3 1 MGH, Woburn, USA; 2 MGH, USA; 3 MGH, Boston, USA Introduction : Blood contains different bio‐nanoparticles that can be exploited for clinical diagnoses. Purifying these particles according to type, however, remains technically challenging. EVs are outnumbered >104‐fold by low density lipoproteins (LDLs), yet similar in size and density. These fundamental disadvantages often cause LDL spillover into EV isolates, thus confounding assay results. Here, we report that surface charge can be an effective parameter to differentiate EVs from LDLs. To address this, we refined an all‐in‐one system which performed i) size‐exclusion to remove particles smaller than EVs and LDLs and ii) cation‐exchange in an acidic elution to retain LDLs longer than EVs. Methods : We built an enhanced dual‐mode device (DMD) which performed i) size‐exclusion to remove particles smaller than EVs and LDLs and ii) cation‐exchange in an acidic elution to retain LDLs longer than EVs. The performance of the DMD, in comparison to size‐exclusion only, was evaluated by analyzing the yield and purity of the isolated EVs. Results : We estimated the surface charge densities of EVs (‐6.2 mC/m2) and LDLs (‐3.6 mC/m2) by measuring zeta potentials at different buffer pH, revealing that EVs are more negatively charged than LDLs. In addition, we found that the charge difference between EVs and LDLs was maximal at a weak acidic condition (pH = 6.4). By applying this, we optimized eDMC operation to enrich EVs directly from plasma, depleting >99.8% of LPPs within 30 min, Minimizing LDL contamination improved analytical signals in EV molecular assays, including single vesicle imaging, bulk protein, and mRNA detection. Summary/Conclusion : We have demonstrated a fast and non‐biased way for EV isolation from blood plasma, promoting the translational value of the dual‐mode separation. Funding : This work was supported by NIH Grants R01CA229777, R21DA049577, R01CA239078, R01CA237500, U01CA233360, R01CA264363; and MGH Scholar Fund. Keywords : extracellular vesicles, lipoproteins, surface charge modulation, size‐exclusion, cancer

Exosome‐Mediated

Soyeon Won 1 ; Ji Eun Lee 2 1 Sungkyunkwan university, Republic of Korea; 2 SungKyunKwan University, Republic of Korea Introduction : Due to the large range of genetic problems relating to muscles, muscle tissue is a crucial target for genetic engineering. However, gene targeting frequently had poor effectiveness and had transient effects. The toxicity and immune response were amplified by the large vector doses required to achieve the desired target effect. Furthermore, there are issues with the delivery strategy because the gene‐carry virus tends to target the liver rather than muscle cells in high doses, which can have extremely harmful side effects. Exosomes, naturally released nanovesicles, are the next generation of delivery tool because of their inherent biocompatibility, transportation capability, circulatory stability and engineerability. Exosome secretion and uptake into by skeletal muscle cells through an autocrine pathway have both been demonstrated. As a result, we created a technique to deliver Cas9 RNP with myoblast‐derived exosome employing the ability to target homologous tissues. Methods : For myoblast‐secreted exosome isolation, exosome‐depleted FBS was prepared by ultracentrifugation at 100,000g, 4°C for 20 hours. C2C12 myoblast were cultured in DMEM containing exosome‐depleted FBS. Then, exosomes were isolated from the culture media using differential centrifugation. The exosomal protein was quantified by BCA assay and the presence of exosome was determined by western blot analysis. Cas9 proteins were and sgRNA were thoroughly combined to create Cas9 RNP complexes. Then, RNP complexes were loaded onto exosome using electroporation. Exosome RNP complexes were added into C2C12 culture medium to investigate the delivery of exosomal RNP. After incubation, the effectiveness of gene targeting was confirmed by western blot. For tissue targeting, exosome RNP complexes were injected into muscle and skeletal muscle tissue were collected for further gene targeting analysis. Results : We identified the lysate from C2C12‐derived exosomes expressed exosome specific markers but not the Golgi apparatus‐associated protein. The delivery of exosome RNP resulted in the targeted gene indel frequency in vitro and in vivo. The targeting property of exosome RNP was demonstrated sequencing and target protein quantification in skeletal muscle. In addition, we observed that the deletion caused a failure in myogenic tube fusion. Summary/Conclusion : In this study, we created an RNP‐based CRISPR‐Cas9 genome editing delivery tool that allows the generation of genetic disease models. RNP can be successfully electroporated into exosomes secreted from C2C12 and transported to skeletal muscle. Funding : This work has been done by Molecular & Medical Genomics lab of Ji Eun Lee in Sungkyunkwan University and supported by the National Research Foundation of Korea government's MSIP (2021R1A4A2001389 and 2021R1A2C3004572 to J.E.L., 2021R1A6A3A13039817 and 2022K1A3A1A12080469 to S.W.).

Mir‐92A‐1‐5P

Lijuan Yu 1 ; Bing‐Dong Sui 2 ; Xin Zhang 3 ; jiayun Liu 4 ; Xiaoke Hao 5 ; Lei Zheng 6 1 Nanfang Hospital, Southern Medical University, Gothenburg, China (People's Republic); 2 Fourth Military Medical University: Air Force Medical University, Xi'an, China (People's Republic); 3 Nanfang Hospital, Southern Medical University, Guangzhou, China (People's Republic); 4 Air Force Medical University, China (People's Republic); 5 Air Force Medical University, xi'an, China (People's Republic); 6 Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China (People's Republic) Introduction : We have previously reported that extracellular vesicles (EVs) derived from osteoblastic, osteoclastic and mixed prostate cancer cells promote osteoclast differentiation and inhibit osteoblast differentiation via transferring miR‐92a‐1‐5p. In the present study, we focused on engineering miR‐92a‐1‐5p into EVs and determining any therapeutic role and mechanism of the engineered EVs. Methods : A stable prostate cancer cell line (MDA PCa 2b) overexpressing miR‐92a‐1‐5p was constructed by lentivirus, and EVs were isolated by ultracentrifugation. The overexpression of miR‐92a‐1‐5p in both cells and EVs was tested using qPCR. Osteoclast function was evaluated by Trap staining, mRNA expression of osteoclastic markers ctsk and trap, immunolabeling of CTSK and TRAP and microCT using either in vitro and in vivo assays. Target gene of miR‐92a‐1‐5p was proved by a dual‐luciferase reporter assay system. siRNAs were designed and used for transient expression in order to determine the role of downstream genes on osteoclast differentiation. Results : Stable overexpression of miRNA‐92a‐5p in cell line was associated with increased expression in EVs, as confirmed by qPCR. Further, miR‐92a‐1‐5p enriched EVs promote osteoclast differentiation in vitro by reducing MAPK1 and FoxO1 expression, associated with increased osteoclast function as shown by TRAP‐staining and mRNA expression of osteoclast functional genes. siRNA targeting MAPK1 or FoxO1 resulted in similar increase in osteoclast function. In vivo, the miR‐92a‐1‐5p enriched EVs given via i.v. injection promote osteolysis, which was associated with reduced expression of MAPK1 and FoxO1 in bone marrow. Summary/Conclusion : Our results suggest that the engineered miR‐92a‐1‐5p enriched EVs regulate osteoclast function via reduction of MAPK1 and FoxO1. Funding : This work was supported by the National Key R&D Program of China (under Grant No. 2021YFA1300604) and the National Science Funds for Distinguished Young Scholars (under Grant No. 82025024) to L.Z, the National Natural Science Foundation of China to X.H. [under Grant No. 81872347], the National Natural Science Foundation of China to L.Y. [under Grant No. 82203711] and the China Postdoctoral Science Foundation to L.Y. [under Grant No. 2021M701631]. Keywords : miR‐92a‐1‐5p, miR‐92a‐1‐5p enriched EVs, bone diseases, bone metabolism, osteoclast differentiation

Multi‐Parametric

Karl Normak 1 ; Marcell Papp 2 ; Michael Ullmann 2 ; Carolina Paganini 1 ; Paolo Arosio 3 1 ETH Zurich, Zurich, Switzerland; 2 ETH Zurich, Switzerland; 3 ETh Zurich, Zurich, Switzerland Introduction : The complex and heterogeneous nature of EVs poses challenges for their analytical characterization, which requires a multi‐parameter analysis. The characterization of the necessary attributes required by the MISEV2018 guidelines can be only achieved by combining multiple different analytical methods, that require different equipment, significant hands‐on time and different degrees of sample preparation. Here, we demonstrate how liquid chromatography coupled to multi‐angle light scattering (MALS) and fluorescence detection (FLD) can provide multidimensional characterization of EVs with limited hands‐on time. Methods : EVs samples were fractionated on a HPLC system via either size (SEC) or anion exchange (AIEX) chromatography and analyzed by in‐line scattering and fluorescence detectors. EV size and particle amount were characterized by multi‐angle light scattering and compared to NTA. The total protein amount in the sample was characterized by native fluorescence of proteins. The lumen of the EVs was labelled with CalceinAM, the lipid membrane with DiO and CD81 with antiCD81 antibody. GFP was loaded exogenously and an average per particle loading was measured by combining the fluorescence and light scattering data. Results : The combination of these in‐line detections tools with chromatography enables the analysis of sample purity and of EVs directly in crude mixtures such as conditioned media. MALS detection provides average size and number of particles down to 107 particles. Fluorescence detections enables the label‐free and non‐destructive measurement of the total protein amount and, when combined with MALS, the protein to particle ratio. Moreover, by labelling the EVs with fluorescent dyes, fluorescence detection can report on the composition of particles also in terms of specific EV properties, such as the presence of EV specific surface markers, a lipid membrane or a hollow morphology. Finally, exogenous cargo loading methods were compared. Electroporation was the most successful loading method, leading to an average of 56 GFP per particle. Summary/Conclusion : We have described the use of liquid chromatography coupled to in‐line multi angle light scattering (MALS) and fluorescence detection for the characterization of EVs. With this combination, many necessary attributes required by the MISEV2018 guidelines have been characterized. The core of the approach relies on the multi‐dimensional analysis of the sample on the same platform, similarly to microfluidic platform we have shown previously. Funding : H2020‐EU.1.2.2‐FET Proactive programme via the BOW Grant agreement 952183. Keywords : SEC, AIEX, liquid chromatography, light scattering, MALS, fluorescence detection, MISEV guidelines, exogenous loading

Phosphatidylserine

Jan Kranich 1 ; Lisa Rausch 1 ; Lavinia Flaskamp 2 ; Ashretha Ashokkumar 1 ; Anne Trefzer 3 ; Christine Ried 1 ; Veit Buchholz 4 ; Reinhard Obst 1 ; Tobias Straub 5 ; Thomas Brocker 1 1 Institute for Immunology, Biomedical Center (BMC), Faculty of Medicine, LMU Munich, Munich, Germany, Planegg‐Martinsried, Germany; 2 Institute for Immunology, Biomedical Center (BMC), Faculty of Medicine, LMU Munich, Munich, Germany, Germany; 3 Roche Innovation Center Zurich, Roche Glycart AG, 8952 Schlieren, Switzerland, Zurich, Switzerland; 4 Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich (TUM), Munich 81675, Germany, München, Germany; 5 Core Facility Bioinformatics, Biomedical Center (BMC), Faculty of Medicine, LMU Munich, Munich, Germany, Planegg‐Martinsried, Germany Introduction : While it has been shown in many in vitro studies that extracellular vesicles (EVs) can either directly or indirectly prime naïve T cells, it is still controversial whether EVs play a relevant role in T cell responses in vivo. To study naturally occurring EVs and their target cells in vivo, we have recently developed a novel method, using Ca2+‐independent phosphatidylserine (PS) binding reagents based on MFG‐E8 and imaging flow cytometry (Kranich, JEV, 2020, Rausch et al., JEV 2021). We use this approach to analyze and characterize EV‐T cell interactions during acute viral infections in mice and to assess if T cell stimulation by EVs occurs during immune responses. Methods : To visualize EV‐T cell interactions, we administer fluorescent MFG‐E8 intravenously into mice during an acute LCMV infection to label naturally occurring PS+ EVs in situ. We then analyze and characterize T cells carrying PS+ EVs either directly by imaging flow cytometry or perform downstream analyses after FACS‐sorting of EV+ T cells. Downstream analyses include RNAseq and dSTORM superresolution microscopy. Furthermore, we use in vitro generated EVs from activated, antigen pulsed bone marrow derived dendritic cells and compare their interaction with antigen‐specific and non‐specific T cells in vivo. Results : We observed a strong increase in the abundance of EV+ cells during viral infections in the spleen. There, PS+ EVs interact especially with activated, but not with naïve CD8+ T cells. EV‐binding induced antigen‐specific TCR signaling and increased nuclear translocation of the transcription factor NFATc1 in vivo. Single molecule superresolution microscopy showed direct interaction of EVs with the TCR complex on T cells. Furthermore, RNAseq analysis showed that EV‐decorated but not EV‐free CD8+ T cells are enriched for gene signatures associated with T‐cell receptor signaling, early effector differentiation and proliferation. Summary/Conclusion : Our results demonstrate that PS+ EVs stimulate activated CD8+ T cells in vivo by directly interacting with the TCR complex and thus provide an antigen‐specific adjuvant effect to T cells during acute viral responses. The absence of EVs on naïve T cells argues against direct priming of T cells by EVs in vivo. As EV‐binding by T cells only occurs during the effector phase, EVs could act as danger signal, indicating to the cells that virus is still present and that the presence of effector cells is still required. Funding : This project was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research foundation) – Project‐ID 210592381 ‐ SFB 1054 (TP B03; TP B07; TP B15; TP Z02). Keywords : extracellular vesicles, phosphatiylserine, LCMV, T cells, dSTORM, imaging flow cytometry, MFG‐E8, lactadherin

Plasmon‐Enhanced

Jae‐Sang Hong 1 ; Taehwang Son 2 ; Ursula A. Winter 1 ; Mi Ho Jeong 2 ; Satoru Komatsu 3 ; Hyungsoon Im 1 1 Massachusetts General Hospital, Boston, USA; 2 Massachusetts General Hospital, USA; 3 Canon, USA Introduction : Molecular analysis of extracellular vesicles (EVs) represents a new paradigm for early cancer detection and treatment monitoring through liquid biopsies. In particular, single EV analyses are important to understand the heterogeneity of EVs, identify the cellular origins of individual EVs, and detect key biomarkers in the subpopulations. Here, we introduce a nanoplasmonic sensing platform for single EV analysis. Specifically, we used a low‐cost plasmonic substrate that can be fabricated on a wafer scale to amplify EVs’ fluorescence signals without the need for additional chemical or enzymatic signal amplification processes. Methods : We isolated EVs from breast cancer cell lines with different molecular subtypes using size exclusion chromatography and measured the sizes and concentrations by nanoparticle tracking analysis. The isolated EVs were fluorescently labeled by using tetrafluorophenyl with Alexa Fluor 555. The labeled EVs were captured on the plasmonic substrate functionalized by SH‐PEG‐COOH with EDC/NHS activation. We labeled the captured EVs with QUAD cancer markers (MUC1, EGFR, EpCAM, and HER2) for multi‐channel single EV analysis. Results : We showed that the approach could significantly improve the EV detection sensitivity and enable multi‐channel single EV analysis. We applied the assay to detect tumor‐derived EVs using the QUAD marker signature and interrogating molecular subtypes. We also showed molecular profiles of tumor‐derived EVs present similar molecular patterns to originating cells by flow cytometry. Summary/Conclusion : The plasmon‐enhanced sensing enables sensitive, multiplexed EV analysis through a simple assay procedure. The multiplexed single EV analysis could improve the detection accuracy for early cancer detection and treatment monitoring. Funding : A sponsored research agreement between Canon and Massachusetts General Hospital (agreement # 2020A013232). Keywords : nanoplasmonics, multiplexing, breast cancer, sensing

Adipocyte‐Derived

Laura R. Cechinel 1 ; Robert J. Freishtat 1 ; Irene Zohn 1 ; Rachael Batabyal 2 1 Children's National Hospital, Washington, USA; 2 Children's National Hospital, Columbia, USA Introduction : Obesity among women of childbearing age can alter fetal development and increase the risk of metabolic disease in the offspring. The mechanisms underlying the adverse effects of maternal obesity on post‐implantation embryonic development before the establishment of the placental circulation remain poorly understood. We previously demonstrated that adipocyte‐derived small extracellular vesicles (ad‐sEVs) reflect maternal adipose tissue dysfunction. For example, ad‐sEVs microRNA from pregnant women target PTEN and STAT3 and are associated with increased baby adiposity. We hypothesize that ad‐sEVs from individuals with adipose tissue dysfunction (i.e. obesity and diabetes) will alter metabolic pathways during early embryonic development. Methods : Cultured murine embryos at E8.5 were exposed to 3 μg/mL of visceral adipose tissue ad‐sEVs from patients with obesity and insulin resistance (OIR; n = 5), or with obesity and diabetes (OD; n = 5). After 24 hours, embryo growth was measured, and development parameters were recorded using an adapted validated scoring system. Embryos, yolk sacs, and ad‐sEVs were isolated for RNASeq. Results : The embryos exposed to ad‐sEVs from OIR, and OD had similar head size (1.14 ± 0.2mm; 1.07 ± 0.2mm), dorsal length (11.9 ± 1.6mm; 11.01 ± 2.3mm), and yolk‐sac diameter (5.01 ± 0.15mm; 4.5 ± 0.7mm), when compared to controls (1.25 ± 0.2mm, 12.8 ± 3.4mm, 5.3 ± 0.5mm; p>0.05). In addition, developmental scores for heart development, embryo flexion, yolk sac circulation, and somite numbers were similar in all embryos. RNAseq from embryos and yolk sacs, in addition to small RNAseq from ad‐sEVs is currently being performed. Summary/Conclusion : As expected, a 24‐hour exposure of post‐implantation murine embryos to ad‐sEVs from OIR and OD does not significantly alter morphology and developmental scores. However, we do expect the imminent molecular analyses will define that ad‐sEVs microRNAs target genes in the embryo and yolk sacs. This analysis will identify changes in key pathways (i.e., IGF2 and PPAR‐γ) that alter embryonic metabolism increasing the risk for metabolic disease in the offspring. Funding : Children's National Research Institute, Center of Genetic Medicine ‐ Collaborative Pilot Award.

Astrocyte‐Derived

Samantha F. Friend 1 ; Dylan Delmar 2 ; Katy Torres 3 ; Caroline Nievergelt 3 ; Victoria B. Risbrough 1 1 VA San Diego Healthcare System, San Diego, USA; 2 University of California, San Diego, San Diego, USA; 3 University of California, San Diego, USA Introduction : Growing evidence suggests inflammation plays a role in trauma‐related psychiatric disorders. Studies suggest PTSD is associated with altered immune protein levels. However, little is known about the relationship between central and peripheral inflammation in driving PTSD. Methods : To specifically probe the relationship between the central nervous system (CNS) and peripheral inflammation, we isolated astrocyte‐derived extracellular vesicles (ADEs) from peripheral blood plasma samples from subjects with and without PTSD (N = 32). After vesicle lysis, cargo protein cytokines were quantified using multiplex enzyme‐linked immunosorbent assay plates. These cytokines were compared to clinical measures of PTSD using the PCL‐5 and PHQ9. Results : As has been previously demonstrated in the literature, we found that participant plasma correlated with PTSD clinical symptoms measured by PCL‐5, using BMI and age as covariates (IL‐1b, R = 0.35, p = 0.0062; IL‐6, R = 0.30, p = 0.019; TNFa, R = 0.34, p = 0.0071). However, most cytokines from ADEs did not correlate significantly with plasma cytokine levels (ρ ranging from 0.059 ‐ 0.570), except for IL‐2 measured from both plasma and ADEs (ρ = 0.498, = 0.170, p =.004). Notably, we detected a significant relationship between anhedonia as measured and ADE IL‐1b and IL‐2 levels (ρ = 0.493, p< 0.01; ρ = 0.489, p< 0.01). Summary/Conclusion : Our findings that few plasma cytokines correlate with cytokines isolated from ADEs suggest that ADE cytokines may represent a tissue‐specific signature of CNS immune dysregulation. Furthermore, these exploratory findings highlighting the association between ADE cytokine levels and anhedonia underscore that ADE's may hold promise to identify immune dysfunction in neuropsychiatric disorders. Funding : This work was supported in part by Career Development Award Number IK2 CX002343 from the United States (U.S.) Department of Veterans Affairs Clinical Sciences R&D (CSRD) Service, as well as by a Merit Award BX002558 ‐01 from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service. Keywords : immune, astrocyte, cytokine

High‐Productivity

Hyun jae Kim 1 ; Seokhwan Yun 2 ; Seung‐Jin Kim 1 ; Keunsun Ahn 3 1 Sphebio, USA; 2 Sphebio Co.Ltd, Seoul, Republic of Korea; 3 Sphebio, Seoul, USA Introduction : The traditional models of cell culture are two‐dimensional (2D) as monolayers, but this has a problem of not mimic the actual natural environment. 3D cell culture techniques have been studied recently because they might provide more accurate environment of human tissues. Among 3D culture methods, spheroids are 3D cell aggregates that can mimic tissues and microtumors, and are in the spotlight because they can be applied to industrial mass production. Methods : In this study, uniform spheroids were mass‐produced by precisely controlling and printing bio‐ink mixed with human adipose‐derived stem cells (ADSC). The spheroids are formed through static culture for 48 hours within the printed core shell structure. Subsequently, the exosomes from the culture media of the spheroids dynamically cultured for 3 days were purified by the TFF method. Results : Through the our mass production method, it was confirmed that the production of exosomes of spheroids produced to a predetermined size by controlling the concentration of bio‐ink was significantly increased compared to spheroids produced to be non‐uniform in size. In addition, there were differences in cell proliferation and regeneration between these two exosomes. Summary/Conclusion : In conclusion, the technology of maintaining a constant size of spheroids using bio‐ink showed high productivity and high quality in exosome production. In short, the exosome production method using this technology can overcome the qualitative and quantitative limitations of the existing exosome production method by producing exosomes from controlled‐sized spheroids. Funding : This research was funded by TIPS(Tech Incubator program for startup) by Korean Goverment (Grant number S3197974, 20105847). Keywords : spheroid, dynamic culture, exosome

Membrane‐Anchored

Seong‐eon Cho 1 ; In‐San Kim 2 1 Korea Institute of Science and Technology, Republic of Korea; 2 Korea Institute of Science and Technology, Seoul, Republic of Korea Introduction : In this study, we found that exosomes with epidermal growth factor (EGF) on their membrane have cell proliferative effects than those of recombinant EGF proteins (rEGF). Furthermore, mesenchymal stem cell exosomes expressing EGF exhibited regenerative ability compared with either rEGF or MSC exosoms. This suggest that exosome provides the optimal membrane environment for EGF. Methods : Preparation & characterization of exosome Cells were maintained in DMEM with 10% FBS and 1% antibiotic‐antimycotic at 37°C in 5% CO2. The supernatants of transfected HEK293T cells by PEI and transduced ASC52telo cells were centrifuged followed by concentration with tangential flow filtration. The concentrates were ultracentrifuged at 150,000 g for 3 h and were re‐suspended in PBS. Exosomes were analyzed by DLS, NTA, and cryo‐TEM. Exosomal proteins were analyzed by Western blot. In vitro cell proliferation & migration assay To assess cell proliferating ability of exosomes, Cell Counting Kit‐8 assay was performed. Serum‐starved cells for 24 h, were incubated with rEGF or exosomes. After 48 h, CCK‐8 solution was added and the optical density was measured. To assess the ability of exosomes on cell migration, cells were scratched and incubated with rEGF or exosomes and observed. In vivo wound healing assay 8 mm of wounds were made on the back of Balb/c mice with biopsy punch. PBS, and PBS containing rEGF or exosomes were injected subcutaneously. The tissues were analyzed by H&E staining and IHC. Results : Exosomes characterization 3 constructs of EGF were prepared. The EGF precursor (preEGF); EGF domain inserted after transmembrane domain of PDGFR (pEGF); lacking prepro domain and juxtamembrane stalks region of preEGF (tEGF). All types of exosomes expressed exosomal markers and EGF. The treatment of ADAM17 induced the loss of the ectodomain only with preEGF. tEGF‐Exosomes have the regenerative effect The efficacy of tEGF‐Exo was evaluated by Cell Counting Kit‐8 and scratch assay. tEGF‐Exo treatments significantly increased cell viability and migration of HaCaT and Balb/3T3 cells compared with non‐treated group. stEGF‐Exosomes promoted migration & proliferation in vitro Stem cell exosomes with tEGF (stEGF‐Exo) treatments promoted HaCaT and Balb/3T3 cells proliferation and migration compared with control groups. tEGF‐Exo efficiently promoted wound healing in vivo stEGF‐Exo significantly enhanced wound closure in vivo. Furthermore, H&E and IHC detection of KI67 showed that stEGF‐Exo promoted wound healing. Summary/Conclusion : The treatment tEGF‐Exo promoted cell proliferation compared with rEGF. Further, stEGF‐Exo treatment showed effective wound healing. Collectively, exosome enhanced the biologic effect of secretory proteins if they are anchored to exosome membrane. Our study suggests that exosomes can be a promising therapeutic option to overcome limitations of growth factors.

Neuronal‐Enriched

Leandra K. Figueroa‐Hall 1 ; Kaiping Burrows 2 ; Ahlam M. Alarbi 3 ; Chibing Tan 3 ; Rayus Kuplicki 2 ; Jennifer L. Stewart 2 ; Robin L. Aupperle 4 ; Bethany N. Hannafon 5 ; Rajagopal Ramesh 6 ; Jonathan B. Savitz 2 ; Victoria B. Risbrough 7 ; T. Kent Teague 3 ; Sahib S. Khalsa 2 ; Salvador Guinjoan 8 ; Martin P. Paulus 2 1 Laureate Institute for Brain Research, Tulsa, USA; 2 Laureate Institute for Brain Research, TULSA, USA; 3 The University of Oklahoma, TULSA, USA; 4 Laureate Institute for Brain Research, USA; 5 The University of Oklahoma, USA; 6 The University of Oklahoma Health Sciences Center, USA; 7 University of California, San Diego, USA; 8 Laureat Institute for Brain Research, Tulsa, USA Introduction : Suicide is one of the top ten causes of death in the US. For several reasons, it has been difficult to identify biological markers of the underlying process or predictive measures of suicide: (1) underlying neurobiology of suicidal behavior (SB) is still poorly understood; and (2) peripheral blood measures may not adequately assess the brain state preceding SB. The aim of this pilot study was to determine whether alterations in miRNA expression in neuronal‐enriched extracellular vesicles (NEEV) could serve as biological markers of SB in individuals with Major Depressive Disorder (MDD). Methods : This study was approved by the Western Institutional Review Board and carried out in accordance with the Declaration of Helsinki. Subjects gave informed consent. Healthy comparisons (HC = 18); non‐SB (MDD‐ = 18) and SB (MDD+ = 18) were matched for age, sex, and percent body fat. Total EVs were isolated from plasma with a polymer‐based kit; and NEEV immunocaptured with a biotinylated neuronal adhesion marker antibody. NEEV were characterized with flow cytometry and immunoblot analysis to validate EV‐positive markers, and microfluidic resistive pulse sensing to determine NEEV size and particle concentration. miRNA was isolated from NEEV with a small miRNA kit, purity verified with a bioanalyzer, and sent for Next Generation Sequencing. For statistical analysis, data were log‐transformed and non‐parametric Kruskal‐Wallis and Dunn's tests were performed. Gene Set Enrichment Analysis (GSEA) was performed with the miRWalk software to identify genes and relevant biological pathways (BP). EV‐TRACK ID EV210507 . Results : MDD+ versus MDD‐ differed on 1) miR‐182‐5p (H = 9.16, p = 0.01; MDD+ lower than MDD‐ (Cohen's d (d = 1.04); and 2) miR‐486‐3p (H = 9.16, p = 0.01; MDD+ higher than MDD‐ (d = 0.99). HC did not differ between MDD+ nor MDD‐ for either miRNA. For each miRNA, GSEA revealed 120 genes and 15 BP (miR‐182‐5p) and 322 genes and 26 BP (miR‐486‐3p). Bioinformatics analyses identified several shared pathways with both miRNAs including FC gamma receptor signaling involved in phagocytosis and viral process. Summary/Conclusion : MDD+ versus MDD‐ differed on 2 miRNAs. Findings provide evidence that dysregulation of NEEV miRNAs could offer possible molecular targets for predicting SB in individuals with MDD. Funding : This work was supported by the William K. Warren Foundation, the National Institute of Mental Health (R01MH123652 to JS, K99MH126950 to LFH) and the National Institute of General Medical Sciences Center Grant Award (P20GM121312). Keywords : neuronal‐enriched extracellular vesicles, suicide, major depressive disorder, hsa.miR.182.5p, hsa.miR.486.3p

Probiotic‐Derived

Graciela L. Lorca 2 ; Danilo da Silva 1 ; Reagan Beliakoff 2 ; Claudio F. Gonzalez 3 1 University of Florida, USA; 2 University of Florida, Gainesville, USA; 3 University of Florida, G, USA Introduction : Lactobacillus johnsonii N6.2 is a novel probiotic bacterium that was shown to mitigate the onset of type 1 diabetes (T1D) in in rodent models. Our working hypothesis is extracellular vesicles (EV) released in the gut can mitigate the autoimmune response at distal locations. Using SEM, we observed EV produced by L. johnsonii 90–125 nm in size. Untargeted lipidomic and proteomics revealed that L. johnsonii N6.2 EV are enriched in phospholipids and have a unique protein composition. Using the Sdp differentially enriched protein as biomarker, the host response to L. johnsonii EV was evaluated in blood samples of volunteers that ingested L. johnsonii. The individuals consuming the whole probiotic showed increased amounts of IgA and IgG for EV or the Sdp protein. In vitro, EV can induce a tolerogenic M2 phenotype in macrophages, reduce apoptosis in βlox5 human beta cells and stimulate insulin secretion in human islets. RNAseq analysis showed that EV induced the expression of genes associated with the aryl carbon receptor (AHR) as well as the OAS RNA sensing pathways. Methods : Treatments of βlox5 cell lines with uptake inhibitors or endosome markers were visualized and quantified by confocal fluorescence microscopy. Enzymatic, chemical, and physical methods were used to disrupt EV. RNAseq and qRT‐PCR was utilized to quantify gene expression. Results : L. johnsonii EV are internalized by the clathrin/dynamin mediated endocytosis pathway. Co‐localization experiments with the endosome markers Rab5, Rab7 and Lamp as well as calcein indicated that EV escape the endosome shortly after Rab7 fusion. Using the expression of the OAS host pathway, we found that the host cellular responses to the EV are dependent on the integrity of the external components of the EV as well as on the RNA cargo. It was found that the RNA transcripts found within the EV largely represent the most abundantly transcribed genes in the bacterial cells such as those associated with protein synthesis and glycolysis. Summary/Conclusion : Ongoing experiments are evaluating host‐related physiological conditions that affect vesicle biogenesis in L. johnsonii. The shifts in protein, lipid and RNA composition of the EV cargo are expected to have a strong impact in host cellular transit and, potentially, their mechanistic targets. Funding : This study is funded by NIH NIDDK R01DK121130. Keywords : probiotic, OAS, RNA cargo, endosome escape

Radiation‐Induced

Gregory Berumen 1 ; Marjan Rafat 2 1 Vanderbilt University, USA; 2 Vanderbilt University, Nashville, USA Introduction : Although primary tumors are managed through a combination of surgery, radiotherapy, and chemotherapy, triple‐negative breast cancer (TNBC) patients experience relatively high rates of recurrence after treatment. The role of intercellular communication in this process is not well understood. Recent studies have shown that ionizing radiation (IR) activates several systemic biological responses, which largely depend on interactions between healthy and injured cells. Radiation‐induced bystander effects (RIBE) are secondary effects that develop in non‐irradiated cells as a result of complex intercellular signals sent by irradiated cells. Therefore, we hypothesized that extracellular vesicles (EVs)—membrane‐delimited structures containing all major classes of biomolecules—function as mediators of RIBE, leading to TNBC progression. In this study, the microenvironmental consequences of radiation‐induced EV secretion were examined. This work represents a critical step toward determining how cell‐cell crosstalk after IR contributes to breast cancer recurrence. Methods : Human fibroblasts were used to model the key cell type in the wound healing response. Fibroblasts were irradiated to a dose of 10 Gy. We used nanoparticle tracking analysis (NTA) to characterize EV secretion 48 hours post‐IR in control and irradiated cells. We also determined the ability of irradiated fibroblast‐derived EVs to alter the phenotype of recipient cells. We examined fibroblast morphology and cytoskeletal dynamics following EV treatment through visualization and quantification of actin fiber reorganization. Furthermore, we used western blot and immunofluorescence analysis to analyze fibroblast activation and shift to a cancer‐associated phenotype. Finally, we utilized mass spectrometry to evaluate the differentially regulated proteins in EVs after IR. Results : IR enhances EV secretion but does not alter the size of the EVs. Additionally, the treatment of fibroblasts with EVs from irradiated cells led to morphology changes and a re‐distribution of F‐actin. This trend follows changes observed in directly irradiated cells. Our findings suggest the ability of irradiated cells to induce bystander effects through EVs. Finally, mass spectrometry results suggest differential packing of key EV proteins after IR causes EV‐induced phenotypic shifts. Summary/Conclusion : Our results establish changes arising from interactions between irradiated and non‐irradiated cells through the transfer of EVs, suggesting a connection to local and systemic RIBE. Notably, EVs derived from irradiated fibroblasts resulted in morphological and phenotypic changes in unirradiated fibroblasts. This work will further our understanding of EV‐mediated communication after radiotherapy and may lead to novel therapeutic strategies for preventing TNBC recurrence.

Tumour‐Associated

Caitlin Boyne 1 ; Silvia Synowsky 2 ; Sally Shirran 3 ; Simon J. Powis 4 1 University of St Andrews ‐ School of Medicine, Cupar, United Kingdom; 2 Biomedical Sciences Research Complex ‐ University of St Andrews, United Kingdom; 3 Biomedical Sciences Research Complex ‐ University of St Andrews, St Andrews, United Kingdom; 4 School of Medicine ‐ University of St Andrews, St Andrews, United Kingdom Introduction : Melanoma is the form of skin cancer associated with the highest rate of mortality and the incidence of the disease is increasing more rapidly than any other form of cancer. Extracellular vesicles (EVs) are small membrane vesicles that have been indicated as key players in the communication that exists between cancer cells and the host microenvironment, both locally and distally. The MHC class I immunopeptidome present on EVs released by cancer cells may be the defining feature of EVs that allows them to have a significant impact on the anti‐tumour immune response. Thus, in this study we isolated EVs from melanoma patient blood samples to characterise their peptide ligandome. Methods : Twelve stage III and IV melanoma patient blood samples and twelve control blood samples were run through size exclusion columns to collect EVs, which were then lysed. MHC class I peptides were isolated and eluted using the immunoprecipitation technique before being processed by mass spectrometry analysis. Results : MHC‐class I bound peptides derived from blood EVs were successfully eluted and identified. Peptides present on the HLA‐I ligandome of EVs derived from melanoma patient plasma stratified into two different peptidomic profiles with varying functional characterizations. Additionally, ten known T‐cell epitopes/immunogenic peptides were identified on the HLA ligandome of EVs isolated from melanoma patient blood samples. Summary/Conclusion : This study indicates the possibility of a peptidomic stratification between the blood EVs of melanoma patients into two distinct groups. Further, the data achieved supports the possibility of identifying peptides on the HLA‐I of EVs that can be recognized by T‐cells and mediate a reaction. The discovery of these key antigens would be a major step toward the development of a novel immunotherapy for melanoma, as well as possibly providing a novel diagnostic or prognostic tool for the disease. Funding : The Melville Trust ‐ For the Care and Cure of Cancer. Keywords : extracellular vesicles, HLA ligandome, T‐cell epitopes, tumour associated antigen (TAA), melanoma

Macrophage‐Derived

Stefano Tacconi 1 ; Anna Maria Giudetti 2 ; Emmanuelle Meugnier 3 ; Serena Longo 2 ; Federica Angilé 2 ; Francesco Paolo Fanizzi 2 ; Audrey JALABERT 4 ; Rienk Nieuwland 5 ; Sophie Rome 3 ; Luciana Dini 1 1 Department of Biology and Biotechnology “C. Darwin”, Sapienza University of Rome, Rome, Italy, Rome, Italy; 2 Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, 73100 Lecce, Italy, Lecce, Italy; 3 CarMeN Laboratory (UMR INSERM 1060/INRA 1397), Lyon‐Sud Faculty of Medicine, University of Lyon, Pierre‐Bénite, France, Lyon, France; 4 CarMeN Laboratory (UMR INSERM 1060/INRA 1397), Lyon‐Sud Faculty of Medicine, University of Lyon, Pierre‐Bénite, FRANCE, Lyon, France; 5 (2) Laboratory of Experimental Clinical Chemistry and Vesicle Observation Center, Amsterdam; The Netherlands;, Amsterdam, Netherlands Introduction : Obesity‐induced diabetes is associated with systemic inflammation, increased numbers of skeletal muscle (SkM) resident macrophages, and SkM insulin‐resistance (SkM‐IR). Here, we determined whether lipoglucotoxicity induced by high‐fat diets affects the release and function of macrophage‐released extracellular vesicles (MEVs) and contributes to SkM‐IR. Methods : To mimic in vivo lipoglucotoxicity, THP‐1‐derived macrophages were treated with free fatty acids (FFA = palmitate+oleate) with/without 15 mM D‐glucose (FFA/G15). Polarization markers, lipid profile, insulin‐induced AKT phosphorylation (IIAP) and oxidative stress were quantified in treated‐THP‐1. Small and large EVs (sEV and lEV), collected by differential centrifugation, were used to treat C2C12 SkM cells. Lipid composition, insulin‐sensitivity and RNA sequencing were performed on recipient C2C12. Results : FFA‐polarized THP‐1 into a M2‐CD163+phenotype. M2‐CD163+ expressed pro‐/anti‐inflammatory cytokines, accumulated triacylglycerols (TAG), FFA, and had altered IIAP vs untreated‐THP‐1. C2C12 treated with lEV‐FFA accumulated TAG, FFA and had reduced insulin‐sensitivity vs untreated C2C12, mimicking FFA action on THP‐1. lEV‐FFA also modulated component from extracellular matrix in C2C12. sEV‐FFA triggered TAG accumulation without affecting muscle insulin‐sensitivity, reduced lipid oxidation and mitochondrial respiration in recipient C2C12. Thus, in a context of lipotoxicity, MEVs participate in maintaining muscle integrity. In the presence of FFA/G15, THP‐1 exhibited a more pro‐inflammatory phenotype, altered lipid composition, and increased oxidative stress. In addition, the beneficial action of MEVs was lost as MEVs from FFA/G15‐treated THP‐1 induced SkM‐IR. Summary/Conclusion : MEVs mirror macrophage phenotypic plasticity during the transition of lipo‐ to lipoglucotoxicity which is associated with the development of SkM‐IR. Funding : This work is supported by the FRENCH AGENCY OF RESEARCH (# ANR‐MEXID‐21‐CE14‐0081). Keywords : high‐fat diet, lipoglucotoxicity, macrophage‐derived extracellular vesicles, muscle homeostasis

Crispr/Cas13A‐Based

Jae‐Sang Hong 1 ; Taehwang Son 2 ; Hyungsoon Im 1 1 Massachusetts General Hospital, Boston, USA; 2 Massachusetts General Hospital, USA Introduction : Extracellular vesicle (EV) microRNAs (miRNAs) play important roles in cancer initiation and progression via cell‐to‐cell communication. Thus, quantitative measurements of EV miRNAs is critical for cancer diagnosis and longitudinal monitoring. Traditional PCR‐based methods, however, require an RNA extraction process and remained as bulk analysis. Here, we introduce an amplification‐free and extraction‐free EV miRNA detection method by delivering liposome‐encapsulated CRISPR/Cas13a components into the EVs through EV‐liposome fusion. Methods : We isolated EVs from 5 different ovarian cancer (OVCA429, SkOV3, ES‐2, CaOV3, and OV90) and 1 benign (TIOSE4) cell lines using size exclusion chromatography. The isolated EVs were characterized by nanoparticle tracking analysis and western blot analysis. Next, we incorporated CRISPR sensing components (Cas13a, CRISPR RNA/crRNA, and fluorescence‐quencher probes) in liposomes and delivered them to EVs through EV‐liposome fusion to detect miR‐21‐5p. We also applied the method for EpCAM‐positive tumor‐derived EVs captured on gold disk arrays for multiplexed analysis. Results : We showed that miR‐21‐5p‐positive EV counts are in the range of 2∼10% in ovarian cancer EVs (OV90, ES‐2, OVCA429, SkOV3, CaOV3,), which is significantly higher than the positive EV counts from the benign cells (< 0.65%, TIOSE4). The result showed an excellent correlation between bulk analysis with the gold standard method, RT‐qPCR. We also found that EpCAM‐positive EVs showed significantly higher abundances of miR‐21‐5p in OV90 compared with the TiOSE4. Summary/Conclusion : The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and the possibility of multiplexed single EV analysis for protein and miRNA markers. Funding : NIH R21CA217662 and R01GM138778. Keywords : extracellular vesicle, microRNA, CRISPR, Cas13a, liposome, ovarian cancer

Ultrasound‐Mediated

Matheus A. Chaves 1 ; Alessandra Bridi 2 ; Mariani Fiorenza 2 ; Felipe Perecin 3 ; Flávio Meirelles 3 ; Samantha Pinho 1 ; Juliano C. Da Silveira 4 1 University of São Paulo, Pirassununga, Brazil; 2 University of São Paulo, Brazil; 3 University of São Paulo, 13635900, Brazil; 4 University of São Paulo, Pirassununga, Brazil Introduction : Nowadays, the use of extracellular vesicles (EVs) as drug delivery systems remains challenging, mainly due to the lack of standardized isolation methods, limited drug loading efficiency, and insufficient clinical grade production. In turn, nanoliposomes (NLPs) are lipid carriers that have been efficiently produced in several large‐scaled industrial practices, as well as easily characterized by well‐established techniques. One can say that EVs can be considered as more complex forms of NLPs due to their biological origin. Therefore, we intended with this study to produce NLPs containing miRNAs usually found in EVs and of interest during oocyte in vitro maturation, in order to design a synthetic vesicle with increased endogenous properties and possible to be scaled‐up. Methods : Unloaded NLPs were produced using cholesterol and commercial phospholipids at a 20:80 ratio. Dispersions were sonicated for 5 s on and 2 s off cycles (50‐75 in total) at a 60% amplitude using an ultrasonic tip homogenizer, and then extruded at 12 bar through 100‐nm polycarbonate membranes. Samples were characterized in respect to their average mean diameter, size distribution and zeta potential using dynamic light scattering, and to concentration using nanoparticle tracking analysis. Morphology of vesicles was observed using atomic force microscopy. NLPs staining with rhodamine‐labeled lipids or PKH26 fluorescent dye was confirmed by flow cytometry. NLPs were incorporated into bovine cells in culture to verify possible uptake mechanisms, which were analyzed using fluorescence microscopy. Results : Results showed that NLPs were spherical with diameters lower than 100 nm, electrically stable with zeta potential values ranging from ‐60 and ‐50 mV, and homogeneous in size with polydispersity index values lower than 0.3. Fluorescence microscopy showed that NLPs appeared to remain in the cytoplasm, around the nuclei of bovine cells. Summary/Conclusion : Further studies regarding the engineering of NLPs in respect to the amount and transfection of incorporated miRNAs are needed to validate their potential as genetic delivery systems. The hybridization of EVs with NLPs is also proposed to produce endogenous nanocarriers with more flexible membranes, easier to interact with cumulus cells. In addition to that, the NLPs did not present any harming effect to the cells and would possibly allow their use during oocyte in vitro maturation. Funding : FAPESP #2022/01236‐7; #2021/06645‐0; #2022/01433‐7; #2022/01505‐8; #2015/21829‐9. Keywords : lipid carriers, engineered extracellular vesicles, reproduction

Age/Senescence‐Related

Sandip Kumar Patel 1 ; Jacob Rose 1 ; Rebecca Beres 2 ; Joanna Bons 1 ; Roland Bruderer 3 ; Lukas Reiter 4 ; Erin Baker 2 ; Judith Campisi 1 ; Birgit Schilling 1 1 Buck Institute for Research on Aging, Novato, USA; 2 University of North Carolina at Chapel Hill, Chapel Hill, USA; 3 Biognosys AG, Schlieren, Swaziland; 4 Biognosys AG, Schlieren, Switzerland Introduction : Cellular senescence is a predominant factor causing aging by triggering profound secretory phenotype (SASP), including sEVs. sEVs have recently emerged as drivers and promising therapeutic targets for multiple age‐related conditions. Thus have tremendous potential for diagnostics of age‐related diseases, which is largely unexplored. Methods : sEVs multi‐omics analysis identifies multicomponent biomarkers for aging/senescence in plasma (Old vs. young) and senescent lung fibroblasts (induced by three stimuli; IR, Doxo, and MiDAS). sEVs are isolated and enriched by size‐exclusion chromatography/ultrafiltration and antibody‐based methods. sEVs intactness and quality were confirmed based on median size and CD9 and TSG101 western blot. Results : We report the first comprehensive high‐throughput analytical platforms to screen sEVs‐specific proteins, lipids, and miRNA for age/senescence and health biomarkers. Deep profiling of sEVs‐specific proteins (∼2,300), lipids (∼350), and miRNA (331) are generated as a resource for the fast‐growing gerontology community. Based on machine learning models, sEVs components independently classify aging/senescent and control samples. sEVs proteomics using DIA‐MS resulted in 144 differentially regulated proteins in aged plasma and ∼1,300 changing proteins from the senescent fibroblasts. sEVs lipidomics on a timsTOF ion mobility MS platform identified >300 lipid species, 23 were differentially regulated in young and old plasma, and 156 lipids were differentially expressed in senescent fibroblasts. In addition, 88 sEVs miRNAs are unique to old plasma. Summary/Conclusion : Ten potential aging/senescence biomarkers emerged; proteins (Peroxidasin, Hemopexin, Plasminogen activator inhibitor 1, SPARC, Transforming growth factor beta‐1‐induced transcript1 protein), lipids (ceramide, sphingolipids), and miRNA (miR‐532, miR‐654‐3p, miR‐409‐3p). In the future, the biomarkers will be validated in aging and age‐related disease cohorts. Funding : U01 AG060906 (Schilling), U54 AG075932 (Campisi/Schilling), S10 OD028654 (Schilling), P01AG017242 and R01AG051729 (PI: Campisi), Glenn Fellowship (Patel). Keywords : sEVs, biomarker, cellular senescence, aging, multi‐omics

Cardiomyocytes‐Derived

Marta Prieto‐Vila 1 ; Yusuke Yoshioka 2 ; Naoya Kuriyama 3 ; Akihiko Okamura 1 ; Asao Muranaka 1 ; Takahiro Ochiya 4 1 Tokyo Medical University, Tokyo, Japan; 2 Department of Molecular and Cellular Medicine, Tokyo Medical Univesity Institute of Medical Science, Shinjuku‐Ku, Japan; 3 Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku‐ku, Japan; 4 Department of Molecular and Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shimjuku‐ku, Japan Introduction : Cardiac fibrosis, found in most cardiovascular diseases, including COVID‐19 patients, is the result of an erroneous hyperactivation of fibroblasts. This dysfunction evokes excessive fibroblast cell proliferation and extracellular matrix (ECM) accumulation, decreasing cardiac function and eventually causing death. It is known that cardiomyocytes (CM) communicate and affect the surrounding cells through extracellular vesicles (EVs). Therefore, CM‐derived EVs may be a promising cell‐free system for fibrosis treatment. Methods : A defined conjunct of chemicals was used to improve human CM culture and ensure a high collection of EVs. Terminal differentiation, as well as senescence markers emergence, were delayed in comparison to the predetermined culture medium without apparent malignant alteration. EVs were isolated by ultracentrifugation, and their characteristics (morphology, particle number, membrane markers, and internalization) were analyzed in accordance with MISEV2018 guidelines. Finally, their effect on fibrosis was tested. Results : EV secretion and their characteristics were unaffected in chemically‐treated CM. Interestingly, CM‐derived EVs were specifically internalized by cardiac fibroblasts compared to other corporal fibroblasts, while no apparent differences were observed in mesenchymal stem cell‐derived EVs. Treatment of EVs on TGFβ‐activated cardiac fibroblasts showed a decrease of fibroblast activation markers at mRNA and protein levels. Furthermore, ECM secretion was also reduced. Consequently, intracardiac injection of EVs reduced the fibrotic area and induced angiogenesis, which translated to an improved cardiac function in a hypertension mouse model. Because the EV content was comprised of not only anti‐fibrotic but also anti‐inflammatory microRNAs, their use in COVID‐19‐like infection is also being studied. To date, the anti‐inflammatory effect of EVs on macrophages activated by SARS‐CoV2 Spike protein was corroborated by cytokine secretion. Correspondent animal experiments are being processed to elucidate the anti‐inflammatory and further anti‐fibrotic protection effect in SARS‐CoV2 infection‐like mouse model. Summary/Conclusion : Our findings indicate that, due to the anti‐fibrotic effects and the specificity of the EV cargo, the use of EVs derived from CM is a promising treatment for several types of cardiac fibrosis. In addition, their study may help to understand the biological meaning of CM‐derived EVs in the cardiac microenvironment. Funding : AMED. Keywords : EVs, cardiomyocytes‐derived EVs, cardiac fibrosis, EV therapy, specific internalization

Rhabdomyosarcoma‐Derived

Paula R. Quaglietta 1 ; Ashby Kissoondoyal 1 ; Ethan Malkin 2 ; David Malkin 1 ; Reto Baertschiger 1 1 The Hospital for Sick Children, Toronto, Canada; 2 The Princess Margaret Cancer Centre, Toronto, Canada Introduction : Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. It is classified into 2 primary histological subtypes: alveolar (ARMS) or embryonal (ERMS), and by presence (FP) or absence (FN) of a PAX3/7‐FOXO1 gene fusion. Novel biomarkers are needed for improved and less invasive diagnosis. Extracellular vesicles (EVs) are present in many biological fluids and play key roles in intercellular communication and the pathophysiology of tumorigenesis and metastasis. This study aimed to identify correlations between RMS‐derived EV protein cargo and clinical characteristics. Methods : We isolated and analyzed EVs from 4 RMS cell lines (RH4, RH18, RH30, RD) in triplicate, in accordance with MISEV 2018. Cells were grown to 70% confluency and EV‐depleted FBS media was conditioned for 48 hours, then concentrated (120mL to < 10mL). EVs were isolated through differential ultracentrifugation. EV quality and characterization was performed by Western blot, transmission electron microscopy and nanoparticle tracking analysis. EV protein cargo was evaluated by LC‐MS/MS. Proteomics, compared to Vesiclepedia, and gene ontology was examined using FunRich (v 3.1.3). Protein interaction networks were generated using the stringApp within Cytoscape (v 3.9.1). Results : We identified 1,657 RMS‐derived EV proteins. We also identified protein patterns correlating with PAX3/7‐FOXO1 fusion status, histological subtype and TP53 mutation status. ARMS EV proteins were associated with collagen fibril organization, cell communication, DNA replication, and striated muscle development, whereas ERMS EV proteins were linked to cell‐matrix adhesion, extracellular matrix organization, and binding various ligands. FP EV contained proteins involved in protein binding, fatty acid metabolism, and DNA damage repair regulation. FN EV proteins were associated with cytoskeleton organization, positive regulation of cell migration, satellite cell maintenance in skeletal muscle regeneration, non‐canonical Wnt signaling, and negative regulation of angiogenesis. Summary/Conclusion : RMS cells secrete EVs with unique protein cargo based on clinical characteristics, demonstrating a potential liquid biopsy method for diagnostics or surveillance. Further validation of these expression patterns in patient‐derived biological fluids is necessary prior to clinical translation. Funding : This study was funded by scholarships granted to P. R. Quaglietta by the Canadian Institutes of Health Research (Canadian Graduate Scholarship‐Masters) and The Hospital for Sick Children (SickKids RESTRACOMP Graduate Award), and support from the Research Institute, The Hospital for Sick Children to R. M. Baertschiger. Keywords : pediatric cancer, rhabdomyosarcoma, diagnosis, liquid biopsy, biomarker