Abstract Book: ISEV2017

Helen Vallhov 1 , Henrik Johansson 2 , Ulf Gehrmann 3 , Tina Holm 3 , Janne Lehtiö 2 and Annika Scheynius 1 1 Department of Clinical Science and Education, Karolinska Institutet, and Sachs’ Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden; 2 Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden; 3 Department of Medicine Solna, Translational Immunology Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden Introduction: Malassezia is the dominant commensal fungi in the human skin mycobiome but is also associated with common skin disorders including atopic eczema (AE). More than 50% of AE-patients have specific IgE and T-cell reactivity towards Malassezia sympodialis , which is one of the most frequently isolated species from both AE patients and healthy individuals. Malassezia releases nanosized exosome-like vesicles, designated MalaEx, which carry allergens and can induce inflammatory cytokine responses (1). Recently, we detected several small RNAs in MalaEx and interestingly, bioinformatic analyses indicated that MalaEx have an RNAi-independent route for biogenesis (2). We did not find any significant difference concerning the levels of these RNAs or the production and the morphology of the MalaEx when comparing MalaEx, which have been isolated from M. sympodialis cultured at normal skin pH versus the higher pH present on the skin of AE patients. Our aim is now to further understand how MalaEx is involved in host-microbe interactions, by comparing protein content of MalaEx and the whole yeast cells, and by investigating interactions of MalaEx with cells in the skin. Methods: MalaEx are collected from M. sympodialis cultures by serial ultracentrifugation and when needed by sucrose gradient. The particle size is estimated by NanoSight and transmission electron microscopy (TEM). The protein content of MalaEx ant the whole yeast cells is assessed with quantitative proteomic analysis. Human primary cells are isolated from skin taken care after cosmetic surgery and cultured together with MalaEx. Results: We have identified 2714 proteins in whole yeast cells and approximately 300 in MalaEx. 34 proteins are enriched in MalaEx and among those two of the major M. sympodialis allergens, Mala s 1 and s 7. Preliminary functional experiments suggest an active binding of MalaEx to human keratinocytes using confocal microscopy. Conclusion: Our results support an active involvement of MalaEx in host-microbe interactions, by binding to host cells, and by the spreading of allergens, thereby contributing to the allergic inflammation. By understanding the role of MalaEx in the sensitisation and maintenance phases of AE, novel prevention strategies and potential therapeutic targets may arise. Franklin W.N. Chow 1 , Cesare Ovando Vazquez 2 , Georgios Koutsovoulos 3 , José Roberto Bermúdez Barrientos 2 , Tuhin Maity 4 , Mark Blaxter 3 , Julie Claycomb 4 , Cei Abreu-Goodger 2 and Amy H. Buck 1 1 Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom; 2 Langebio–Cinvestav; 3 University of Edinburgh, United Kingdom; 4 University of Toronto, Canada Introduction: Extracellular RNA has been proposed as a means of cell-to-cell communication within an organism and a mechanism of cross-species communication. We previously showed that an Argonaute protein (HpWAGO) and small RNAs (miRNAs and Y-RNAs) are secreted in extracellular vesicles produced by Heligmosomoides polygyrus , a gastrointestinal nematode that infects mice. Some of these miRNAs can suppress mouse immune-related host genes in a reporter assay. Here we describe the biochemical properties of the secreted Argonaute protein including its potential interaction partners inside the nematode as well as the mouse host. Methods: Sucrose gradient purification of the ultracentrifuged excreted-secreted material from H. polygyrus followed by western blot analysis was used to determine the association of the nematode Argonaute protein with extracellular vesicles. Associated protein binding partners of the Argonaute protein were then identified by immunoprecipitation followed by LC MS/MS in H. polygyrus as well as a C. elegans strain in which it has been introduced (HpWAGO::GFP). Small RNA sequencing analysis was used to identify the class of Argonaute-associated RNAs in the secreted material and modified nucleotide analogues were used to label nematode RNA to examine uptake into mouse cells using CLICK chemistry. Results: The H. polygyrus co-fractionates with extracellular vesicles based on sucrose gradient purification and co-fractionates with a class of secreted secondary small siRNAs based on size exclusion chromatography. Consistent with this, immunoprecipitation experiments of the Argonaute protein suggest it associates with factors involved in secondary siRNA biogenesis inside of nematodes. Most of these siRNAs come from unannotated regions within the H. polygyrus genome, not clearly associated with protein-coding genes. The nematode RNA can be visualised in the cytoplasm of mouse cells following incubation in transwell assays. Conclusion: These results suggest further diversity in extracellular RNA from a nematode parasite and lay a foundation for understanding the origin of these exRNAs inside the parasite and the mechanisms by which they mediate cross-species communication through interaction with mouse genes. Julia Tandberg 1 , Leidy Lagos 2 , Mona Gaarder 1 , Petter Langlete 1 , Erik Ropstad 2 and Hanne Winther-Larsen 1 1 University of Oslo, Norway; 2 Norwegian University of Life Sciences, Oslo, Norway Introduction: Bacterial membrane vesicles (MVs) are 50–250 nm spherical structures secreted from the surface of many bacteria. Proteomic and biochemical characterisation has revealed that the vesicles contain a variety of bacterial components, including proteins, lipopolysaccharides, DNA and RNA. This makes MVs interesting as potential vaccine candidates, as they represent several aspects of the bacteria, but in a non-replicative form. MV-based vaccines have, furthermore, been successfully used for epidemic control in against serogroup B meningococcal disease, but there are still little known regarding the use of MV-based vaccines in other animals. We recently identified and characterised MVs from the fish pathogen Piscirickettsia salmonis , which showed that the isolated MVs share several similarities with the bacteria. Thus, the present study focused on evacuating the use of MVs from P. salmonis as a vaccine candidate using an adult zebrafish model. Methods: Adult zebrafish were immunised with a concentration of 20 µg MVs or phosphate buffer by i.p. injection. The fish were then challenge by i.p. injection after an immunisation period of 28 days with a challenge dose of 10 8 CFU P. salmonis . Serum and organ sampling for immunoblot analysis and RT-qPCR was performed 1, 14 and 28 days post-immunisation and 1, 3, 5 and 28 days post-challenge. Fish for histology was sampled at 28 days post-immunisation and 3 and 7 days post-challenge. All zebrafish experiment was approved by The Norwegian Animal Research Authority. Results: Immunisation with MVs protected zebrafish against a lethal dose of P. salmonis , and histology showed a reduced formation of granulomas compared to the control group. Immunised fish also displayed an increased macrophage response and reduced inflammatory response after challenge, as well as an increased IgM response after vaccination. Summary: Our data suggest an immunogenic potential of P. salmonis MVs and indicate an important immune response associated with P. salmonis pathogenesis and protection. Yong Cheng and Jeff Schorey University of Notre Dame, IN, USA Introduction: Mycobacterium tuberculosis , the causative agent of tuberculosis (TB), is an intracellular pathogenic bacterium which primarily infects pulmonary macrophages. Approximately one third of the world’s population is infected with M. tuberculosis of which 5–10% develop active TB at some point in their lives. In 2015 this resulted in an estimated 10.4 million new active TB cases and 1.8 million deaths. Our studies aim to better understand how this pathogen intersects with our immune system with the primary focus being on the release of extracellular vesicles (EVs) and their role during an M. tuberculosis infection. The current study addresses the presence of mycobacterial RNA in EVs and their function as modulators of an immune response. Methods: Next-generation sequencing (NGS) technique (Illumina MiSeq) and a subsequent RNA analysis pipeline was used to reveal mycobacterial transcript profile in exosomes isolated from the serum of mice infected with M. tuberculosis . Mycobacterial genetic manipulation, quantitative real-time PCR and ELISA were performed to determine M. tuberculosis components that contribute to the trafficking of mycobacterial transcripts into exosomes. Type I interferon (IFN-β) was measured by both quantitative RT-PCR and ELISA. Results: Sixteen potential mycobacterial transcripts were originally identified from serum exosomes of mice infected with M. tuberculosis using Illumina MiSeq data. RT-PCR and DNA sequencing further determined the existence of mycobacterial transcripts in these exosomes that include mce1B, rpoC, rv0730, rv1629 and rv0453 . The abundance of these mycobacterial transcripts was markedly diminished in exosomes released by macrophages infected with a Δ secA2 mutant of M. tuberculosis in which the secA2 gene was inactivated by a transposon insertion. Consistent with RNA viruses, exosomes isolated from M. tuberculosis -infected macrophages induced a dose-dependent expression of IFN-β in primary murine macrophages. Conclusion: M. tuberculosis transcripts are delivered into exosomes of host cells via a SecA2-dependent pathway, and these mycobacterial transcripts may induce expression of type I interferon in neighbouring cells, potentially increasing mycobacterial survival in TB patients. Withdrawn at author’s request. Matthew Hendricks 1 , Jeffrey Melvin 1 , Yingshi Ouyangi 2 , Donna Stolz 1 , Yoel Sadovsky 2 and Jennifer Bomberger 1 1 University of Pittsburgh, PA, USA; 2 Magee Womens Research Institute, PA, USA Clinical observations link respiratory virus infection and chronic Pseudomonas aeruginosa infection in chronic lung disease patients, including cystic fibrosis, but the mechanism underlying this interaction is not well understood. The development of chronic P. aeruginosa infections often involves the development of highly recalcitrant biofilm communities in the lung. We have recently shown that respiratory syncytial virus (RSV) co-infection significantly increases P. aeruginosa biofilm growth on airway epithelial cells (AECs) through a mechanism that is dependent on the induction of antiviral innate immune response and apical release of the host iron-binding protein transferrin, suggesting that RSV dysregulates nutritional immunity in the airway epithelium (1). However, the mechanism by which transferrin is released from AECs during respiratory viral infection remains undefined. We hypothesised that respiratory viral infection causes a mislocalisation of transferrin within AECs and allows its apical secretion, thereby promoting P. aeruginosa biofilm biogenesis. In the current study, we show that extracellular vesicles released apically from AECs during RSV co-infection enhanced P. aeruginosa biofilm growth. The extracellular vesicles had significantly increased levels of iron and chelation of iron from the extracellular vesicles reduced their ability to stimulate P. aeruginosa biofilm growth. Interestingly, RSV infection enhanced transcytosis and apical secretion of transferrin loaded onto extracellular vesicles. Together these results suggest RSV infection redirects transferrin trafficking in AECs, resulting in the loading of transferrin onto extracellular vesicles, which are secreted from AECs and can be utilised as an iron source by P. aeruginosa to form biofilms. Interferon signalling, which is a key component of antiviral immunity, replicates the enhanced biofilm formation observed during viral co-infection. We are currently investigating mechanisms by which interferon signalling induces transferrin packaging and secretion in extracellular vesicles to stimulate P. aeruginosa biofilm growth. Our data suggest a novel nutrient acquisition pathway for bacteria and provide mechanistic insight into nutritional immunity in the lung.

Martijn J.C. van Herwijnen 1 , Marijke I. Zonneveld 2 , Soenita Goerdayal 3 , Arianne van Bruggen – de Haan 4 , Esther N.M. Nolte-’t-Hoen 1 , Johan Garssen 5 , Maarten A.F. Altelaar 3 , Gerbrich N. van der Meulen 4 , Ruurd M. van Elburg 6 , Frank A. Redegeld 7 and Marca H.M. Wauben 1 1 Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; 2 Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands and Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherl; 3 Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences; 4 Department of Paediatric Allergy, Martini Hospital, Groningen, The Netherlands; 5 Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands and Nutricia Research Centre for Specialised Nutrition, Utrecht, The Netherlands; 6 Department of Pediatrics, Emma Children’s Hospital/Academic Medical Centre, Amsterdam, Netherlands and Nutricia Research Centre for Specialised Nutrition, Utrecht, The Netherlands; 7 Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands Introduction: Breast milk is nature’s first functional food which contains various bioactive components that modulate the infant’s immune system. Early life nutrition is vital for health later in life and breastfeeding may aid in the prevention of allergies. Nevertheless, it has been suggested that the breast milk from allergic mothers can negatively influence the infant’s immunity, possibly caused by an altered milk composition. However, due to the complex structure of milk, the molecular mechanism underlying this effect has not been solved. Recently, we and others have identified milk-derived extracellular vesicles (EVs) as an immune modulatory component in milk. In this study, we compared the protein composition and functional T cell modulatory capacity of milk-EV derived from allergic and non-allergic mothers. Methods: Milk-derived EVs were isolated via differential centrifugation followed by density gradient-based separation of human milk from allergic or non-allergic mothers. Functionality was tested in vitro by co-culturing EVs with αCD3/αCD28-stimulated CD4+ T cells. Additionally, LC-MS/MS proteomic analysis was performed to compare the milk-EV proteomes, followed by pathway analysis of proteins that were differentially expressed using MetaCore and ImmuNet. Results: T cell proliferation, upregulation of activation markers and overall cytokine production were inhibited in the presence of milk-derived EVs, in contrast to T cells that were cultured with milk supernatant depleted of EVs. Remarkably, milk-derived EV from allergic mothers inhibited T cell activation to a lesser extent than EVs from non-allergic mothers. By comparing the proteomes of milk-derived EVs from allergic and non-allergic mothers we found quantitative differences in key proteins between these two groups. These individual proteins linked specifically to the Rac1 and CDC42 signalling pathways, affecting cell proliferation pathways. Conclusion: These data show that milk-derived EVs differ in their T cell modulatory capacity depending on the allergic status of the mother. The reduced T cell inhibition by EVs from allergic mothers might be due to the relative abundance of key proteins in these EVs. Dmitry Ter-Ovanesyan 1 , Emma J.K. Kowal 2 , Aviv Regev 3 and George M. Church 4 1 Harvard University, MA, USA; 2 Massachusetts Institute of Technology, MA, USA; 3 Broad Institute/MIT, MA, USA; 4 Harvard Medical School, MA, USA Introduction: Exosomes contain a variety of RNAs, including both protein-coding messenger RNAs (mRNAs) and non-coding RNAs. Previous reports have found that for extracellular microRNAs, some exist inside vesicles whereas others are contained outside of vesicles in protein complexes. It is unclear what proportion of extracellular RNA resides inside vs. outside of vesicles. Methods: We have used differential ultracentrifugation to isolate exosomes from the K562 leukaemia cell line. We then developed a protocol to get rid of RNA not protected by intact lipid membranes by sequential Proteinase and RNAse treatment, resulting in only the RNA inside of the vesicles. We have also verified that this method does not break vesicles. To characterise the resulting RNA inside of vesicles, we have used various techniques such as Bioanalyzer, qRT-PCR and RNA-Seq. Results: We have found that the majority of RNA (particularly the small RNA fraction) in an exosome pellet isolated by differential ultracentrifugation is not inside vesicles when comparing Bioanalyzer traces of the untreated pellet to the proteinase/RNase treated one. However, our qRT-PCR and RNA-Seq analysis demonstrates that the mRNAs in the exosome pellet are inside the vesicles. Conclusion: The exosome pellet isolated by differential ultracentrifugation contains RNA that is both inside and outside vesicles. We have developed a protocol to distinguish RNA that is inside of vesicles from that which is outside. We have found that the mRNAs are inside vesicles whereas a considerable portion of the small RNAs are outside of vesicles (presumably in free protein complexes). Identifying RNAs that are truly inside vesicle has important implications for studying the role of exosome cargo in intercellular communication. Frederik J. Verweij 1 , Philippe Herbomel 2 , Graça Raposo 3 , Filippo del Bene 4 and Guillaume Van Niel 5 1 Exosomes Research Group Department of Pathology VU University Medical Center Cancer Center Amsterdam (CCA), Amsterdam, The Netherlands; 2 Insitut Pasteur; 3 Centre National de la Recherche Scientifique and Institut Curie, PSL Research University, Paris, France; 4 Institut Curie, PSL Research University, CNRS, Paris, France; 5 Institut Curie, PSL Research University, CNRS, UMR 144, Paris, France /Center for Psychiatry and Neuroscience Introduction: Exosomes are a nano-sized subclass of Extracellular Vesicles (EVs), released by a wide variety of cell types, that have been implicated in many important physiological and pathological processes. Due to the lack of suitable in vivo models, however, the in vivo dynamics and physiology of exosomes are poorly understood. Methods: We developed an animal model to study endogenous exosomes in vivo by (site-specific) expression of a hCD63-based fluorescent reporter for exosome secretion in zebrafish and used various light- and electron microscopy (LM and EM) techniques for our analysis. Results: A combination of light- and electron microscopy (LM and EM) techniques allowed us to observe exosome release in vivo and track a massive pool of endogenous exosomes in the blood flow of zebrafish embryos. Site specific expression confirmed that these exosomes originated from the Yolk Syncytial Layer (YSL), a multinucleate cell layer in-between the yolk and the developing embryo with essential nutrient transport functions, sharing functional homologies with the mammalian placenta. By Electron Microscopy we observed massive release of EVs from the apical side of the YSL into the blood flow, further confirming the YSL as major source of (CD63+ve) exosomes in the developing embryo. Next, we used live imaging to track endogenous EVs in the blood flow to identify their main targets. CD63+ EVs where preferentially interacting with endothelial cells in the caudal vein and plexus compared to the caudal artery. EM revealed endocytosis of these EVs in endosomal compartments of endothelial cells. We detected another major fraction of exosomes in the interstitial fluid, suggesting extravasation outside of the vasculature of YSL derived EVs. We finally observed active and specific endocytosis and storage of CD63+ EVs by scavenging macrophages of the caudal plexus. Summary/Conclusion: Functionally, our data could support a role for YSL derived EVs in nutrient delivery during development, which is our current focus. Altogether, these data reveal for the first time the release, journey and target of endogenous exosomes in vivo . We propose the zebrafish embryo as a new model to study endogenous EVs in vivo that will open new avenues to unravel fundamental aspects in EV biology. Funding: EMBO ALTF 1383-2014; ARC PDF20160604167
; Labex CelTisPhyBio post-doc & project grants; FRM AJE20160635884

Paula Saá 1 , Tracey Díaz 2 , Felicia Santa María 2 , Anoop Pal 3 , Gary Holley 1 , David Krysztof 1 , Adonis Stassinopoulos 2 and Susan Stramer 1 1 American Red Cross; 2 Cerus Corporation; 3 Izon Science Introduction: New strategies have been developed to inactivate infectious agents in blood. Cerus’ INTERCEPT™ Blood System for platelet components combines amotosalen with Ultraviolet (UVA) illumination to specifically target nucleic acids and irreversibly cross-link or form adducts on these molecules, thereby blocking pathogen replication. The efficacy and safety of amotosalen/UVA treated platelet and plasma components has been broadly investigated, the effects on EVs and their biological activity have not been extensively explored. Methods: Apheresis platelet concentrates (PCs) in 100% plasma were collected from healthy donors and pathogen inactivated following standard procedures. To evaluate changes in EV size and concentration, a control sample aliquot was removed from the PCs after amotosalen addition, but before UVA illumination. PCs were then illuminated and a post-treatment aliquot collected. EVs were isolated from pre- and post-illumination samples by centrifugation and size exclusion chromatography (SEC) and their presence was assessed by biochemical and biophysical methods. Results: EVs were detected by electron microscopy imaging in all samples regardless of treatment. The size and morphology of these vesicles (30-50 nm) was consistent with literature reports. No substantial qualitative changes were observed between pre- and post-illumination samples. Comprehensive analyses of pooled SEC fractions 9-11 by tunable resistive pulse sensing confirmed the absence of statistically significant differences in EV concentration (3.39E^10 vs. 3.81E^10) and size (157 vs. 158 nm) between pre- and post-illumination samples, respectively. Likewise, protein analysis of EV lysates by quantitative and qualitative assays did not find major phenotypic changes. Summary/Conclusion: These results suggest that INTERCEPT does not induce significant changes in EV size, concentration and phenotype. Additional studies will evaluate their biological properties and clinical applications. Funding: Cerus Corporation and American Red Cross. Aisling S. Dugan 1 , Bethany O’Hara 2 , Gretchen Gee 2 , Benedetta Assetta 2 , Jenna Morris-Love 3 and Sheila Haley 2 1 Assumption College, MA, USA; 2 Brown University, RI, USA; 3 Pathobiology Graduate Program, Brown University, RI, USA Introduction: Human JC polyomavirus (JCPyV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in AIDS patients. JCPyV utilizes the sialyated glucan, LSTc, and the serotonin 2 subgroup of receptors to gain entry into the human glial cell line, SVG cells. Paradoxically, patient isolates of JCPyV from PML patients have mutations in the major viral capsid protein, VP1, that prevent binding to the serotonin receptor and infection of SVG cells. Moreover, some primary cells without the LSTc receptor can be infected with JCPyV. These observations suggest that there may be an alternative route for JCPyV infection in humans that does not involve the canonical receptors. Exosomes are small (30-100 nm) vesicles released by cells shown to be critical for cell-cell communication and important in the spread of some viruses. Methods: Exosomes were isolated from JCpyV infected SVG cells and examined for exosome number, infectivity, and visualized using transmission electron microscopy. Results: Our data showed that exosomes isolated and purified from the supernatant of JCPyV infected SVG cells contain VP1 and are infectious. JCPyV infection increased the number of exosomes released in media compared to uninfected cells. Exosome inhibitors block JCPyV spread. Transmission electron microscopy revealed that exosomes isolated from JCPyV infected cells were found within vesicle-like sacs consistent with exosomes. Summary/Conclusion: Together, these data suggest a role for exosomes in the spread of infectious JCPyV. Funding: NIH funded: 2R01NS043097-15A1. Stephanie Jung 1 , Yuchen Xia 1 , Michaela Gack 2 and Ulrike Protzer 1 1 Helmholtz Center Munich, Munich, Germany; 2 University of Chicago, IL, USA Introduction: Hepatitis B Virus (HBV) infection is a global health issue with 240 million chronical carriers and a major driver of liver cirrhosis and hepatocellular carcinoma. Coinfection of HBV-positive patients with Hepatitis D Virus (HDV) enhances the risk of severe fulminante hepatitis with liver failure and high mortality rates. Current therapies against HBV prevent the progression of the disease but are ineffective in mediating viral clearance. We aim to understand the immunomodulatory role of EVs in HBV and HDV infection. HBV is considered a stealth virus, fundamentally blocking cytokine production in the infected cell by extensive immune evasive mechanisms. HDV is a satellite virus that requires HBV for virus production and propagation. Methods: We hypothesize that HBV/HDV infected cells shuttle exosomes to non-infected cells, herewith modulating the innate immune response. To test this hypothesis, we purify and transfer exosomes produced by virus-infected cells to non-infected immune cells and quantify cytokine production by both qRT-PCR and ELISA. Results: Preliminary results indicate an immuno-modulatory effect of exosomes released by HDV-infected cells. Additionally, we observe that both intracellular HBV-DNA and HBV transcription levels are diminished in response to transfer of supernatant derived from IFN-α pretreated cells. It can be shown that not interferon itself but heparin-binding particles of high molecular weight released by pretreated cells are responsible for this effect. These very particles inhibit virus entry into hepatoma cells and interact with the HBV receptor heparan glycosaminoglycan. Summary/Conclusion: Resulting data shall elucidate mechanisms of HBV and/or HDV pattern recognition by the immune response. Not only the mode of signal transmission, but also detected pathogen-associated molecular patterns and their corresponding receptors can be identified. These results might give insight into additional HBV-detecting pattern recognition receptors. Funding: SJ is funded by the Helmholtz Association’s Initiative and Networking Fund, YX is partly sponsored by The International Liver Cancer Association and MG is funded by NIH. Elie Beit-Yannai , Sofia Schreiber-Avissar and Natalie Lener Ben-Gurion University, Israel Introduction: Extracellular vesicles (EVs) mediated signaling attract researcher in many biological disciplines, and many research are conducted in-vitro. How much EVs are required to mimic the physiological condition is unclear. EVs calibrated according to their protein content were used in the range of 1µg to 50µg per couple of millions targeted cells. In most of the cases EVs dose response was not addressed. In the present research we examine the effects of different concentrations of EVs derived from the aqueous humor producing cells (NPCE) on the trabecular meshwork (TM) cells. Communication between these tissues in-vivo is considered important for maintaining the intra ocular pressure and have an important role in glaucoma disease. Changes in gene, protein expression and activity of the Wnt signaling pathway members, known to be involved in the pathology of glaucoma disease, were examined according the tested EVs doses. Methods: Human NPCE cell line was grown in exosome depleted medium and EVs were extracted using the PEG precipitation method followed by ultra-centrifugation. The exosomes were incubated for 8h, at three different concentrations (6.8*10 9 (X1), 13.6*10 9 (X2) or 60.8*10 9 (X10) with 0.5*10 6 TM cells. EVs were determined using Bradford and FRAP methods. Retinal pigment epithelium cells derived EVs were used as control and incubated at the same concentrations with 0.5*10 6 TM cells. Quantitative PCR, Western Blot analysis and Zymography were used. Results: Exposure of confluent TM cells for 8h to various concentrations of EVs, lead to significant changes in the expression of the measured proteins. Exposure to low exosome concentration was associated with decreased expression of β-Catenin, GSK-3β, as compared to exposure to high exosomal concentrations (p < 0.01). When exosomes were incubated with TM cells for 2h, a tendency of decrease was found in β-catenin, Axin2 and LEF1 mRNA levels in low concentrations of exosomes compared to high ones. Summary/Conclusion: Cross talk between the ocular drainage tissues via EVs exist . NPCE derived EVs specifically target the TM cells resulting in changes in the TM ECM Our findings suggest that EVs concentration plays a major role in the NPCE-TM communication in-vitro. Furthermore, a bimodal TM response to exosome concentration exposure was found. Lynn Pulliam 1 , Bing Sun 2 and Pranjali Dalvi 3 1 University of California, San Francisco, CA, USA; 2 Veterans Affairs Medical Center, USA; 3 Veterans Affairs Medical Center, USA Introduction: A subset of HIV-infected subjects continues to have cognitive impairment in spite of effective therapy suppressing viral load. This may be due to the high probability of a persistent viral reservoir and ongoing injury. Finding an inexpensive, noninvasive, peripheral biomarker for cognitive impairment has been a high priority. Exosomes (exos) are shed from most cells including neural cells. We isolated neuron-derived exosomes (NDE) in plasma from controls and HIV-infected subjects with varying degrees of cognitive impairment. We examined these NDE for markers of neuronal damage. Methods: Total exos were isolated from plasma of 12 healthy control subjects and 23 HIV-infected subjects using Exoquick. Institutional informed consent was obtained from all subjects. All HIV positive subjects were on antiretroviral therapy with none to varying degrees of cognitive impairment; most had controlled viral load. Further isolation to NDE was performed by immunoadsorption using antibody to the neuron specific cell marker L1CAM. NDE were characterized by NSE, NF-L and synaptophysin (SYP). We looked at exo numbers, CD81 and protein content by ELISA for 2 targets associated with neurodegeneration, Aβ and HMGB1. Results: HIV-infected subjects had significantly fewer total plasma exos compared to controls but there was no difference in NDE numbers; NF-L and SYP were elevated in NDE. Neuropsychologically impaired (NPI) subjects had significantly fewer NDE. HMGB1, Aβ and NF-L were increased in NDE from impaired compared to normal subjects. Summary/Conclusion: We demonstrate for the first time that the number of total exos in the plasma of individuals with HIV infection is decreased. Subjects with cognitive impairment had decreased NDE suggesting fewer neurons. However, within NDE, there is a significant increase in several targets including an alarmin, HMGB1, from impaired subjects. NDE cargo reflects neuronal damage and may predict a cognitive impairment profile. Funding: National Institutes of Health, MH085538 and MH112483 (to L.P.). Withdrawn at author’s request. Leidy Lagos 1 , Sabina Leanti La Rosa 2 , Julia Tandberg 3 , Hanne Winther-Larsen 3 and Margareth Øverland 2 1 Norwegian University of Life Sciences, Oslo, Norway; 2 University of Life Sciences, Oslo, Norway; 3 University of Oslo, Oslo, Norway Introduction: Extracellular vesicles (EVs) with lipid bilayers consist of apoptotic bodies, ectosomes, microparticles, microvesicles and exosomes. Exosomes represent endosome-originated small membrane vesicles, about 30–100 nm in diameter that are secreted by cells and contain several molecules, including proteins and nucleic acids. The secretion of MVs is a common feature of most cells, including eukaryotic cells and bacteria. Piscirickettisa salmonis is a facultative intracellular pathogen that affects farmed salmonids around the world. This Gram-negative fastidious bacteria produces a systemic infection characterized by colonization of several organs in the fish and is able to infect, survive, and replicate inside macrophages, however little is known about its mechanisms of pathogenesis. Recently, we have proven that P. salmonis is able to secrete MVs as strategies to deliver virulence factors to the eukaryotic host cells. Methods: MVs were isolated from salmon ( Atlantic salmon ) plasma that were infected with the fish pathogen P. salmonis using a commercial kit and their presence was confirmed by transmission electron microscopy, Nanosizer, flow cytometry and its protein content was analyze by mass spectrometry. Results: The proteomics results shown 113 protein present in both, healthy and infected fish. However, 106 proteins were present just in the group infected with P. salmonis . Among these proteins are heat shock protein 90, proteasomes subunit, complement protein, autophagy and major histocompatibility class (MHCI), which are key to overcome intracellular infection. Interestingly, we were able to identify bacterial proteins, such as phenylalanine–tRNA ligase, porin and diguanylate cyclase in the plasma of infected animals. Our results suggest that the protein present in MVs depend of the health status of the animal, as well as confirming the ability of P. salmonis to secrete MVs in vivo . Summary/Conclusion: Our results suggest that the protein present in MVs depend of the health status of the animal, as well as confirming the ability of P. salmonis to secrete MVs in vivo . Audrey Hubert 1 , Caroline Subra 2 , Julien Vitry 1 , Myriam Vaillancourt 1 , Anne Bergeron 1 , Frédéric Barabé 1 , Mohammad-Ali Jenabian 3 , Cecile Tremblay 4 , Jean-Pierre Routy 5 , Provost Patrick 6 , Sylvie Trottier 1 and Gilbert Caroline 6 1 Université Laval, Quebec, Canada; 2 U.S. Military HIV Research Program; 3 UQAM; 4 CHUM; 5 McGill University, Montreal, Canada; 6 Department of Microbiology-Infectious Disease and Immunity and Faculty of Medicine, Université Laval, Quebec, Canada Introduction: Extracellular vesicles (EVs) mediate cellular communications and transformation, and contain genetic materials. The abundance of EVs in plasma (pEVs), and their microRNA content may thus be used as biomarkers of inflammatory diseases such as HIV infection. We reported that pEVs isolated from antiretroviral-therapy-naïve HIV-1 patients are enriched in miR-155. However, the type of microRNA-containing EVs and their relevance to HIV-1 pathogenesis remains unknown. Methods: pEVs were precipitated from plasma ART-treated and untreated patients, elite controllers and healthy individuals (n=8/group) by using ExoQuick TM and separated by velocity gradients. Selected microRNAs were detected by qPCR, and the impacts of EVs enriched in miR-155 were tested in vitro and in vivo by using relevant HIV-1 infection models. Results: We observed an increased abundance of acetylcholinesterase-positive pEV (exosomes) in first fractions, and concentrations of EV-borne miR-155 in mischaracterized denser fractions in the case of ART-naïve subjects. Peripheral blood mononuclear cells or NOD/Scid/IL2rγnull (NSG) humanized mice responded to miR-155-bearing vesicles with a marked decrease in the CD4+/CD8+ T lymphocyte ratio resulting from an increase in CD8 T cells, and with the expression of exhaustion marker PD-1 and increased viral production. Summary/Conclusion: This study confirms that the pEV population increases in heterogeneity during infection with HIV-1 and those ART-naïve patients appear to have uncharacterized pEVs that are larger than exosomes and enriched in miR-155. This study showed that velocity gradient remains the most effective method of resolving the pEV population. More importantly, we provide evidence that miR-155-enriched EVs affect HIV-1-associated pathogenesis by promoting activation of CD8 T cells and possibly exhaustion on the long term. Funding: This study was funded through grants MOP-267056 (HIV/AIDS initiative) to C.G., a FRQ-S AIDS and infectious Diseases Network grant to C.G. and S.T, grant MOP-03230 to J.P.R. and C.T. (for cohort establishment) and by the FRQ-S AIDS and infectious Diseases Network. This work was supported in part from a grant awarded to Drs Barabé and Gilbert through a donation of Merck Sharpe & Dohme Corp. to the Faculty of Medicine via the Fondation de l’Université Laval. Olga González-López 1 , Kevin L. McKnight 1 and Stanley M. Lemon 2 1 University of North Carolina, NC, USA; 2 University of North Carolina at Chapel Hill, NC, USA Hepatitis A virus (HAV) is an important cause of enterically transmitted hepatitis in humans and a unique picornavirus that is released from cells non-lytically enclosed in 50-110 nm membranous vesicles. These infectious, quasi-enveloped virions (eHAV) are the only form of virus found in blood from infected humans. Release of eHAV from infected Huh-7.5 hepatoma cells is ablated by siRNA knockdown of ALIX or VPS4B, ESCRT-associated proteins required for exosome biogenesis via the multi-vesicular body (MVB) pathway. Consistent with this, a proteomics analysis of gradient-purified eHAV identified numerous host proteins normally localizing to endolysosomes. Among these, NDFIP2 (Nedd4-family interacting protein 2) is an adaptor protein that binds and activates HECT domain Nedd4 family E3 ubiquitin ligases and that has been implicated in sorting of cargo for delivery to MVBs. Ryan Yu and Benjamin D. Zeitlin University of the Pacific Arthur A. Dugoni School of Dentistry, CA, USA Introduction: Nanovesicles have been identified in preparations of edible plants and certain fruit although the exact cellular origin of these vesicles remains unclear. Cross-kingdom biological effects have been demonstrated for nanovesicles isolated from certain fruit - notably lemons and grapes - that are absent from normal, dietary intake of the whole fruit, this suggests a therapeutic potential for isolated fruit vesicles. Anthocyanins and other polyphenolic anti-oxidant compounds are known to accumulate in plant vesicles as are specific proteins, lipids and microRNAs species. Here we investigate the presence of nanovesicles in antioxidant-rich blueberry fruit. Methods: Fresh blueberries were manually crushed and the pulp passed through a course sieve. Pulp was diluted with phosphate buffered saline and subject to differential centrifugation and ultracentrifugation. The resulting pellet was insoluble and highly resistant to disruption. A jelly-like consistency of the pellet suggested precipitation of soluble structural polysaccharide such as pectin common to soft fruits. To examine the pellet, a sample was fixed in formaldehyde/glutaraldehyde, dehydrated in acetone and embedded in resin. The sample was sectioned and subject to transmission electron microscopy (TEM). Results: We observed sections with numerous vesicle-like structures - approximately 30-100 nm - in addition to highly fibrous areas but generally not in the same field. Summary/Conclusion: In summary we report we believe for the first time the presence of nanovesicle-like structures in extracts from fresh blueberries. We also highlight a previously unreported challenge to vesicle isolation from berry fruit in the form of a fibrous matrix. Funding: University of the Pacific Dugoni School of Dentistry intramural funds. Withdrawn at author’s request. Eline Palm Hansen 1 , Kasper Lind Andersen 1 , Antonio Marcilla 2 , Aaron Jex 3 , Robin Gasser 3 , Neil David Young 3 , Ross Stephen Hall 3 , Merete Fredholm 1 , Stig Milan Thamsborg 1 and Peter Nejsum 4 1 University of Copenhagen, Denmark; 2 Universitat de Valencia, Valencia, Spain; 3 University of Melbourne, Melbourne, Australia; 4 Aarhus University, Denmark Introduction: Parasitic worms have developed an amazing ability to evade or modulate the host immune response and recent studies indicate that extracellular vesicles (EVs) are crucial for host-parasite interactions. In this study, we examined EVs released from three gastrointestinal nematodes of pigs, Ascaris suum, Trichuris suis and Oesophagostomum dentatum . These parasites have different patterns of migration and location in the host, as well as the immunological response they evoke. They could therefore represent suitable candidates to explore unique as well as common modes of host adaptation and immune modulation. Methods: Adult worms were incubated in RPMI under sterile conditions and EVs were purified by differential centrifugations, including two ultracentrifugations at 110,000 x g, and identified by transmission electron microscopy. EV RNA was isolated and sequenced, and the miRNAs for each of the three species were identified using miRDeep2. Predicting miRNA targets and potential functions is part of an on-going analysis, but preliminary results indicate immune-related properties of highly expressed miRNAs. In order to visualize the uptake of EVs and the subsequent transfer of EV RNA into host cells, EVs were treated with RNase, labelled with EV membrane stain as well as RNA stain, transferred to intestinal epithelial cells (Caco-2) and examined by confocal microscopy at 37°C. Results: We found that EVs of all three species were taken up by intestinal epithelial cells followed by a release of RNA, which had a tendency to accumulate in the cell nuclei. We are currently undertaking in vitro studies to functionally test the role of EVs on immune cells. Summary/Conclusion: The findings of this study contribute to unraveling the complex interplay between parasites and their hosts, which may provide new targets for diagnostic tests and novel ways for intervention and disease control in the future. Funding: The University of Copenhagen, Denmark; Direktør Ejnar Jonasson kaldet Johnsen og hustrus mindelegat: Danish Council for Independent Research (DFF-6111-00521) Jose Roberto. Bermúdez 1 , Franklin W.N. Chow 2 , Amy H. Buck 3 and Cei Abreu-Goodger 4 1 LANGEBIO, CINVESTAV, IRAPUATO MX; 2 Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom; 3 Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, United Kingdom; 4 Langebio–Cinvestav Introduction: Evidence for a cell-to-cell communication system mediated by vesicles loaded with small RNAs (sRNAs) is accumulating. However, we still ignore if sRNA-loaded vesicles could be used for communication between different species. Heligmosomoides polygyrus is a nematode that infects mice and is a valuable model to study the immune response to chronic infections. During its adult phase, H. polygyrus secretes vesicles while residing in the mouse intestine. We have already shown that these vesicles have immune-modulation capacity, and that they contain proteins as well as small RNAs. We ultimately aim to determine if these H. polygyrus small RNAs (Hp-sRNAs) have a role during interaction with the host. Nematode vesicles have the capacity to enter MODE-K epithelial intestine mouse cells in vitro , as revealed by fluorescently labelling the nematode vesicles. Methods: To explore in an unbiased manner the capacity of Hp-sRNAs to enter mouse cells we cultured mouse cells together with H. polygyrus vesicles or total secretion and performed Small RNA sequencing (sRNA-Seq). To determine if there is specificity in Hp-sRNAs internalization, we used two different cell types: MODE-K and bone marrow derived macrophages. To assess the stability of Hp-sRNAs inside mouse cells we applied two incubation times (4 and 24 hours), prior to sequencing. Results: We can use bioinformatics to disentangle nematode and mouse sRNAs with mapping strategies and differential expression analyses. We identify 75 high confidence Hp-sRNAs inside MODE-K cells after treatment. These sRNAs are found in vesicles and tend to belong to the 22G siRNA class (nematode specific). We are currently exploring the targeting capacity for these Hp-sRNAs in mouse, and looking for over-represented functions among putative targets. Summary/Conclusion: Our project explores the possibility of sRNA-loaded vesicles being used as a means for inter-species communication.

George Daaboul 1 , David Freedman 2 , Mani Sadredini 3 , Steven Scherr 4 , Paola Gagni 5 , Marcella Chiari 5 , John Connor 6 and Selim Unlu 7 1 nanoView Diagnostics Inc.; 2 nanoView Diagnostics; 3 Oslo University Oslo, Norway; 4 Boston University College of Mechanical Engineering, MA, USA; 5 Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare; 6 Boston University School of Medicine, MA, USA; Department of Microbiology; 7 Boston University College of Electrical and Computer Engineering, MA, USA Introduction: Exosomes are currently characterized by running non-specific nanoparticle analysis followed by proteomic analysis to confirm the existence of exosome markers. This approach usually requires exosomes to be isolated through ultracentrifugation (UC) to separate interference from soluble markers in the biological fluid. Recently, flow cytometers have been adapted to combine light scatter measurements from nanoparticles with fluorescent detection of exosome markers. The combination of the light scatter with specific markers improves the reliability and specificity of exosome detection. However, the small-size of exosomes makes specific detection above background levels difficult because these diameters (50-200 nm) are too small for traditional visualization technologies. Methods: We have applied a label-free microarray imaging technique for enumeration, sizing and phenotyping of exosomes. The technique is termed Single Particle Interferometric Reflectance Imaging Sensor (SPIRIS) that allows visualization of individual nanovesicles captured on the sensor surface, which is functionalized with a non-fouling polymer arrayed with antibodies against surface markers. The sensor is comprised of a silicon substrate with a thin silicon dioxide layer forming a common path interferometer. The spectrally reflected light from the sensor surface interferes with the scattered light from captured nanoparticles enhancing their visibility. Results: We have verified the sizing sensitivity of the sensor using viral particles from cell culture media spanning diameters from 40 nm (Zika Virus) to 360 nm (Vaccinia Virus). We have also demonstrated direct-from-sample extracellular vesicle phenotyping from cell culture media and human plasma. To validate specificity of direct-from-sample detection, results were compared to detection post-isolation using UC. Summary/Conclusion: A detection limit of 5 x 10 5 particles/ml or 0.5 zepto moles when using 5 µl of sample was demonstrated. Direct detection of exosomes from cell culture and human plasma is shown without the need for isolation. SPIRIS direct-from-sample high-throughput technique could improve standardization of exosome preparations and facilitate translation of exosome-based liquid biopsies. Anoop Pal , Robert Vogel, Julien Muzard and Murray Broom Izon Science Introduction: Extracellular Vesicles (EVs) are heterogeneous in size, number, membrane composition and contents. A thorough understanding of this diversity and the linkage of biophysical properties to EV biological role and function is necessary. Real, validated, repeatable measurement data are required for the biomedical adoption of EV based diagnostics and therapeutic developments. These have not always been prominent in EV research. We also believe that normalization of any biochemical analyses back to the EV particle properties will become a standard requirement. Methods: An integrated, standardizable and very practical methodology for EV purification and measurement has been developed. SEC columns provide clean EVs from biological fluids and cell culture, 99% free of non-vesicular proteins. TRPS provides detailed, calibrated measurement of EV particle number, size, concentration of each size fraction, and individual particle surface charge. Continual improvements by users in the usability and reproducibility of TRPS have reduced the time required while improving quality. Results: In the first case study, EVs purified from BAL fluid were quantified and analyzed for difference in size, concentration over a defined size range and surface charge post lung exposure to nanoparticles. In second case study tissue factor bearing microparticles from two different cell lines were separated, characterized, and functionally evaluated by tissue factor activity assay. The assay responses were normalized to the TRPS data to assess the impact of particle size, surface area and volume on tissue factor activity. Further, quantification of EV surface markers (CD63 and CD142) and phenotyping of specific EVs captured via antibody conjugated to magnetic beads was achieved. Our results showed a proportional increase in size, volume and surface charge of the EV-Magnetic bead complex (immunoprecipitated) over a defined dose-range. Secondary measurements confirmed these findings as well. Summary/Conclusion: Thus, the proposed integrated methodology provides a simple, rapid, reliable, and cost efficient approach for EV purification and biophysical characterization amenable for diagnostic and therapeutic proposes. Oliver Kenyon Apogee Flow Systems Ltd Introduction: The complex relationship between particle size and the amount of light scattered at different collection angles makes it difficult to infer particle size from a flow cytometer’s light scatter data. A population may be described as scattering an amount of light equal to a reference particle (e.g. a latex or silica bead of known size) but same sized particles of different refractive index give different signal strengths. When comparing data between flow cytometers the difficulties are compounded by differences in light scatter illumination and collection angles Methods: A particle suspension containing a continuum of particle sizes of well-defined and known refractive index may be used to characterize the light scatter optics of any flow cytometer. Once the light scatter optics have been characterized in this way, data from biological samples (e.g. virions, extracellular vesicles) can be transformed from light scatter space (e.g. small, medium and large angle dimensions) to size and refractive index dimensions. Results: It is possible to convert light scatter data into particle size and refractive index information. This may be thought of as a conversion from three (or more) dimensional light scatter space to 2-dimensional space with dimensions ‘size’ and ‘refractive index’. Summary/Conclusion: Size and refractive index parameters allow comparison of data among flow cytometers and other particle analyzers in a way not possible with light scatter data. For this reason it is well suited to studies of submicron particles such as bacteria, virus and extracellular vesicles. The new size and refractive index parameters can be stored in FCS format, compatible with widely available software. Funding: Apogee Flow Systems Ltd Oleg Guryev 1 , Tatyana Chernenko 1 , Majid Mehrpouyan 1 , Gulam Shaikh 1 , Pam Canaday 2 , Claudia Lopez 3 , Terry K. Morgan 4 and Marybeth Sharkey 1 1 BD Biosciences; 2 Department of Pathology, OHSU; 3 Multiscale Microscopy Core, Center for Spatial Systems Biomedicine, OHSU; 4 OHSU Introduction: Liposomes are nano/micro-size sphere-shaped lipid vesicles of single or multiple lipid bilayers. They are often used as a model objects in study of biological microvesicles (MVs). However, there is no standardized method for preparation of liposomes of different sizes. The goal of our project was to develop reliable and reproducible procedure for on-site liposome preparation when researcher can make liposomes and use them immediately in their experiments. Using liposomes as reference standards, we propose a technique based on side-scattered light analysis on a flow cytometer to characterize MVs from human serum. Methods: Liposomes where prepared via new centrifugation technology. They were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and flow cytometry. Results: Flow cytometry was used to study fluorescein labeled or blank liposomes of defined dimensions. Their size and structure was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Linear dependence of side scattering (SSC) from liposome size was established in the range from 200 nm to 700 nm. However, it was found that liposome light scatter depends on their lipid composition. We use liposomes and polystyrene microparticles for flow cytometry instrument calibration and MV size determination. Summary/Conclusion: 1. A new technology was utilized to produce a set of liposomes of different sizes ranging from 100 to 700 nm. 2. Dependence of SSC from liposome dimensions has a linear correlation. 3. This set of liposomes can be used for size determination of MVs from human serum. Jonathan Shaffer 1 , Martin Schlumpberger 2 , Karolin Spitzer 2 , Verena Schramm 2 and Markus Sprenger-Haussels 2 1 QIAGEN Sciences; 2 QIAGEN GmbH Introduction: Fast and reproducible isolation of exosomes and other extracellular vesicles presents a major challenge in exosome research and further hinders downstream analysis. Here, we demonstrate a complete and reproducible workflow from rapid isolation of vesicular-specific RNA, including miRNA and other small RNAs, using a membrane affinity-based procedure in spin column format [1] to efficient profiling and analysis of vesicular miRNA content by next-generation sequencing. This workflow was applied to colorectal cancer (CRC) patients. [1] Enderle D, Spiel A, Coticchia CM, Berghoff E, Mueller R, Schlumpberger M, et al. (2015) Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method. PLoS ONE 10(8): e0136133. Methods: Vesicular RNA from plasma of CRC patients was isolated using a spin column-based approach. Libraries for miRNA-Seq were prepared using a novel ligation-mediated approach for library prep which assigns unique molecular indices (UMIs) to each miRNA. Next-generation sequencing was then performed using a benchtop sequencer. Reads were mapped to miRbase and identical reads were collapsed based on the UMI sequences. Results: Using the novel workflow, EV-specific miRNA content from serum, plasma and other biofluids can be profiled efficiently with total hands-on time of less than four hours for the complete workflow from isolation of vesicular RNA to miRNA-seq libraries. 35-40% of all reads consistently map to known miRNAs annotated in miRbase. This high percentage of mapped reads results from efficient removal of Y-RNAs and other small RNAs during the library preparation. We find EV-specific miRNAs to be highly abundant among all sequenced miRNAs proving the isolation of EV-specific RNA content. Summary/Conclusion: We conclude that the combination of a spin-column based EV-specific RNA isolation and miRNA-seq library preparation optimized to remove Y-RNAs is highly suited to accurately profile miRNAs from CRC patients. This approach maximizes the amount of interpretable data to specifically profile miRNAs inside of EVs without background from miRNAs outside of EVs. Funding: This research was funded by QIAGEN GmbH, Hilden, Germany. CRC patient samples were provided by our collaborator Prof. Dockhorn at Zentrum für Pathologie, Kempten, Germany Dapi Chiang 1 , Dominik Buschmann 2 , Benedikt Kirchner 2 and Michael Pfaffl 2 1 Biovesicle Inc.; 2 Division of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University Munich Introduction: Exosomes are small vesicles (30-150 nm) secreted from different cell types and found in various biofluids, such as blood, urine, saliva and CSF. Exosomes contribute to cell-cell communication, antigen presentation or tumor progression by carrying cellular proteins, RNA/DNA, glycans and lipids. Differential ultracentrifugation (UC) is still regarded the ‘Gold Standard’ for exosome isolation. However, UC is a laborious and time-consuming method that requires specialized equipment and operational expertise. Several alternative methods such as antibody-conjugated beads were developed to isolate exosomes without UC. Isolation based on antibody-conjugated beads, however may damage exosomes by using acidic or alkaline reagent to break antigen-antibody interactions. Methods: To solve these issues, we create a “non-antibody” coated bead, called EXÖBead, that is able to isolate exosomes. We incubated EXÖBead with pre-cleared cell culture medium (CCMs) or serum and analyzed the pulled-down fraction by FACS, western blot, Bioanalyzer 2100 capillary electrophoresis, Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Results: Our result showed that CD63 can be detected by FACS in exosome-bead complexes from 100 µl to 1 ml human serum (MFI: 40.7% to 76.2%). Additionally, the expression of Alix and Rab5 was substantiated by western blot using the exosome-EXÖBead complex from 200 µl mouse serum or 200 µl B16F10 CCMs. The pattern of vesicular RNA and its cDNA was found to be similar for exosomes isolated by EXÖBead and differential UC (120,000 g pellet). For the RT-qPCR study, U6 (28.9 cycles) and miR-33 (34.7 cycles) can be detected in exosomes isolated from 10 ml THP-1 derived macrophage CCMs. Furthermore, we designed an exosome elution buffer without using any acidic or alkaline reagents. To test its ability to release exosomes from beads, we performed NTA and TEM to assess vesicle size and morphology. The size of exosomes from NTA (mode of diameter: 154.3 +/- 4.9 nm) and TEM (diameter: 50 nm to 110 nm) eluted from beads is similar to exosomes isolated by UC. Summary/Conclusion: In conclusion, EXÖBead can capture exosomes from biofluid samples without ultracentrifugation and exosomes can be successfully eluted from the beads. Alessandra Fierabracci 1 , Valeria La Marca 1 , Kelly Van Wemmel 2 , Sally Snyman 2 , Silvia Balosso 3 , Laura Papetti 4 , Maurizio Muraca 5 , Annamaria Vezzani 6 , Federico Vigevano 7 and Marcin Jurga 2 1 Children’s Hospital Bambino Gesù, Infectivology and Clinical Trials Area, Type 1 Diabetes Centre; 2 Esperite Cell Factory; 3 Dept of Neuroscience, IRCCS Ist. Ricerche Farmacologiche Mario Negri; 4 Dept of Neurosciences, Children’s Hospital Bambino Gesu’; 5 Department of Women’s and Children’s Health, University of Padua, Padua, Italy; 6 Dept of Neuroscience, IRCCS Ist Ricerche Farmacologiche Mario Negri; 7 Children’s Hospital Bambino Gesù, Dept of Neuroscience Introduction: MSCs exert their biological effects through secretion of extracellular vesicles (EV). We previously showed that MSC-EV have significant immunomodulatory properties. MSC-EV inhibit B cells proliferation/differentiation upon PBMC CpG stimulation, similarly to parent MSCs. Furthermore, MSC-EV induce Treg proliferation/apoptosis and IL-10 secretion, following antiCD3/CD28 PBMC stimulus. In this study we show that clinical grade (CG) EV exert similar immunomodulation to research grade (RG) counterparts. CG EV could be produced with higher efficiency if compared to RG EV and MSCs manufacturing. Currently our group is preparing MSC-derived EV for clinical tests in treatment of epilepsy, a disorder resistant to anti-epileptic drugs in 40% of children due to neuroinflammation. A novel anti-inflammatory strategy, based on CG EV, is proposed. Methods: A method of CG EV production is based on human umbilical cord derived (UC) MSCs cultured in a closed, scalable stirred-tank bioreactor system in fully defined GMP culture media. EVs are purified by sequential filtration/sterilization. The final product is analyzed by NTA to evaluate size and quantity, and EVs are characterized by MACSPlex immunophenotyping (FACS), to identify specific CD markers. The immuno-modulatory activity of the CG product is evaluated in comparison with RG EVs and MSCs by specific in vitro B and T cells assays. Results: The CG EV isolation method, has been optimized to obtain at least 1.5 * 10^9 EV/mL in 24 h from 0.1 * 10^6 MSCs. EV diameter cut off is 300 nm. MACSPlex exosome assay revealed that EV are CD9, CD63 and CD81 positive, but HLA-ABC and HLA-DRPQ negative. T and B potency assays, performed on PBMC, indicate immunosuppression by CG EV, similarly to the RG EV obtained from the same MSCs. This effect is revealed by Treg increase, counteracting T eff, upon T cells activation, and by reduction of B cells proliferation and plasma cell differentiation, following B cells activation. Summary/Conclusion: We have developed and standardized a reproducible method for the production, quantification and immunophenotyping of CG EV, starting from human UC MSCs, with similar immunomodulation if compared to RG EV counterparts. Our data indicate that the use of CG EV could be effective in the treatment of a wide range of immunological diseases, and provide a more accessible alternative for allogenic MSCs. Funding: Esperite (B) John J. Cadwell 1 , John Ludlow 2 and Tony Rutt 3 1 FiberCell Systems Inc.; 2 Zen Bio; 3 KD Bio Introduction: Current methods for collecting EVs from conditioned medium use large numbers of tissue culture flasks. EVs are secreted in small quantities and a standard preparation can entail a final stage of 200 T225 flasks or more. This method is wasteful, time consuming, space consuming and the cells are growing in a non-physiologic environment. Hollow fiber bioreactors have been used to produce large quantities of exosomes from human adipose derived MSCs in culture for over two months with no change in phenotype. The harvest volume is 40mls and harvests can be performed 2-3 times a week. Functional assays have shown the bioreactor exosomes were at least as active as the ones obtained from flasks. Methods: Cryopreserved ASCs (1 x 10 9 ) were used to seed a 20Kd MWCO PS cartridge (C2011, FiberCell® Systems Inc.) After 48 hours the non-adhered cells were removed from the system. Circulating PM-1 medium was changed when the starting glucose level of 0.45mg/mL was depleted by 50%. A time course of monitoring revealed medium changes need to occur every 3-5 days, which coincided with harvesting the conditioned serum-free medium from the bioreactor cartridge for EV isolation. Cultures were maintained in this manner for three months. In vivo biological activity was determined using a model whereby 2cm diameter wounds were made in the back skin of rats and then treated with vehicle control or EVs (25 x10 6 particles). The wounds received a single treatment by topical application onto the surface of the wound and allowing to air-dry for 10 min before covering with a bandage. Results: A total of 8.60 X 10 11 EVs were harvested in a total volume of 120 ml over the course of 6 weeks. 130 T225 flasks yielded a total of 1.6 X 10 9 EVs in a volume of 4000 ml. The hollow fiber bioreactor produced the equivalent of nearly 7,000 T225 flasks but in a much smaller volume. The rat wound healing model demonstrated a significant acceleration of the wound healing process. Summary/Conclusion: Hollow fiber bioreactors represent a more in vivo like way to produce exosomes of both the quality and quantity required for clinical relevance in a closed, single use system. MSC phenotype remains unchanged over the course of culture and can produce EVs in a continuous process at a high concentration. EVs produced in this manner demonstrate significant wound healing and biological activity. Christine M. Coticchia 1 , James Hurley 1 , Anand Srivastava 2 , Esilida Karecci 2 , Vasisht Tadigotla 1 , Mia Sher 1 , Siawosh Eskandari 2 , Albana Mihali 2 , Jamil Azzi 2 and Johan Skog 1 1 Exosome Diagnostics; 2 Transplant Research Center, Brigham and Women’s Hospital and Harvard Medical School, MA, USA Introduction: Approximately 15% of patients with end stage renal disease (ESRD) who undergo kidney transplant will suffer from acute rejection, negatively impacting long-term graft survival and function. Current methods for monitoring rejection, such as serum creatinine and urine protein secretion are not specific and cannot detect subclinical rejection. Kidney biopsy is therefore routinely used to assess acute rejection, increasing both risk to the patient and cost. An accurate, non-invasive method to determine the presence of early, acute kidney rejection would minimize the amount of immunosuppression needed to manage these patients. Exosomal mRNA (exoRNA) and proteins are an ideal source for such biomarker studies. In the transplanted kidney, exosomes originate from glomerular podocytes, renal tubular cells and from immune cells, generated during rejection. Using these exosomes we previously reported the discovery and validation of a 23-gene urinary exoRNA signature for the diagnosis of human kidney transplant rejection. Here we asked if urine exosomal proteins could increase the accuracy, and reduce the number of genes required for the detection of kidney transplant rejection. Methods: Urine samples were collected from patients undergoing a transplant kidney biopsy for clinical indications. A total of 21 urine samples (10 rejections, 11 non-rejections) were collected from 21 individual patients. Total exosomes were isolated from 10 mL of patient urine and the presence of 92 exosome proteins was determined by Proseek® Multiplex Inflammation, an immunoassay using Olink Proteomics proximity extension assay (PEA). Results: Among the 92 proteins examined, CXCL9 and CXCL10 were identified to be differentially expressed in both rejection versus non-rejection urine exosome protein and urine exoRNA. Receiver-operating-characteristic (ROC) area under the curve (AUC) analysis determined that urine exosome-associated proteins CXCL9 and CXCL10 could distinguish patients with kidney transplant rejection from those without rejection with an accuracy of 0.827, ( P ≤ 0.01). Summary/Conclusion: We additionally identified 3 independent exosome proteins that are differentially expressed in patients with and without kidney transplant rejection, demonstrating that urine exosome proteins are a promising source of biomarkers for organ rejection. Anna Markowska, R. Scott Pendergrast, J. Stephen Pendergrast and P. Shannon Pendergrast Ymir Genomics LLC Introduction: Urine offers many advantages over blood as a source of the diagnostic and prognostic biomarkers contained in extracellular vesicles (EVs). Its collection is easy and less invasive. It is itself less of a biohazard and does not generate biohazards such as used needles and specimen vials. These advantages suggest the potential for At-Home donation of urine samples for clinical studies via mail. At-home donation would dramatically increase the convenience and thus compliance from patients and decrease costs for clinicians. However, although urine contains far less cells than blood, the number contaminating white blood cells, red blood cells, epithelial cells and podocytes is not negligible and is also highly variable from sample to sample. Delivery of urine samples to a clinical and/or research laboratory via the mail introduces the possibility of cellular rupture and contamination of the extracellular fraction with cellular biomolecules. Methods: Here we investigate the degree of cellular contamination of EV preparations from “natural” urine samples and from samples spiked with red and white blood cells. We look at both protein and RNA contamination under a variety of shipping, storage, and experimental conditions. Storage/transport temperatures investigated include Room Temperature, Refrigeration, and Freezing for 0-3 days. Experimental conditions include filtration, cell preservatives, and different low speed spins. Results: We find that natural samples can contain very significant contamination from proteins and RNA that are highly expressed in blood cells. For instance, the red blood cell miRNA mir-451a can increase >50-fold in samples from women during menstruation. Also, no storage or shipping condition completely protects samples from cellular contamination, including commercial preparations advertised to protect biofluids from cellular degradation. Furthermore, some standard methods for removing cells can actually introduce cellular contamination. Summary/Conclusion: These findings strongly encourage researchers working with urine samples to take precautions towards preparing truly cell free fractions of vesicles. Possible solutions to this problem will be discussed. Funding: This study was funded entirely by Ymir Genomics LLC Nikki Heath 1 , Lois Grant 2 , Xabier Osteikoetxea 1 , Niek Dekker 1 , Lorenz Mayr 2 and Ross Overman 2 1 Astrazeneca; 2 AstraZeneca Introduction: Extracellular vesicles (EVs) have a unique and natural ability to deliver functional cargoes to recipient cells. Exploitation of EVs to deliver therapeutic cargoes such as nucleic acids, small molecules or proteins, to diseased cells is becoming an increasingly interesting and feasible notion. For this to become a reality and enter the clinic, a rapid, scalable and reproducible method of EV isolation will need to be developed. There are some caveats surrounding the current methods for EV isolation. For example the gold standard protocol of differential centrifugation is not readily scalable, and cross flow filtration requires additional subsequent clean-up procedures to isolate EVs in a pure form. Methods: Here we develop a method by which we use column-based chromatography to isolate EVs in a single step protocol. EVs were isolated by ultracentrifugation, cross flow filtration and ion exchange chromatography from HEK293T cells. Collected EVs were analysed by Western blotting for EV markers, nanoparticle tracking analysis and cryoelectron microscopy. Results: We have demonstrated that ion exchange chromatography can reproducibly isolate CD63, CD81, ALIX and TSG101 containing EVs from conditioned media. The size distribution of EVs isolated by ion exchange chromatography (mean 179 nm) was similar to that of EVs isolated by ultracentrifugation (mean 160 nm) but not EVs isolated by filtration (mean 123 nm). Although the yield from ion exchange isolation was lower than achieved by filtration (IEX 183 EVs/cell vs. filtration 748 EVs/cell), it was higher than for ultracentrifugation-derived EVs (125 EVs/cell). In addition, unlike cross flow filtration, the isolated EVs did not require further downstream processing to purify the vesicles away from contaminating proteins such as BSA. Summary/Conclusion: Ion exchange chromatography provides an ideal compromise as an efficient and scalable method for the isolation of clean preparations of EVs in a single step. Further analysis of EVs isolated by ion exchange at a larger scale, together with a better understanding of their in vivo characteristics, will be beneficial to determine the extent to which this isolation method could be used within a clinical setting. Funding: Postdoctoral research scientist AstraZeneca Julia Gavrilova 1 , Jekaterina Muhhina 2 , Triin Oja 2 , Davide Zocco 3 , Giorgia Radano 3 , Natasha Zarovni 4 and Paolo Guazzi 2 1 HansaBioMed Life-Sciences; 2 HansaBioMed Life Sciences; 3 Exosomics Siena; 4 Exosomics Siena SpA Introduction: Size Exclusion Chromatography (SEC) is emerging as one the most promising methods for isolating and purifying extracellular vesicles (EVs) from different matrices. SEC technique is very efficient for separating EVs from the circulating proteins and does not affect the original shape and functionality of the vesicles, but its use is applicable only to small sample volume (maximum 2 ml, due to the volume capacity of the columns commercially available) limiting negatively the EV recovery from diluted matrices as urine or cell media. HBM-LS has developed a new SEC column for isolating EVs from a large volume of sample and adapted it to different matrices. Additionally, the column separated efficiently the different EV sizes from a single sample. Methods: EVs isolation was performed from 20 ml of bodily fluids (urine) and cell medium, using ultracentrifugation or SEC. Isolation efficiency, EV size and shape have been assayed with different common techniques (NTA, TEM, ELISA quantification). Results: SEC had several advantages over ultracentrifugation, including reduced hands-on time and cost, improved ease of use, and higher yield from the same sample volume. Remarkably, the SEC column allowed the separation of EVs of different sizes from the same sample, subsequently characterized by nanotracking analysis and electron microscopy Summary/Conclusion: The novel SEC column allows EVs isolation from large volume of diluted matrices with higher yield than ultracentrifugation. The protocol enables the separation of EVs of different size suitable for phenotyping or molecular analysis. Clemens Helmbrecht and Hanno Wachernig PARTICLE METRIX GmbH Introduction: Nanoparticle Tracking Analysis (NTA) measures size and concentration in the size range from 10 nm to 1 µm. Physical techniques such as NTA detect particles, however, cannot discriminate whether the detected particles are biological or inorganic particles such as e.g. dust, nano-bubble, metal-oxide particles or precipitates from buffer. To overcome this limitation, NTA is equipped with fluorescence detection capabilities combining the advantages of fluorescence detection and nanoparticle characterization to form fluorescence NTA (F-NTA). Quantification of fluorescent nanoparticles by F-NTA has proven to be challenging, but recently credible results have been obtained as researchers examine what is required to obtain reliable results with exosome samples. Particle Metrix GmbH (PMX) expanded the options available by proper choice of photo-stable dyes as well as instrument design to limit photo bleaching. Methods: Rapid, reliable and fast acquisition has been performed by short acquisition at several positions by scanning-NTA to avoid photo bleaching of standard fluorophores such as Alexa 488. By scanning through the sample volume, significant statistics can be achieved in a short acquisition time. Results: Performance of NTA fluorescence detection was verified by means of fluorescent nanoparticle size standards < 40 nm. With quantum nano-dots (Q-dots), lower sizes are achievable. The dynamic range of detection was expanded to the detection of ONE fluorescent PS particle (100 nm) in the presence of 10000 unlabeled particles. Evaluation of methods using membrane dyes such as PKH67, DiO, DiL and CMO are shown on EVs and Liposomes. Quantification of EVs selectively tagged by means of specific antibody labelled with Alexa-Fluor dyes is also shown. Summary/Conclusion: Although F-NTA was first introduced 6-8 years ago, it has been slow to develop due to challenges tagging with quantum dots, and photo bleaching of standard fluorophores. PMX GmbH has designed an F-NTA instrument that in large part negates the issue of photo bleaching with many fluorophores by quickly scanning through the sample volume with 1-2 second acquisition times. Agnieszka Siupa, Clayton Deighan, Sonja Capracotta and Duncan Griffiths Malvern Instruments Introduction: As interest in extracellular vesicles (EV) continues to grow, the Nanoparticle Tracking Analysis (NTA) technique has proven to be a valuable and effective tool for EV characterization, commonly used for the detection and measurement (size and concentration) of EV’s after isolation. By introducing a fluorescence label and using fluorescence mode NTA (fNTA), researchers are able to confirm that the isolated particles are vesicles or identify a particular biomarker to expand upon the current EV characterization methods. To date, fNTA experiments have met with varying degrees of success. Methods: This paper discusses a critical variable for successful fNTA measurements, the minimum number of fluorophore molecules needed per particle for detection and example experiments to show how to ascertain this value for different fluorophores. Detection of a fluorescently labeled particle is a multifaceted problem related to the intrinsic properties of the dye molecule, the optical arrangement of the instrument, and method of sample preparation. To quantify in specific terms the number of fluorophores required for detection in different systems three model experiment results are presented. Results: Three separate model systems were evaluated: Liposomes (~120 nm) loaded with Atto 550 incorporated at different concentrations. Cationic lipoplex nanoparticles (~60 nm) formed with various loadings of Cy3 labeled short RNAs. Titration of biotinylated 80 nm gold nanoparticles labeled with streptavidin labelled with NorthernLights™ 557 dye Liposomes (~120 nm) loaded with Atto 550 incorporated at different concentrations. Cationic lipoplex nanoparticles (~60 nm) formed with various loadings of Cy3 labeled short RNAs. Titration of biotinylated 80 nm gold nanoparticles labeled with streptavidin labelled with NorthernLights™ 557 dye These model systems provide easily quantifiable approaches to determining number of fluorophores per particle and give results of 160, 35, and 20 fluorophores/particle respectively. Summary/Conclusion: We discuss these results in the context of exosome labeling experiments, providing the reader with important considerations and experimental design points. Funding These experiments were funded as regular work duties of the authors in developing new applications. Julia Luciano-Chadee Beckman Coulter Inc. Introduction: Research involving exosomes is rapidly expanding with a vast increase in the quality and quantity of publications. An improved and more efficient isolation protocol for exosomes is critical to advancing this exciting filed. Methods: Challenges to researchers working with exosomes include setting up density gradients by hand, because it is tedious, time consuming and subject to user, lab, and method analysis. At the same time, experts in the field have called for the establishment of standard protocols. This poster focuses on solutions to those challenges through cost-effective, large-scale purification and fast analysis of exosomes. Results: Specifically, the Beckman Coulter product portfolio helps to overcome human variables and cost while maintaining reliability, reproducibility, and high-throughput. Summary/Conclusion: Beckman Coulter is a complete exosomes workflow solution that combines methods of centrifugation, automation, flow cytometry, and particle analysis. Funding: Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners. Room: Harbour Ballroom   Networking Event 8:00 p.m. Scientific Program ISEV2017 Sunday, May 21, 2017 Experts Workshop Morning SessionsRoom: Metropolitan Ballroom West and Centre EW- Session-I Workshop on exRNA biology and the analytical methods organised by the ERCC, NIH   Moderator: Louise Laurent 7:45–8:45 a.m. Room: Metropolitan Ballroom East   EW- Session II EV-TRACK   Moderator: An Hendrix 7:45–8:45 a.m. Room: Harbour Ballroom   EW-Session III Demonstration workshop data analysis with FunRich   Moderator: Suresh Mathivanan 7:45–8:45 a.m. Room: Metropolitan Ballroom West and Centre   Session 28 – EVs in Cardiovascular Diseases and Vascular Disorders   Chairs: TBD and Jason Fish 9:00–10:00 a.m.LBO.24 to LBO.27 Room: Metropolitan Ballroom East   Session 29 - EVs in Immune System and Inflammation   Chairs: TBD and Eric Boilard 9:00–10:00 a.m.LBO.28 to LBO.31 Room: Harbour Ballroom   Session 30 - Novel Developments in EV Biogenesis and Characterization   Chairs: An Hendrix and Jeff Franklin 9:00–10:00 a.m.LBO.32 to LBO.35 Ke Cheng UNC-Chapel Hill & NC State University, NC, USA Introduction: Stem cell therapy faces a number of challenges. It is difficult to expand, store, and transport stem cells before they are administered to the patient. Synthetic analogs for stem cells and acellular approaches are promising to overcome these hurdles and hold the potential to revolutionize regenerative medicine. We sought to fabricate synthetic analogs of stem cells and test their therapeutic potential for treatment of acute myocardial infarction in mice. Methods: We packaged secreted factors from human stem cells into Poly(lactic-co-glycolic acid) PLGA microparticles and then coated them with stem cell membranes. We named these therapeutic particles “synthetic stem cells”. Results: Synthetic stem cells exhibited a factor release profile and surface antigens similar to those of genuine cells. They promoted cardiomyocyte functions and displayed cryopreservation and lyophilization stability in vitro and in vivo . In a mouse model of acute myocardial infarction, direct injection of synthetic stem cells promoted angiogenesis and mitigated left ventricle remodeling. Summary/Conclusion: The synthetic stem cell strategy may provide novel insight into tissue engineering for treating multiple diseases. In my talk I will cover the results recently published (Circ Res 2017; Nature Commun 2017) as well as some unpublished data regarding the vascular delivery and targeting aspect of microparticles and stem cells. Funding: NIH of USA. Anastasia Kyselova 1 , Ingrid Fleming 1 and Voahanginirina Randriamboavonjy 2 1 Institute for Vascular Signaling, Goethe University, Frankfurt, Germany; 2 Institute for Vascular Signaling, Goethe University, Frankfurt, Germany Introduction: The morbidity and mortality associated with diabetes is related to micro-and macro-vascular complications. The Ca 2+ -activated proteases or calpains have been implicated in the platelet hyperactivation associated with diabetes. Since calpains are known to be carried by platelet-derived microparticles (PMPs), the aim of the present study was to determine the effect of platelet-derived calpain on the vascular wall. Methods: Mass spectrometry and ELISA were used to analyse proteins in the culture medium from calpain-treated endothelial cells. Protein levels on the surface of endothelial cells were measured by FACS and en-face immunostaining was used to assess protein expression levels on intact aorta while Western-blot was used to investigate intracellular signaling. Results: In vitro treatment of endothelial cells with PMPs or recombinant calpain 1(CAPN1) led to a decrease in endothelial protein C receptor (EPCR) levels on the cell surface and an increase in its levels in the culture medium. EPCR levels were also increased in plasma from diabetic patients and correlated with plasma calpain activity. Diabetes induction in mice led to increased EPCR levels in the plasma which was prevented by the treatment of the mice with the calpain inhibitor A-705253. Mechanistically, calpain cleaved the protease-activated receptor 1 (PAR-1) on endothelial cells leading to the activation of an intracellular signaling i.e. protein kinase C and extracellular signaling-regulated kinase (ERK) phosphorylation causing on one hand the activation of the Tumor necrosis factor-α converting enzyme and EPCR shedding and on the other hand the increase in the expression levels of Intercellular cell adhesion molecule-1 and leukocytes adhesion as well as cell permeability. All of the calpain effects could be mimicked by PMPs from wild-type but not from CAPN1 −/- mice and were abolished in PAR-1 −/- endothelial cells. Summary/Conclusion: These data demonstrate that platelet-derived calpains contribute to diabetes-associated vascular inflammation by targeting the PAR-1 receptor and suggest calpain as a therapeutic target for the prevention of cardiovascular complication of diabetes. Funding: Deutsche Forschungsgemeinschaft-RA 2435/3-1. Avash Das 1 , Olivia Ziegler 2 , Shulin Lu 3 , John Tigges 3 , Vasilis Toxavidis 3 , Kirsty Danielson 4 , Saumya Das 2 and Ionita C. Ghiran 5 1 Massachusetts General Hospital, MA, USA; 2 Mass General Hospital, MA, USA; 3 Beth Israel Deaconess Medical Hospital, MA, USA; 4 University of Otago, Dunedin, New Zealand; 5 Beth Israel Deaconess Medical Center; Harvard Medical Hospital, MA, USA Introduction: Extracellular vesicles (EVs) function as novel mediators of intercellular communication. Here, we describe a fluorescence switch-based, experimental model to study EV-mediated communication between RBCs and the heart as well as other organs that permits characterization of cross-talk between RBCs and cardiomyocytes at homeostasis and after myocardial infarction. Methods: Mice with RBC-specific expression of Cre (Erythropoietin Receptor (EpoR) Cre) were crossed with reporter mTmG Rosa26 mice to yield EpoRCre/mTmG off-springs with membrane GFP expression in RBCs and RBC-derived EVs. Cultured dermal fibroblasts from mTmG mice and a mT/floxed/mGFP HEK 293 reporter cell line were used to assess transfer of functional Cre in RBC-derived EVs. To determine targets of RBC-EVs, organs from i) EpoRCre/mTmG (n=3), ii) mTmG (n=3) or iii) mTmG mice transfused with RBC-EVs from EpoR-Cre mice and targets of RBC-EVs (determined by mGFP expression due to Cre-recombination) were assessed by confocal microscopy. Finally, ischemia-reperfusion-infarction (30 min. LAD ligation) was done in EpoRCre/mGmT mice (n=3) and their blood and organs harvested after a span of 4 weeks to analyze changes in quality and quantity of RBC-EV targets following MI. Results: 1. RBC-EVs (mGFP positive) in plasma accounted for about 9% of total fluorescent EVs as detected by nano-flow cytometry and microscopy. 2. In vitro dermal fibroblasts from mTmG mice or mT/floxed/mGFP HEK 293 reporter cells showed mGFP expression with EpoRCre RBC-EVs, suggesting EV-mediated transfer of functional Cre. 3. Cre-mediated recombination was noted in diverse organs in EpoRCre/mTmG mice and mTmTG mice transfused with EpoRCre- EVs with the bone marrow, heart, lungs, kidney and spleen showing the largest degree of recombined cells. 4. Target profile of RBC-EVs demonstrates a distinct pattern of EV-mediated communication among the organs at baseline that was altered following MI, with increased recombination events in the brain, spleen and peri-infarcted heart. Summary/Conclusion: We show proof-of-concept for a novel model to study origin and targets of EV-mediated intercellular communication with significant EV-mediated communication between RBCs and cardiomyocytes under homeostatic conditions and following myocardial infarction. Funding: NIH (NHLBI R01 HL122547 to Saumya Das). Marta Adamiak 1 , Guangming Cheng 2 , Sylwia Bobis-Wozowicz 1 , Elżbieta Karnas 3 , Robert Vincent 2 , Michał Sarna 4 , Zbigniew Madeja 1 , Buddhadeb Dawn 2 and Ewa K. Zuba-Surma 1 1 Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 2 Division of Cardiovascular Diseases, Cardiovascular Research Institute, University of Kansas Medical Center, KS, USA; 3 Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; Malopolska Centre of Biotechnology, Krakow, Poland; 4 Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Introduction: Extracellular vesicles (EVs) from different kinds of stem cells have been effectively used to promote cardiac function under pathological conditions. One of the most fascinating characteristics of EVs is their ability to carry cell-type specific mRNAs, miRNAs and proteins. Thus, EVs derived from induced pluripotent stem cells (iPSCs) may provide an interesting alternative to harness the salutary effects of iPSCs and circumvent concerns associated with iPSC research. Here, we aimed to determine the effect of murine iPSC - derived EVs (miPSC-EVs) on the repair of ischemic myocardium in vivo . Methods: EVs were purified by sequential centrifugation of conditioned media collected from the serum- and feeder-free culture of miPSCs. EV morphology, molecular content as well as the influence of EVs on target cell function in vitro were carefully characterized. In the in vivo study, C57BL/6 mice underwent a 30-minute coronary occlusion followed by reperfusion, and 48 hours later, received intramyocardial injection of vehicle (PBS, n=14), miPSCs (n=15) or miPSC-EVs (n=13). Echocardiography was performed 4 days prior to coronary occlusion/reperfusion and at 48 hours and 30 days after injection. Mice were euthanized at 30 days post injection, heart tissue was harvested and histological analyses were performed. Results: Cardiac endothelial cells treated with miPSC-EVs exhibited greater angiogenic activity in vitro and were more resistant to apoptosis. At day 32 after coronary occlusion/reperfusion, mice injected with iPSC-EVs exhibited significantly improved left ventricular ejection fraction and end-systolic volume compared with vehicle-treated mice (P < 0.05). Although iPSC-injected hearts showed improved function and structure, several mice in this group grew teratomas at myocardial injection sites. Summary/Conclusion: Our data show that intramyocardial injection of miPSC-EVs after myocardial infarction/reperfusion produced similar improvement in cardiac structure and function compared with miPSCs. Thus, we conclude that miPSC-EVs may represent a safer therapeutic alternative to whole cell-based therapy for cardiovascular repair. Carole Parent 1 , Ritankar Majumdar 2 and Paul Kriebel 1 1 Lab Cellular & Molecular Biology, CCR, NCI, NIH; 2 Lab Cellular & Molecular Biology, CCR, NCI. NIH Introduction: The property of sensing and initiating directional migration in response to external cues or chemotaxis is a fundamental property of biological systems. How cells detect and respond to external chemotactic signals and, in particular, how the spatial and temporal relay of chemotactic signals between cells impact single and group cell migration are key questions in the chemotaxis field. Methods: Using the social amoebae Dictyostelium discoideum , where cAMP acts as a chemoattractant, we have shown that the relay of chemotactic signals between cells is mediated through the release of extracellular vesicles that contain the enzyme responsible for synthesizing cAMP, the adenylyl cyclase ACA. We purified the extracellular vesicles from chemotactic cells and showed that they are exosomal, contain and release cAMP and attract cells in an ACA-dependent fashion. Indeed, mass spectrometry analyses identified many canonical exosomal proteins as well as upstream regulators of ACA. We further show that cAMP is released through specific ABC transporters expressed in exosomes. Results: We extended our studies to neutrophils and show that LTB4, a key secondary chemoattractant in neutrophils, and its synthesizing enzymes localize to intracellular multi-vesicular bodies that, upon stimulation, release their content as exosomes. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We also investigated the mechanism by which LTB4 synthesizing enzymes, which are primarily localized on the nuclear envelope, are repackaged in exosomes and provide evidence that lipid homeostasis is involved in LTB4 vesicular packaging. Summary/Conclusion: We envision that the packaging of chemoattractants in exosomes provides a means of maintaining highly diffusible signals available for long-range cell-cell communication. We foresee that this newly uncovered mechanism is used by other signals to foster communication between cells in harsh extracellular environments. Funding: This research was supported by the Intramural Research Program of the Center for Cancer Research, NCI, NIH. Hyun Taek Park , Kim Oh Youn, Nhung Thi Hong. Dinh, Gyeongyun Go, Lee Changjin and Yong Song Gho POSTECH Introduction: Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria is spherical nano-scale membrane vesicles filled with periplasmic contents and currently shed new light on possibility of non-living complex vaccines or delivery vehicles. However, there was no attempt to treat cancer using OMVs. In this study, we investigated bacterial OMVs as a therapeutic agent for treating cancer. Methods: CT26 (murine colon adenocarcinoma) were grafted subcutaneously to wild type and interferon- γ knockout mice. Following intravenous administration of bacterial OMVs derived from Δ msbB E. coli to those mice, tumor volume was measured every 3 days. Tumor tissues obtained are subsequently examined. IVIS analyses are used to monitor targeting of OMVs to tumor tissue. Cytokine levels in blood and tumor tissue lysates are measured by ELISA. Results: Treatment of ∆msbB E. coli OMVs reduced tumor volume in dose-dependent manner and completely eradicated the tumor tissue when 5 μg of ∆msbB OMVs are injected (P < 0.001, versus no-treated mice; total n = 14 mice per group, two independent experiments). Interestingly, IVIS imaging analyses showed that OMVs were highly enriched in tumor tissue rather than spreading out the other organs. Cytokine analyses have revealed that IL-12p40, IFN-γ and CXCL10 cytokines increased in blood and tumor tissue upon OMVs treatment. In addition, OMV treatments of mice with IFN-γ deficiency failed to induce such anti-tumor effect (P < 0.001, versus wild type mice; n = 6 mice per group). Summary/Conclusion: We here demonstrated that administration of bacterial extracellular vesicles, especially Gram-negative bacterial OMVs, resulted in a remarkable anti-tumor effect without noticeable side effects. Moreover, we found that anti-tumor effect of OMVs is mediated by interferon-γ dependent manner since tumor in IFN-γ deficient mice were not affected by OMV treatment. Therefore, we here suggest that bacterial OMVs are promise immunotherapeutic agent to treat various cancers and these could bring a new insight in the development of novel immunotherapy in the future. Ole Østergaard 1 , Julia T. Tanassi 2 , Christoffer T. Nielsen 3 , Jesper V. Olsen 4 and Niels H. H. Heegaard 5 1 Department of Autoimmunology and Biomarkers, Statens Serum Institute + The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark; 2 Department of Autoimmunology and Biomarkers, Statens Serum Institut; 3 Copenhagen Lupus & Vasculitis Clinic, Centre for Rheumatology and Spine Diseases, Rigshospitale, Copenhagen, Denmark; 4 The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark; 5 Department of Autoimmunology and Biomarkers, Statens Serum Institute + Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Copenhagen, Denmark Introduction: The pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) may be linked to aberrant microparticle (MP) generation and removal leading to inflammatory signaling and subsequent autoimmune reactions and tissue damage. While the removal of normal MPs appears to work efficiently in SLE we previously found increased numbers of Annexin V (AnxV) non-binding MPs in the plasma of SLE patients compared to healthy controls (HC) and other autoimmune diseases. We here characterize the proteomes of MPs and AnxV non-binding MPs from SLE and HC to get insights to the origin and uniqueness of SLE associated MPs (luposomes). Methods: MPs were isolated from HC (n=4) and SLE (n=4) citrate plasma by repeated centrifugation at 18,890 x g. AnxV non-binding MPs were enriched by negative selection using AnxV-coupled microMACS beads. The total MPs and the AnxV non-binding MPs were then subjected to tryptic digestion and resulting peptides were analyzed by LC-MSMS to identify and quantitate proteins by label free quantitation using MaxQuant. MP groups were compared after normalization using β-actin levels and results were analysed further in Perseus and Excel. Results: In the total MPs samples 2,500-3,500 unique proteins were identified while the AnxV non-binding MPs yielded 1,700-3,000 proteins. Hierarchical clustering showed separation between HC and SLE MPs and between HC and SLE AnxV non-binding MPs. Overall, collagen-derived peptides were increased in the AnxV non-binding MPs. Specifically for the SLE AnxV non-binding MP there were increased amounts of complement factors, immunoglobulins, galectin-3 binding protein and other proteins. Conversely, platelet integrins and mitochondrial proteins were increased in total MPs compared to the AnxV non-binding MPs. Summary/Conclusion: Deep proteome profiling of MPs from SLE patients and HC confirm the presence of a sub-fraction of abnormal MPs in SLE. Furthermore, for the first time we here characterize the extremely different proteome profile of AnxV non-binding MPs from SLE patients that seems to be responsible for the differences in the total MP profiles that are unique for SLE. The characterization of these MPs (luposomes) is of importance for understanding the pathogenesis of SLE, for development of new diagnostic tools, and also provide avenues for the development of new therapeutic concepts. Craig P. Hasilo 1 , Sarita Negi 2 , Isabelle Allaeys 3 , Nathalie Cloutier 3 , Alissa K. Rutman 4 , Marco Gasparrini 2 , Eric Bonneil 5 , Pierre Thibault 5 , Eric Boilard 6 and Steven Paraskevas 2 1 McGill University, Montreal, Canada; 2 McGill University Health Centre, Research Institute of the MUHC, Montreal, Canada; 3 Centre de Recherche du CHU de Québec, CHUL; 4 Research Institute of the McGill University Health Centre, McGill University, Montreal, Canada; 5 Institute for Research, Immunology and Cancer, Université de Montreal, Canada; 6 Department of Microbiology-Infectious Disease and Immunity and Faculty of Medicine, Université Laval, Quebec, Canada Introduction: The role of extracellular vesicles (EV) in the etiology of type 1 diabetes is poorly understood. We set out to determine if human islets, isolated using clinical transplant protocols, produce EV in culture and if they contain diabetes autoantigens (DAA). Methods: Islet conditioned media (ICM) was collected from human islets (≥80% purity; cultured up to 72hrs) isolated from non-diabetic donors (n=10) under research consent. Nanoparticle tracking analysis (NTA) and flow cytometry (FC) were used to determine particle concentration and size distribution. ICM (n=10) were labeled with CellTracker Deep Red (CT), annexin V, DAA GAD65, IA-2, and ZnT8, βcell markers Glut2 and Hsc70, EV markers CD9 and CD14 on a FACS Canto II with small particle analyzer. Serial centrifugation at 50 000 g and 200 000 g generated ICM-50K and ICM-200K fractions, respectively. Double-labeled immunogold TEM was performed for AnnV plus GAD65, ZnT8 or Glut2. Proteomic analyses were performed by mass spectrometry on ICM-50K and ICM-200K (n=5 donors), and islet cell lysate (ICL) on a LTQ-Orbitrap Elite. Values obtained in lot-matched unconditioned media were subtracted from experimental values. Results: Varying levels of EV between donor ICM were detected with a consistent size distribution, and varying antigen levels. Proteomics of ICM-50K and ICM-200K revealed over 56% homology with ICL, but not with an endothelial cell line. Enrichment analyses identified proteins of exosome lineage, associated with diabetes pathways, and capable of eliciting an immune response. GAD65, ZnT8 and Glut2 presence on EV was confirmed with immunogold-EM. Summary/Conclusion: Morphologically diverse ICM EV, containing varying levels of DAA GAD65, ZnT8, and Glut2, were detected with a consistent size profile between non-diabetic donors. Proteomic evaluation reflected an islet-specific protein signature capable of promoting an immune response. These biomarkers may be strong targets for early diagnostic markers of β-cell injury and interventional strategies in diabetes and islet transplantation. Funding: Canadian National Transplant Research Program (CNTRP) and the McGill University Health Centre Foundation. C.P.H. is the recipient of the Astellas CNRTP Training Award, and E.B. is the recipient of a new investigator salary award from the Canadian Institutes of Health Research. Julia C. Gross 1 , Kerstin Menck 2 , Can Sönmezer 3 , Thomas Worst 4 , Matthias Schulz 3 , Gry Dihazi 3 , Frank Streit 3 , Gerrit Erdmann 5 , Simon Kling 6 , Michael Boutros 7 and Claudia Binder 3 1 University Medical Center Gottingen, Germany; 2 INSERM, U1068, Centre de Recherche et Cancérologie de Marseille, France; 3 University Medical Center Gottingen, Germany; 4 Mannheim Medical Center, University of Heidelberg, Germany; 5 NMI TT Pharmaservices; 6 NMI Natural and Medical Sciences Institute at the University of Tubingen, Germany; 7 German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, Heidelberg, Germany, Department of Cell and Molecular Biology, Faculty of Medicine Mannheim, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany; German Cancer Con Introduction: Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Methods: Using differential centrifugation EVs pelleting at 14,000 g (“microvesicles” (MV)) and 100,000 g (“exosomes”) are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. Results: How nSMases (also known as SMPD2 and SMPD3) affect the secretion of MVs is unknown. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically shifted onto MVs under these conditions. Summary/Conclusion: Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggests that - despite the different vesicle biogenesis - the routes of vesicular export are adaptable. Hyun Jeong Oh 1 , Seok Chung 2 , Do Won Hwang 1 and Dong Soo Lee 1 1 Department of Nuclear Medicine, Seoul National University, Seoul, Republic of Korea; 2 School of Mechanical Engineering, Korea University, Seoul, Republic of Korea Introduction: Exosomes are cell-derived vesicles that shuttle miRNAs involved in regulation of cellular process including cell proliferation and differentiation. Neurogenic microRNA (miRNA) such as miR-124 or miR-9 can be transferred and plays essential roles in differentiation of neural stem cells (NSCs) and neural progenitor cells (NPs) to neuronal cells. In this study, we proposed a mode of exosomal miRNA-mediated cell-non-autonomous neurogenesis and visualized the migration of exosomes to neighboring cells using a customized microfluidic assay. Methods: Real-time imaging of exosomal transport was performed using the GFP-tagged CD63 plasmid vector and GFP-tagged exosomes were monitored by confocal microscopy on microfluidic device. NE-4C, neural stem cells and F11, neural progenitor cells were used to examine exosomes. The pRV-effLuc/3xPT_miR-193a vector was used to detect miR-193a expression which luciferase signal could be turned off by binding of miR-193a to the triplicates of miRNA binding site in the 3’ UTR of effLuc. Results: The miR-193a was highly expressed in differentiated cells and exosomes secreted from those cells after neurogenesis. The miR-193a facilitated neurogenesis in neural progenitor cells and neural stem cells by blocking proliferation-related target genes. Luciferase activity of undifferentiated (UD) recipient F11 cells/effLuc/3×PT_miR-193a was decreased only co-culture with differentiated (D) donor cells after neurogenesis using a microfluidic device. Time-lapse live-cell imaging using microfluidics clearly visualized the convective transport of exosomes from D-donor to UD-recipient cells. Exosomes containing miR-193a from D-donor cells were taken up by UD-recipient cells and lead them to neurogenesis. Summary/Conclusion: In this study, we established exosome-tracing microfluidic platform to visualize convective exosomal transport from differentiated to undifferentiated cells and validated that exosomes and neurogenic miRNA within these exosomes propagate cell-non-autonomous neurogenesis to neighboring progenitors. Sriram Balusu 1 , Charysse Vandendriessche 1 , Caroline Van Cauwenberghe 1 , Marjana Brkic 1 , Bart De Strooper 2 , Claude Libert 1 and Roosmarijn Vandenbroucke 1 1 VIB-UGent; 2 VIB-KULeuven Introduction: Increasing evidence indicates that extracellular vesicles (EVs), including exosomes, play an important role in Alzheimer’s disease (AD) pathology. We recently reported that the choroid plexus epithelial cells, present at the interface between blood and cerebrospinal fluid (CSF), show increased EV secretion into the CSF upon peripheral inflammation. Moreover, these EVs were able to enter the brain parenchyma thereby spreading a pro-inflammatory message. Here, we studied the importance of choroid plexus-derived EVs in AD pathology. Methods: We made use of two mouse models of Alzheimer’s disease: transgenic APP/PS1 mice and intracerebroventricular (icv) injection of Aβ oligomers (AβO) in wild type mice. EVs were analyzed using NanoSight, electron microscopy and western blot analysis. Several immunostainings with EV markers were performed on brain sections. To assess cognition, we made use of the novel object recognition test. Results: Analysis of CSF of transgenic APP/PS1 mice revealed that early on in disease progression, there was an increase in amount of EVs compared to age-matched controls. In contrast, no difference in amount of EVs could be observed later on during disease progression. Interestingly, this correlated with an early increase in CSF Aβ. Next, we studied the effect of icv AβO injection and this revealed a significant increase in amount of EVs in CSF. Moreover, we observed that the choroid plexus epithelial cells are an important source of CSF EVs based on in vitro analysis of AβO stimulated primary choroid plexus cells and in vivo immunostainings and transmission electron microscopy analysis of choroid plexus tissue. Importantly, we could link the choroid plexus-mediated EV secretion with AβO-induced cognitive decline. Summary/Conclusion: In conclusion, our results show that AβO induces EV secretion by the choroid plexus and that these EVs play a role in disease spreading and loss of cognition. These data suggest that inhibition of EV production by the choroid plexus might be an interesting therapeutic approach to prevent or treat AD. Funding: SAO-FRA (Stichting Alzheimer Onderzoek), Research Foundation - Flanders (FWO) and MouseAge COST action. Siyang Zheng 1 , Yuan Wan 2 , Gong Cheng 2 , Xin Liu 3 , Si-Jie Hao 2 , Merisa Nisic 4 , Chuan-Dong Zhu 5 , Yi-Qiu Xia 2 , Wen-Qing Li 2 , Zhi-Gang Wang 2 , Wen-Long Zhang 2 , Shawn J. Rice 3 , Aswathy Sebastian 6 , Istvan Albert 7 and Chandra P. Belani 3 1 Department of Biomedical Engineering, Micro & Nano Integrated Biosystem (MINIBIO) Laboratory; Dept of Biochemistry and Molecular Biology, University Park, PA; Penn State Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA, USA; 2 Department of Biomedical Engineering, Micro & Nano Integrated Biosystem (MINIBIO) Laboratory, University Park, PA; Penn State Materials Research Institute, University Park, PA, USA; 3 Penn State Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA; Penn State Hershey Cancer Institute, The Pennsylvania State University, Hershey, PA, USA; 4 Department of Biomedical Engineering, Micro & Nano Integrated Biosystem (MINIBIO) Laboratory, University Park, PA; The Huck Institutes of the Life Sciences, University Park, PA, USA; 5 Department of Biomedical Engineering, Micro & Nano Integrated Biosystem (MINIBIO) Laboratory, University Park, PA, USA; The Second Hospital of Nanjing, Affiliated to Medical School of Southeast University, Nanjing, China; 6 Department of Biochemistry and Molecular Biology, University Park, PA, USA; 7 The Huck Institutes of the Life Sciences, University Park, PA; Department of Biochemistry and Molecular Biology, University Park, PA, USA Introduction: Extracellular vesicles (EVs) released by various cells are lipid bilayer-enclosed entities that can mediate intercellular communication by transferring cargo proteins and nucleic acids. Recently, the pathophysiological roles and clinical values for EVs are under intense investigation. However, most studies are limited by technical challenges in isolating EVs. Methods: We report a new method for EV isolation that uses a nanoprobe system for spontaneous membrane labeling and magnetic enrichment of EVs in 15 minutes. We use EVs derived from cancer cell line as a model system and compare the isolation performance between the nanoprobe system and ultracentrifugation. Furthermore, we isolate EVs from plasma samples of 19 non-small lung cancer (NSCLC) patients and perform DNA mutation detection. Results: A comparison study demonstrates the nanoprobe system offers comparable isolation efficiency, similar cargo composition compared with ultracentrifugation. In addition, it can be readily modified to meet the requirements for various EV and cargo analyses such as EV amount, morphology, cargo contents (DNA, RNA and protein). Further, aided with this system we isolated nEVs in blood plasma from NSCLC patients and successfully identified EGFR and KRAS mutations. Summary/Conclusion: The efficiency and versatility make this nanoprobe system a new method for EV isolation. Analyses based on this method are conducive to future cancer diagnostics.