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      Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop

      research-article
      a , b , a , c , d , e , f , g , h , i , j , k , l , m , n , a , o , p , q , r , s , t , u , c , a , l , v , w , x , a , y , z , aa , bb , cc , dd , bb , ee , ff , gg , hh , ii , jj
      Journal of Extracellular Vesicles
      Taylor & Francis
      Extracellular vesicles, Exosomes, membranes, ISEV workshop, position paper, biogenesis, uptake, fusion, technology

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          ABSTRACT

          Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.

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          Most cited references82

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          Exosomal microRNA: a diagnostic marker for lung cancer.

          To date, there is no screening test for lung cancer shown to affect overall mortality. MicroRNAs (miRNAs) are a class of small noncoding RNA genes found to be abnormally expressed in several types of cancer, suggesting a role in the pathogenesis of human cancer. We evaluated the circulating levels of tumor exosomes, exosomal small RNA, and specific exosomal miRNAs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer (AJCC) disease stage to validate it as an acceptable marker for diagnosis and prognosis in patients with adenocarcinoma of the lung. To date, 27 patients with lung adenocarcinoma AJCC stages I-IV and 9 controls, all aged 21-80 years, were enrolled in the study. Small RNA was detected in the circulating exosomes. The mean exosome concentration was 2.85 mg/mL (95% CI, 1.94-3.76) for the lung adenocarcinoma group versus 0.77 mg/mL (95% CI, 0.68-0.86) for the control group (P < .001). The mean miRNA concentration was 158.6 ng/mL (95% CI, 145.7-171.5) for the lung adenocarcinoma group versus 68.1 ng/mL (95% CI, 57.2-78.9) for the control group (P < .001). Comparisons between peripheral circulation miRNA-derived exosomes and miRNA-derived tumors indicated that the miRNA signatures were not significantly different. The significant difference in total exosome and miRNA levels between lung cancer patients and controls, and the similarity between the circulating exosomal miRNA and the tumor-derived miRNA patterns, suggest that circulating exosomal miRNA might be useful as a screening test for lung adenocarcinoma. No correlation between the exosomal miRNA levels and the stage of disease can be made at this point.
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            High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

            Background Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. Methodology/Principal Findings We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. Conclusions/Significance We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.
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              Cellular internalization of exosomes occurs through phagocytosis.

              Exosomes play important roles in many physiological and pathological processes. However, the exosome-cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non-phagocytic cells. Most exosomes were observed attached to the plasma membrane of non-phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate-modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3-kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant-negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin-coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome-cell interactions and the exosome intracellular trafficking pathway.
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                Author and article information

                Journal
                J Extracell Vesicles
                J Extracell Vesicles
                ZJEV
                zjev20
                Journal of Extracellular Vesicles
                Taylor & Francis
                2001-3078
                2019
                8 November 2019
                : 8
                : 1
                : 1684862
                Affiliations
                [a ]Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine , Baltimore, MD, USA
                [b ]Department of Chemical and Biomolecular Engineering, Johns Hopkins University , Baltimore, MD, USA
                [c ]Department of Neurology, Johns Hopkins School of Medicine , Baltimore, MD, USA
                [d ]Department of Molecular and Translational Medicine, Università degli Studi di Brescia, CSGI and INSTM , Brescia, Italy
                [e ]Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology , Kolkata, India
                [f ]Cardiff University, School of Medicine , Cardiff, UK
                [g ]Division of Pharmaceutics and Pharmacology, College of Pharmacy , Columbus, OH, USA
                [h ]Comprehensive Cancer Center, The Ohio State University , Columbus, OH, USA
                [i ]University of Virginia , Charlottesville, VA, USA
                [j ]Exosomes laboratory and Metabolomics Platform, CIC bioGUNE, CIBERehd , Bizkaia, Spain
                [k ]IKERBASQUE, Basque Foundation for Science , Bizkaia, Spain
                [l ]Laboratory of Epidemiology and Population Science, National Institute on Aging, National Institutes of Health , Baltimore, MD, USA
                [m ]Department of Microbiology and Immunology, Loyola University Chicago , Chicago, IL, USA
                [n ]School of Biological and Healthy Sciences, Technological University Dublin , Dublin, Ireland
                [o ]Department of Clinical Laboratory Medicine, Nanfang Hospital, Southern Medical University , Guangzhou, Guangdong, China
                [p ]Fischell Department of Bioengineering, University of Maryland , College Park, MD, USA
                [q ]Department of Psychiatry and Behavioral Neurobiology, The University of Alabama at Birmingham School of Medicine , Birmingham, AL, USA
                [r ]INSERM U932, Institut Curie, PSL Research University , France
                [s ]Department of Pediatrics, University of California San Diego , La Jolla, CA, USA
                [t ]Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital , Oslo, Norway
                [u ]Program in Molecular and Integrative Physiological Sciences Departments of Environmental Health, Genetics & Complex Diseases Harvard T.H. Chan School of Public Health , Boston, MA, USA
                [v ]INBIRS Institute, UBA-CONICET School of Medicine University of Buenos Aires , Buenos Aires, Argentina
                [w ]CNR Institute of Neuroscience , Milan, Italy
                [x ]Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai , New York, NY, USA
                [y ]K. G. Jebsen - Thrombosis Research and Expertise Center (TREC), Department of Clinical Medicine, UiT - The Arctic University of Norway , Tromsø, Norway
                [z ]Department of Pathology, University of Washington School of Medicine , Seattle, WA, USA
                [aa ]Department of Chemistry, Shaw University , Raleigh, NC, USA
                [bb ]Institute for Psychiatry and Neuroscience of Paris, INSERM U1266, Hopital Saint-Anne, Université Descartes , Paris, France
                [cc ]VIB Center for Inflammation Research , Ghent, Belgium
                [dd ]Department of Biomedical Molecular Biology, Ghent University , Ghent, Belgium
                [ee ]Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC) CSIC-Universidad de Cantabria and Departamento de Fisiología y Farmacología, Universidad de Cantabria , Santander, Spain
                [ff ]Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University , Utrecht, The Netherlands
                [gg ]Rudolf Virchow Center, Julius-Maximilians-Universität Würzburg , Würzburg, Germany
                [hh ]School of Pharmaceutical Sciences, Tsinghua University-Peking University Joint Center for Life Sciences, Tsinghua University , Beijing, China
                [ii ]Department of Biological and Medical Sciences, Oxford Brookes University , Oxford, UK
                [jj ]Laboratory of Clinical Chemistry and Haematology & Department of Experimental Cardiology, University Medical Center Utrecht , Utrecht, The Netherlands
                Author notes
                CONTACT David Raul Francisco Carter dcarter@ 123456brookes.ac.uk Department of Biological and Medical Sciences, Oxford Brookes University , Gipsy Lane, Oxford, Oxon OX3 0BP, UK
                Pieter Vader pvader@ 123456umcutrecht.nl Laboratory of Clinical Chemistry and Haematology & Department of Experimental Cardiology, University Medical Center Utrecht , Heidelberglaan 100, Utrecht 3584 CX, The Netherlands
                [*]

                These authors contributed equally to this work.

                Author information
                http://orcid.org/0000-0003-1664-4233
                http://orcid.org/0000-0002-4652-2168
                http://orcid.org/0000-0002-0088-9845
                http://orcid.org/0000-0002-3827-5988
                http://orcid.org/0000-0002-1683-3838
                http://orcid.org/0000-0002-8327-620X
                http://orcid.org/0000-0001-9826-4132
                http://orcid.org/0000-0002-7059-8920
                Article
                1684862
                10.1080/20013078.2019.1684862
                6853251
                31762963
                a0bd2008-ae26-45b4-b5d3-a361e7537b25
                © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 03 June 2019
                : 23 August 2019
                : 04 October 2019
                Page count
                Figures: 15, Tables: 5, References: 172, Pages: 34
                Funding
                Funded by: US NIH 10.13039/100000002
                Award ID: R01 DA047807
                Funded by: US NIH 10.13039/100000002
                Award ID: R01 DA040385 and R56 AG057430
                Funded by: National Science Foundation Graduate Research Fellowship
                Award ID: 1746891
                Funded by: National Interuniversity Consortium of Materials Science and Technology INSTM and the Center for Colloid and Surface Science (CSGI)
                Funded by: Intramural Research Program of the National Institutes of Health, National Institute on Aging 10.13039/100000049
                Funded by: NIH 10.13039/100000002
                Award ID: (HL141611
                Award ID: GM130923); NSF (1750542)
                Funded by: NIH 10.13039/100000002
                Award ID: R01 MH113645
                Funded by: Fondation ARC 10.13039/501100004097
                Award ID: PJA20171206453
                Funded by: Cancéropôle Île-de-France (Emergence 2018) and French National Research Agency
                Award ID: ANR-10-IDEX-0001-02 PSL*
                Award ID: ANR-11-LABX-0043 and ANR-18-CE15-0008
                Funded by: The South-Eastern Norwegian Regional Health Authority, The Research Council of Norway, The Norwegian Cancer Society
                Funded by: NIH 10.13039/100000002
                Award ID: R01HL139496
                Award ID: R01ES029097
                Award ID: R01ES030302 and P30ES000002
                Funded by: Maryland Stem Cell Research Fund 10.13039/100012443
                Award ID: 2016‐MSCRFE‐2739
                Funded by: National Institutes of Health 10.13039/100000002
                Award ID: R56 AG‐057430
                Funded by: NIH/NHLBI
                Award ID: R01 HL124187
                Award ID: R01 HL140469
                Award ID: R01HL145644
                Funded by: Fondation pour la Recherche Medicale
                Award ID: contract AJE20160635884
                Funded by: Deutsche Forschungsgemeinschaft 10.13039/501100001659
                Award ID: WE5719/2-1
                Funded by: National Key R&D Program of China
                Award ID: 2017YFA0505200
                Funded by: the National Natural Science Foundation of China
                Award ID: 20151300651 and No.20181371453
                Funded by: Beijing Outstanding Young Scientist program
                Award ID: BJJWZYJH01201910003013
                Funded by: BBSRC 10.13039/501100000268
                Award ID: BB/P006205/1
                Funded by: Cancer Research UK 10.13039/501100000289
                Award ID: A28052
                KWW and AER are supported in part by US NIH R01 DA047807; KWW and YH were supported in part by US NIH R01 DA040385 and R56 AG057430, AS is supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1746891; PB is supported by the National Interuniversity Consortium of Materials Science and Technology INSTM and the Center for Colloid and Surface Science (CSGI) through evFOUNDRY (Horizon 2020- Future and emerging technologies (H2020-FETOPEN), ID: 801367); UE is supported by NIH 1K23HL126101-01A1; DWF and NNH are supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging; SMJ is supported by NIH (HL141611, GM130923); NSF (1750542); SK is supported by NIH R01 MH113645; GL is supported by Fondation ARC (PJA20171206453), Cancéropôle Île-de-France (Emergence 2018) and French National Research Agency (ANR-10-IDEX-0001-02 PSL*, ANR-11-LABX-0043 and ANR-18-CE15-0008); AML is supported by The South-Eastern Norwegian Regional Health Authority, The Research Council of Norway, The Norwegian Cancer Society; QL is supported in part by the US NIH R01HL139496, R01ES029097, R01ES030302 and P30ES000002. VM is supported in part by grants from Maryland Stem Cell Research Fund (2016‐MSCRFE‐2739) and National Institutes of Health (R56 AG‐057430); CM received support through NIH T32 OD011089. SS is supported by NIH/NHLBI: R01 HL124187, R01 HL140469, R01HL145644; NYSTEM: C32562GG; AHA:17GRNT33460554; Foundation Leducq: 17CVD04; GvN and FV are supported by Fondation pour la Recherche Medicale (contract AJE20160635884), to GvN, the Fondation ARC pour la Recherche sur le Cancer fellowship (PJA 20161204808) to FJV; AVV is supported by BIO2015-72124-EXP. RTI2018-095214-B-I00 MICINN Ministerio de Ciencia Innovación y Universidades. INNVAL17/22 University Hospital Marqués de Valdecilla-IDIVAL.; AMW is supported by Deutsche Forschungsgemeinschaft (DFG) grant WE5719/2-1; HY is supported by the National Key R&D Program of China Grand No. 2017YFA0505200, the National Natural Science Foundation of China Grand No.20151300651 and No.20181371453, Beijing Outstanding Young Scientist program Grand No. BJJWZYJH01201910003013; DRFC is supported by BBSRC (BB/P006205/1), Cancer Research UK (A28052).
                Categories
                Position Paper

                extracellular vesicles,exosomes,membranes,isev workshop,position paper,biogenesis,uptake,fusion,technology

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