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Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. Extracellular vesicles (EVs) play a role in intercellular communication, however the precise biogenesis of different populations of EVs are not clear. Here, the authors follow the intracellular trafficking of two proteins before their secretion in EVs and report the biogenesis and protein markers of EV subtypes: ectosomes budding from the plasma membrane as well as exosomes from late endosomes.

In the past decade, cell-to-cell communication mediated by exosomes has attracted growing attention from biomedical scientists and physicians, leading to several recent publications in top-tier journals. Exosomes are generally defined as secreted membrane vesicles, or extracellular vesicles (EVs), corresponding to the intraluminal vesicles of late endosomal compartments, which are secreted upon fusion of multi-vesicular endosomes with the cell's plasma membrane. Cells, however, were shown to release other types of EVs, for instance, by direct budding off their plasma membrane. Some of these EVs share with exosomes major biophysical and biochemical characteristics, such as size, density and membrane orientation, which impose difficulties in their efficient separation. Despite frequent claims in the literature, whether exosomes really display more important patho/physiological functions, or are endowed with higher potential as diagnostic or therapeutic tools than other EVs, is not yet convincingly demonstrated. In this opinion article, we describe reasons for this lack of precision knowledge in the current stage of the EV field, we review recently described approaches to overcome these caveats, and we propose ways to improve our knowledge on the respective functions of distinct EVs, which will be crucial for future development of well-designed EV-based clinical applications. This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.This Review describes the state of the art in imaging extracellular vesicles in animals to study their release, biodistribution and uptake, and covers labeling strategies, microscopy methods and discoveries made in model organisms.

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion. Extracellular vesicles mediate cell–cell communication, however, their capacity to deliver their content within acceptor cells is unclear. Here, the authors develop a quantitative assay and show that release of extracellular vesicle contents requires endosomal acidification and may involve membrane fusion.

The ability of exosomes to transfer cargo from donor to acceptor cells, thereby triggering phenotypic changes in the latter, has generated substantial interest in the scientific community. However, the extent to which exosomes differ from other extracellular vesicles in terms of their biogenesis and functions remains ill-defined. Here, we discuss the current knowledge on the specificities of exosomes and other types of extracellular vesicles, and their roles as important agents of cell-to-cell communication. In this Perspective, Théry and co-authors discuss our current understanding of the biogenesis, secretion and uptake of exosomes and extracellular vesicles.

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the \"ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)\", 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

[...]most survey participants are in favour of standards, with a majority of respondents supporting MISEV2014. Only minimal overlap between the \"volunteers\" and \"volunteered\" was observed. [...]in total, approximately 85 unique individuals were identified as possible contributors. Functional studies should include: a) Dose-response studies b) Process controls to rule out influence of serum components/ other possible contaminants c-i.) Density gradients to show activity is intrinsic to EVs, not just associated or c-ii.) EV depletion to remove activity or c-iii.) EV/cell labelling (e.g., fluorescent labelling, with careful interpretation) Text Box 2. Available form: https://www.ncbi.nlm.nih.gov/pubmed/26799652 Kenneth W. Witwer Departments of Molecular and Comparative Pathobiology and Neurology, The Johns Hopkins University School of Medicine, Baltimore, USA © kwitwer1@jhmi.edu ©http://orcid.org/0000-0003-1664-4233 Carolina Soekmadji Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia ©http://orcid.org/0000-0002-6920-6627 Andrew F. Hill Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia ©http://orcid.org/0000-0001-5581-2354 Marca H. Wauben Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Edit I. Buzás Department of Genetics, Cell- and Immunobiology, Semmelweis University and MTA-SE Immunoproteogenomics Extracellular Vesicle Research Group, Budapest, Hungary Dolores Di Vizio Division of Cancer Biology and Therapeutics, Departments of Surgery, Biomedical Sciences, and Pathology and Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Juan M. Falcon-Perez Exosomes Laboratory & Metabolomics Platform, CIC bioGUNE, CIBERehd, Derio, Spain IKERBASQUE Research Science Foundation, Bilbao, Spain Chris Gardiner Research Department of Haematology, University College London, London, UK Fred Hochberg Neurosurgery, University of California at San Diego, San Diego, CA, USA The Scintillon Institute, La Jolla, CA, USA Igor V. Kurochkin Central Research Laboratories, Sysmex Co, Kobe, Japan Jan Lötvall Codiak BioSciences, Cambridge, MA, USA Krefting Research Centre, University of Gothenburg, Gothenburg, Sweden ©http://orcid.org/0000-0001-9195-9249 Suresh Mathivanan Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia Rienk Nieuwland Department of Clinical Chemistry and Vesicle Observation Centre, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands Susmita Sahoo Department of Medicine, Cardiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Hidetoshi Tahara Department of Cellular and Molecular Biology, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan Ana Claudia Torrecilhas Departamento de Ciencias Farmacéuticas, UNIFESP, Diadema, Brazil Alissa M. Weaver Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA ©http://orcid.org/0000-0002-4096-8636 Hang Yin Center of Basic Molecular Science, Department of Chemistry, Tsinghua University, Beijing, China Department of Chemistry and Biochemistry and the BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, USA Lei Zheng Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China Yong Song Gho Department of Life Sciences, POSTECH (Pohang University of Science and Technology), Pohang, South Korea Peter Quesenberry Brown Medical School, Rhode Island Hospital, Providence, RI, USA Clotilde Théry Institut Curie, INSERM U932, PSL Research University, Paris, France © clotilde.thery@curie.fr Received 29 September 2017; accepted 20 October 2017

To communicate, cells are known to release in their environment proteins which bind to receptors on surrounding cells. But cells also secrete more complex structures, called membrane vesicles, composed of a lipid bilayer with inserted transmembrane proteins, enclosing an internal content of hydrophilic components. Exosomes represent a specific subclass of such secreted membrane vesicles, which, despite having been described more than 20 years ago by two groups studying reticulocyte maturation, have only recently received attention from the scientific community. This renewed interest originated first from the description of exosome secretion by antigen-presenting cells, suggesting a potential role in immune responses, and very recently by the identification of the presence of RNA (both messenger and microRNA) in exosomes, suggesting a potential transfer of genetic information between cells. In this review, we will describe the conclusions of 20 years of studies on the immune properties of exosomes and the most recent advances on their roles and potential uses as markers or as therapeutic tools during pathologies, especially in cancer.