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.

Extracellular vesicles, including exosomes, are naturally derived nanovesicles generated in and released by numerous cell types. As extracellular entities they have the capacity to interact with neighbouring cells and distant tissues and affect physiological processes as well as being implicated in numerous diseases including tumorigenesis and neurodegeneration. They are also under intense investigation as delivery vectors for biotherapeutics. The ways in which EVs interact with recipient cells to influence cell physiology and deliver a macromolecular payload are at the early stages of exploration. A significant challenge within these studies is the ability to label EVs directly or indirectly with fluorescent probes to allow visualization without compromising functionality. Here, we present a thiol-based fluorescence labelling method allowing comprehensive analysis of the cellular uptake of prostate cancer derived EVs in live cells using confocal microscopy. Labelling of the EVs in this way did not influence their size and had no effect on their ability to induce differentiation of lung fibroblasts to myofibroblasts. For endocytosis analyses, depletion of key endocytic proteins and the use of chemical inhibitors (Dynasore, EIPA, Rottlerin and IPA-3) indicated that fluid-phase endocytosis and/or macropinocytosis was involved in EV internalisation. Over a period of six hours EVs were observed to increasingly co-localise with lysosomes, indicating a possible termination point following internalisation. Overall this method provides new opportunities for analysing the cellular dynamics of EVs as biological entities affecting cell and whole body physiology as well as investigating their potential as drug delivery vectors.

Extracellular vesicles (EVs) mediate the cross-talk between cancer cells and the cells of the surrounding Tumour Microenvironment (TME). Professional killer cells include Natural Killer (NK) cells and CD8+ Cytotoxic T-lymphocytes (CTLs), which represent some of the most effective immune defense mechanisms against cancer cells. Recent evidence supports the role of EVs released by NK cells and CTLs in killing cancer cells, paving the road to a possible therapeutic role for such EVs. This review article provides the state-of-the-art knowledge on the role of NK-and CTLderived EVs as anticancer agents, focusing on the different functions of different subtypes of EVs. We also reviewed the current knowledge on the effects of cancer-derived EVs on NK cells and CTLs, identifying areas for future investigation in the emerging new field of EV-mediated immunotherapy of cancer.