The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell‐culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high‐quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

We propose a straightforward method to estimate the purity of vesicle preparations by comparing the ratio of nano-vesicle counts to protein concentration, using tools such as the increasingly available NanoSight platform and a colorimetric protein assay such as the BCA-assay. Such an approach is simple enough to apply to every vesicle preparation within a given laboratory, assisting researchers as a routine quality control step. Also, the approach may aid in comparing/standardising vesicle purity across diverse studies, and may be of particular importance in evaluating vesicular biomarkers. We herein propose some criteria to aid in the definition of pure vesicles.

There is a growing interest in the cell-cell communication roles in cancer mediated by secreted vesicles termed exosomes. In this study, we examined whether exosomes produced by cancer cells could transmit information to normal stromal fibroblasts and trigger a cellular response. We found that some cancer-derived exosomes could trigger elevated a-smooth muscle actin expression and other changes consistent with the process of fibroblast differentiation into myofibroblasts. We show that TGF-b is expressed at the exosome surface in association with the transmembrane proteoglycan betaglycan. Although existing in a latent state, this complex was fully functional in eliciting SMAD-dependent signaling. Inhibiting either signaling or betaglycan expression attenuated differentiation. While the kinetics and overall magnitude of the response were similar to that achieved with soluble TGF-b, we identified important qualitative differences unique to the exosomal route of TGF-b delivery, as exemplified by a significant elevation in fibroblast FGF2 production. This hitherto unknown trigger for instigating cellular differentiation in a distinctive manner has major implications for mechanisms underlying cancer-recruited stroma, fibrotic diseases, and wound-healing responses. Cancer Res; 70(23); 9621-30. Ó2010 AACR.

NKG2D is an activating receptor for NK, NKT, CD8+, and γδ+ T cells, whose aberrant loss in cancer is a key mechanism of immune evasion. Soluble NKG2D ligands and growth factors, such as TGFβ1 emanating from tumors, are mechanisms for down-regulating NKG2D expression. Cancers thereby impair the capacity of lymphocytes to recognize and destroy them. In this study, we show that exosomes derived from cancer cells express ligands for NKG2D and express TGFβ1, and we investigate the impact of such exosomes on CD8+ T and NK cell NKG2D expression and on NKG2D-dependent functions. Exosomes produced by various cancer cell lines in vitro, or isolated from pleural effusions of mesothelioma patients triggered down-regulation of surface NKG2D expression by NK cells and CD8+ T cells. This decrease was rapid, sustained, and resulted from direct interactions between exosomes and NK cells or CD8+ T cells. Other markers (CD4, CD8, CD56, CD16, CD94, or CD69) remained unchanged, indicating the selectivity and nonactivatory nature of the response. Exosomal NKG2D ligands were partially responsible for this effect, as down-modulation of NKG2D was slightly attenuated in the presence of MICA-specific Ab. In contrast, TGFβ1-neutralizing Ab strongly abrogated NKG2D down-modulation, suggesting exosomally expressed TGFβ as the principal mechanism. Lymphocyte effector function was impaired by pretreatment with tumor exosomes, as these cells exhibited poor NKG2D-dependent production of IFN-γ and poor NKG2D-dependent killing function. This hyporesponsiveness was evident even in the presence of IL-15, a strong inducer of NKG2D. Our data show that NKG2D is a likely physiological target for exosome-mediated immune evasion in cancer.

Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4 + T cells, CD8 + T cells, and NK cells revealed that CD8 + T-cell proliferation was not inhibited in the absence of CD4 + T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2-mediated CD25 up-regulation, affecting all but the CD3 + CD8 À T-cell subset. IL-2-induced Foxp3 expression by CD4 + CD25 + cells was not inhibited by tumor exosomes, and the suppressive function of CD4 + CD25 + T cells was enhanced by exosomes. In contrast, exosomes directly inhibited NK cell killing function in a T-cell-independent manner. Analysis of tumor exosomes revealed membrane-associated transforming growth factor B 1 (TGFB 1 ), which contributed to the antiproliferative effects, shown by using neutralizing TGFB 1 -specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated ''double hit'' to cellular immunity strongly implicates the role of exosomes in tumor immune evasion. [Cancer Res 2007;67(15):7458-66]

No abstract