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.

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Minimal information for studies of extracellular vesicles 2018 (MISEV2018) : a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Cancers develop in complex tissue environments, which they depend on for sustained growth, invasion and metastasis. Unlike tumor cells, stromal cell types within the tumor microenvironment (TME) are genetically stable and thus represent an attractive therapeutic target with reduced risk of resistance and tumor recurrence. However, specifically disrupting the pro-tumorigenic TME is a challenging undertaking, as the TME has diverse capacities to induce both beneficial and adverse consequences for tumorigenesis. Furthermore, many studies have shown that the microenvironment is capable of normalizing tumor cells, suggesting that re-education of stromal cells, rather than targeted ablation per se, may be an effective strategy for treating cancer. Here we discuss the paradoxical roles of the TME during specific stages of cancer progression and metastasis, as well as recent therapeutic attempts to re-educate stromal cells within the TME to have anti-tumorigenic effects.

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Microenvironmental regulation of tumor progression and metastasis.

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.

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Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

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Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles

In multicellular organisms, communication between cells mainly involves the secretion of proteins that then bind to receptors on neighbouring cells But another mode of intercellular communication - the release of membrane vesicles - has recently become the subject of increasing interest Membrane vesicles are complex structures composed of a lipid bilayer that contains transmembrane proteins and encloses soluble hydrophilic components derived from the cytosol of the donor cell These vesicles have been shown to affect the physiology of neighbouring recipient cells in various ways, from inducing intracellular signalling following binding to receptors to conferring new properties after the acquisition of new receptors, enzymes or even genetic material from the vesicles This Review focuses on the role of membrane vesicles, in particular exosomes, in the communication between immune cells, and between tumour and immune cells

Membrane vesicles as conveyors of immune responses

There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1–2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52–665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors

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A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

Production and characterization of clinical grade exosomes derived from dendritic cells.

The present invention relates to methods of preparing biological material, for use in various experimental, diagnostic or therapeutic uses, including immunotherapy treatment or prophylaxy of tumors. More particularly, the present invention relates to methods of preparing membrane vesicles (in particular exosomes) released by various types of mammalian cells, comprising diafiltration and/or density cushion centrifugation. The invention also provides novel methods for characterizing and analyzing exosome preparations, which can be used in quality control assay for the purpose of pharmaceutical product production. The invention is suitable to produce pharmaceutical grade preparations of such membrane vesicles and to fully characterize said preparations, for use in human beings.

Methods of producing membrane vesicles

The present invention relates to compositions comprising vesicles released' from activated T lymphocytes, as well as to methods for their production and uses. Said vesicles contain a set of bioactive molecules which confer remarkable properties, such as antigen recognition, antigen presentation and other regulatory and effector functions. This invention also relates to methods for transferring or delivering antigenic molecules (e.g., peptides, peptide/MHC complexes, TCR or subunit thereof, etc.) to antigen presenting cells (APCs) using said vesicles, to induce specific immune responses, particularly specific CTL responses. The invention further relates to methods of delivering molecules selectively or specifically to target cells using said vesicles.

Di-Hwei Hsu

Papers

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

Production and characterization of clinical grade exosomes derived from dendritic cells.

Methods of producing membrane vesicles

Functionalization of t cell derived vesicles and use thereof for the preparation of immunogenic pharmaceutical compositions