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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Exosomes, membrane vesicles of 40–100 nm in diameter, are derived from endosomes in various cells. The bioactive molecules specifically packed into exosomes can be horizontally transferred into recipient cells changing their biological properties, by which tumour cells continuously modify their surrounding microenvironment and distant target cells favouring cancer metastasis. It has been suspected for a long time that exosomes participate in the whole process of tumour metastasis. Although there is much unknown and many controversies in the role of cancer exosome, the major contribution of tumour-associated exosomes to different steps of cancer metastasis are demonstrated in this review. Mainly because these exosomes are easily accessible and capable of representing their parental cells, exosomes draw much attention as a promising biomarker for tumour screening, diagnosis and prognosis. Currently, researchers have found numerous biomarkers in exosomes with great potential to be utilized in personalized medicine. In this article, we summarize the roles of biomarkers, which are validated by clinical samples. Even though many conundrums remain, such as exosome extraction, large multicentre validation of biomarkers and data interpretation, exosomes are certain to be used in clinical practice in the near future as the field rapidly expands.
The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-β–bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.
Microvesicle (MV) release from tumour cells influences drug retention, contributing to cancer drug resistance. Strategically regulating MV release may increase drug retention within cancer cells and allow for lower doses of chemotherapeutic drugs. The contribution of exosomes to drug retention still remains unknown. Potential exosome and MV (EMV) biogenesis inhibitors, tested on human prostate cancer (PC3) cells for their capacity to inhibit EMV release, were also tested on PC3 and MCF-7 (breast cancer) cells for improving chemotherapy. Agents inhibiting EMV release most significantly, whilst maintaining cell viability, were chloramidine (Cl-amidine; 50 µM) and bisindolylmaleimide-I (10 µM). Apoptosis mediated by the chemotherapy drug 5-fluorouracil (5-FU) was significantly enhanced in PC3 cells in the presence of both these EMV inhibitors, resulting in a 62% (Cl-amidine + 5-FU) and 59% (bisindolylmaleimide-I + 5-FU) decrease in numbers of viable PC3 cells compared to 5-FU alone after 24 h. For MCF-7 cells, there were similar increased reductions of viable cells compared to 5-FU treatment alone ranging from 67% (Cl-amidine + 5-FU) to 58% (bisindolylmaleimide-I + 5-FU). Using combinatory treatment, the two EMV inhibitors further reduced the number of viable cancer cells tested. Neither inhibitor affected cell viability. Combining selected EMV inhibitors may pose as a novel strategy to enhance the efficacy of chemotherapeutic drug-mediated apoptosis.
IntroductionProtein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs) and has been linked to various cancers. Cellular microvesicle (MV) release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression.BackgroundWe have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am). We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment.MethodsProstate cancer cells (PC3) were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation.ConclusionWe report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3) to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX) treatment of prostate cancer cells increased the cytotoxic effect of MTX synergistically. Refined PAD inhibitors may form part of a novel combination therapy in cancer treatment.
Glioblastoma multiforme (GBM) is the most aggressive form of adult primary malignant brain tumour with poor prognosis. Extracellular vesicles (EVs) are a key-mediator through which GBM cells promote a pro-oncogenic microenvironment. Peptidylarginine deiminases (PADs), which catalyze the post-translational protein deimination of target proteins, are implicated in cancer, including via EV modulation. Pan-PAD inhibitor Cl-amidine affected EV release from GBM cells, and EV related microRNA cargo, with reduced pro-oncogenic microRNA21 and increased anti-oncogenic microRNA126, also in combinatory treatment with the chemotherapeutic agent temozolomide (TMZ). The GBM cell lines under study, LN18 and LN229, differed in PAD2, PAD3 and PAD4 isozyme expression. Various cytoskeletal, nuclear and mitochondrial proteins were identified to be deiminated in GBM, including prohibitin (PHB), a key protein in mitochondrial integrity and also involved in chemo-resistance. Post-translational deimination of PHB, and PHB protein levels, were reduced after 1 h treatment with pan-PAD inhibitor Cl-amidine in GBM cells. Histone H3 deimination was also reduced following Cl-amidine treatment. Multifaceted roles for PADs on EV-mediated pathways, as well as deimination of mitochondrial, nuclear and invadopodia related proteins, highlight PADs as novel targets for modulating GBM tumour communication.
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