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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The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis.

Glioblastomas shed large quantities of small, membrane-bound microvesicles (MVs) into the circulation. While these hold promise as potential biomarkers of therapeutic response, their identification and quantitation remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating MVs directly from blood samples of glioblastoma patients. MVs, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity, and can differentiate glioblastoma multiforme (GBM) MVs from non-tumor host cell-derived MVs. We also show that circulating GBM MVs can serve as a surrogate for primary tumor mutations and a predictive metric of treatment-induced changes. This platform could provide both an earlier indicator of drug efficacy and a potential molecular stratifier for human clinical trials.

Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reflecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal fluid.

Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumors is often not a clinical option. MGMT (O6-alkylguanine DNA alkyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult and when done, primarily relies on promoter methylation studies of tumor biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyze mRNA levels of MGMT and APNG in enriched tumor exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.