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.

Extracellular vesicles (EV), including exosomes and microvesicles, are nano-sized intercellular communication vehicles that participate in a multitude of physiological processes. Due to their biological properties, they are also promising candidates for the systemic delivery of therapeutic compounds, such as cytokines, chemotherapeutic drugs, siRNAs and viral vectors. However, low EV production yield and rapid clearance of administered EV by liver macrophages limit their potential use as therapeutic vehicles. We have used a hollow-fiber bioreactor for the efficient production of bioactive EV bearing the heterodimeric cytokine complex Interleukin-15:Interleukin-15 receptor alpha. Bioreactor culture yielded ∼40-fold more EV per mL conditioned medium, as compared to conventional cell culture. Biophysical analysis and comparative proteomics suggested a more diverse population of EV in the bioreactor preparations, while serum protein contaminants were detectable only in conventional culture EV preparations. We also identified the Scavenger Receptor Class A family (SR-A) as a novel monocyte/macrophage uptake receptor for EV. In vivo blockade of SR-A with dextran sulfate dramatically decreased EV liver clearance in mice, while enhancing tumor accumulation. These findings facilitate development of EV therapeutic methods.

Extracellular vesicles (EVs), including exosomes and microvesicles, are 30–800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.

Myeloid-derived suppressor cells (MDSC) that block antitumor immunity are elevated in glioblastoma (GBM) patient blood and tumors. However, the distinct contributions of monocytic (mMDSC) versus granulocytic (gMDSC) subsets have yet to be determined. In mouse models of GBM, we observed that mMDSCs were enriched in the male tumors, whereas gMDSCs were elevated in the blood of females. Depletion of gMDSCs extended survival only in female mice. Using gene-expression signatures coupled with network medicine analysis, we demonstrated in preclinical models that mMDSCs could be targeted with antiproliferative agents in males, whereas gMDSC function could be inhibited by IL1β blockade in females. Analysis of patient data confirmed that proliferating mMDSCs were predominant in male tumors and that a high gMDSC/IL1β gene signature correlated with poor prognosis in female patients. These findings demonstrate that MDSC subsets differentially drive immune suppression in a sex-specific manner and can be leveraged for therapeutic intervention in GBM. Significance: Sexual dimorphism at the level of MDSC subset prevalence, localization, and gene-expression profile constitutes a therapeutic opportunity. Our results indicate that chemotherapy can be used to target mMDSCs in males, whereas IL1 pathway inhibitors can provide benefit to females via inhibition of gMDSCs. See related commentary by Gabrilovich et al., p. 1100. This article is highlighted in the In This Issue feature, p. 1079

The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high‐throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size‐exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP‐compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine‐fusion protein, heterodimeric IL‐15 (hetIL‐15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow‐fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL‐15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100‐fold reduction in ferritin concentration, a major protein‐complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL‐15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL‐15/lactadherin, which may be useful in targeted cancer immunotherapy approaches.

Hantaviruses comprise an emerging global threat for public health, affecting about 30,000 humans annually. Infection may lead to Hantavirus pulmonary syndrome (HPS) in the Americas and hemorrhagic fever with renal syndrome (HFRS) in the Europe and Asia. Humans are spillover hosts, acquiring infection primarily through the inhalation of aerosolized excreta from infected rodents and insectivores. Risk factors for infection include involvement in outdoor activities, such as rural- and forest-related activities, peridomestic rodent presence, exposure to potentially infected dust and outdoor military training; prolonged, intimate contact with infected individuals promotes transmission of Andes virus, the only Hantavirus known to be transmitted from human-to-human. The total number of Hantavirus case reports is generally on the rise, as is the number of affected countries. Knowledge of the geographical distribution, regional incidence and associated risk factors of the disease are crucial for clinicians to suspect and diagnose infected individuals early on. Climatic, ecological and environmental changes are related to fluctuations in rodent populations, and subsequently to human epidemics. Thus, prevention may be enhanced by host-reservoir control and human exposure prophylaxis interventions, which likely have led to a dramatic reduction of human cases in China over the past decades; vaccination may also play a role in the future.