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.

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Extracellular vesicles (EVs) have emerged as pivotal mediators of communication in the tumour microenvironment. More specifically, nanosized extracellular vesicles termed exosomes have been shown to contribute to the establishment of a premetastatic niche. Here, we sought to determine what role exosomes play in medulloblastoma (MB) progression and elucidate the underlying mechanisms. Metastatic MB cells (D458 and CHLA-01R) were found to secrete markedly more exosomes compared to their nonmetastatic, primary counterparts (D425 and CHLA-01). In addition, metastatic cell-derived exosomes significantly enhanced the migration and invasiveness of primary MB cells in transwell migration assays. Protease microarray analysis identified that matrix metalloproteinase-2 (MMP-2) was enriched in metastatic cells, and zymography and flow cytometry assays of metastatic exosomes demonstrated higher levels of functionally active MMP-2 on their external surface. Stable genetic knockdown of MMP-2 or extracellular matrix metalloproteinase inducer (EMMPRIN) in metastatic MB cells resulted in the loss of this promigratory effect. Analysis of serial patient cerebrospinal fluid (CSF) samples showed an increase in MMP-2 activity in three out of four patients as the tumour progressed. This study demonstrates the importance of EMMPRIN and MMP-2-associated exosomes in creating a favourable environment to drive medulloblastoma metastasis via extracellular matrix signalling.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon‐induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS‐CoV‐2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS‐CoV‐2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS‐CoV‐2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS‐CoV‐2 and highlight the multiple distinct mechanisms by which SARS‐CoV‐2 subverts tetherin function.

For cancer cells to escape from the primary tumour and metastasize, they must degrade and navigate through the extracellular matrix (ECM). The transmembrane protease MT1‐matrix metalloprotease (MMP) plays a key role in localized matrix degradation, and its overexpression promotes cancer invasion. In this study, we demonstrate that MT1‐MMP is trafficked to the intraluminal vesicles of multivesicular endosomes, and subsequently released from cells on exosomes, a subtype of extracellular vesicle that can be retained to the surface of the originating cell by the anti‐viral restriction factor, tetherin. Although tetherin overexpression is linked to increased cell migration and invasion in various cancers, its role in these processes remains unclear. Our findings reveal that expression of tetherin by breast cancer cells promotes the retention of MT1‐MMP‐positive exosomes at their cell surface, while tetherin loss enhances exosome escape and impairs ECM degradation. Thus, tethered exosomes promote the retention of MT1‐MMP at the surface of cells, aiding the degradation of the ECM and promoting cancer cell invasion.

Three of the hallmarks of poor prognosis in paediatric ependymoma are drug resistance, local invasion and recurrence. We hypothesised that these hallmarks were due to the presence of a sub-population of cancer stem cells expressing the multi-drug efflux transporter ABCB1. ABCB1 gene expression was observed in 4 out of 5 paediatric ependymoma cell lines and increased in stem cell enriched neurospheres. Functional inhibition of ABCB1 using vardenafil or verapamil significantly (p ≤ 0.05–0.001) potentiated the response to three chemotherapeutic drugs (vincristine, etoposide and methotrexate). Both inhibitors were also able to significantly reduce migration (p ≤ 0.001) and invasion (p ≤ 0.001). We demonstrate that ABCB1 positive patients from an infant chemotherapy-led trial (CNS9204) had a shorter mean event free survival (EFS) (2.7 versus 8.6 years; p = 0.007 log-rank analysis) and overall survival (OS) (5.4 versus 12 years; p = 0.009 log-rank analysis). ABCB1 positivity also correlated with reduced event free survival in patients with incompletely resected tumours who received chemotherapy across CNS9204 and CNS9904 (a radiotherapy-led SIOP 1999-04 trial cohort; p = 0.03). ABCB1 is a predictive marker of chemotherapy response in ependymoma patients and vardenafil, currently used to treat paediatric pulmonary hypertension in children, could be repurposed to reduce chemoresistance, migration and invasion in paediatric ependymoma patients at non-toxic concentrations.