Cancer‐derived extracellular vesicles (EVs) are membrane‐enclosed structures of highly variable size. EVs contain a myriad of substances (proteins, lipid, RNA, DNA) that provide a reservoir of circulating molecules, thus offering a good source of biomarkers. We demonstrate here that large EVs (L‐EV) (large oncosomes) isolated from prostate cancer (PCa) cells and patient plasma are an EV population that is enriched in chromosomal DNA, including large fragments up to 2 million base pair long. While L‐EVs and small EVs (S‐EV) (exosomes) isolated from the same cells contained similar amounts of protein, the DNA was more abundant in L‐EVs, despite S‐EVs being more numerous. Consistent with in vitro observations, the abundance of DNA in L‐EV obtained from PCa patient plasma was variable but frequently high. Conversely, negligible amounts of DNA were present in the S‐EVs from the same patients. Controlled experimental conditions, with spike‐ins of L‐EVs and S‐EVs from cancer cells in human plasma from healthy subjects, showed that circulating DNA is almost exclusively enclosed in L‐EVs. Whole genome sequencing revealed that the DNA in L‐EVs reflects genetic aberrations of the cell of origin, including copy number variations of genes frequently altered in metastatic PCa (i.e. MYC, AKT1, PTK2, KLF10 and PTEN). These results demonstrate that L‐EV‐derived DNA reflects the genomic make‐up of the tumour of origin. They also support the conclusion that L‐EVs are the fraction of plasma EVs with DNA content that should be interrogated for tumour‐derived genomic alterations.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high‐throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture “what the community needs in a tool”. Here, we describe a novel community‐driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type–based diagnosis and antithrombotic intervention.

We have previously uncovered the impact of oncogenic and differentiation processes on extracellular vesicles (EVs) in cancer. This is of interested in the context of glioma stem cells (GSC) that are responsible for recurrent nature of glioblastoma multiforme (GBM), while retaining the potential to undergo differentiation and self renewal. GSCs reside in vascular niches where they interact with endothelial cells through a number of mediators including bioactive cargo of EVs. GSCs can be classified as proneural (PN) or mesenchymal (MES) subtypes on the basis of their gene expression profiles and distinct biological characteristics. In the present study we investigated how GSC diversity and differentiation programmes influence their EV‐mediated communication potentials. Indeed, molecular subtypes of GBMs and GSCs differ with respect to their expression of EV‐related genes (vesiculome) and GSCs with PN or MES phenotypes produce EVs with markedly different characteristics, marker profiles, proteomes and endothelial stimulating activities. For example, while EVs of PN GSC are largely devoid of exosomal markers their counterparts from MES GSCs express ample CD9, CD63 and CD81 tetraspanins. In both GSC subtypes serum‐induced differentiation results in profound, but distinct changes of cellular phenotypes including the enhanced EV production, reconfiguration of their proteomes and the related functional pathways. Notably, the EV uptake was a function of both subtype and differentiation state of donor cells. Thus, while, EVs produced by differentiated MES GSCs were internalized less efficiently than those from undifferentiated cells they exhibited an increased stimulatory potential for human brain endothelial cells. Such stimulating activity was also observed for EVs derived from differentiated PN GSCs, despite their even weaker uptake by endothelial cells. These findings suggest that the role of EVs as biological mediators and biomarkers in GBM may depend on the molecular subtype and functional state of donor cancer cells, including cancer stem cells.Abbreviations: CryoTEM: cryo‐transmission electron microscopy; DIFF: differentiated GSCs; EGF: epidermal growth factor; DUC: differential ultracentrifugation; EV: extracellular vesicle; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; GO: gene ontology; GSC: glioma stem cells; HBEC‐5i: human brain endothelial cells; MES: mesenchymal cells; MTS ‐ [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt; PMT1: proneural‐to‐mesenchyman transition cell line 1; PN: proneural cells; TEM: transmission electron microscopy; WB: western blotting

Non-coding RNA (ncRNA) species have emerged in as molecular fingerprints and regulators of brain tumor pathogenesis and progression. While changes in ncRNA levels have been traditionally regarded as cell intrinsic there is mounting evidence for their extracellular and paracrine function. One of the key mechanisms that enables ncRNA to exit from cells is their selective packaging into extracellular vesicles (EVs), and trafficking in the extracellular space and biofluids. Vesicular export processes reduce intracellular levels of specific ncRNA in EV donor cells while creating a pool of EV-associated ncRNA in the extracellular space and biofluids that enables their uptake by other recipient cells; both aspects have functional consequences. Cancer cells produce several EV subtypes (exosomes, ectosomes), which differ in their ncRNA composition, properties and function. Several RNA biotypes have been identified in the cargo of brain tumor EVs, of which microRNAs are the most studied, but other species (snRNA, YRNA, tRNA, and lncRNA) are often more abundant. Of particular interest is the link between transforming oncogenes and the biogenesis, cargo, uptake and function of tumor-derived EV, including EV content of oncogenic RNA. The ncRNA repertoire of EVs isolated from cerebrospinal fluid and serum is being developed as a liquid biopsy platform in brain tumors.