Extracellular vesicles (EVs) are lipid‐membrane enclosed nanoparticles that play significant roles in health and disease. EVs are abundant in body fluids and carry an array of molecules (proteins, lipids, nucleic acids and glycans) that reflect the identity and activity of their cell‐of‐origin. While the advent of high throughput omics technologies has allowed in‐depth characterisation of EV compositions, how these molecular species are spatially distributed within EV structures is not well appreciated. This is particularly true of the EV surface where a plethora of molecules are reported to be both integral and peripherally associated to the EV membrane. This coronal layer or ‘atmosphere’ that surrounds the EV membrane contributes to a large, highly interactive and dynamic surface area that is responsible for facilitating EV interactions with the extracellular environment. The EV coronal layer harbours surface molecules that reflect the identity of parent cells, which is likely a highly valuable property in the context of diagnostic liquid biopsies. In this review, we describe the current understanding of the mechanical, electrostatic and molecular properties of the EV surface that offer significant biomarker potential and contribute to a highly dynamic interactome.

Exosomes are nano-sized extracellular vesicles released by many cells that contain molecules characteristic of their cell of origin, including microRNA. Exosomes released by glioblastoma cross the blood–brain barrier into the peripheral circulation and carry molecular cargo distinct to that of “free-circulating” miRNA. In this pilot study, serum exosomal microRNAs were isolated from glioblastoma (n = 12) patients and analyzed using unbiased deep sequencing. Results were compared to sera from age- and gender-matched healthy controls and to grade II–III (n = 10) glioma patients. Significant differentially expressed microRNAs were identified, and the predictive power of individual and subsets of microRNAs were tested using univariate and multivariate analyses. Additional sera from glioblastoma patients (n = 4) and independent sets of healthy (n = 9) and non-glioma (n = 10) controls were used to further test the specificity and predictive power of this unique exosomal microRNA signature. Twenty-six microRNAs were differentially expressed in serum exosomes from glioblastoma patients relative to healthy controls. Random forest modeling and data partitioning selected seven miRNAs (miR-182-5p, miR-328-3p, miR-339-5p, miR-340-5p, miR-485-3p, miR-486-5p, and miR-543) as the most stable for classifying glioblastoma. Strikingly, within this model, six iterations of these miRNA classifiers could distinguish glioblastoma patients from controls with perfect accuracy. The seven miRNA panel was able to correctly classify all specimens in validation cohorts (n = 23). Also identified were 23 dysregulated miRNAs in IDH^MUT gliomas, a partially overlapping yet distinct signature of lower-grade glioma. Serum exosomal miRNA signatures can accurately diagnose glioblastoma preoperatively. miRNA signatures identified are distinct from previously reported “free-circulating” miRNA studies in GBM patients and appear to be superior.

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM) biology and represent novel sources of biomarkers that are detectable in the peripheral circulation. Despite this notionally non-invasive approach to assess GBM tumours in situ, a comprehensive GBM EV protein signature has not been described. Here, EVs secreted by six GBM cell lines were isolated and analysed by quantitative high-resolution mass spectrometry. Overall, 844 proteins were identified in the GBM EV proteome, of which 145 proteins were common to EVs secreted by all cell lines examined; included in the curated EV compendium (Vesiclepedia_559; http://microvesicles.org). Levels of 14 EV proteins significantly correlated with cell invasion (invadopodia production; r² > 0.5, p < 0.05), including several proteins that interact with molecules responsible for regulating invadopodia formation. Invadopodia, actin-rich membrane protrusions with proteolytic activity, are associated with more aggressive disease and are sites of EV release. Gene levels corresponding to invasion-related EV proteins showed that five genes (annexin A1, actin-related protein 3, integrin-β1, insulin-like growth factor 2 receptor and programmed cell death 6-interacting protein) were significantly higher in GBM tumours compared to normal brain in silico, with common functions relating to actin polymerisation and endosomal sorting. We also show that Cavitron Ultrasonic Surgical Aspirator (CUSA) washings are a novel source of brain tumour-derived EVs, demonstrated by particle tracking analysis, TEM and proteome profiling. Quantitative proteomics corroborated the high levels of proposed invasion-related proteins in EVs enriched from a GBM compared to low-grade astrocytoma tumour. Large-scale clinical follow-up of putative biomarkers, particularly the proposed survival marker annexin A1, is warranted.

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES⁺/CD133⁺) and their differentiated (diff) progeny cells (NES⁻/CD133⁻). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.

Glioblastoma, WHO‐grade IV glioma, carries a dismal prognosis owing to its infiltrative growth and limited treatment options. Glioblastoma‐derived extracellular vesicles (EVs; 30–1000 nm membranous particles) influence the microenvironment to mediate tumor aggressiveness and carry oncogenic cargo across the blood–brain barrier into the circulation. As such, EVs are biomarker reservoirs with enormous potential for assessing glioblastoma tumors in situ. Neurosurgical aspirates are rich sources of EVs, isolated directly from glioma microenvironments. EV proteomes enriched from glioblastoma ( n = 15) and glioma grade II–III ( n = 7) aspirates are compared and 298 differentially‐abundant proteins ( p‐ value < 0.00496) are identified using quantitative LC–MS/MS. Along with previously reported glioblastoma‐associated biomarkers, levels of all eight subunits of the key molecular chaperone, T‐complex protein 1 Ring complex (TRiC), are higher in glioblastoma‐EVs, including CCT2, CCT3, CCT5, CCT6A, CCT7, and TCP1 ( p  < 0.00496). Analogous increases in TRiC transcript levels and DNA copy numbers are detected in silico; CCT6A has the greatest induction of expression and amplification in glioblastoma and shows a negative association with survival ( p = 0.006). CCT6A is co‐localized with EGFR at 7p11.2, with a strong tendency for co‐amplification ( p  < 0.001). Immunohistochemistry corroborates the CCT6A proteomics measurements and indicated a potential link between EGFR and CCT6A tissue expression. Putative EV‐biomarkers described here should be further assessed in peripheral blood.

Glioblastoma multiforme (GBM) tumor invasion is facilitated by cell migration and degradation of the extracellular matrix. Invadopodia are actin-rich