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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-016-2298-3) contains supplementary material, which is available to authorized users.

Activin-A is a transforming growth factor-␤ (TGF-␤) superfamily member that plays a pivotal role in many developmental and reproductive processes. It is also involved in neuroprotection, apoptosis of tumor and some immune cells, wound healing, and cancer. Its role as an immuneregulating protein has not previously been described. Here we demonstrate for the first time that activin-A has potent autocrine effects on the capacity of human dendritic cells (DCs) to stimulate immune responses. Human monocyte-derived DCs IntroductionDendritic cells (DCs) form sentinel networks within the body sampling the microenvironment for pathogens, tissue injury, and inflammation via an array of pattern recognition receptors. [1][2][3] Pathogen encounter induces DC maturation, resulting in profound alterations in DC function. Antigen uptake is reduced, antigen processing is enhanced, and proinflammatory mediators are released. [4][5][6][7][8] The class, magnitude, and timing of cytokine or chemokine release are under exquisite control through both autocrine and paracrine signals as well as the signal strength and magnitude of the initiating stimulus. 8 DC cytokine and chemokine production can be induced by specific classes of stimuli. These include CD40 ligand (CD40L) and pathogen signals, such as toll-like receptor (TLR) agonists (eg, lipopolysaccharide [LPS] or intact bacteria). Appropriate release of cytokines, chemokines, and other soluble mediators by DCs and neighboring cells induces and moderates inflammation, recruits innate effectors, and regulates T-cell functions. [9][10][11][12] Many cytokines/chemokines produced by DCs at the epicenter of infection and inflammation, such as interleukin-6 (IL-6), 13,14 IL-8, 4,15,16 IL-10 17,18 as well as the potent T helper 1 (Th-1) cytokine, 19,20 have pleiotropic effects ranging from enhancing to inhibitory depending on the context and target cell type. However, uncontrolled cytokine/chemokine release within this microenvironment can also result in inappropriate T-and B-cell responses and subsequent immunopathology. [21][22][23] In this regard, the immune system has evolved to coordinately express mediators that attenuate exaggerated or inappropriate responses so as to minimize tissue damage and immunopathology (eg, prostaglandin E 2 (PGE 2 ), adenosine triphosphate (ATP), transforming growth factor-␤ [TGF-␤]). 24 Activin-A is a homodimer of activin-␤A subunits and was first described as a reproductive factor that accentuates the release of follicle-stimulating hormone. 25 It is a member of the TGF-␤ superfamily of cytokines and intimately shares with TGF-␤ the Smad intracellular signaling proteins. 26 The signaling, however, occurs through separate and distinct serine threonine kinase receptor subunits, and its release into the circulation during acute systemic inflammation differs from TGF-␤. 27 Activin-A signals through heteromeric receptor complexes consisting of both type I (ALK 2, 4, or 7) and type II (ActRIIA and ActRIIB) receptors. In addition, it is known to be pivotal in ...

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 p53 β 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. Electronic supplementary material The online version of this article (10.1007/s12035-018-1385-1) contains supplementary material, which is available to authorized users.