Comprehensive proteome profiling of glioblastoma-derived extracellular vesicles identifies markers for more aggressive disease

Abstract

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.

Extracellular Vesicles from Neurosurgical Aspirates Identifies Chaperonin Containing TCP1 Subunit 6A as a Potential Glioblastoma Biomarker with Prognostic Significance

“…We then compared proteomes between EVs isolated from cultured cells with those captured directly from the CUSA fluid and observed 329 proteins identified in both preparations, ≈63% of total proteins identified in CUSA‐cell EVs (Figure e). We observed a 79% (114) overlap between CUSA cell EV proteins and our previously reported in vitro GBM EV proteomic signature, which is composed of 145 proteins shared by EVs derived from six established GBM cell lines . Funrich annotated the biological processes associated with identified proteins that were, on the whole, similarly enriched in CUSA fluid and CUSA cell EV preparations (Figure f).…”

“…d) NTA measured modal EV population sizes of 85–100 nm. e) Venn diagram shows overlap between LC–MS/MS analysis of EVs isolated from expanded CUSA cells, directly from CUSA fluid and previously reported GBM EV signature proteins . f) Funrich annotations of proteins profiled in EVs isolated from CUSA fluid and cultured cells shows similarly enriched biological processes.…”

“…This necessitates enrichment steps, which are still in the process of being standardized, precluding comprehensive and collaborative analysis . We recently identified cavitron ultrasonic surgical aspirator (CUSA) fluid as a rich, clinical source of glioma‐derived EVs useful for biomarker discovery . CUSA is a surgical tool designed to fragment and aspirate solid tumors of the CNS .…”

“…To confirm that CUSA fluid is a suitable source of glioma‐derived EVs for biomarker discovery, we cultured cells isolated from a GBM CUSA washing in vitro and compared the proteome profiles of EVs released by these cells to those captured directly from CUSA fluid, as well as to our previously reported in vitro GBM EV proteome signature . Using an optimized density‐gradient ultracentrifugation EV enrichment protocol, we performed quantitative MS‐based proteomics on CUSA‐EVs captured from GBM and grade II–III glioma surgeries to identify biomarkers associated with more aggressive disease.…”

The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells

“…Molecular regulators of tumor invasion are often detectable in the cellular secretome (44), EVs (20), and cellular plasma membrane in GBM and other cancers (45). EVs also act as regulators of cellular motility and dissemination (16,46).…”