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.
Highlights
The need for clinically useful biomarkers is becoming more apparent as the clinical management of glioblastoma (GBM) moves towards individualised therapy and adaptive trial designs
This is the first account of osteonectin (SPARC; Vesiclepedia ID, VP_6678) and laminin subunit alpha-4 (LAMA4, VP_3910) proteins in cancer Extracellular vesicles (EVs), corresponding mRNA species were documented in GBM EVs [3]
This is the first account of SPARC and LAMA4 proteins in EVs secreted by cancer cells, with previous observations restricted to normal saliva [22], bloods from healthy donors [23] or from patients with stable coronary artery disease [24], as well as cultured endothelial cells [25, 26] and embryonic stem cell-derived mesenchymal stem cells
Summary
The need for clinically useful biomarkers is becoming more apparent as the clinical management of glioblastoma (GBM) moves towards individualised therapy and adaptive trial designs. Extracellular vesicles (EVs) are stable, membrane-enclosed particles released from either the cell surface (microvesicles, 100–1000 nm) or from an endosomal route (exosomes, 40–100 nm). EVs are composed of an array of proteins, nucleic acids, lipids, and other metabolites that often reflect the cell of origin [1, 2], meaning they are excellent reservoirs of biomarkers. GBMderived EVs can cross the brain–blood-barrier and are. J Neurooncol (2017) 131:233–244 detectable in the peripheral circulation. Profiling the composition of GBM-derived EVs may, offer a noninvasive means of assessing tumours in situ, e.g., to identify molecular signatures indicative of tumour progression, recurrence and treatment failure. A ‘liquid biopsy’ would be especially valuable for patients with primary brain tumours, where radiological findings can be ambiguous, i.e., pseudoprogression and neurosurgery carries a very real risk of complication
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