Extracellular Vesicles as Surrogates for Understanding and Monitoring Glioblastoma Progression

Abstract

Glioblastoma (GBM; WHO grade IV glioma), is the most common and lethal adult primary brain tumour. Lack of sensitive monitoring methods contributes to poor patient outcomes. Extracellular vesicles (EVs) are stable nanoparticles that carry molecules from their cell-of-origin and cross the blood-brain barrier into the circulation. EV release is upregulated in neoplasia, offering access to GBM molecular phenotypes by blood sampling. While blood-EVs are attractive for biomarker discovery, highly abundant serum proteins complicate proteomic studies. SWATH-MS allowed deep coverage of glioma plasma-EV proteins, with 4909 proteins identified using a custom spectral library; 463 proteins significantly changed across glioma cohorts and controls, including TRiC subunits. This work is the first to analyse glioma blood-EVs by SWATH-MS and shows promise for patient stratification. A rich clinical source of GBM-EVs was identified to substantiate blood-based studies. Neurosurgical aspirates, isolated directly from tumour microenvironments, are a novel and abundant source of tumour-EVs. EVs were enriched from the aspirates for protein and miRNA analyses. Comparison of EVs from GBM and glioma II-III surgeries identified 298 significant proteins, including all TRiC subunits, and 68 miRNAs, of which 21 were detected in a GBM serum-EV study. Profiling rich clinical EVs enhances our understanding of EV molecules released into the periphery and verifies GBM biomarkers identified in blood-EVs. GBM-EVs also play critical signaling roles and can influence non-neoplastic cells to support GBM invasion. Astrocytes internalise GBM-EVs and induce podosome formation. Bioinformatics of proteome changes in GBM-EV treated astrocytes predicted changes to MYC and TP53 signaling, confirmed by qPCR. Measured p53-isoform changes implicated a shift towards a pro-inflammatory, tumour-promoting senescence-associated secretory phenotype, shedding light on GBM-astrocyte interactions in assisting GBM progression.