Faculty of Science, School of Molecular Bioscience
Abstract
Glioblastoma (GBM) is the most common, primary malignant brain tumours in adults. A better understanding of GBM biology is required to identify targets and develop new therapeutics. Tumour invasion is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich organelles that degrade the ECM and thereby actively re-modelling the surrounding tumour microenvironment. We have characterised the invasiveness of nine established GBM cell lines using an invadopodia assay and performed quantitative MS-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia to some degree, with U87MG the most and LN229 the least invasive cells. Overall, 1,141 proteins were identified and the abundance levels of 49 proteins correlated with invasiveness, many of which were previously linked to invadopodia formation, epithelial-mesenchymal transition and GBM cell invasion. Invadopodia act as multi-vesicular endosome docking sites and have been shown to be a site of exosome release. Overall, 844 proteins were identified in the extracellular vesicles (EVs) protein profiles, where approximately 50% were also identified in the membrane dataset. The abundance levels of 14 proteins correlated with the invasiveness. GBM-derived EVs can cross the blood brain barrier and are detectable in peripheral blood, the profiles and significant markers presented here could be of interest to diagnostics. Bioinformatics, Western blot analysis, co-localisation immunofluorescence and a siRNA knockdown substantiated some of interesting membrane and EV proteomics findings. In silico analysis of publically available gene databanks demonstrated the clinical prognostic significant of several invasion-related targets identified in these analyses. GBM invasion is regulated by a dynamic cross-talk between tumour cells and the brain microenvironment. After confirming the uptake of fluorescently labelled GBM-EVs, astrocytes were observed to assume a more invasive phenotype, using the same invadopodia assay. Whole cell proteome analysis of astrocyte before and after exposure to GBM EVs provides insight into the intercellular communication between GBM cells and surrounding normal astrocytes. This study highlights the signalling pathways that contribute to GBM invasion and may help to understand the aggressiveness of this disease
