Identification of Immunosuppressive Mechanisms in Glioblastoma Mulltiforme Brain Tumours

Abstract

Glioblastoma Multiforme (GBM) is the most common form of brain tumours with poor prognosis. The current treatment failure is a result of high heterogeneity and tumour-drivenimmunosuppression. GBM tumours may suppress T cell responses by elevating the effect of the infiltrating regulatory T cells or through secreting soluble proteins and extracellular vesicles (EVs) in the tumour microenvironment. However, the key receptor-ligand interactions responsible are not yet known. In this project, we hypothesised that GBM-derived soluble factors and EVs are immunosuppressive via the protein-protein interactions and regulate the CD4+ T cell function in the tumour microenvironment (TME). Mass Spectrometry-based Proteomic analysis was performed on tumour samples from GBM patients, after separation of the EVs from the soluble proteins, in order to scan the whole secretome. Optimisation protocols for sample preparation for Mass Spectrometry (MS) were developed using U251 glioblastoma cell-derived conditioned medium, in order to eliminate the model errors along the procedure steps. MS revealed the holistic protein profile in the GBM TME, and potential immunosuppressive candidates were identified following bioinformatics analysis. Furthermore, the data was stratified via molecular subtyping. Finally, the effects of EVs/soluble proteins and immunosuppressive candidates on CD4+ T cells were studied and the expression of cell surface markers and cytokine output was assessed. Proteomic analysis in GBM secretome, identified more than 1300 proteins in both EVs and soluble protein fractions. THBS1, ICAM1, CD47 were the proteins selected as EV-related mediators of immunosuppression. The samples’ grouping into molecular subtypes revealed overlapping between them due to the high heterogeneity of GBM. For the first time, potential protein biomarkers, linked to each molecular subtype, were identified here, such as RPLP2, ATP2B1 or C9. Extracellular vesicles and soluble proteins contained in the secretome of GBM tumour samples inhibited T cell proliferation and viability, when added in high doses. The CD4+ T cell production of IFNγ , IL-21 and TNFα was impaired, especially as a response to GBM soluble proteins.