FAK-dependent regulation of TME vascular phenotype impacts on tumour hypoxia and immune infiltration in GBM

Abstract

Glioblastoma is the most common and aggressive malignant primary brain tumour in adults. GBM’s standard of care is largely ineffective and significant improvements in survival rates remain limited. Vascular abnormality and hypoxia are considered hallmarks of these highly angiogenic and immunologically “cold” tumours. Focal Adhesion Kinase (FAK) has key roles in proliferation, survival, and invasion in cancer. Its involvement in angiogenesis and immunosuppressive signalling has been widely acknowledged previously, yet its impact on GBM’s vascular phenotypes and the tumour microenvironment is not understood. In this study, we aim to investigate the role of FAK in modulation of tumour vasculature, and subsequent impact on hypoxia and immune infiltration in a murine glioma stem cell-like model.

METHODS: Tumours were induced via intra-cranial implantation of murine 005-glioma stem cell (GSC) line either expressing FAK (FAK-WT) or with genetic deletion of FAK (FAK-KO). Survival studies and end-point experiments analysed tumour progression. Ex vivo analysis from extracted tumours using immunohistochemical and immunofluorescent staining assessed the presence of endothelial cells, T cells and HIF1a-positive cells. In vitro 3D-culture of 005 cells measured impact of FAK-KO and pharmacological inhibition with AMP945 and VS4718 inhibitors in spheroid viability and proliferation as well as in expression of angiogenic and hypoxic-associated mouse genes.

RESULTS: FAK depletion in GSCs did not significantly affect survival or tumour growth at different in vivo timepoints. However, FAK-KO tumours exhibited lower vascular density, increased vessel radius and a trend towards greater immune infiltration compared to FAK-WT tumours. In one of the survival cohorts, a 2.5-fold increase in HIF1a-positive cells was observed in FAK-WT tumours. Correlations between vessels, immune cells, and HIF1a-positive cells, were analysed across tumour regions. In vitro, neither FAK-KO nor pharmacological inhibition significantly affected 005 proliferation, viability, or gene expression, although AMP945 treatment notably increased expression of angiogenic markers.

CONCLUSIONS: Previous research highlights the role FAK plays in different hallmark pathways of GBM. This study provides evidence of FAK as a regulator of key events happening in the tumour microenvironment involving angiogenesis, hypoxia, and immune infiltration. The mechanisms underlying the role of FAK in this context need to be further studied.