The role of stromal cells in the establishment of Glioblastoma tumour microenvironment immunosuppression

Abstract

Glioblastoma (GBM) is the most aggressive, fatal, yet most common tumour to affect the central nervous system in adults. With a median survival time of only 15 months following diagnosis, it is a disease of high morbidity and mortality. Immunotherapies have shown great promise in the treatment of other malignancies but have failed to translate to the treatment of GBM. This is in part due to one of GBM’s key hallmarks - its profound immunosuppression. This is established through the immunosuppressive phenotypes and secretions of cells within the tumour microenvironment (TME). The TME comprises a mixture of malignant tumour cells, stroma, blood vessels and infiltrating immune cells. Most attempts to alleviate immunosuppression focus on the contribution of T cells, microglia, and tumour associated macrophages (TAMs). However, the potential for targeting other inflammation-modulating cell types, such as vascular stromal cells and pericytes, has not been thoroughly investigated in GBM. Using a patient-matched, primary human model, this thesis aimed to elucidate the inflammatory profile of GBM stromal cells compared to their non-neoplastic (NNP) stromal cell counterparts. Pericytes derived from patients undergoing surgery for epilepsy were also used for comparison. Firstly, the stromal cells were characterised by examining their marker expression both in situ and in vitro. Once in culture, their inflammatory responses were induced using a pro-inflammatory cytokine, IL-1β. Interestingly, the patient-matched GBM stromal cells responded with lower expression levels of adhesion molecules, ICAM-1 and VCAM-1, and chemokines such as CX3CL1, compared to their NNP counterparts. In contrast, GBM stromal cells also showed an increase in the expression of the cytokine, IL-6. To investigate the changes in signalling pathways resulting in these differing responses, key inflammatory mediators such as NFκB and TGFβ/SMAD were interrogated using activators and pharmacological inhibitors. Despite the lowered ICAM-1, VCAM-1, and CX3CL1 expression, the expression of pro-inflammatory transcription factor, NFκB, was significantly increased in GBM stromal cells compared to their NNP counterparts. Interestingly, aberrant anti-inflammatory SMAD2/3 signalling following IL-1β treatment was also observed in GBM stromal cells. Taken together, these results indicate that GBM stromal cells may be inherently immunosuppressive in nature and therefore contribute to the immunologically cold tumour microenvironment described in GBM. This may be due to the ability of GBM stromal cells to reprogram canonically inflammatory pathways into anti-inflammatory pro-tumourigenic states. This highlights stromal cells as potential targets for alleviating GBM immunosuppression and may enhance the chance of success of immune-based therapies for the treatment of GBM.