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BACKGROUND: Glioblastoma multiforme (GBM) is the most common and fatal type of primary malignant brain cancer in adults. Tumour micro-environment immunosuppression is a hallmark of the disease and largely contributes to its recalcitrance to conventional cancer therapies. Current research to better understand GBM-mediated immunosuppression has focused on glioma cells as well as tumour associated microglia and macrophages, leaving pericytes - perivascular cells with demonstrated immune functions - largely unstudied. In a field of research plagued by poor translational validity of animal models, we studied immunologically relevant differences in patient-matched GBM and normal brain pericytes, focusing on proteins involved in immune system activation. MATERIAL AND METHODS: Patient-matched GBM pericytes and normal brain pericytes were isolated and cultured from surgically excised samples donated by consenting patients undergoing tumour resections at Auckland City Hospital. Following treatments with differential concentrations of pro-inflammatory cytokines, we studied the expression and secretion of adhesion molecules ICAM-1 and VCAM-1 and chemoattractant molecule MCP-1 through a variety of molecular studies. RESULTS: Our research reveals that patient and passage matched GBM and normal brain pericytes have different immune phenotypes. Specifically, GBM pericytes have an immunosuppressive propensity characterized by a curbed expression and secretion of ICAM-1, VCAM-1 and MCP-1 in response to the common pro-inflammatory trigger IL-1β. CONCLUSION: Our study is the first to characterise differences in the immune profiles of patient-matched normal and GBM primary human brain pericytes. Our results support the involvement of human pericytes in GBM tumour immune escape. A better understanding of tumour micro-environment immunosuppression in GBM would present immense potential in the development of new immunotherapeutic agents.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary brain tumour in adults. Despite the current clinical management of surgical resection, followed by radiation and chemotherapy, the median survival time is still a dismal 15 months. This may be due to treatment-resistant GBM stem cells (GSCs) that survive and migrate, resulting in tumour recurrence. Therefore, elucidating the mechanisms of GSC migration and studying ways of limiting this process will be beneficial in the treatment of GBM. A key molecule of interest is polysialylated neural cell adhesion molecule (PSA-NCAM). During normal development and adult neurogenesis, its presence on the cell surface reduces interactions with the extracellular matrix and aids in cellular migration. However, it has also been associated with malignant brain tumours and with negative patient prognosis. METHODS: We investigated PSA-NCAM’s role in tumour recurrence by immunohistologically evaluating over 160 surgically resected brain tumour specimens for PSA-NCAM expression and correlating this with patient outcomes. We also utilized GSCs isolated from the same patient specimens to investigate the role of PSA-NCAM in tumour cell migration. RESULTS: Univariate and Cox proportional hazard analysis showed that PSA-NCAM expression was a strong predictor of rapid tumour progression, even more so than the cell proliferation marker Ki67, which is often used to assess tumour grade. In addition, high-content image analysis revealed that PSA-NCAM was highly expressed by migrating patient-derived GSCs in vitro, and drugs that limited their migration also decreased PSA-NCAM expression. This led to investigations that aimed to elucidate the effects of PSA-NCAM removal on GSC migration. SUMMARY: Our work highlights the potential role of PSA-NCAM in GSC migration and tumour progression. This opens up avenues to pharmacologically and genetically manipulate PSA-NCAM to reduce GBM cell migration and curb tumour recurrence.

BACKGROUND Glioblastoma Multiforme (GBM) is the most aggressive, fatal, yet most common form of brain malignancy in adults. Despite advances in immune-based treatments for other modes of cancer, GBM remains a challenge due to its ability to dampen immune responses via mechanisms not yet fully understood. With a median survival time of only 15 months following diagnosis, there is a strong push to find new targets for therapy. The microenvironment comprises a mixture of malignant tumour cells, stroma, blood vessels and infiltrating inflammatory cells. Despite advances in understanding the contribution of these cells in establishing an anti-inflammatory microenvironment, the contribution of pericytes, an important neurovascular mural cell that forms the blood-brain barrier, has been inadequately studied. Therefore, we investigated the differences in immune profile between patient-matched non-neoplastic brain- and GBM-derived pericytes under basal and induced conditions. MATERIAL AND METHODS Primary patient-matched non-neoplastic brain and GBM tumour derived pericytes were isolated from specimens excised from consenting patients undergoing GBM surgical resection at Auckland City Hospital. Pericytes were treated with inflammatory cytokines including IL-1β, IFN-γ, TNFα and TGFβ for up to 24 hours. Inflammatory profile changes were probed for using fluorescent immunocytochemistry, qRT-PCR and spectral flow cytometry. Media was also collected for secretome analysis via cytometric bead array. RESULTS GBM pericytes show decreased expression of CX3CL1, both basally and following IL-1β treatment, via qRT-PCR and CBA. In contrast, increased gene expression and secretion of IL-6 and IL-8 by GBM pericytes were observed. GBM pericytes also basally express CD90 and anti-inflammatory molecule PD-L1 compared to their normal counterparts. In terms of activated pathways, basal SMAD2/3 activation is increased in GBM pericytes, while also showing greater activation following treatment with IL-1β, IFN-γ but not TNFα. C/EBPδ is activated and translocated following inflammatory stimulation; however, shows localised expression within the cytoplasm only observed in GBM pericytes. CONCLUSION This immunological screen of GBM pericytes highlights them as key players in the establishment of the tumour microenvironment. With data suggesting the activation of pathways such as the SMAD2/3 pathway in an unconventional manner, it suggests the potential for pericytes to manipulate pathways towards a more immunosuppressive outcome. Further immune characterisation of such cells is required to fully understand how they might contribute to the immunosuppressive nature of GBM.

Glioblastoma Multiforme (GBM) is the most aggressive, fatal, yet most common form of brain malignancy in adults. Despite advances in immune-based treatments for other modes of cancer, GBM remains a challenge due to its ability to dampen immune responses via mechanisms not yet fully understood. With a median survival time of only 15 months following diagnosis, there is a strong push to find new targets for therapy. An emerging aspect of GBM’s pathogenesis is the idea of the tumour’s immunosuppressive microenvironment which comprises a mixture of malignant tumour cells, stroma, blood vessels and infiltrating inflammatory cells. These cells can alter their phenotypes to express less pro-inflammatory and more anti-inflammatory mediators. Despite advances in understanding the contribution of these cells in establishing an anti-inflammatory microenvironment, the contribution of pericytes, an important neurovascular mural cell that forms the blood-brain barrier, has been inadequately studied. Therefore, we investigated the changes in immune responses between the non-neoplastic brain and GBM-derived pericytes isolated directly from the same patient’s tissues obtained during neurosurgery. Our results show that cellular responses to pro-inflammatory stimuli such as Interleukin 1 beta (IL-1ß) were dampened in GBM-derived pericytes when compared to pericytes isolated from non-neoplastic brain tissue from the same patient. More specifically, GBM-derived pericytes showed lower induction of pro-inflammatory molecules, Intracellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule (VCAM-1), and Monocyte Chemoattractant Protein 1 (MCP-1) when compared to normal brain pericytes. Due to the involvement of these molecules in immune cell recruitment and infiltration, this decreased response may contribute to the maintenance of GBM microenvironment immunosuppression. This highlights potential targets for alleviating such immunosuppression, which could enhance the success of immunotherapy-based treatments for GBM.