Surgery (RMH) - Theses
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ItemDeconstructing the brain tumour microenvironment using multimodal analysis( 2023-06)Gliomas are a type of astrocytoma and are the most prevalent type of primary brain cancer, with the most aggressive form being glioblastoma (GBM), with a median survival of only 15 months. Rapid tumour cell invasion and progression is a significant challenge for patients and their oncologists and neurosurgeons, reducing treatment efficacy and inevitably leading to tumour recurrence. Cancer cells thrive by responding and adapting to cellular and non-cellular cues in the tumour microenvironment, including the extracellular matrix (ECM). However, little is known about ECM composition in brain tumours and how the ECM evolves during disease progression, and the impact of the ECM on immune cell localisation, cancer cell signalling and the functional activity of tumour cells. The PI3K and MAPK signalling pathways are typically dysregulated in GBM, and can activate the downstream transcription factor, CREB, which has been reported to regulate GBM malignancy. By integrating multiplex immunohistochemistry, histopathological staining, and spatial tissue analysis, as well as in vitro 3D GBM models, I investigated ECM composition in low- and high-grade glioma, and the spatial relationship between neoplastic cells, immune cells and the ECM in GBM tissue. My results demonstrated a grade-dependent increase in ECM deposition and an upregulation of type I and type IV collagen mRNA expression, which is associated with poor survival in patients with GBM. GBM cells and vascular cells were identified as key contributors of ECM protein deposition in GBM. Spatial analysis demonstrated that T-cells were predominantly located in perivascular niches in ECM-rich regions, while macrophages exhibited more efficient infiltration into tumour cell-rich regions. Extensive tissue remodelling contributes to cellular compartmentalisation in the tumour microenvironment and this compartmentalisation correlates with PI3K, MAPK and CREB activity, and histopathological hallmarks, including angiogenesis, tumour cell density and cell invasion. Inhibiting the PI3K and MAPK signalling pathways reduced 3D cell invasion and also facilitated a shift in the ECM composition, from a more fibrotic to a less fibrotic state. Taken together, the results suggest that the accumulation of ECM plays an important role in GBM progression, affecting both immune cell distribution and cancer cell signalling. These findings suggest that targeting the PI3K and MAPK pathways to ‘normalise’ the ECM could serve to enhance the efficacy of existing and novel therapies for GBM.
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ItemDeveloping novel therapies using glioma stem cells( 2016)Glioblastoma multiforme (GBM) is a heterogeneous and malignant brain tumour with poor survival. Despite current best therapies most patients die within 15 months of diagnosis. These aggressive tumours are thought to recur after initial therapy due to their infiltrative nature. GBM recurrence and heterogeneity is also thought to be due to glioma stem cells (GSC). A panel of patient-derived cell lines (PDCL) isolated at The Royal Melbourne Hospital were previously subjected to in vitro analysis to confirm self-renewal and differentiation capacity. The stemness of the PDCLs was further evaluated with elevated mRNA expression of stem cell markers (nestin, SOX2, Oct4 and prominin-1) identified. The stem-like PDCLs were then tagged with luciferase recapitulated in vivo bioluminescent tumours within the brains of balb-c nude mice. The epidermal growth factor receptor (EGFR) pathway and its downstream phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signalling are often dysregulated in GBM and have been shown to be involved in cell proliferation, survival and migration. A selection of patient-derived glioma stem cells (GSC) were chosen for their mutant expression of EGFR, PI3K and PTEN proteins. The expression of the mutations was assessed as being stable over multiple passages in cell culture. The efficacy of PI3K inhibition was then assessed (in vitro and in vivo), but irrespective of the EGFR and/or PTEN status, standalone PI3K inhibition lacked cytotoxicity in GSC despite demonstrating target inhibition of phospho-Akt. Complex interactions within and between signalling pathways exist. Lysophosphatidic acid (LPA) signalling has been reported to interact with PI3K signalling and has been shown to stimulate migration and invasion. LPA signalling components have also been shown to be dysregulated in GBM and our GSC demonstrate a stable increase in mRNA expression of LPA receptor 1 (LPAR1). GSC expressing elevated levels of LPAR1 mRNA migrated in response to stimulation with LPA and this response was ameliorated using a selective LPA1/3 receptor antagonist (Ki16425). LPA appeared to stimulate migration in the GSC via a combination of PI3K and ROCK signalling. This interaction between LPA and PI3K signalling may help explain treatment resistance to PI3K inhibitors and future studies investigating the combined use of EGFR/PI3K and LPA inhibitors may be promising.