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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ItemThe role of IL-11 signaling in glioblastoma progression( 2022)Glioblastoma is the most common and lethal brain tumour in adults with a mean survival rate of only 12-15 months with current treatment. The microenvironment of a tumour is becoming increasingly more important to current research, with many findings suggesting that the transcription factors driving oncogenic processes are more often due to cytokine stimulation than gene mutation. There have been multiple signalling molecules and corresponding receptors identified as key role-players in the development of glioblastoma, its severity and ability to evade treatment. Cytokines are molecules that initiate and mediate a range of cellular activities essential to the homeostasis of a heathy person but also to tumour growth, invasion and survival. This includes the critical growth factors and cytokines that activate signalling pathways controlling many pro-oncogenic cellular functions. The interleukin-11 (IL-11) cytokine has become increasingly recognised as a driver of the pathogenesis of a wide range of cancers, however, very little is known regarding its role in glioblastoma. Considering this, we hypothesized that IL-11 would contribute to glioblastoma cell viability, migration, invasion and overall tumour progression. We initially identified that IL-11 and its receptor (IL-11RA) inversely correlate with tumour grade and glioblastoma survival. To study the role of IL-11 in glioblastoma, we next determined the expression of endogenous IL-11RA in a range of cell lines and transfected those expressing very little of the gene with the IL-11RA (cell lines #20 and #28). Proteomic analysis was conducted to reveal changes in protein expression after transfection. A large number of proteins involved in proliferation, migration and invasion were seen to be upregulated in the IL-11RA transfected cells. Indeed, the IL-11RA transfected cells displayed significantly greater growth, migration and invasion in proliferation, wound healing, transwell and spheroid invasion assays. This was reversed with IL-11RA knockdown. The proteomic analysis also highlighted the upregulation of proteins involved in metabolism, particularly glutaminolysis and inhibition of apoptosis. Metabolomic analysis revealed the IL-11RA transfected cells displayed increased levels of glutamine oxidation, as well as increased proliferation and survival of these cells in conditions of depleted glucose or glutamine. Similarly, IL-11RA transfected cells displayed no significant difference in invasion rate in the presence or absence of glucose, when glutamine was available. Alternatively, blocking both glucose and glutamine metabolism with a number of drugs significantly reduced the proliferation, migration and invasion of these cells. Our findings suggest that the IL-11RA transfected cells are able to utilise alternative metabolites such as glutamine, in the absence of glucose, in order to proliferate, migrate and survive. Overall, the results of this thesis suggest that the IL-11RA plays an important role in proliferation, migration, invasion, survival and metabolism in glioblastoma.
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ItemEvaluating the role of invadopodia in glioma invasion and response to therapeutics( 2021)Glioblastoma (GBM) is the most prevalent and aggressive form of glioma, and is associated with an extremely poor prognosis, with a low median patient survival time of just 15 months post-diagnosis with the current therapeutic approach known as the Stupp protocol, consisting of surgical resection, followed by radiotherapy (RT) and concomitant chemotherapy with temozolomide (TMZ). A significant contributing factor that impacts the survival of GBM patients is the highly infiltrative nature of GBM cells, which prevents complete tumour resection and also limits the capacity of targeted therapies to effectively reach the infiltrating tumour cells. Consequently, these tumours can exhibit high rates of recurrence, appearing within months following the completion of the first round of treatment and can also demonstrate minimal response to further rounds of RT/TMZ treatment. Evidence suggests that the efficacy of current therapeutic approach may be compromised by an enhanced invasive phenotype that is displayed by the GBM cells that survive the current treatment protocol (Wild-Bode, Weller et al. 2001, Cordes, Hansmeier et al. 2003, Hegedus, Zach et al. 2004, Trog, Fountoulakis et al. 2006, Trog, Yeghiazaryan et al. 2006, Steinle, Palme et al. 2011). The targeting of the enhanced invasive abilities exhibited by RT/TMZ treated GBM cells could provide a potential therapeutic approach for improving patient outcome, however the mechanisms utilised by invasive GBM cells following the current treatment are not well understood. As GBM cells have been shown to form actin-rich membrane protrusions known as invadopodia that can facilitate invasion by degrading the surrounding ECM via highly localised proteolytic activity (Stylli, Kaye et al. 2008, Mao, Whitehead et al. 2017, Petropoulos, Guichet et al. 2018), it is possible that the enhanced invasive capabilities of GBM cells post- RT/TMZ treatment may be mediated by invadopodia. In this thesis, the role of invadopodia in GBM cell invasion and response to RT/TMZ treatment was investigated. Using clinically relevant doses of RT and TMZ, it was demonstrated that the enhanced invasive capabilities of GBM cells post-RT/TMZ treatment may be attributed to an increase in invadopodia formation and activity. The role of intracellular communication between GBM cells via small extracellular vesicles (sEVs) was also investigated, highlighting the ability of GBM cell line secreted sEVs to transfer a pro-invadopodia phenotype to recipient GBM cells, as well as their potential to facilitate an enhanced pro-invadopodia phenotype following RT/TMZ treatment. Demonstrating the potential to dualistically target invadopodia activity and sEV secretion to overcome RT/TMZ-induced GBM invasion, the addition of the microtubule-targeting agent Vinorelbine Tartrate (VT) alongside RT/TMZ reduced the enhanced secretion of sEVs, in accordance with previous data from our laboratory showing VT also reduces invadopodia activity in GBM cells surviving RT/TMZ (Whitehead, Nguyen et al. 2018). Lastly, GBM cell lines and their corresponding secreted sEVs were subjected to comprehensive proteomic profiling to identify proteins that may facilitate invadopodia formation and activity following exposure to RT/TMZ treatment, thereby contributing to enhanced GBM invasion. Collectively, this work highlights the contributing role of invadopodia and sEVs to the pro-invasive abilities of GBM cells, and provides insight into the dysregulated proteomic landscape of GBM cells and sEVs following exposure to RT/TMZ treatment that may contribute to enhanced invasive capacity, which may ultimately assist in the development of novel adjuvant therapeutic strategies to improve the clinical efficacy of RT and TMZ treatment.
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ItemGlioblastoma: Treatment Stagnation and Cellular and Molecular Mechanisms( 2020)Glioblastoma, a WHO grade IV primary brain tumour, remains as one of the most aggressive forms of human cancer. Despite intensive research efforts into understanding the key drivers of tumour progression, few therapeutic advances have been made, with the current standard of care (the Stupp protocol) remaining unchanged for 15 years. The overall improvement to glioblastoma survival in the real-world population has been attributed to the use of the Stupp protocol, yet evidence suggests that survival outcomes were already significantly improving in the years prior to the introduction of this standard of care questioning the overall veracity of this claim. Using the Surveillance, Epidemiology and End Results (SEER) registry data we analysed the survival outcomes for real-world glioblastoma patients diagnosed from 2000 – 2016. Our findings show a consistent incremental survival improvement that preceded the introduction of the Stupp protocol and continued to increase at the same rate till 2009, stagnating afterwards. Significantly, however, this survival improvement is short-term for patients, with no survival improvement observed in patients surviving more than 2 years. Additionally, with the exception of complete tumour resection, all treatment modalities did not improve survival beyond 2 years for glioblastoma. These findings highlight the clinical stagnation of glioblastoma treatment and highlight the inability of current treatments to target the underlying causes of tumour progression. Following the introduction of the Stupp protocol attempts to develop new treatment options have universally been disappointing with a close to 0% success rate for over 1000 phase II and above clinical trials. Conflictingly, many of the therapeutic agents tested have shown promising results in preclinical trials. Current preclinical models, however, test therapies against the primary tumour, which does not recapitulate the biology or targets of tumour recurrence. We therefore developed a highly sensitive luciferase-based glioblastoma mouse model capable of single cell detection in mouse tissue. Analysis of mouse brain tissue implanted with luciferase-labelled human glioblastoma U87MG or MU20 tumours revealed the presence of tumour cells ubiquitously spread across the supratentorial regions of the brain, and distally located from the primary tumour. These tumour cells were observed as single cells in U87MG implanted mice and clusters in MU20 glioblastoma cells. Remarkably, U87MG tumours did not exhibit invasive margins and were contained within an expansive growth phenotype, suggesting invasion-independent dissemination. Our model is consistent with reports of glioblastoma as a systemic brain disease and is capable of sensitive detection of disseminated tumour cells, a model of recurrence potential. Furthermore, this model can be utilised to investigate new mechanisms of glioblastoma infiltration. Targeting aberrant angiogenesis in glioblastoma has been the major focus for glioblastoma treatment since the Stupp protocol. Yet after over a decade of basic research and clinical trials, antiangiogenic inhibitors have failed to translate into improved patient outcome. The discovery of abnormalities in the tumour vasculature suggest that there may be alternate mechanisms driving tumour progression. Vasculogenic mimicry has been observed in glioblastoma and presents as a novel aspect of tumour biology, yet the mechanisms and functional relevance of these structures remain unknown. Our study has confirmed the ability of some glioblastoma cell lines to undergo endothelialisation, forming lattice structures similar to endothelial cells when seeded onto Matrigel in vitro. One lattice forming cell line, U87MG, was also found incorporate into the tumour vasculature in an in vivo orthotopic mouse model. This behaviour was found to be regulated by an expanded TGF-beta-ALK1-Smad1/5 signalling pathway. In vivo inhibition of the Smad1/5 signalling pathway via intracranial treatment with Ad-Smad6 resulted in reduced endothelialisation in the tumour vasculature and inhibited whole brain infiltration in the U87MG mouse model. Since U87MG xenograft tumours are non-invasive, these results suggest that endothelialisation may lead to haematogenous dissemination and distal brain infiltration, providing a novel mechanism for glioblastoma progression.
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ItemThe role of receptor tyrosine kinases in mediating glioblastoma resistance to radiotherapy and temozolomide( 2020)Glioblastoma is the most common and aggressive form of malignant glioma. Currently, despite treatment with surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ), mean patient survival time is approximately 12 months and the 5-year survival rate is close to 0%. A key factor for the dismal prognosis is tumour recurrence post-treatment which is largely due to: 1) the infiltrative nature of glioblastoma rendering complete resection impossible and 2) glioblastoma cell resistance to radio-chemotherapy. In this thesis we aimed to investigate the cellular mechanisms of receptor tyrosine kinases in conferring resistance to therapy. We first performed a literature search and found that almost all studies that advocated for the utility of targeting RTKs in overcoming treatment resistance did not employ both therapeutic agents comprising standard therapy – radiotherapy and TMZ. We next generated an in vitro glioblastoma resistant model via short-term treatment with radiotherapy and TMZ and found that these cells had down-regulated RTK activity in addition to down-regulated protein and gene expression of the commonly altered and studied epidermal growth factor receptor (EGFR) and MET receptor. After generating an in vitro glioblastoma recurrent model via long-term treatment we demonstrated that the surviving sub-population of cells also displayed down-regulated EGFR and MET expression compared to treatment naive cells. Furthermore, we also showed that the resistant cell population already pre-exists within the parental population which suggests the possibility of pre-emptively targeting the inherently resistant population. Interestingly, we also observed differential microRNA expression in radiotherapy- and TMZ-treated cells and, specifically, found that miR-221 confers resistance to glioblastoma cells and is capable of down-regulating EGFR expression. We validated this relationship in a human cohort of 105 primary and 36 recurrent glioblastoma patients, showing a significant inverse relationship between miR-221 and EGFR. Consistently, we showed that high miR-221 and low-EGFR expression at recurrence is associated with a poorer prognosis. Lastly, we investigated the relevance of epithelial to mesenchymal transition markers after observing that migration rates were maintained in resistant cells despite low EGFR and MET. Both N-Cadherin and CD44 were found to be highly expressed in treatment-resistant cells and the down-regulation of AKT activity with wortmannin led to reduced levels of EMT markers, suggesting that AKT is a regulator of key EMT transcription factors that are specific to N-Cadherin and CD44. The thesis gains it significance by providing an explanation to the failure of RTK inhibitors in the glioblastoma clinic by suggesting that standard radio-chemotherapy down-regulates RTK activity and expression, thereby diminishing any theorised benefit of targeting RTKs. Furthermore, the thesis advocates for microRNAs to be crucial regulators of therapy resistance, potential biomarkers and targetable molecules for the clinic.