Surgery (RMH) - Theses
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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.