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Item3D Imaging and Cellular Barcoding: Novel Tools for Exploring Cancer Heterogeneity( 2023-09)Breast cancer affects 1 in 7 Australian women, and the risk of death from metastatic (stage 4) disease remains high. Progression to advanced disease is difficult to treat, especially when the availability of targeted treatments is limited for some cancer subtypes. Metastases form when cancer cells shed from the primary tumour, enter the blood and lymphatic vessels, exit and proliferate in distant organs. Understanding the interactions between these heterogeneous lesions and the vessels that facilitate their spread, will enable a better understanding of this process and potentially lead to improved cancer treatments. Not all tumour cells have the same ability to generate metastases. Specific clones (defined as cancer cells that have derived from the same ancestral cell) differ in terms of the organs they target, their behaviour in specific microenvironments, and how they cooperate with other clones. To date, the methods used to study the clonality and heterogeneity of cancer metastases often involve tissue dissociation or 2D imaging. Consequently, the spatial resolution of clones in their native microenvironment is lost. New methodologies and technologies are required to facilitate spatial discoveries, to advance our understanding of cancer heterogeneity, metastasis, and the tumour microenvironment. Here, I developed a novel pipeline for three-dimensional whole organ imaging of human-in-mouse models of metastatic breast cancer. Light-sheet microscopy was used to capture large volumetric datasets, reducing the information loss observed in 2D tissue sections. I used lentiviral gene ontology (LeGO) vectors, an optical barcoding method, to identify seven individual clones. In combination with vessel casting (a perfusion-based method that enables vasculature imaging), tissue clearing, and an analysis pipeline, I reveal the relationship of aggressive breast cancer clones and the blood vasculature in murine lungs and brain. This represents a method with unprecedented detail and clonal resolution at large volume scales. My results indicate that large vessels may be correlated with enhanced metastatic growth. I also show that metastases that wrap around blood vessels are more likely to be polyclonal (containing multiple clonal populations), which are more aggressive than monoclonal (single clone) metastases, with potential implications for treatment targets. Underlying the clones’ specific behaviours, are differences in gene expression. Based on these results, I propose that transcriptional information, in combination with clonal identity and spatial tissue context, is required to reveal the molecular pathways that are responsible for these clonal behaviours, which may represent novel therapeutic targets. To simultaneously track a higher number of cancer clones (i.e. thousands) and their gene expression in situ, I developed a novel smFISH and RNA barcoding method (FISHcodes). I show that the detection of hundreds of transcripts alongside dozens of clones (scalable to thousands) is feasible and will enable novel insights regarding clonal behaviour in cancer biology. Together, the results presented throughout this thesis have demonstrated that novel methodologies enabling the study of cancer cell heterogeneity and its interplay with the microenvironment, can address new questions about in situ cancer clone metastasis and growth.
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ItemCRISPR/Cas9 Engineering of Next Generation Armoured CAR T Cells( 2023-08)Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable successes in the treatment of certain B cell haematological malignancies; however, its efficacy in the solid tumour setting remains limited. This is attributed to a number of factors, including immunosuppression in the tumour microenvironment (TME), T cell exhaustion, tumour antigen heterogeneity and limited trafficking of CAR T cells into the tumour. A promising strategy to overcome these limitations is by engineering, or “armouring”, CAR T cells to express factors that have the potential to enhance therapeutic efficacy, such as proinflammatory cytokines, chemokines, transcription factors, cell surface molecules and synthetic constructs. This strategy has been assessed in the context of various forms of adoptive cellular therapy (ACT), including tumour-infiltrating lymphocyte (TIL) therapy, as well as transgenic TCR and CAR T cell therapy. Preclinical studies exploring a vast range of armouring genes have demonstrated promising results, with armoured T cell products mediating enhanced anti-tumour efficacy through the capacity to modulate the TME, engage host anti-immune responses and directly enhance T cell function. However, a key limitation of this approach is the potential toxicities caused by peripheral expression of the armouring gene by the engineered T cells, highlighted by the life-threatening side effects observed in an early clinical trial assessing this approach with IL-12-engineered TILs. While strategies have been developed to limit transgene expression to the tumour, such as the synthetic NFAT promoter system, none thus far have successfully demonstrated the ability to prevent clinical toxicities. The advent of CRISPR/Cas9 gene editing, including recent advancements in the engineering of primary T cells, has introduced unique opportunities to enhance the design of armoured T cells. In particular, CRISPR-mediated homology-directed repair (CRISPR-HDR) enables the precise modification of specified genomic sites, including the introduction of a complementary DNA (cDNA) sequence into a target gene locus. Hence, in the context of armoured T cells, CRISPR-HDR can be used to insert armouring genes into a specific endogenous site, creating the possibility to leverage endogenous gene regulatory mechanisms to drive transgene expression. By targeting the armouring gene into the site of an endogenous gene that is exclusively upregulated in intratumoural CAR T cells, this should lead to tumour-restricted expression of the armouring gene. Additionally, integration of the armouring gene can concurrently disrupt expression of the endogenous target gene. Thus, by selecting a target gene that is not only specifically expressed in CAR T cells within the tumour, but also regulates inhibitory T cell functions, this would enable knock-in of a transgene that can enhance therapeutic efficacy while simultaneously knocking out an endogenous inhibitory gene. Therefore, this study assessed the overarching hypothesis that that engineering armoured T cells in accordance with this “knock-in, knock-out” approach would lead to enhanced anti-tumour efficacy while maintaining a favourable safety profile. To explore this hypothesis, a CRISPR-HDR protocol was first developed to engineer primary murine and human transgenic TCR and CAR T cells at high editing efficiencies. This protocol was subsequently applied to generate armoured transgenic TCR and CAR T cells expressing various proinflammatory cytokines under the control of endogenous tumour-specific promoters, and assessed for therapeutic efficacy, mechanism and safety in syngeneic murine models and human xenograft models of ACT. T cells engineered to express TNF, IFN-gamma, IL-2 and IL-12 in a tumour-restricted manner mediated significantly enhanced anti-tumour efficacy; however, their therapeutic effect was largely dependent on the endogenous promoter used. The PD-1 promoter was found to support potent transgene expression in the tumour, which was required for the therapeutic efficacy of TNF, IFN-gamma and IL-2. However, its low level of peripheral expression led to toxicities when used to drive IL-12 expression. Instead, the NR4A2 promoter, which supported highly tumour-restricted transgene expression, was capable of regulating IL-12 production with minimal toxicities while mediating a robust therapeutic response. Hence, these data demonstrate that this CRISPR-HDR strategy is highly customisable, enabling the use of different endogenous promoters to achieve the optimal expression pattern for specific transgenes. In summary, the current study details the development of a novel CRISPR-HDR approach for engineering next generation armoured T cells for ACT, and presents promising preclinical data demonstrating the efficacy, safety, feasibility and translational potential of this approach.
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ItemThe state of data sharing in oncology research: an examination of policies, practices and perspectives of key research stakeholders( 2023-08)Over the last decade or more, there have been increasing efforts to evaluate and improve the transparency of scholarly research across many fields of science. Part of these efforts include lobbying for greater investment in data stewardship, as well as increased public and private access to researchers’ data. In the context of cancer research, this would bring numerous benefits to the research community. For example, greater access to data provides researchers with opportunities to validate discovered findings, answer questions not originally considered by the data creators, and accelerate research through the synthesis of existing datasets. However, transparency also brings challenges, such as the navigation of privacy legislation, increased demands on time and resources, development of infrastructure and expertise, and substantial concerns among researchers such as fears about misinterpretation and misuse of shared data. In this thesis, I present research from five empirical studies that have explored the state of data sharing in oncology. These constitute Chapters 2 to 7. In Chapter 2, I report findings from a survey of journal editors, observing that journals report a wide variety of policies and practices on peer review and data sharing; even within different disciplines, norms are far from fixed. In Chapters 3 and 4, I present the protocol and the findings of a systematic review and individual participant data meta-analysis of over 2.1 million medical publications. At the end of these studies, I estimate that only 8% of medical articles published between 2016 and 2021 declared that the data were publicly available, and only 2% actually shared data. I also estimate that only a third of researchers comply with mandatory data sharing policies of journals, and even fewer – only a fifth – comply with policies requiring researchers to share with others on request. In Chapter 5, I narrow my focus down to data sharing in oncology research, and report that while one in five articles declare that data are publicly available, less than 1% share in accordance with the FAIR Guiding Principles. In this chapter, I also observed similar levels of non-compliance with mandatory data sharing policies of journals to Chapter 4. In Chapter 6, I report the results of a survey of cancer researchers, asking them about their experience with, and thoughts on data sharing. In my final empirical chapter, Chapter 7, I turn to individuals affected by cancer to ask their opinions about whether data from cancer research should be shared, and with whom. Together my work indicates that while both cancer researchers and patients support sharing data, the practice is still uncommon, and that current policies which aim to increase sharing are often wanting. A stronger commitment from key research policymakers such as publishers, funders, and research institutions are needed if we are to improve both the frequency and quality of data sharing in oncology.
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ItemUncovering epigenetic regulators of bivalent MHC class I genes in cancer( 2023-06)Recent breakthroughs in cancer immunotherapy have transformed the management of many malignancies and renewed interest in the molecular understanding of tumour antigen presentation. However, despite the success of these therapies, resistance remains a significant challenge for many patients. One prominent resistance mechanism involves the disruption of major histocompatibility complex class I (MHC-I) antigen presentation, which has been demonstrated to occur through inactivating mutations or transcriptional silencing in the MHC-I antigen presentation pathway, with the latter presenting as a potentially reversible and, therefore, targetable mechanism of resistance. In this thesis, I investigate the epigenetic mechanisms underlying MHC-I resistance in cancer. Firstly, I elucidate the significance of the polycomb repressive complex 2 (PRC2) in maintaining transcriptional repression of MHC-I, which is conserved across different species, including humans, mice, and Tasmanian devils. The silencing of MHC-I by PRC2 in cancer cells facilitates the evasion of T-cell killing. However, I demonstrate that this can be overcome through genetic or pharmacological depletion of PRC2. By conducting ChIP-sequencing, I identify that silenced MHC-I genes in cancer cells exhibit bivalent modifications, specifically repressive H3K27me3 and activating H3K4me3 marks, which is a developmental process seen in embryonic stem cells and maintained during neural progenitor differentiation. Collectively, these findings reveal how cancer cells can co-opt an evolutionarily conserved, lineage-specific function of PRC2 to silence MHC-I antigen presentation and evade immune surveillance. Driven by the observation that bivalency is often dysregulated in cancer, I set out to identify the regulators of bivalent chromatin. Building upon the previous observation that MHC-I is bivalently modified, I leveraged this characteristic as a readout and conducted whole genome CRISPR/Cas9 screens to pinpoint key regulators involved. I uncover specific roles of the PRC2.1 and PRC1.1 sub-complexes in maintaining silencing of bivalent gene expression. Unexpectedly, I make the intriguing discovery that genetic depletion or pharmacological inhibition of Menin, traditionally known as a co-activator and a component of the KMT2A/B H3K4me3 methyltransferase complexes, phenocopies the effects of polycomb disruption. This results in the derepression of bivalent genes in cancer and human pluripotent and embryonic stem cells, findings which challenge the existing paradigm whereby disruption of the KMT2A/B and polycomb complexes is expected to have opposing effects on bivalent gene regulation. Furthermore, my research reveals an essential role of KMT2A/B in MHC-I gene expression following Menin inhibition and, therefore, highlights the existence of Menin-independent and Menin-dependent functions of KMT2A/B. Finally, I demonstrate that targeting the Menin-KMT2A interaction leads to the release and redistribution of KMT2A from active genes to bivalent genes, which creates a permissive chromatin environment that facilitates gene activation. My research has uncovered previously unknown roles for specific components of the KMT2A/B and polycomb complexes in regulating bivalency. Moreover, these findings have significant implications for cancer therapy. By identifying strategies to overcome transcriptional MHC-I repression, my work provides a compelling rationale for utilising inhibitors targeting PRC2 and Menin in the treatment of difficult-to-treat malignancies. Additionally, these insights offer potential avenues for developing novel therapeutic approaches to effectively treat cancers characterised by low MHC-I expression.
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ItemImproving the diagnosis and treatment of cancer of unknown primary using genomic profiling and patient-derived organoids( 2023-04)Cancer of unknown primary (CUP) is a metastatic cancer for which a standard clinical investigation fails to identify a primary tissue of origin (TOO). Clinical decision-making is impaired without a primary TOO diagnosis since this is typically used to inform optimal patient care. Most CUP patients are treated with empirical chemotherapy leading to poor survival outcomes. Therefore, improved diagnostic and treatment options for CUP patients are needed. The diagnostic utility of targeted RNA and DNA profiling was investigated in a cohort of 215 CUP patients. A retrospective clinicopathology review was performed to assign a putative TOO diagnosis. Seventy-seven percent (166/215) of the cohort had insufficient clinicopathological evidence to support a TOO diagnosis, while the remaining 23% were putatively resolved and had a clinicopathology diagnosis assigned. A custom gene expression profiling (GEP) TOO classifier was applied. The clinicopathology-unresolved CUPs were more challenging to classify than the resolved CUPs, and only 56% had a high confidence TOO classification. Cancer-type specific DNA features, including mutations and mutational signatures, were investigated and provided diagnostic evidence to support a TOO in 31% of unresolved CUPs, while GEP classifications supported a diagnosis in 13%. A genomics-informed clinicopathology review could direct an unconfirmed TOO diagnosis in one-third of CUPs. The RNA and DNA profiled CUP cohort were also investigated for immune and genomic biomarkers that could direct immune checkpoint inhibitor (ICIs) treatment for CUPs. Sixteen percent of CUPs had a high tumour mutation burden, 33% had high expression of genes associated with immunotherapy response, and 47% of the TOO-resolved CUPs belonged to an ICI-responsive cancer type. Of a subset of 28 CUPs treated with ICIs, 8/28 (29%) responded to treatment, and all of the responders had a high gene expression score (7/8) and/or high tumour mutation burden (3/8), and most (5/8) belonged to ICI-responsive cancer types. Therefore, many CUP patients have molecular features indicative of ICI susceptibility. Whole genome sequencing (WGS) was explored in 30 CUPs with targeted DNA sequencing to identify additional diagnostic and therapeutic informative features. An 82% concordance of variants was detected between WGS and panel sequencing. WGS could detect additional genomic features, including mutational signatures. Actionable mutations and biomarkers were detected in 48% of cases by targeted DNA sequencing, while WGS increased this to 59%. Furthermore, 72% of CUPs could have a TOO assigned following genomics-informed iii histopathology assessment, of which a third resulted from WGS-identified features only, and all diagnostic features detected by targeted sequencing were supported by WGS. Although only assessed in a small subcohort, tumour DNA extracted from archival tissue and cell-free material could be sequenced to detect clinically relevant findings. Patient-derived organoids (PDOs) were attempted from fresh CUP tumours. PDOs were successfully derived from six of 20 tumour samples (30%). The PDOs were heterogenous, genomically and morphologically representative of the tumour and could be used for WGS. Anticancer compounds were selected for PDO drug screening based on molecular and clinically directed treatment. The PDO drug responses frequently concurred with genomic biomarkers, but multiple drug sensitivities were also unanticipated. By incorporating targeted DNA sequencing and GEP testing, many CUP patients had a putative TOO diagnosis assigned. The genomic tests were also used to identify molecular biomarkers for ICI treatment. When compared with WGS, there were specific diagnostic scenarios whereby WGS could improve the diagnosis and identify additional potentially useful therapies for patients. Additionally, CUP PDOs were used to test genomic-directed therapies through in vitro drug testing. Overall, the study provides insights into how genomic tests can improve the diagnosis and treatment of CUP.
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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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ItemNo Preview AvailableInterleukin-6 family cytokines contribute to pancreatic cancer pathogenesis and can be targeted therapeutically( 2023-01)Pancreatic cancer is a malignancy of significant unmet need. There have been negligible improvements in survival outcomes over the past four decades, with poor prognosis driven by late-stage diagnosis, which is in turn driven by multiple factors including a lack of early screening options, non-specific symptoms, and an aggressive tumour phenotype. The inflammatory and fibrotic nature of the disease presents many difficulties as the dense stroma and associated immune exclusion limit therapeutic efficacy, while the complex cellular network is highly heterogenous and drives pro-tumorigenic programmes through a multitude of cell-type and context dependent signalling pathways. The inherent intertumoral and intratumoral heterogeneity, in combination with the intricate and intertwining signalling pathways occurring within the pancreatic tumour microenvironment has complicated the delineation of pathogenic signalling mechanisms and identification of reliable therapeutic targets. The IL-6 family of cytokines and associated downstream mediators JAK and STAT3 have, however, been implicated in several pro-tumorigenic processes that drive tumour initiation and disease progression, though additional work is required to disentangle the mechanistic complexities of these signalling pathways. Clinical patient samples and associated data were analysed with the aim to understand the value of IL 6 family cytokine expression in predicting disease stage or survival outcomes. Though several associations were identified, our data did reflect the variation often observed in the literature between different patient cohorts, highlighting the need to consider multiple features during analysis. Of the IL-6 family cytokines, pancreatic IL-11 was associated with advanced disease stage, while IL-11, LIF, an OSM were individually associated with survival outcomes. Importantly, an additional analysis considering simultaneous expression of multiple IL-6 family cytokines demonstrated a significant relationship between elevated cytokine expression and reduced recurrence-free and overall survival. This represented an opportunity to increase the prognostic value of IL-6 family cytokines in pancreatic cancer through combinatorial approaches rather than individual analysis. Previous work highlighted a role of IL-11 in solid tumours, with IL-11 also being implicated in pancreatic ductal adenocarcinoma (PDAC). We utilised a PDAC mouse model crossed with IL-11R knockout mice to investigate the contribution of IL-11 signalling to disease pathogenesis. Analysis revealed that STAT3 was activated in PDAC tumours, with this reduced in mice lacking IL 11R. However, this reduction in STAT3 activation was not significantly associated with reduced disease progression or alterations to the tumour microenvironment. While the current data suggests that abrogation of IL-11 signalling is not sufficient to drive meaningful biological change, additional analyses are required. There are currently limited therapeutic options available for pancreatic cancer patients. Recently, the use of chemotherapy/immunotherapy in combination with drugs targeting components of the tumour microenvironment have shown promise. In this study, we generated IL-11, IL-11R, and gp130 nanobodies as a potential new therapeutic opportunity targeting these pro-tumorigenic signalling pathways within PDAC tumours. Characterisation of these nanobodies revealed one IL-11 and two gp130 nanobodies that were able to inhibit downstream STAT3 activation and reduce PDAC cell proliferation and migration in vitro. These nanobodies represent a novel approach by which IL-11 or multiple IL-6 family cytokine signalling pathways could be inhibited. In particular, the use of gp130 nanobodies may provide additional therapeutic benefit by simultaneously targeting multiple IL-6 family members, and thereby limiting potential compensatory mechanisms. Overall, this work has contributed to understanding the associations and roles of IL-6 family cytokines in pancreatic cancer, and characterised a novel therapeutic with which to target these pathways. The complexity of the tumour microenvironment poses obstacles in terms of our ability to understand and outline the mechanisms underlying tumorigenesis, but each additional piece of evidence is vital in informing the development of new and innovative therapies that are crucial to improving clinical outcomes for pancreatic cancer patients.
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ItemCharacterising the molecular heterogeneity of neuroendocrine prostate cancer( 2023-04)Prostate cancer is primarily made up of androgen receptor-driven adenocarcinoma, but some patients may progress to a more aggressive and lethal neuroendocrine prostate cancer (NEPC) phenotype lacking AR expression. NEPC is highly heterogeneous, posing a challenge in detection and treatment. While mutations such as RB1, PTEN, and TP53 have been identified as genomic alterations in NEPC progression, they do not fully explain the heterogeneity and varied response to treatment. Understanding the molecular drivers behind the heterogeneity and therapy resistance of NEPC is vital in improving detection and treatment. Therefore, the main goal of the thesis is to characterise the transcriptional heterogeneity of tumour cells of NEPCs and the cells of the tumour microenvironment (TME), to understand the biology of these tumours and determine whether there are any consistent therapeutic targets. I hypothesise that by characterising the different levels of tumour heterogeneity in NEPCs, new insights into how to treat these tumours can be detected. To do so, I focus on three main questions 1) What is the level of intra-tumour heterogeneity of NEPC pathologies? 2) Are there common and/or distinct transcriptional profiles among NEPC pathologies? 3) How does the tumour microenvironment (TME) vary across different pathologies in NEPC? To extensively analyse the transcriptional heterogeneity of NEPC, single-cell RNA sequencing technology was used on a novel cohort of nine patient-derived xenografts (PDX) models that recapitulate the pathological and clinical heterogeneity of NEPC. I analysed scRNA-seq data from 18,632 cells from 9 patient-derived xenografts (PDX) models of NEPC to detect transcriptional heterogeneity between sub-populations. PDXs are a powerful tool to analyse the heterogeneity of NEPC due to their ability to retain the genomic and phenotypic features of the original tumour in vivo. The opportunity to study rare types of NEPC using PDX models is particularly valuable, as it allows for a more comprehensive understanding of the disease. Additionally, the feasibility of using PDX models for NEPC research is enhanced by the availability of fresh tissue with more viable cells for single-cell experiments. This makes PDX models an attractive option for studying the disease's biology and potential treatment options in a more comprehensive and detailed way. The thesis is divided into three distinct studies. The first study comprehensively characterises the degree of intra-tumour heterogeneity in each NEPC sample. This is achieved through a rigorous quality control process and standardisation of a single-cell RNA sequencing pipeline, which accurately assesses the levels of heterogeneity. Analysis revealed the presence of 3-8 subpopulations in each sample. The degree of heterogeneity changed depending on the pathology; small cell NEPC showed more heterogeneity than any other pathology. One key finding is that at least one chemo-resistant cluster was detected in each pathology, and those clusters showed a unique transcriptional profile. These findings suggest that the intra-transcriptional heterogeneity of NEPC could be a critical factor in identifying potential therapeutic targets. The second section of the thesis aims to examine the inter-patient heterogeneity of NEPC. The analysis of NEPC cells revealed that certain pathologies exhibit a similar transcriptional profile, while others show a closer resemblance to adenocarcinoma. Although these pathologies share the expression of neuroendocrine markers, there is evident heterogeneity in the underlying biology driving each phenotype. Both small and large cell neuroendocrine pathologies exhibit enriched LEF1, YAP, Notch and NMYC signalling pathways and hallmarks of aggressiveness, which are associated with poor prognosis for patients. Notably, the NED cell populations revealed a distinct profile of enriched markers and pathways, such as TNFA via NFKB and KRAS signalling. Unexpected cell co-expression of both neuroendocrine and adenocarcinoma markers was observed in NED pathologies. The co-expression analysis has uncovered this unexpected cell plasticity in NEDs, exposing that they retain adenocarcinoma features and exhibit a transcriptional profile similar to adenocarcinoma. The data indicate the presence of two molecular profiles of NEPC; it is important to consider these molecular profiles in developing personalised therapeutic strategies, as they can potentially improve patient outcomes and response to treatment. The third section of the thesis is dedicated to the investigation of the TME of NEPC. The study focused on murine non-cancerous cells extracted from tumours collected from PDXs. The analysis revealed the presence of heterogeneous subpopulations of fibroblasts, including the novel antigen-presenting CAF, as well as the inflammatory and myofibroblasts CAF populations. Based on their gene expression profile, these cells may represent a dynamic state of cancer-associated fibroblasts (CAFs), whose role is likely to be influenced by cell-to-cell interactions within the TME. Notably, a significantly increased proportion of myofibroblasts (myCAFs) and inflammatory CAFs (iCAFs) were observed in the small and large cell pathologies. This suggests that the TME may play a crucial role in the progression and aggressiveness of NEPC and highlights the importance of considering the TME when developing therapeutic strategies for these patients. The detection of transcriptional sub-populations in NEPC has provided insights into the underlying molecular mechanisms that make finding effective treatments for these tumours so challenging. These sub-populations exhibit unique gene expression profiles, resulting in a high degree of heterogeneity within NEPC. This heterogeneity is likely a key contributor to the failure of standard prostate cancer treatments in NEPC, as the different sub-populations may respond differently to various therapies. Furthermore, these sub-populations may play a role in the resistance to existing treatments, making them potential targets for the development of new therapeutics. Overall, the characterisation of the transcriptional heterogeneity of NEPC represents a significant step towards developing more personalised and effective treatments for this aggressive cancer.
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ItemNo Preview AvailableA Quantitative Analysis of Natural Killer Cell Homeostasis, Competition, and Collaboration( 2022-12)Contemporary Immunology views Natural Killer (NK) cells as critical facilitators of immune protection in various pathological settings. Still, this has not always been the case; a somewhat challenging history of NK cell research has delayed full scientific appreciation of their importance and modus operandi, which rendered NK cells a mysterious and misunderstood immune cell subset for several decades. In more recent years, NK cells are receiving a resurgence in clinical attention owing to characterisation of their potent anti-tumour and immunomodulatory properties; however, as modern Immunology remains in the aftermath of an uncertain era for NK cells, harnessing this revolutionary therapeutic potential has proven difficult. NK cells are key inducers of early inflammation and systemic immune activation, as well as expert decision makers in the destruction of harmful cells versus protection of healthy tissue. As may be expected, catastrophic consequences can occur to a host if these processes are not properly regulated. There is growing appreciation in the research community regarding the sheer complexity and redundancy in regulatory processes that maintain NK cell homeostasis and functions, as well as the plethora of cytokines and cell-cell interactions that govern this regulated behaviour. As a means of dissecting these complex processes, we have applied a reductionist approach to study how various individual signals are integrated into the internal machinery of an NK cell to produce different outcomes. To this end, we applied quantitative methods previously established in adaptive T and B lymphocytes to delineate and quantify parameters relating to survival and proliferation. In this work, we uncovered that stimulatory proliferative signals from the cytokines IL-15, IL-18, and IL-12 are offset by enhanced propensity for NK cell death, which limits the overall efficiency of their expansion during stimulation. These responses were largely dependent on direct interactions between NK cells via Fas and FasL, which induce fratricidal killing of each other. These competitive relationships between fellow NK cells were heavily dependent on the type and dose of cytokine present. Further, our investigation of NK cell interactions led us to identify that NK cells also facilitate advantageous interactions with other NK cells in more homeostatic contexts, which were dependent on IL-15. We discovered that these homotypic collaborative interactions are the result of complex interactions and bidirectional signalling events between SLAM family receptors 2B4 and CD48, which together facilitate IL-15 responsiveness and education events, thereby enhancing NK cell fitness and function, respectively. This work offers valuable insights to improve in vitro culture protocols in the clinical cultivation of NK cells for immunotherapies, such as Adoptive Cell Therapy, as well as indicating broader and nuanced roles of immune and target cell interactions in the stimulation and regulation of NK cell fitness, function, and homeostasis.
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ItemThe role of USP9X in Low-Grade Serous Ovarian Cancer( 2023)Low-grade serous ovarian carcinoma (LGSOC) is a rare histotype of epithelial ovarian cancer (EOC), and accounts for approximately 3-5% of diagnosed EOC cases. LGSOC is characterised by wildtype TP53 expression, frequent aberrance in the RAS/RAF signalling pathway, and relative genomic stability which in part explains LGSOC resistance to current standard-of-care platinum-based chemotherapeutics. Current chemotherapy strategies for LGSOC have predominantly been driven by that of the far more common high-grade serous subtype. Optimal cytoreductive surgery is challenging, given that the majority of LGSOC diagnoses are at a late stage where the cancer has metastasised from the primary site. Recently, alternative therapy strategies including targeted therapy of the RAS/RAF pathway have shown efficacy against tumours, but further characterisation into potential novel drivers of this disease is required to expand the treatment repertoire for patients suffering from this disease. Previous sequencing studies elucidated USP9X as one such potential driver of LGSOC. USP9X is a deubiquitinase involved in protein turnover. The gene has been implicated as both oncogenic and tumour-suppressive depending on the cancer type being investigated. In the context of LGSOC, little is known as to the role that this gene has in disease development. This thesis evaluated 121 LGSOC cases, 71 sequenced via targeted sequencing, 49 through whole exome sequencing, and 1 by whole genome sequencing. Sequencing results identified USP9X mutations at a frequency of 14%, and as the most frequently mutated non-RAS/RAF gene in the assessed cohort. Interrogation into the allelic status of these mutations revealed more than half of the mutations were inactivating, suggesting a tumour-suppressive function; USP9X was elucidated to follow a classical two-hit tumour suppressor model. Gene knockdown and knockout experiments on LGSOC cell lines highlighted a potential perturbance to clonogenic survival, but not to migration and proliferation. Mass spectrometry analysis on USP9Xnull LGSOC cell lines identified the molecular chaperone BAG3 as a likely direct substrate of USP9X, and the deubiquitinase as a potential regulator of the mTORC signalling pathway. Assessment of the global proteomic perturbations as a result of USP9X downregulation suggested the downstream consequences of USP9X suppression are likely to be decreased cell adhesion, and potentially increased cell migration and invasion.