Sir Peter MacCallum Department of Oncology - Theses
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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.