Theses - Cancer Research UK Cambridge Institute
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Item Embargo Investigating the impact of ageing on the tumour microenvironment and its crosstalk with malignant cells in pancreatic cancerAraos Henriquez, JoaquinPancreatic ductal adenocarcinoma (PDAC) is characterised by an extensive tumour microenvironment (TME), which contributes to disease progression and therapy response. PDAC primarily affects elderly individuals as patients are typically diagnosed when 70 years old or older. While ageing has been shown to impact the TME and progression of other malignancies, most pre-clinical studies of PDAC use young mouse models that may not mimic the physiological state and TME interactions of the majority of PDAC patients. Thus, ageing could affect the PDAC TME and aged mouse models may reveal therapeutic targets to improve the treatment of elderly PDAC patients. To investigate this, I established (~18-month-old) orthotopically grafted KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) organoid-derived mouse models of PDAC and compared them to young (~3-month-old) mouse models. Single- cell transcriptomics, graph network-based machine learning, flow cytometry and histological analyses showed that aged models have a more inflammatory TME relative to young models. Moreover, tumours from aged models showed a higher abundance of senescent cells, suggesting that senolytic therapy could be a potential therapeutic strategy against aged PDAC. Using a recently developed natural language processing and graph network-based machine learning approach, I also demonstrated that aged mouse models of PDAC better recapitulate features of old PDAC patients. This approach identified the Toll-like receptor signalling cascade and IRAK4 activation in aged mouse models as a potential age-dependent therapeutic vulnerability. Finally, while senolytic therapy failed to show beneficial results, inhibition of IRAK4 significantly and selectively reduced tumour growth in aged mouse models of PDAC. This work highlights how ageing shapes the TME and therapy response in PDAC and how aged mouse models of PDAC could be leveraged to tailor therapies for specific groups of patients.Item Embargo Deciphering Genomic Evolution: From Early Breast Cancer Stages to Invasive CarcinomaFranco Alvarez, JoseThis dissertation investigates the evolution of ductal carcinoma in situ (DCIS) to invasive forms such as invasive ductal carcinoma (IDC) at the individual patient level. DCIS is considered a non-obligatory precursor of IDC, being the predominant form of breast cancer detected and comprising approximately 80% of all diagnosed breast cancers. This project presents an opportunity to investigate the initial stages of breast cancer development and its progression from normal milk duct cells to invasive neoplastic forms. Through computational analyses and re-processing of a cohort of 394 broadly distributed genome samples from 77 patients, including normal milk duct, benign, atypical, DCIS, and IDC, this project aims to illuminate the genomic diversity, mutational exposures, and evolution of normal human duct milk cells and to characterize the major evolutionary forces that operate in a short space of time to develop invasive forms. This dissertation is structured in five chapters. The first chapter introduces the concepts of evolution and clonality, among the different classifications, and the early history of breast cancer treatment, ending with a discussion of the necessity to advance through early prediction. The second chapter describes the sample collection, processing, and sequencing process. Identification of issues with the sequencing data led to the development of a new computational approach to curating the data, aiming to achieve a gold standard for the processed samples. This chapter also investigates preliminary exploratory analyses of the discovered somatic variants and discusses the potential for missing data. The third chapter explores the mutational patterns assigned to mutational processes in the early stages of breast cancer development, including the exploration of potential endogenous and exogenous sources of mutation. The fourth chapter presents the methodology and results of somatic phylogenetic analyses, including reconstruction of the relationship between annotated pathological tissue types and the phylogenetic distribution of previously identified mutational signatures. The last and final chapter presents the conclusions and future directions of the research. Overall, this work traces the genomic evolution of early breast cancer stages, giving a snapshot of the complex forces, mutagenic exposures, and driver genes at the patient level and highlighting the need for larger cohorts, personalized treatment and evolution-assisted prediction.Item Embargo Clinical applications of quantitative photoacoustic imagingElse, Thomas; Else, Thomas [0000-0002-2652-4190]Photoacoustic imaging is a medical imaging tool increasingly used in clinical research. It enables the imaging of molecules, including melanin, haemoglobin, lipids, collagen and water deep inside the body, with high resolution and high sensitivity. This molecular information has made it an appealing tool for detecting and monitoring a range of diseases, from degenerative muscular disorders to cancer. Here, I take three steps towards the clinical translation of photoacoustics: tackling skin colour bias, applying it to a new disease and improving the transparency of computational methods. To characterise the effects of skin colour on photoacoustic imaging, I carried out a technical study using simulations, tissue-mimicking phantoms, and pigmented mice. Linear unmixing and a machine learning approach were compared to estimate blood oxygenation (sO2). A consistent trend of increasing sO2 with increasing skin pigmentation was observed in all three settings, suggesting that spectral colouring was leading to decreased light penetration at shorter wavelengths compared to longer wavelengths. The results, however, hinted that further effects were at play. While the machine learning unmixing approach worked well for the simulations and phantoms, it did not work well in the mice due to substantial regions of negative pixels in the pigmented mice. To explore this phenomenon further, I carried out a healthy volunteer study. Data from a diverse cohort of subjects was acquired, and photoacoustic measurements of several major blood vessels and muscles were made. Blood oxygenation was then estimated using linear unmixing, revealing a similar trend to that observed in models. However, rather than seeing a consistent increase in sO2 with skin pigmentation, the estimated sO2 appears to reach a peak and then decrease again beyond a specific limit. I proposed a possible mechanism: the reflection of photoacoustic waves originating in the epidermis. I used simulations to explore this possibility and revealed that the reflected epidermis signal can be detected in the measured photoacoustic time series and may contribute to this bias. If true, this poses a severe problem to existing correction methods, as most are based purely on the optical component of the photoacoustic problem and neglect acoustic artefacts. Notwithstanding the challenges of applying photoacoustics equitably in people with different skin colours, the technology has been successfully applied previously to various disease types. Here, I analysed photoacoustic data from a clinical study of patients with mitochondrial disease. The patients were given 60 % oxygen to assess its effects. Photoacoustic imaging and pulse oximetry were conducted at three timepoints: before, during, and after oxygen delivery. I showed that photoacoustic imaging is sensitive to changes in the blood oxygenation induced by the changing breathing gas and that characteristic multispectral differences between affected patients and healthy volunteers may be associated with lipid content. To improve transparency in photoacoustic imaging, I developed a Python data analysis toolkit called PATATO. It provides open-source implementations of commonly used image reconstruction algorithms and a convenient interface for technical and non-technical users. The parameters of the reconstruction algorithm were optimised, showing that model-based image reconstruction improves the image quality and the photoacoustic spectra derived from clinical data. Overall, I showed that photoacoustic imaging is a promising clinical tool but shows a clear bias due to melanin in the epidermis. Transparency, openness, and improved reporting will all be required to ensure equity in photoacoustic clinical trials going forward. More advanced hardware, software and standardisation protocols will be required to eliminate skin colour bias.Item Open Access Polyclonal interactions in intestinal tumorigenesisSadien, Iannish; Sadien, Iannish [0000-0002-3367-1252]The adenoma-carcinoma sequence posits that colorectal cancers result from the stepwise accumulation of mutations in a number of cancer driver genes, with mutations in the tumour suppressor gene \textit{APC} thought to be necessary and sufficient for transformation. However, large-scale sequencing efforts have uncovered significant intratumoural genetic heterogeneity which is inconsistent with such a linear model of tumour progression. Alternative models of tumour evolution have been put forward but they all assume an initial clonal expansion originating from a single transformed cell. This contrasts with reports of polyclonality in a number of neoplastic lesions. In the first part of this thesis, I combine multicolour lineage tracing with a mouse model of intestinal tumorigenesis to show that a significant proportion of tumours have a polyclonal origin following the loss of one allele of \textit{Apc} and subsequent mutagenesis. These polyclonal tumours cannot be explained by the random collision of independent tumours but instead, appear to be the result of recruitment of a poorly- or partially-transformed clone. Next, using a highly multiplexed targeted amplicon sequencing approach, the nature of the mutagen-induced variants in Apc is characterised in monoclonal and polyclonal tumours. By microdissecting polyclonal tumours based on their lineage marker status, I show that mutations landing in the N-terminus of Apc are depleted in monoclonal tumours and are instead selected for in minor clones. This suggests that certain Apc mutations are unable to lead to transformation via the monoclonal route but instead have to be rescued through clonal interactions. In the final part of this thesis, I go on to show that clones within polyclonal tumours are complementary in their Kras and Myc pathway activation levels, which suggests that the well-established phenomenon of oncogenic cooperation between these two pathways can sometimes be non-cell autonomous. These transcriptomic differences are reflected in an observed variation in cell phenotypes, with pronounced differences in cancer stem cell identities with an Lgr5 and Anxa1 dichotomy. Importantly, this intratumoural heterogeneity in cancer stem cell identity corresponds to lineage marker territories, indicating that founder cell multiplicity can contribute to intratumoural heterogeneity that persists during the life of a tumour. In summary, this work adds to a growing literature that proposes an alternative to the predominant monoclonal theory of tumour formation.Item Embargo Evolutionary conservation of unistrand piRNA clusters and characterisation of the piRNA precursor export machinery in Drosophilavan Lopik, JasperThe PIWI-interacting RNA (piRNA) pathway prevents endogenous genomic parasites, transposable elements, from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, are thought to define each species’ piRNA repertoire and therefore its capacity to recognize and silence specific transposon families. The *Drosophila melanogaster* ovary contains two distinct types of piRNA clusters: unistrand and dual-strand clusters. While there is only one major unistrand cluster, dual-strand clusters are plentiful with more than 100 reported to be active. The major unistrand cluster *flamenco* (*flam*) is essential in the somatic compartment of the *Drosophila* ovary to restrict *Gypsy*-family transposons from infecting the neighbouring germ cells. Disruption of *flam* results in transposon de-repression and sterility, yet it remains unknown whether this silencing mechanism is present more widely amongst *Drosophila* species. During my PhD we found that unistrand, *flamenco*-like, clusters are a conserved feature across the *Drosophila* genus. Importantly, not only did we illustrate that *flamenco* itself is conserved via synteny beyond the *melanogaster* species subgroup, but through systematic characterisation of 119 *Drosophila* species an additional five distinct and entirely new clusters with *flamenco*-like characteristics were found in species that diverged from *D. melanogaster* up to 45 million years ago. Remarkably, through bioinformatic approaches and deep sequencing of somatic piRNAs, me and my colleagues showed that these loci act as bona-fide unistrand piRNA clusters and are specifically organised to selectively silence *Gypsy*-family transposons, a class of endogenous retroviruses, in the soma. Notable, these transposons are able to invade germ cells from the surrounding somatic tissue and our study therefore provides strong evidence of a function of these clusters in transposon control, similar to *flamenco*. Together, this study provides important insight into the co-evolution between virus-like *Gypsy*-family transposons and a host defence mechanism. The other type of piRNA producing loci, dual-strand clusters, are non-canonically transcribed loci requiring specialised machinery for both their transcription and nuclear export. Their export process hinges on the nuclear export factor Nxf3, its co-factor Nxt1, and the orphan protein Bootlegger (Boot). Through interaction studies, I established that Boot and Nxf3 are multivalent interaction partners. Orthogonally, XL-MS data indicates that there are interacting residues between these two proteins throughout their entire protein length. In addition, by means of recombinant protein complex purification I showed that the association of the ATP-dependent RNA helicase UAP56 with Boot is required to form a stable export complex comprising of Boot, Nxf3, UAP56 and Nxt1. At the same time, I showed that the interaction of Boot with the chromatin bound protein Deadlock prevents binding to Nxf3, thus the export complex appears to form when Boot has dislodged from the chromatin bound piRNA transcription machinery. Furthermore, mapping of the cross-linked residues of UAP56 onto the structure embedded in the human transcription export (TREX) complex revealed that all residues are located on the surface and are facing away from the TREX complex, thus possibly allowing an interaction between Boot-Nxf3 and UAP56 whilst in complex with TREX. Lastly, preliminary low-resolution structure reconstruction from Cryo-EM images, performed by our collaborator, suggests that the export complex forms a hand-like structure. Overall, this work has laid a basis for in-detail structural and biochemical characterisation of the export complex. In summary, my research improved our understanding of how this complex facilitates the export of dual-strand cluster transcripts.Item Embargo Effect of platinum chemotherapy on tumour infiltrating immune cells in metastatic ovarian cancer: Mechanistic insights and therapeutic applicationsBoyles, ChristabelPlatinum chemotherapeutics remain standard of care for many cancer patients, including high grade serous ovarian cancer. It is becoming clear that chemotherapy can initiate adaptive immune responses to the cancer, however, it remains unclear to what extent innate Natural Killer (NK) cells contribute. Using two syngeneic models (UPK10 and ID8p53-/-) of metastatic ovarian cancer this thesis found that treatment with carboplatin upregulates NK cell activating receptor ligands, induced a pro-inflammatory phenotype and sensitised tumour cells to NK cell mediated cytotoxicity. *In vivo*, carboplatin therapy was dependent on the presence of NK cells and CD8+ T cells and treatment with carboplatin resulted in an enrichment for NK cells and CD8+ T cells within the tumour. This effect however was model dependent. Therapeutically, Il-15/IL-15Rα treatment increased the numbers of cytotoxic NK cells at sites of metastasis, which significantly reduced tumour burdens in mice when used in combination with carboplatin. Collectively, these findings demonstrated the importance of NK cells as mediators of standard cancer therapy and represents an attractive and relatively safe therapeutic target for combination therapy.Item Open Access Notch activation and clone fate in intestinal homeostasis and inflammationLines, GeorgeThe Notch signalling pathway is a highly conserved pathway that has important and often differing roles and functions depending on the tissue. In the intestinal epithelium Notch signals are important for determining cell fate between the absorptive and secretory lineage and regulating the proliferation of intestinal stem cells (ISCs). As a result, Notch is also an important pathway during disease such as inflammation and previous literature has reported conflicting arguments on the role of Notch during inflammation. So far there is a lack of understanding of the effect Notch signals play on ISC dynamics during homeostasis and a disease setting, with most studies focussed on the small intestinal epithelium rather than the colon. In this study I aim to determine the effect of Notch signals on the cell cycle and ISC clone dynamics in the colonic epithelium. Using double nucleotide labelling and pulse-chase lineage tracing experiments along with immunofluorescence and immunohistochemistry methods with a transgenic Notch active mutant mouse model (N1ICDΔIEC), cell and clonal dynamics were determined in the normal and inflamed colon. Nucleotide analogue labelling data found N1ICDΔIEC crypts had an increased number of proliferative cells with shorter cell cycle times whilst inhibiting secretory cell differentiation. Subsequent pulse-chase experiments determined N1ICDΔIEC clones were negatively biased proximally and neutral, distally. This highlighted a disconnect between cell cycle and clone dynamics. Whilst long-term lineage tracing results show these N1ICDΔIEC clones are all but lost from the proximal and distal colonic epithelium owing to their increased rate of proliferation and lack of secretory cells. These same dynamics were then explored in a DSS-induced inflammation model. Here, DSS was found to directly inhibit cell proliferation in N1ICDΔIEC crypts proximally and following a post-DSS chase period this phenotype was reversed, with rapid N1ICDΔIEC clone expansion. In the distal colon, DSS caused N1ICDΔIEC clone expansion through increased cell proliferation. Following a post-DSS chase period there was further cell proliferation increases that did not yield further N1ICDΔIEC expansion. These data find N1ICDΔIEC crypts drive repair and regeneration of the intestinal epithelium during and after inflammation to maintain homeostasis. Therefore, Notch activation on its own in the normal colon does not confer a competitive advantage and requires a disease setting for clonal expansion. Important differences exist between the proximal and distal colon and this information should be considered before using unspecific Notch targeted drugs as these could potentially have varying outcomes and degrees of success.Item Embargo The chronic response to centrosome loss-induced cell cycle exitThomas, DanisThe centrosome is a membrane-less organelle which primarily functions as a microtubule organising centre in eukaryotic cells, to allow normal progress through the cell cycle. While centrosome amplification in the absence of p53 is a well-established feature of cancers, more recently centrosome loss was also detected in tumour tissues. The development of a small molecule (called centrinone) that blocks centrosome duplication revealed that cells with centrosome loss undergo a p53-dependent cell cycle arrest that appears to show some senescence-like features with long-term treatment. However, the nature of cell cycle arrest and its relationship with senescence are still not well understood. The aim of this project is to investigate the temporal dynamics of p53 in centrinone-treated cells and to determine whether long-term centrinone treatment induces bona fide senescence. I have used a novel fluorescent reporter of p53 transcriptional activity in RPE1 cells and combined it with live cell lineage analysis to reveal that p53 activation upon centrinone treatment is insufficient to commit cells to cease proliferation immediately. Instead, long-term centrinone treatment is required to induce cell cycle arrest. This state is reminiscent of senescence, but the phenotype is modest compared to classical DNA damage-induced senescence. To examine whether this phenotype is due to cellular heterogeneity or a globally slow-cycling state, I have taken a single-cell transcriptomics approach and found that long-term centrinone treatment results in two distinct sub-populations, one exhibits typical senescence features, the other appears to maintain proliferative capacity. These findings extend our understanding of the line between senescence and centrosome biology, highlighting a unique heterogeneous senescence response.Item Open Access Measuring ongoing chromosomal instability in single-cell DNA sequencing dataSchneider, MichaelChromosomal instability is a characteristic hallmark of cancer. It is providing part of the genomic variation that makes cancer so heterogeneous and difficult to treat. Consequently, better insights into the mechanism and dynamics of chromosomal instability are fundamental to understanding and treating cancer. First, we introduce scAbsolute – a computational tool to correctly estimate single-cell ploidy and replication status. Cancer cells often exhibit DNA copy number aberrations and can vary widely in their ploidy as a consequence. Correct estimation of the ploidy of single cell genomes is crucial for many aspects of downstream analysis, such as copy number calling and inference of cell phylogenies. Based only on single-cell DNA sequencing information, scAbsolute achieves accurate and unbiased measurement of single-cell ploidy and replication status, including whole-genome doublings. We demonstrate scAbsolute’s capabilities using experimental cell multiplets, a FUCCI cell cycle expression system, and a benchmark against state-of-the-art methods. scAbsolute provides a robust foundation for single-cell DNA sequencing analysis across different technologies and for various downstream analyses. Second, we introduce scUnique and the notion of recent copy number aberrations – a way to measure ongoing chromosomal instability using single-cell whole-genome DNA sequencing. We demonstrate scUnique’s effectiveness in a number of simulations and investigate the read depth required to reliably call copy number aberrations at the level of individual cells. scUnique detects differences in the activity of mutational processes in cell lines derived from treatment naive and relapse tumour samples. By measuring chromosomal instability at the level of individual cells, we provide quantitative, scalable and whole-genome level information about the dynamics of chromosomal instability, not available in previous studies relying on bulk DNA sequencing or single-cell imaging. The temporal and whole-genome resolution of scUnique offers the opportunity to gain novel insights into the generative process underlying chromosomal instability. Ultimately, this thesis contributes to a better understanding of the dynamics and fundamental mechanisms of chromosomal instability, and has the potential to improve treatment decisions in the future.Item Embargo Investigating DNA G-quadruplex structure function through genome and epigenome engineeringEsain Garcia, IsabelThe importance of DNA secondary structures, such as four-stranded DNA G-quadruplexes (G4s), in genome function remains a largely unanswered question. G4s are implicated in transcriptional regulation; however, earlier studies are mostly correlative and do not directly address the role of an individual G4 within its endogenous cellular context. Using CRISPR genome and epigenome engineering, I performed a series of genetic and epigenetic perturbations in human cells to specifically modulate G4 formation within the upstream regulatory region of the *MYC* oncogene. In combination with chromatin profiling, whole-genome and locus-specific sequencing, and biophysical assays, I investigated how G4 folding in cells regulates the local chromatin environment and gene expression. I found that the *MYC* G4 structure positively regulates *MYC* transcription. In particular, the *MYC* G4 regulates promoter choice by promoting transcription from the P1 promoter. I also found that G4s shape the local chromatin architecture to coordinate multiple molecular processes and regulate transcription. First, G4s act as anchors for the binding of transcription factors, including SP1 and CNBP. Second, G4s recruit histone modifiers which in turn dictate the local histone methylome. Third, G4s organise nucleosome positioning to influence chromatin accessibility and recruitment of RNA polymerase II. Cells edited to lack the *MYC* G4 lead to loss of *MYC* transcription from the P1 promoter and the deposition of a *de novo* nucleosome that interferes with normal RNA polymerase recruitment. Confirming the importance of structure rather than primary sequence, I demonstrate that replacing the endogenous *MYC* G4 with a different G4 sequence restores G4 folding and *MYC* transcription. Furthermore, through epigenome engineering, I show how cytosine methylation can modulate G4 formation in cells. My findings suggest a mechanism whereby G4s are central features that coordinate regulatory protein recruitment and establishment of epigenetic and nucleosome landscapes to modulate gene expression. Overall, the evidence presented in this thesis provides direct and robust support of the importance of DNA secondary structure rather than primary sequence in genome regulation.Item Open Access Graph Representation Learning to Study the Tumour MicroenvironmentMartin Gonzalez, PaulaThe progression and treatment response of cancer is influenced by the intricate tissue structure where cancer cells are embedded, known as the tumour microenvironment (TME). Recent technical advances allow the acquisition of highly multiplexed biomedical images (HMBI) that generate spatial tissue maps of dozens of proteins capturing the intricacies of the TME. Combining the multidimensional cell phenotypes acquired with their spatial organization to predict clinically relevant information is a challenging computational task. Inspired by the opportunities that artificial intelligence offers to capture the language of biology, this thesis focuses on using graph representation learning on HMBI to highlight unknown biological patterns in a data-driven manner. Here I propose MULTIPLAI, a novel framework to predict clinical biomarkers from HMBI data using Graph Neural Networks (GNNs) that integrate both the phenotypic and spatial dimensions of HMBI images while learning the best representation of the whole slide for each task and providing a framework to explore feature attribution. By the time of publication, it was the first application of GNNs to this type of data. For the first case study of MULTIPLAI, I carry out a proof-of-concept study to predict oestrogen receptor (ER) status, a key clinical variable for breast cancer patients. The results suggest that MULTIPLAI successfully captures TME features with clinical importance. For the second case study of MULTIPLAI, I explore the relationship between TME patterns and different definitions of chromosomal instability (CIN). The results indicate that a combination of immune cells and markers associated with breast cancer, as well as cellular birth and death processes, play significant roles in different CIN metrics. These results support the potential of MULTIPLAI for data-driven analysis of HMBI to enhance our comprehension of spatial tumour biology. This will become particularly valuable in a multi-modal research context, where it can shed light on the interconnections between various data sources.Item Embargo Imaging Glioblastoma metabolism using mass spectrometry imagingTsyben, Anastasiya; Tsyben, Anastasiya [0000-0001-9659-3127]Glioblastoma (GB) is an inherently heterogenous and invasive primary brain tumour. Genetic and transcriptomic studies have attempted to classify GB into subtypes that can identify therapeutic vulnerabilities and predict survival, these include metabolic subtypes. An outstanding question and technical challenge is to visualize the metabolism of a tumour within its native microenvironment and understand to what extent its metabolism is driven by the microenvironment. Using isotope tracing and mass spectrometry imaging (MSI), I identified three metabolic signatures in patient GB tumours: glycolytic, oxidative and a mixed glycolytic/oxidative phenotype. These phenotypes do not correlate with microenvironmental characteristics such as proliferation rate, immune cell infiltration, hypoxia, or vascularisation. Growing cells outside the primary tumour as primary cell lines and then implanting them orthotopically into rodent brains did not change their metabolic phenotype. In addition, modulation of the microenvironment *in vitro* (including hypoxia, growth factors addition and immune signalling), did not alter these metabolic phenotypes suggesting that they represent cell intrinsic states. The three metabolic phenotypes showed differential drug sensitivity. One drug, MM315, an apelin receptor antagonist that is known to play a role in angiogenesis and differentiation of neural progenitor cells, significantly prolonged the survival of orthotopically implanted patient-derived xenografts and led to apoptosis of primary GB cells in culture. Taken together, the data suggests that metabolic imaging may be used clinically to identify tumour phenotypes and stratify patients for personalised therapy trials.Item Open Access Computational profiling of the tumour immune microenvironmentCast, OliverA tumour comprises not only cancer cells but other populations, including immune and stroma, collectively termed the tumour microenvironment (TME). Single-cell RNA sequencing has offered unparalleled insights in different and rare cell populations. However, bulk RNA-seq remains a cost-effective and widely-used approach to analyse large clinically annotated cohorts. Given the ever growing volume of transcriptomics data, computational methods for estimating immune cell proportions and studying tumour immune dynamics have gained prominence. This thesis presents novel bioinformatic approaches for studying the TME, yielding insights into how the complex cellular landscape can be altered by cancer and therapeutic intervention. Central to this work is the development and refinement of ConsensusTME, a novel ensemble computational method that estimates immune and stromal cell proportions in the TME from bulk transcriptomics data. This method outperforms existing approaches, ranking in the top three for all cancer-related benchmarks. A key contribution of this research is the comprehensive comparison of various computational tools used in TME analysis, establishing ConsensusTME as a robust method. Enhancements to this tool include refined human gene sets and the ability to estimate immune infiltration in mouse models, broadening its applicability. The dedicated web resource, www.ConsensusTME.org, and R package, “ConsensusTME”, facilitate its use. The web resource further provides additional deconvolution resources to aid development and vigorous benchmarking of new methods. The biological impact of this work is showcased through case studies, particularly in the context of pancreatic and ovarian tumours. The findings reveal how treatments, such as chemotherapy or hedgehog inhibition, dynamically alter the TME. Additionally, the application of the 10x Visium platform provides new spatial perspectives on TME features, underlining both the utility and limitations of current methodologies. In summary, this thesis advances our understanding of the TME through bioinformatic innovation but also offers practical tools and insights for future cancer research. Potential therapeutic implications of these findings underscore the importance of understanding the TME for continued development cancer treatment strategies.Item Embargo Exploration of cell-free DNA’s biological properties for better understanding and improved circulating tumour DNA detectionVijayaraghavan, AadhitthyaBackground: Liquid biopsies, which are sampling from bodily fluids, are minimally-invasive techniques that can be used for cancer profiling. Cell-free DNA (cfDNA) are fragmented DNA shed into circulation from various types of cells. The cfDNA derived from cancer cells, circulating tumour DNA (ctDNA), forms a subset of cfDNA. It can be challenging to detect ctDNA as it often constitutes only a minor fraction of the cfDNA. Recent studies suggest that understanding the biological properties of ctDNA can improve its detection in a tumour-naive way. Aim: To develop novel data analysis approaches and tools to explore the biological properties of cfDNA sequencing data, aiming to improve cancer detection and diagnosis. Methods: Two methods FRENDS (FRagment ENDS Sequence context) and MIDS (fragMent dIstance to the miDpoint of genomic elementS) were developed from biological properties sequence content of fragment ends and the fragment positioning. FRENDS method extracts nucleotide frequency and motif frequency at fragment start and ends. The MIDS method assesses the distance of the fragment midpoints to the interested region’s centre in 66 different genomic lists ranging from nucleosome positions, transcription start sites, to DNASE1 hypersensitive regions. Results: The FRENDS and MIDS signals showed differences between cancer patients and healthy donors in both plasma and urine cfDNA data and between analytes. FRENDS model (26 features), MIDS model (94 features) and ensemble stacked model (built from FRENDS, MIDS and fragment length features obtained from previous publications) showed an AUC of 0.98, 0.94 and 0.99 in train (n=279), and 0.97, 0.86 and 0.96 in test (n=183) data from in-house sWGS plasma data (0.4X depth), with AUC of >0.97, >0.84 and >0.96 in early stage (stages I and II) disease and AUC of >0.99, >0.91 and >0.99in late stage disease. FRENDS scores derived from the 26 FRENDS features showed a significant difference in patient outcomes. The same feature sets from FRENDS and MIDS showed good performance in external datasets (DELFI (n=504, 1.5X; FRENDS AUC - 0.96; MIDS AUC - 0.94; Ensemble AUC - 0.98); Heitzer (n=401, 0.2X; FRENDS AUC - 0.99; MIDS AUC - 0.98, Ensemble AUC - 0.99)) and in-house urine sWGS cfDNA data (n=92, 0.4X FRENDS AUC - 0.97; MIDS AUC - 0.99; Ensemble AUC - 0.99). All stages (including stage I) in the DELFI dataset show > 0.92 AUC. Conclusions: Using fragment end and position properties for detecting cancer has shown promising results. An ensemble model built from these properties can be a valuable tool for early cancer detection, detection of minimal residual disease, and monitoring of patients in clinical settings.Item Open Access Tissue adaptations to colitis influence neoplastic risk through clonal interactionMoutin, ElisaDespite a significant fraction of worldwide cancer cases being linked to chronic inflammation, full understanding of the neoplastic process in this context is lacking. The profound tissue remodelling described in chronic inflammatory diseases suggests that alteration of reciprocal signalling between the epithelium, stroma and extracellular matrix participates in the pathogenesis of such illnesses. With the work presented in this thesis, I aimed to determine the impact of tissue alterations on neoplasia in the context of Inflammatory Bowel Disease. Tissue adaptations were described in the *Muc2KO* model of colitis, including profound remodelling of the extracellular matrix compartment pre-pathology and during active disease. Upregulation of small-leucine-rich proteoglycans was found to be an early adaptation to colitis, whilst this class of proteins was seen downregulated in advanced disease. At this later stage, modulated interactions were identified between matrisomal ligands and receptors mapping to recruited cell populations, revealing the extent of matrisomal orchestration of tissue remodelling and highlighting their potential as treatment targets in the context of Inflammatory Bowel Disease. Colitis in the *Muc2KO* model triggered epithelial adaptation in the form of neutral clonal expansion necessary for tissue regeneration. Introduction of cancer-driver mutations in *Trp53* and *Kras* in a *Muc2KO* background had limited impact on survival and pathology, which drew interesting parallels between tissue regenerative processes and cancer initiation. Such processes were explored in depth after chemical mutagenesis in the *Muc2KO* model, where both anti-inflammatory and cancer-driver mutations were shown to benefit from the permissive chronically inflamed environment for expansion. Regenerative expansions did not seem to compete with cancer-drivers for space, and evidence was presented for potential synergy between the two types of expansions. Therefore, early intervention in IBD is key to prevent tissue adaptations that will lead to colitis-associated-cancer. In this context, remodelling of the extracellular matrix such as upregulation of small-leucine-rich proteoglycans could be an indicator to start treatment immune modulating treatment, whilst the switch to their downregulation could indicate the need for more aggressive intervention, such as surgery.Item Embargo Characterising the crosstalk between pancreatic fibroblasts and group 2 innate lymphoid cells in pancreatic homeostasis, inflammation, and neoplasiaYip, ChiGroup 2 innate lymphoid cells (ILC2) are important orchestrators of type 2 inflammation with established roles in anti-helminth immunity and allergic disease. More recent studies have shown both pro- and anti-tumourigenic functions, indicating our incomplete understanding of their role in cancer. Another emerging notion within the field is that ILC2 interactions with cells of mesenchymal origin, including fibroblasts, are critical for their functions in homeostasis and inflammatory states. Furthermore, immune niches enriched in fibroblasts and type 2 immune cells have recently been described in various homeostatic and disease contexts. Given the advancements in our appreciation of fibroblast heterogeneity in steady and perturbed states, as well as the importance of fibroblast contributions to pancreatic pathologies, in this thesis I characterised fibroblast and ILC2 populations in the pancreas and defined the mechanisms of their bi-directional communication in homeostatic, inflamed, and neoplastic states. The majority of pancreatic ILC2s were localised together with a subset of *Pi16+Ly6c1+Dpp4+Il33+* fibroblasts within an unappreciated interstitial niche beneath the submesothelial surface and within the interlobular septae of the exocrine pancreas. Computational and experimental evidence suggested that these *Pi16+* fibroblasts serve as progenitors for parenchymal *Col15a1+* fibroblasts that bear resemblance to pancreatic stellate cells. In homeostatic conditions, ILC2s were associated with increased fibroblast abundance; upon activation, ILC2s were found to promote proliferation of *Pi16+* fibroblasts while restraining *Col15a1+* fibroblast numbers. Mechanistic profiling using *in vitro* and *in vivo* models suggested that IL-13 and other ILC2-derived factors promoted fibroblast proliferation, while oncostatin M production by activated ILC2s might be responsible for inducing apoptosis in *Col15a1+* fibroblasts. This ILC2-dependent signalling axis was found to promote fibroblast progenitor proliferation in mouse models of caerulein-induced acute pancreatitis, potentially to restore fibroblast numbers after acute injury. I next investigated how ILC2-fibroblast signalling was affected in mouse models of pancreatic adenocarcinoma. In preneoplastic pancreatic lesions, ILC2 numbers were found to be substantially increased, with a dramatic local co-expansion of *Dpp4+Ly6c1+* fibroblasts in the desmoplastic peritumoural stroma. Using orthotopic implantation models of pancreatic cancer, I demonstrated that the presence of ILC2s resulted in increased abundance of inflammatory cancer-associated fibroblasts (CAFs) that may promote tumour progression. Together, these data define a role for ILC2-fibroblast interactions for their mutual support in homeostatic conditions, that may be abnormally activated in pancreatic cancer.Item Restricted Determining the Role of Hedgehog Signalling in Natural Killer Cell FunctionClark-Leonard, Stephen[Restricted]Item Open Access Exploring the adaptive immune response to Oncogene-Induced-SenescenceGough, Sarah; Gough, Sarah [0000-0001-8697-9659]Clinical studies have shown that chronic liver disease, a major risk factor for hepatocellular carcinoma (HCC), is associated with the accumulation of senescent cells within the liver. Senescence is an acutely tumour-suppressive, but chronically pro-tumorigenic stress-responsive cell pathway. Senescent cells signal to immunocytes through a complex secretome, to trigger their own CD4+ T-cell dependent destruction, termed senescence surveillance. The form and functionality of this adaptive immune reaction and why it fails when cancer develops remain unclear. In this work, I have studied the adaptive immune response to senescence using in vitro and in vivo models. I have used hydrodynamic tail vein (HDTV) delivery of NRASG12V-containing transposons in mice, to model hepatocyte oncogene-induced senescence (OIS) combined with multiple downstream assays to interrogate four elements of adaptive senescence surveillance: functionality of T-helper subsets; their transcriptomic profile; antigen-reactivity; and adaptive cell-in-cell mechanisms which are laid out in separate results chapters of this thesis. We found that OIS hepatocytes were actively cleared at day 9 post NRASG12V-senescence induction. This clearance was dependent on functional adaptive immunity, evident from in vivo studies of immunodeficient Rag2(-/-) mice. Through analyses of lineage commitment of T-helper cells (functional studies of Th1, Th2, Th17 and T-regulatory cells) I demonstrated that Rorγt, a transcriptional regulator of Th17 cells, was more abundant in hepatic CD4+ T-helper subsets during senescence immune surveillance compared to the control. A Rorc(-/-) (the gene encoding Rorγt) murine model, lacking Th17 cells displayed blunted senescence clearance, implicating its functionality in the senescence surveillance of OIS hepatocytes. I used Single-cell RNA sequencing of intrahepatic T-cells during senescence surveillance to understand potential functional and phenotypic correlates of successful anti-cancer immune response. This found that the Rorc+-expressing Th17 cluster of T-cells in the senescent condition downregulated 65 and upregulated 45 gene transcripts. This included Runx3 and Ccl5 involved in tissue residency and HCC-associated hepatic immunosurveillance, respectively. I attempted to utilise the Antigen-Receptor Signalling Reporter (AgRSR) mouse, a novel lymphocyte-activation tracing mouse model that permits dual fluorescence-based lineage tracing of TCR-stimulated lymphocytes to understand adaptive immune cells that are specifically responding to senescence induction. We studied fluorescently labelled cells during the adaptive immune response to OIS hepatocytes in the AgRSR mouse. However, despite functional results linking adaptive immunity to successful senescence surveillance we were not able to identify a significant change in antigen-reactive intrahepatic T-helper cells within the senescent liver compared to the non-senescent liver. Similarly, we were not able to identify a specific enrichment within lineage committed T-helper subsets Th17, Th2, Th1 and T-regs. Unexpectedly, I identified CD4+ punctate regions that co-localised uniquely with senescent hepatocytes. Flow cytometry, immunofluorescence, confocal and live-cell imaging techniques of hepatocyte and T-helper cell co-culture, suggested that senescent cells had the ability of heterotypic engulfment of T-helper cells. Single-cell sequencing of senescent hepatocytes found negligible levels of ectopic Cd4 transcripts. This implied that the Cd4 protein was likely transcribed and translated outside of the senescent cell before uptake into the senescent cell. I conducted further exploratory analyses of sc-RNAseq data from OIS hepatocytes that identified gene transcripts associated with three cell-in-cell mechanisms: cannibalism, emperipolesis, entosis. The results that I have generated here have furthered our understanding of adaptive immunity during surveillance of hepatocyte senescence and led to further questions about the complexity of this process.Item Embargo Functionality of mutant p53 in early tumorigenesisSheekey, Eleanor; Sheekey, Eleanor [0000-0002-1501-550X]TP53, encoding a stress-activated transcription factor, is commonly mutated in human cancers. Most of these mutations are missense mutations which, in the presence of WT-p53 (p53mut/+), can cause loss of function (LOF), dominant-negative (DN) and/or gain of function (GOF) activities. However, mutant p53 is more commonly studied following loss-of-heterozygosity (p53mut/-) which is thought to promote tumorigenesis. The functionality of mutant-p53 in early tumorigenesis (p53mut/+) has been underexplored but it may uncover how the mutation primes a cell for aberrant activities. By modelling the heterozygous expression of p53R175H in unstressed, acute- and chronic-stress conditions (DNA damage induced senescence, DDIS), I have identified potential DN and GOF activities and the underlying molecular mechanism using the latest -omics and chromatin binding assays. Despite the DN activity being strong and p53R175H-dose-dependent in acute stress, the cells show a stress-dependent cell fitness response, with the chronic stress state making the heterozygous p53R175H less fit than WT-cells.Item Embargo Pre-operative ablative radiotherapy in combination with immunotherapy and novel agents in pancreatic cancerBuckley, HannahPancreatic ductal adenocarcinoma (PDAC) is an unmet clinical need and innovative strategies are urgently needed to improve clinical outcome. In pre-clinical models, radiotherapy (RT) is associated with a cascade of immune-modulatory changes within the tumour microenvironment (TME), and combining radiotherapy with immune checkpoint blockade can enhance T cell infiltration and T cell mediated tumour cell death. However, the potential for RT to modulate the tumour immune micro-environment in PDAC, using clinically relevant RT doses is unknown. The aim of this project was to investigate the potential of stereotactic ablative body radiotherapy (SABR) to modulate the pancreatic cancer TME through pre-clinical studies and a window of opportunity clinical study with a view to developing combinations of SABR and immunotherapy for clinical translation. A pre-clinical radiotherapy platform was developed which allowed the precise and accurate delivery of a clinically relevant dose of ablative radiotherapy (35Gy delivered in 5 daily fractions) to mice bearing subcutaneous tumour allografts. In preclinical modes of pancreatic cancer, which varied in the degree and composition of tumour infiltrating immune cells, I demonstrated using flow cytometry and RNA sequencing that the TME is significantly modulated by this dose and schedule of radiotherapy. While radiotherapy appeared to activate an immune response resulting in increased proliferation of cytotoxic CD8+ T cells, this was insufficient to increase intra-tumoural CD8+ T cell infiltration. Instead, immunosuppressive responses dominated in the TME following radiation delivery with an increase in immunosuppressive Tregs, m-MDSCs and ‘M2’ macrophages seen in tumours following ablative radiotherapy. On myeloid cells PD-L1 expression increased following radiotherapy and the expression of CD39 and CD73, key ectoenzymes involved in the conversion of ATP to immunosuppressive adenosine, was increased on Tregs and myeloid cells respectively. However, combining ablative radiotherapy with dual PD-L1 and CTLA-4 blockade or CD73 inhibition with or without PD-L1 inhibition did not improve the anti-tumour effect of radiotherapy. As a prelude to future interventional studies, the PORTICOtrans clinical study in patients with operable PDAC demonstrated the feasibility and safety of taking intra-operative biopsy samples using a trans-duodenal approach prior to complete devascularisation of the tumour and post-devascularisation samples immediately after tumour resection in the majority of patients. There was no systematic difference in immune infiltrate of tumour samples collected at different time points using multiplex immunofluorescence panels. Gene expression analysis using TempO-Seq, a ligation based assay on FFPE tissue, revealed only 0.2% or fewer genes were differentially expressed (p<0.05) in tumour samples when the different sampling time points were compared. The PORTICO-SABR study was designed to evaluate the safety of pre-operative ablative radiotherapy (35Gy in 5 daily fraction) delivered immediately prior to surgery in operable pancreatic cancer and will characterise immunomodulatory changes in the PDAC TME following radiotherapy. This study will provide unique insights into whether the immunomodulatory changes demonstrated in murine models of PDAC are seen in a clinical setting.