Theses - Cancer Research UK Cambridge Institute
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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.Item Embargo Experimental Design of Single Cell Sequencing ExperimentsBaker, AlexanderThis thesis addresses limitations of current single-cell sequencing technologies and proposes alternative experimental designs to increase statistical power. Single-cell sequencing enables high-throughput and high-dimensional studies of bio- logical systems. This is particularly useful in functional genomics screens that introduce perturbations to investigate and reconstruct the regulatory networks within and between cells. CRISPR screens are the leading method of conducting functional genomics screens due to their specificity, precision and ease of use. Single-cell sequencing and CRISPR screens have recently been integrated to create the first generation of high-throughput and high-dimensional functional genomics screens. However, the development of efficient experimental design is lagging behind technical advances for single cell CRISPR (scCRISPR) screens. To address this challenge, I developed two wet-lab-aware statistical simulators to compare various experimental protocols and assess their performances. I specifically studied (1) how different protocols affect the performance of scCRISPR screens and (2) how to reduce overall sparsity in the data. First, to increase the statistical power of scCRISPR screens, new alternative experimental protocols need to be investigated. However, conducting these experiments is time consuming and expensive; therefore, I developed crisprPower, a statistical simulator capable of simulat- ing scRNA-Seq CRISPR screens and allowing researchers to investigate alternative protocols. Simulations showed that the current experimental design of scRNA-Seq CRISPR screens is underpowered, requiring at least 600 cells to observe the effect of a perturbation compared to targeted panels that only need 100 cells. Second, I proposed an improved single-cell experimental protocol that decreases sparsity. I developed a new simulator, Minerva, to simulate the effects that experimental enrichment protocols (e.g. antibody pulldowns or PCR) would have upon the observed counts of single- cell datasets. Using Minerva, I showed that it is possible to reduce the sparsity of single-cell datasets and measure lowly expressed genes that would have been lost otherwise. My research shows that improving the experimental design of single-cell protocols using theoretical analysis and simulation leads to concrete and easily implemented recommendations that improve scRNA-Seq CRISPR screens and single-cell sequencing methods.Item Embargo Imaging Metabolic Signatures in High Grade Serous Ovarian CancerChia, Ming LiHigh Grade Serous Ovarian Cancer (HGSOC) can be classified by gene copy number signatures into 7 subtypes that have differing prognoses and treatment sensitivities and that show differences in the activities of various signalling pathways. An ongoing clinical study has demonstrated the feasibility of imaging hyperpolarized 13C pyruvate metabolism in ovarian cancer and has demonstrated inter and intra-tumoural metabolic heterogeneity, where metabolic differences were observed between patients and between different tumour deposits within the same patient. Here I compared the use of 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized [1-13C]pyruvate metabolism and positron emission tomography (PET) measurements of 2-Deoxy-2-[18F]fluoroglucose ([18F]FDG) uptake for detecting metabolic heterogeneity between HGSOC patient-derived xenografts (PDXs) that had different copy number signatures.
I showed that differences in glycolytic metabolism between the subtypes, as defined by their copy number signatures, could be detected using hyperpolarized [1-13C]pyruvate but not with [18F]FDG PET. Dynamic Contrast Enhanced MRI measurements showed that the metabolic differences between the subtypes were not due to differences in tumour perfusion. I also investigated whether differences in tumour metabolism could help to predict and detect early treatment response. I compared the use of metabolic imaging techniques (hyperpolarized [1-13C]pyruvate imaging, [18F]FDG PET/CT) with cell death imaging techniques (diffusion-weighted 1H MRI (DWI) and 2H MRSI measurements of [2,3-2H2]fumarate metabolism, measurement of circulating tumour DNA (ctDNA)) to detect early evidence of response to standard-of-care chemotherapy (Carboplatin). Both hyperpolarized [1-13C]pyruvate and [18F]FDG-PET detected response to treatment with Carboplatin, in a Carboplatin-sensitive tumour, before there was a change in tumour volume. Both metabolic imaging techniques were successful in discriminating responding from non-responding tumours. The techniques for detecting cell death were not as sensitive for detecting treatment response, which may reflect a slow accumulation of dead cells post treatment, a lack of knowledge of when the rate of cell death increases post treatment and also because of immune clearance of dead cells. These studies have shown that imaging with hyperpolarized [1-13C]pyruvate has the potential to be used in the clinic to detect the early response of HGSOC patients to treatment.
Finally, I explored the potential of imaging glyoxalase-1 (Glo-1) activity with [2-13C]Methylglyoxal for detecting metabolic heterogeneity between breast and ovarian cancer PDXs. However, Glo-1 activity did not differ between ovarian or breast cancer subtypes and therefore while 13C MRSI with [2-13C]Methylglyoxal has the potential to detect the presence of disease it may not be useful for differentiating between different HGSOC or breast cancer subtypes.
Item Open Access Targeting Vasculogenic Mimicry in CancerPearsall, IsabellaLike any tissue, tumours depend on a constant blood supply to deliver oxygen and nutrients. Tumour neovascularization is achieved most frequently through the process of angiogenesis, in which tumour cells secrete extracellular factors to recruit host endothelium to grow into the neoplastic tissue. A tumour’s dependency on a blood supply made angiogenesis an attractive target for cancer therapy, but disappointingly, anti-angiogenic drugs often times show limited efficacy in the context of cancer, with benefits only being transient followed by relapse and resistance. In retrospect, this is not surprising because it is now well-appreciated that some aggressive cancers are able to supply themselves with blood via an alternate mechanism to angiogenesis known as vascular mimicry (VM). In VM capable tumours, pseudo blood vessels are formed by tumour cells that have acquired endothelial-like properties independently of host endothelium. VM is a marker of poor prognosis and metastasis in patients, however it is still poorly understood at the molecular level. Recent advances have empirically demonstrated that VM drives resistance to anti-angiogenic therapy (AAT), which informs a motivation to identify VM-related targets that can be exploited therapeutically in combination with existing AATs as a rational approach to target both routes of tumour neovascularization to effectively starve tumours of the blood supply needed for survival. To this end, this thesis aims to identify drivers of VM across a range of cell types, with the goal of uncovering common mechanisms that VM- capable cancer cells use to maintain their pseudo endothelial phenotype, to identify nodes that can be exploited therapeutically to improve patient response to AAT. To achieve this, first we explored what triggers VM formation, through the manipulation of oxygen levels and a previously identified master regulator of VM, FOXC2. Next, we established a high-throughput method to sort cells for VM propensity using acetylated LDL uptake and leveraged this to conduct a genome-wide CRISPR screen using a bespoke dual gRNA-tRNA expression system. Through these approaches, we identified a shortlist of candidate genes to functionally investigate for VM capabilities to validate already known and uncover previously unknown VM-related targets, which can be leveraged as a promising therapeutic route towards sensitizing tumours to AATs.Item Embargo Tumour ecology of breast cancer patient-derived xenograftsLerda, GiuliaTumours are complex ecosystems where several neoplastic and non-transformed cell populations (the tumour microenvironment – TME) interact in heterogeneous and dynamic ways. Descriptive studies in patient material, although extremely informative, often lack the temporal and spatial resolution needed to fully recapitulate this variability. Mouse models represent a natural complement to these correlative analyses, allowing longitudinal and spatially-extensive studies as well as perturbation experiments. Among pre-clinical models, patient-derived tumour xenografts (PDTXs) have been largely excluded from ecological descriptions due to the absence of a human TME and to the immune-compromised status of the mouse host. Evidence however suggests that PDTXs might be good candidates for ecosystem analyses because they contain composite tumour and TME compartments where cells may form functionally relevant interactions. To demonstrate this, we used the extensive collection of breast cancer PDTXs established in our laboratory and performed the first in-depth description of ecosystem complexity in these model systems, confirming their potential as study tools for tumour ecology. After a global survey of all 100 available models, we thoroughly analysed a set of 15 xenografts using single cell RNA- and DNA-sequencing (scRNA/DNA-seq), three-dimensional imaging with serial two-photon tomography (STPT) and two-dimensional transcriptomic and proteomic profiling (the latter using imaging mass cytometry - IMC). In this thesis, we will present part of this work, focusing in particular on the phenotypic and spatial complexity revealed by scRNA-seq, STPT, IMC and *in situ* transcriptomics. These analyses detail for the first time the features of the PDTX TME, which contains multiple cell types that significantly overlap with phenotypes described in other model systems and clinical samples. These cells have varying frequencies in different PDTXs and include multiple innate immune populations with tumour-reactive phenotypes. Expanding previous descriptions, we also show pervasive inter- and intra-model transcriptional heterogeneity in the PDTX tumour compartment. By applying ligand-receptor analysis, we study how different populations interact with each other and generate complex ecological dynamics. Using STPT, IMC and *in situ* transcriptomics we show that the geographical distribution of different neoplastic and TME cells is largely model-specific, indicating a concerted organisation of PDTX spatial ecosystems by the malignant cell-autonomous compartment. In agreement with this, we predict multiple exclusive interactions between cancer and stroma and show that regional variations in tumour phenotypes result in heterogeneous TME features within specific samples. Overall, this thesis unveils the phenotypic and spatial heterogeneity of PDTX ecosystems, showing their variable organisation between and within models. By providing evidence of potential interactions between different cell populations, our study suggests the functional relevance of this variation. In general, these findings support a wider application of PDTXs for the study of tumour ecology and have strong implications for the interpretation of xenograft studies and for the characterisation of tumour-host interactions *in vivo*. The future integration of other data modalities (scDNA-seq) and extension to patient data will likely confirm these observations and offer complementary information on the processes controlling phenotypic and spatial cellular variation in cancer.Item Open Access Genetic Subclones Identify Phenotypic Variation in Diffuse Large B-Cell LymphomaWang, BoyaDiffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin’s lymphoma. Many efforts have characterised DLBCL subtypes that account for some of the heterogeneity in clinical outcome following standard therapy. However, not much is known regarding intratumoural heterogeneity. Case studies show that subclones can emerge after targeted therapy and have different drug responses. Thus, our question is: what are the genotypes and phenotypes of DLBCL subclones? We hypothesise that DLBCL tumours contain genetic subclones, with phenotypic variation that reflects B cell development. Here, I present the largest single-cell DLBCL dataset to date, consisting of 91 cases with paired single-cell RNA (scRNA) and single-cell ATAC (scATAC) sequencing. This work identified genetic subclones based on scRNA copy number. Interestingly, 85% of tumours have genetic subclones (mean 2, range 1–6). We identified seven clusters of gene expression signatures, or themes, that characterise intratumoural subclone diversity. Four themes (memory B cell, plasma cell, germinal centre identity, and B cell identity) recapitulate B cells blocked in differentiation and account for most of the phenotypic heterogeneity between subclones. Three additional themes (MYC, cell cycle, metabolic) characterise transitory cell states. I used an independent discovery dataset (n = 562 samples) to associate genetic alterations with theme expression. A key observation was that amplification of REL (which encodes c-REL, a NF-κB transcription factor subunit), is negatively associated with expression of the memory B cell theme in the discovery dataset (t-test, p = 1×10−11) and this was validated in scRNA of genetic subclones (t-test, p = 0.03). Amplification of REL is a recurrent alteration in germinal centre B cell subtype (GCB) DLBCL, and its functional consequences have not yet been characterised. NF-κB transcription factors (c-REL, RELA) have unique functions in regulating normal B cell development. I hypothesise that REL amplification is an oncogenic event that restricts B cells to the germinal centre and prevents memory cell differentiation. I have designed cell-line and mouse models that will be used to test this hypothesis. In summary, my work has identified DLBCL genetic subclones with highly variable phenotypes that may have distinct therapeutic vulnerabilities. It offers a multi-platform single cell resource for the DLBCL community.Item Open Access Tracing clonal cell plasticity in the microenvironment of pancreatic ductal adenocarcinomaKoplev, Simon; Koplev, Simon [0000-0002-8586-5614]The pancreas may regenerate without stem cells by induction of cellular plasticity between differentiated cells. Although essential for the remarkable capacity for pancreas regeneration, this property entails a vulnerability to cancer through acinar-to-ductal metaplasia and dedifferentiation by epithelial-mesenchymal transition, which contributes in largely unknown ways to metastasis in pancreatic cancer. As with most solid tumours, pancreatic ductal adenocarcinoma develops at the interface of cellular plasticity, genomic instability, and tumour microenvironments. However, current methodologies of analysing tumour tissue are unable to measure these modalities jointly. Here, by developing a method for epitope barcoding measured by cyclical imaging of antibody binding with confocal microscopes, this thesis explores the utility of multicolour cell labelling to investigate how polyclonal cancer evolution relates to epithelial-mesenchymal transition. The assembled barcodes consist of combinations of epitopes attached to a fluorophore and a nuclear-localisation signal, which enables cell sorting, quantitative image analysis, and effective decoding by statistical analysis. Using subcutaneous mouse models of pancreatic cancer, this thesis demonstrates that isogenic epitope labelling can detect epithelial clonal expansion events co-localised with fibroblasts in the tumour microenvironment. The epitope barcode constructs were stably expressed in mouse tumours for at least 2-3 weeks, proving feasibility of multicolour lineage tracing with cyclical imaging. By amending the barcodes with 8bp tags sequenced with single-cell RNA-seq, transcriptomic scores for epithelial-mesenchymal transition showed evidence for selectional acquisition of epithelial lineages during subcutaneous colonisation. Such labelled isogenic cell populations were amenable to Luria–Delbrück fluctuation analysis detecting lineages with epigenetic inheritance of cell states. Altogether, epitope barcoding and cyclical imaging form a compelling experimental strategy for investigating the clonality of transdifferentiation in the microenvironment of pancreatic cancer.Item Embargo Targeting the adenosine pathway reshapes a myeloid-dependent tumour microenvironment in pancreatic ductal adenocarcinoma. An opportunity for therapeutic interventionGraziano, VincenzoBackground: The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor with less than 10% of patients surviving more than 5 years from the diagnosis. PDAC is considered relatively resistant to conventional therapies and recently lack of efficacy of immunotherapy has been shown for this aggressive malignancy. Recently a novel immunosuppressive pathway, called adenosine pathway, has been suggested to contribute to the ability of PDAC to evade the immune system and its resistance to immunotherapies (IOT) and cytotoxic treatments, by generating extracellular adenosine (eAdo) as a consequence of the conversion of a molecule of ATP by enzymes called CD39 and CD73. The stimulation of the adenosine receptors by extracellular adenosine on the immune cells has been shown to favour a pro-tumorigenic and tolerogenic microenvironment. Results: Using syngeneic, *in vivo* murine models of PDAC with differential immune infiltration and response to IOT and the KPC mouse model, CD39 and CD73 were shown to be highly expressed on tumour-infiltrating immune cells. The distribution of extracellular adenosine appeared heterogeneous in murine PDAC, with high concentrations in hypoxic areas, enriched for myeloid infiltration. M2 macrophages among other myeloid populations, highly expressed the Adora2a receptor, particularly in the IOT-resistant model. Targeting the adenosine pathway (Adoi) delayed tumour growth and reduced the occurrence of lung metastases in IOT-resistant tumours. Further, Adoi improved the efficacy of combinations of cytotoxic agents or immunotherapy. Genes related to immune modulation, hypoxia response and tumour stroma were downregulated following Adoi. Further tumour-infiltrating M2 macrophages were found reduced following Adoi. Finally, an adenosine-related gene dataset generated by RNAseq was shown to have similarities with the squamous subtype of PDAC identified by Bailey in humans (Bailey P. et al. 2016) and a specific adenosine signature was generated associating the adenosine pathway to a poorer prognosis in human PDAC. Conclusions: The formation of eAdo appears to favour the development of the immunosuppressive and pro-tumorigenic TME in PDAC, contributing to its resistance to conventional and novel therapies. The presence of the adenosine pathway in human PDAC seems to associate with biological features of aggressiveness and be related to a poorer prognosis. Therefore, inhibition of the adenosine pathway may represent a strategy to modulate the TME and improve response to therapy in patients with PDAC.Item Open Access Development of nanobodies as tools to investigate G-quadruplex DNA secondary structuresGalli, SilviaGuanine-rich sequences in DNA can fold into four-stranded alternative secondary structures called G-quadruplexes (G4). G4s are suggested to be involved in key genome functions such as transcription, genome stability and epigenetic regulation, together with numerous connections to cancer biology. The detection G4s in cells has been facilitated by the use of structure-specific small molecules or antibody probes and much work has relied on G4 recognition by a single-chain variable fragment antibody, BG4. Other G4 probes have been reported but generally their usage has been limited to certain applications and in many cases the probes have limited selectivity for G4s. The aim of this thesis was to develop an alternative probe with high affinity and good selectivity for G4s for use in applications for which BG4 in not suitable and to provide an important orthogonal validation of results previously obtained with BG4. A phage-display screen was used to select nanobodies, antibody fragments derived from of heavy-chain only antibodies of Camelid family, that have high affinity and selectivity for the G4 structure from the human *MYC* oncogene promoter (MycG4). Biophysical characterisation confirmed that one nanobody (B11) binds with high affinity to a wide range of DNA G4 conformations *in vitro*. B11 successfully confirmed the folding of G4s in chromatin isolated from cancer cells. Furthermore, mutational analysis coupled with molecular dynamics simulation identified specific arginines contributing to the interaction of B11 with MycG4. Lastly, I demonstrated that B11 can be expressed in human cells for use in experiments to map G4 genomic locations *in situ*. The results from these experiments are significant to the field as they have demonstrated the validation of earlier data describing the location of folded G4 structures in the human genome. I have also extended previous studies by gaining molecular insights in the mode of action of an antibody-G4 interaction. Finally, I have provided initial proof-of-principle that a G4 antibody can be deployed in a cellular context to probe G4s in living cells. In sum, I have successfully generated a novel tool for the future study of G4s in key cellular processes, such as transcription, and in diseases such as cancer.Item Embargo Investigating Tumour-Immune Cell Interactions in Paediatric MalignanciesMasih, Katherine; Masih, Katherine [0000-0002-0809-668X]There have been significant successes in the treatment of childhood cancer, with the overall survival rate reaching nearly 80%. However, certain leukaemias and solid tumours, as well as metastatic and relapsed cancers, remain resistant to current therapeutic approaches. These patients endure aggressive therapies and severe treatment-related toxicities, often with little chance of a cure. Therefore, novel, less toxic therapeutics with increased efficacies are greatly needed. Immunotherapies have emerged as potential solutions to these challenges. However, not all patients respond to these treatments, and reliable biomarkers that could guide the accurate use of therapy are lacking. In particular, we lack a complete understanding of interactions between the patient’s immune system and tumour. We hypothesised that biological properties of tumours that do not respond to immunotherapy can be detected prior to treatment and serve as biomarkers for patient selection. Furthermore, we hypothesise that tumour cell phenotype, including the diagnosis and genomic background, and prior exposure to therapy can shape the tumour immune microenvironment (TME) and that deep profiling of the patient TME could unmask clinically relevant biomarkers of response and inform future trials. This thesis aimed to both understand non-response to immunotherapies and to deeply profile the TME using established and novel multi-omic analysis platforms on patient samples. To test these hypotheses, we employed three cancer models from paediatric patients. To identify tumour-intrinsic mechanisms of resistance to cellular immunotherapy, we studied B-cell acute lymphoblastic leukaemia treated with CD19 CAR T-cells. To investigate the TME we utilised bulk RNA-seq from patient medulloblastomas and performed highly multiplexed immune focused imaging of paediatric neuroblastoma. We discovered a distinctive methylation pattern and associated stem-cell epigenome as a novel candidate pre-treatment biomarker of non-response to CD19 CAR T-cell therapy in paediatric acute lymphoblastic leukaemia. We next utilised well established deconvolution methods to investigate the TME in medulloblastoma and discovered five transcriptionally distinct clusters, including one that was composed of WNT, SHH, Group 3, and Group 4 tumours, which were associated with differential TME landscapes. We next developed novel immune focused imaging panels to investigate the spatial TME in neuroblastoma. We found that the frequency and spatial distribution of immune cells is influenced both by tumour genotype and exposure to chemotherapy. Finally, we found that high pre-treatment expression of PTEN, TIM-3, CD127, and markers of NK-cells and M1 macrophages and low expression of VISTA and CD40L were associated with improved overall survival. Collectively, this work provides novel insights into tumour-immune interactions across paediatric malignancies. Furthermore, we identify promising biomarkers to be further validated for risk stratification, for predicting response to immunotherapy, and to be utilised as novel therapeutic targets in the TME, which could improve outcomes for children with cancer.Item Embargo Targeting the DNA damage response in hormone-driven cancersKolyvas, EmilyGenomic instability is a hallmark of cancers including breast cancer (BC) and prostate cancer (PCa). Defects in the DNA damage response (DDR) as well as replication stress are responsible, in part, for driving genomic instability and accumulation of DNA damage. While the resulting alterations can be beneficial for tumour development and progression, it also presents a vulnerability for pharmacologic inhibition. This has been demonstrated previously by PARP inhibitor sensitivity in BRCA-deficient cancers. This has led to the discovery and development of several DDR pathway inhibitors. Understanding the biology and genomic context that could lead to tumour responses to these inhibitors will be instrumental for identifying the patient populations that will benefit most. In this thesis, the DDR is explored as a therapeutic target in hormone-driven cancers. Hormone signalling has been shown to drive DNA repair, and is therefore a worthwhile model to study mechanisms of sensitisation to DNA damaging therapeutics. The androgen receptor (AR) has been linked to radio-resistance in PCa and BC by regulating the expression of DNA repair genes. The interaction between AR and DNA repair cofactors, such as DNA-PKcs, is under active exploration as a potential target for sensitising AR-driven cancers to radiotherapy (RT). Chapter 3 aimed to investigate the intersection between AR and DDR signalling pathways and explore the potential of targeting AR and DNA-PKcs for radio-sensitisation. Results indicate that single-agent treatment with inhibition of AR or DNA-PKcs led to sensitisation, and combination treatment had an additive effect. Proteomics data to determine AR cofactors suggested an interaction between DNA repair proteins including DNA-PKcs and AR. Gene expression analyses following combination treatments revealed various signalling pathways implicated in sensitising these models to radiation. Further analysis will be instrumental in validating these pathways and associated TFs with a mechanism for radio-sensitisation in BC and PCa. Furthermore, in Chapter 4, PTEN loss was assessed in BC and PCa as a sensitiser to DNA damage and DDR inhibitors. Nuclear PTEN has been implicated in maintenance of genome stability, and this was tested in PTEN knock-out cell line models. The results show that PTEN loss results in increased sensitivity to ionising radiation along with impaired recovery from exogenously induced replication stress. When screened against a panel of DDR inhibitors, PTEN loss proved to be a predictor of response to inhibitors that sense and respond to replication stress. Interestingly, when assessing DDR inhibitor response in BC PDXs, nuclear PTEN protein expression, but not mRNA expression, is a predictor of response. This suggests that nuclear PTEN could be a useful biomarker of response to these inhibitors. Additionally, RNA sequencing of a PTEN knock-out model suggested increased immune activation. This could suggest a role for combination of DDR inhibitors with immune checkpoint blockade in this model. To test these hypotheses, a CRISPR knock-out of PTEN in a BC patient derived xenograft (PDX) was developed and characterised. Given the known advantages of PDXs for preclinical drug development, generation of a CRISPR knock-out in a PDX is a valuable tool for studying specific effects of single gene loss. Results showed that a stable knock-out was developed that was maintained with passaging and with metastasis, providing a useful model for downstream analysis of DDR effects of PTEN loss.Item Embargo Mechanisms of piRNA-guided co-transcriptional gene silencingEastwood, Evelyn LouiseEukaryotic genomes are populated by repetitive sequences and transposable elements (TEs), mobile DNA sequences that are able to change their position and replicate within a host genome. Heterochromatin establishment at these sequences prevents ectopic recombination and TE mobilisation. In *Drosophila* ovaries, this depends on Piwi, which is guided by a bound small RNA to nascent TE transcripts where it regulates the formation of transcriptionally repressive chromatin states. As well as numerous chromatin modifying proteins, this mechanism depends on the Panx-induced co-transcriptional silencing (PICTS) complex, which contains Panx, Nxf2 and Nxt1. PICTS operates at the interface between piRNA-guided Piwi and heterochromatin effectors. While the N-terminal region of Panx is known to deliver the transcriptional silencing signal, the molecular mechanism by which PICTS induces heterochromatin formation at TE insertions is not fully resolved. In the first part of this thesis, I identified a fourth component of PICTS, Cut-up (Ctp), which I showed is essential for co-transcriptional transposon repression. While initially identified as a component of cytoplasmic dynein, Ctp has been shown to function as a dimerisation hub protein, mediating the assembly and stabilisation of many dynein-independent protein complexes. I demonstrated that the primary function of Ctp within PICTS is to support the assembly of a higher-order complex through dimerisation of the Panx C-terminus. Next, I sought to further explore the molecular function of PICTS using biochemical and structural approaches. I established the expression and purification of full-length and mutant PICTS complexes and showed that PICTS exists as a constitutive homodimer that is able to associate with single-stranded RNA. While structural studies are still ongoing, together this work has contributed to a detailed understanding of the molecular assembly of PICTS. Understanding transcriptional silencing and chromatin regulation more broadly relies on sophisticated tools to profile the genome-wide distribution of histone modifications. In the final part of this work, I established a modified CUT&RUN (Cleavage Under Targets & Release Using Nuclease) protocol that relies on chromatin reader domains fused directly to Micrococcal Nuclease (MNase) and allows rapid and specific antibody-free profiling of histone marks. Together, this work has contributed to a broader understanding of how small RNAs regulate chromatin structure.Item Open Access Prevalence and fate of pro-oncogenic clones in the human colonSkoufou Papoutsaki, Maria NefeliNormal aged tissues are thought to exist as a patchwork of mutations. The detection of cancer-driver mutations in the normal human colonic epithelium had been so far limited. Using immunohistochemistry and sequencing methods, and by screening a large number of patients and crypts, pro-oncogenic clones were detected for eight colorectal cancer driver genes; ARID1A, APC, CTNNB1, FBXW7, KRAS, PTEN, TP53 and SMAD4. Losses or gain of function in these genes were associated with biases in the processes of clone fixation and expansion, which determined their event burden in the tissue. However, by comparing the event burden in the normal tissue and the frequency of the same mutations in colorectal cancer, it was observed that a high mutational burden does not dictate the Availability of clones for Neoplastic Conversion (ANC). For example, TP53 clones had the highest event burden in the normal tissue but KRAS clones had a higher ANC. Some of the factors that could be contributing to the differential fate of pro-oncogenic clones were investigated. An increase in cell-proliferation seemed necessary to induce biases in clonal dynamics but not sufficient to explain the different fates of clones in terms of their ANC. In addition, larger KRAS patches seemed to possess different characteristics from smaller ones, often exhibiting abnormal crypt morphology and containing a reduced frequency of CD4+ immune cells. Finally, a human colonic KO organoid model was used to study the clonal behaviours in vitro and gain insight into the potential mechanisms and pathways that could be mediating mutation-associated advantages. The normal human colon appears to be more mosaic in terms of cancer-driver mutations than originally thought. This means that the status of certain cancer-associated mutations can be reclassified based on their degree of representation in the normal tissue and mutations with a higher probability of initiating cancer can be identified. Thousands of invisible pro-oncogenic clones are inferred to exist in the aged colon but not all of them may have an equal chance for neoplastic transformation. Differential fates were found to exist between clones with mutations in different genes, different amino acid changes in the same gene or even the same amino acid change. This information could in time be used for early-detection efforts by stratifying individuals based on the risk of the mutations that they might possess.