CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.

Background: Lung cancer is the most common cause of cancer death worldwide and is estimated to account for more than 1,380,000 deaths per year. Lung cancer can be separated into two major histological types: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC), accounting for approximately 15% and 85% of cases respectively. Epidermal Growth Factor Receptor (EGFR) is a tyrosine-kinase receptor. In NSCLC EGFR overexpression is found in over 80% of cases, and EGFR copy number gain (CNG) or amplification is found in nearly 60% of them. Tumours with EGFR mutations can be treated using anti-EGFR drugs; however currently genetic analysis has to be performed on tissue which is obtained by biopsy. Aims: This project aims to investigate alternative methods of obtaining tumour DNA for genetic analysis, to potentially improve or support the current diagnostic process. This project will investigate both new testing methods (molecular assays) and new sources of tumour DNA (cell free DNA from plasma). Methods: A number of methods were employed during this project. Initially EGFR and KRAS mutation detection was attempted using a novel Peptide Nucleic Acid Polymerase Chain Reaction (PNA-PCR) assay devised by GeneFirst Ltd (Oxford, UK). The second approach utilised custom designed TaqMan Array 384 well plate assays for the detection of EGFR, KRAS, NRAS and BRAF mutations. 40 clinical EDTA blood samples were obtained for the investigation of the use cfDNA for oncogenic mutation detection. Plasma DNA extracted using two automated platforms (Qiagen EZ1 and Promega Maxwell). The extracted DNA was analysed using the Ion Torrent Next Generation Sequencing (NGS) platform. Results: The GeneFirst novel PNA PCR assays appeared to tolerate low concentration FFPE DNA samples but had a very high false positive rate and the endogenous control assay failed regularly (0- 33.3% failure rate over different assay versions). The TaqMan Array assay was very successful at detecting EGFR, KRAS, NRAS and BRAF mutations from FFPE tissue, displaying 97.62% and 94.74% concordance with previously used diagnostic assays (Qiagen Therascreen EGFR RGQ PCR and Thermo Fisher KRAS castPCR). For the automated isolation of cfDNA, the Promega Maxwell instrument gave consistently superior results to the Qiagen EZ1. CfDNA was successfully used to detect oncogenic mutations using both PCR and NGS assays. Conclusion: This project has utilised a number of approaches in order to investigate new approaches for the detection of clinically actionable oncogenic mutations, both in FFPE tissue (obtained through surgery or biopsy) and the relatively new cfDNA analyte. Two PCR techniques were compared using DNA from FFPE tissue, and the TaqMan Array assay was shown to be vastly superior. The TaqMan Array was subsequently adopted as the primary diagnostic assay in UHCW Pathology. CfDNA (despite the limited number of samples) showed great potential as an alternative for tissue for detection actionable cancer mutations. The Ion Torrent Next Generation Sequencing system proved to be the most sensitive and powerful technique of the ones utilised here, and will prove an invaluable asset for future development of this work.

Colorectal cancer (CRC) is the one of the commonest causes of cancer and cancer related death worldwide. Its aetiology is linked to a number of reversible and irreversible genetic and environmental factors, including age, sex, genetics, smoking and diet. There has been a drive in recent years for non-invasive biomarkers for many malignant and non-malignant diseases across multiple medical specialties. One of the areas of interest is the detection of volatile organic compounds (VOCs) in various bodily substances by means such as mass spectrometry and electronic noses. CRC patients have been shown to be distinguishable from healthy controls using urinary VOC detection in several studies, including two published by the research group at UHCW and the University of Warwick. There has also been much interest in recent years into the role that the intestinal microbiome plays in health and disease in humans. The aim of this thesis was to characterise the urinary VOC and stool microbiome profiles of CRC patients, their spouses and first degree relatives. The aim being to determine whether the urinary VOC profiles could be distinguished using this technology and to try and better understand the underlying mechanism which lead to CRC carcinogenesis. The first degree relatives and spouses were selected as “common gene pool” and “shared environment” control groups respectively. This work was done using an LC-FAIMS-MS hybrid machine to detect urinary VOCs and 16s RNA sequencing using an Illumina Miseq platform. Comparisons were also made between pre-treatment and post-treatment CRC samples to try and determine if there was any change in either VOC or microbiome profiles after CRC treatment. The urinary VOC profiles of CRC subjects could be distinguished from both sets of healthy controls using a 5-fold cross validation and sparse logistics regression and Random Forrest statistical classifiers, achieving sensitivities of 63-69%, specificities of 64-69% and AUC 0.71-0.72. No statistically significant differences could be found in the urinary VOC profiles of pre-operative and post operative samples. Microbiome analysis revealed over 1300 operational taxonomic units (OTUs), with a similarity of >93% between the CRC samples and the control groups, with significantly different bacterial abundances identified in only 82 OTUs (6.2%), mainly Clostridiales bacteria. Pre-treatment and post-treatment sample analysis revealed differences of 17 (3%) and 22 (4%) OTUs at 3 and 6 months respectively, again principally clostridiales. This thesis provides further data on the microbiome composition in CRC. It also provides further proof of the utility of urinary VOCs, for the first time here using LC-FAIMS-MS technology, a variant of the previously utilised FAIMS technology, as a non-invasive biomarker for CRC.

Central chondroid bone tumours are one of the most common primary bone tumours. Benign central chondroid tumours are termed enchondromas and its malignant counterpart are called chondrosarcomas. Enchondromas are frequently observed on routine imaging. Similarly, chondrosarcomas are the second most common primary bone tumour after osteosarcoma. Imaging is crucial in the diagnosis of central chondroid tumours and in the differentiation of enchondromas from chondrosarcomas. Furthermore, imaging plays a vital role in the staging of chondrosarcomas. In this thesis, the published scientific literature on the role of imaging in the diagnosis of benign chondroid tumours and chondrosarcomas and the role of imaging in the staging of chondrosarcomas is reviewed and summarised. Furthermore, the contribution of the authors’ published work is highlighted in the thesis. The first two articles are review articles which discuss the clinical and imaging features of benign and malignant chondrogenic tumours and the significance of imaging in the diagnosis of these tumours. The third article is an original article which investigates the theory of the pathogenesis of enchondromas. It is widely believed that enchondromas arise from cartilage islands which are displaced from the growth plate during the process of skeletal maturation. However, this theory is unproven, and the origin of this theory was forgotten prior to the authors’ study. Based on the incidental prevalence of enchondromas of the knee in the adult population of 2.9%, the study assesses the prevalence of cartilage islands/enchondromas in skeletally immature patients. In this study, no cartilage islands/enchondromas in skeletally immature patients were identified. The study therefore shows the rarity of enchondromas in skeletally immature individuals which is in contrast to the adult population. Furthermore, in view of the absence of cartilage islands in this study, the study raises doubts about the validity of the unproven theory. Lastly, the very origin of this theory is rediscovered in this thesis which has been forgotten in modern medicine. The fourth article is an original article which evaluates the role of diffusion-weighted MRI (DWI) in the diagnosis of central cartilage tumours. Prior to the authors’ study the role of DWI in the diagnosis of central cartilage tumours was uncertain. The authors’ study demonstrates that DWI cannot be used to differentiate between enchondromas and chondrosarcomas and that DWI does not aid in the distinction of low-grade chondroid tumours from high-grade chondrosarcomas. This is a finding which was not known prior to the study. The fifth article is an original article which assesses the utility of conventional MRI in the differentiation of low-grade from high-grade chondrosarcomas of long bone. Prior to the authors’ study the role of conventional MRI in the differentiation of low- grade from high-grade chondrosarcomas of long bone was unknown. The authors’ study shows that bone expansion, active periostitis, soft tissue mass and tumour length can be used to differentiate high-grade from low-grade chondral lesions of long bone on conventional MRI. Furthermore, the presence of these four MRI features shows a diagnostic accuracy of 95.6%. These findings were not known prior to the study and have significantly furthered the knowledge about the role of conventional MRI in the grading of chondrosarcoma of long bone. The sixth article is an original article which evaluates the role of bone scintigraphy and Computed Tomography of the chest in the staging of chondrosarcoma of bone. Whilst guidelines regarding the staging of bone sarcomas state that bone scintigraphy should be performed to assess for the presence of skeletal metastases and that Computed Tomography (CT) of the chest should be performed to evaluate for possible pulmonary metastases, there has been no research on the utility of bone scintigraphy in chondrosarcoma of bone and on the role of CT-chest in the staging of chondrosarcomas. Furthermore, the prevalence of skeletal and pulmonary metastases of chondrosarcoma at presentation was unknown prior to this study. The authors’ study demonstrated no skeletal metastases on bone scintigraphy in chondrosarcoma of bone at presentation. In contrast, pulmonary metastases were observed in approximately 5% of all patients with chondrosarcoma at presentation on CT-chest. The finding therefore demonstrates the rarity of skeletal metastases in chondrosarcoma of bone at presentation which is in contrast to osteosarcoma and Ewing sarcoma. The study therefore concludes that there is little role for skeletal scintigraphy in the surgical staging of chondrosarcoma. In contrast, the study shows that there is a role for CT-chest in the staging of chondrosarcoma. These above described findings are important new findings and represent a significant contribution to the knowledge base regarding metastatic behaviour of chondrosarcomas at presentation and regarding the staging of chondrosarcoma of bone. In summary, the authors’ publications have significantly enhanced and furthered the understanding of the pathogenesis of enchondromas, the role of functional MRI in the differentiation of enchondromas from chondrosarcomas, the utility of MRI in the grading of chondrosarcomas and the role of skeletal scintigraphy in the staging of chondrosarcomas.

During apoptosis, caspase cleavage of ROCK1 removes an auto-inhibitory region yielding a constitutively active kinase fragment. This results in phosphorylation of downstream targets that promote contractile force generation leading to cell shrinkage, membrane blebbing and nuclear disintegration. To address fundamental questions regarding the purpose of ROCK1 cleavage and consequent apoptotic morphological features, a novel mouse model was generated that carries a single amino acid substitution in the caspase cleavage site (D1113A) that converts ROCK1 to a caspase-resistant non-cleavable (ROCK1nc) form. When apoptosis was induced in ROCK1nc cells, morphological features were significantly impaired, although the biochemical apoptotic program itself was unaffected. To understand the biological role of apoptotic morphological features in the maintenance of tissue homeostasis, acute liver damage was induced in mice with the liver-selective genotoxic compound diethylnitrosamine (DEN). Following DEN treatment, there were increased TUNEL-positive apoptotic cells and increased neutrophil infiltration in ROCK1nc mice. Histologically, ROCK1nc livers were more damaged, paralleled by higher serum alanine transaminase levels. We hypothesized that uncleared apoptotic debris undergoing secondary necrosis may release damage associated molecular patterns (DAMPs) that may aggravate liver damage by recruiting neutrophils to the liver. Indeed, inhibiting the cytokine activities of HMGB1 reduced neutrophil infiltration as well as liver damage in ROCK1nc mice. Furthermore, to determine whether defects in tissue damage responses in ROCK1nc mice would affect tumour development, the ROCK1nc mutation was introduced into two different cancer models, specifically the DEN-induced hepatocellular carcinoma and Eµ-myc lymphoma mouse models. Defective caspase cleavage of ROCK1 promoted increased infiltration of CD8+ T-cells in ROCK1nc liver tumours and had a protective effect against tumour development in both tumour models. Taken together, our results indicate that apoptotic morphological features suppress inflammation which helps to maintain tissue homeostasis but enables tumourigenesis.

Prostate cancer is a significant health problem for men in the western world. Of particular concern are patients who present with aggressive, invasive and metastatic disease, and develop lethal castration-resistant prostate cancer (CRPC) following androgen deprivation therapy. The activation of Wnt/β-catenin signalling is a common event in patients with the poorest prognosis, and frequently associated with the loss of PTEN and activation of the PI3K/Akt signalling pathway. However, the molecular basis for the significant impact of these aberrations in prostate cancer remains unclear. By using pre-clinical transgenic in vivo models, we have demonstrated that β-catenin is a potent proto-oncogene that drives prostate cancer tumourigenesis. Concurrent heterozygous loss of Pten exacerbates β-catenin-driven tumour progression and decreases host survival, while tumours are most aggressive when Pten is deleted. By investigating differential gene and protein expression, we have characterised co-operation between β-catenin activation and Pten loss through a complex network of intrinsic and extrinsic molecular events. These drive survival, growth and proliferation signals, and modulate tumour-immune response interactions to evade anti-tumourigenic processes, resulting in aggressive prostate cancer. Furthermore, by examining novel in vivo models of β-catenin-driven CRPC, we have indicated that β-catenin may promote treatment-resistance through androgen receptor (AR) reprogramming. We propose a mechanism for β-catenin-driven CRPC that is independent of classical AR signalling, and mediated through significant upregulation of canonical and non-canonical Wnt pathway components, which may be effectively targeted by Wnt inhibition. In summary, this thesis highlights a number of potential biomarkers and molecular targets that may be exploited to develop new strategies to manage patients with aggressive prostate cancer, to improve prognosis and avoid progression to lethal castration-resistant disease.

Ambient mass spectrometry (AMS) is a new and versatile method for analysing a multitude of different sample types with the benefit of analysis at ambient pressure and the many other advantages that this entails. However, as these techniques are still in their infancy, metrological development of the techniques is essential. This is a critical step before AMS can be used reliably in the application areas in which it has shown great promise. The research in this thesis addresses the development of AMS sources, in particular plasma-assisted desorption-ionisation, PADI. Optimisation and characterisation is fundamental to understanding and developing the technique. Optimisation of PADI is addressed; this includes understanding the effects of different parameters to maximise signal intensities. The power, and temperature, of the plasma is shown to have a significant effect on the fragmentation observed in the mass spectra. This is an important result that is further explored with the use of thermal desorption to aid the analysis of low volatility molecules. The form of the analyte is also an important consideration for analysis by PADI; characteristic ions from powders are easily detected, whereas for thin film samples an analyte vapour pressure of greater than 10-4 Pa is needed. This result provides an indication of the limitations of PADI and what classes of analyte it will be successful at analysing. It is also shown that we can improve signal intensities using a heated sample stage allowing the analytes to be thermally desorbed before being ionised by the plasma. This is an important result for future work, where ambient plasma sources can be implemented as an ionisation source in conjunction with another mechanism, such as thermal or laser desorption, to generate gas-phase ions. A comparison of different ambient methods for personal care products shows the usefulness and also complementarities of PADI with desorption electrospray ionisation, DESI, one of the most established AMS techniques which utilises a different mechanism for desorption and ionisation. This also demonstrates the chemical information that can quickly be gained from these techniques, with minimal sample preparation. DESI is also compared to secondary ion mass spectrometry, SIMS. Vacuum-based techniques such as SIMS are much more established than ambient techniques; it is insightful to understand the advantages that each source can offer, for the analysis of different types of molecule as well as the mass spectral information that can be gained from SIMS and DESI.

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology. Both genetic and environmental factors are known to contribute to disease development. Pathogenesis involves the production of autoantibodies, and the formation of immune complexes, leading to inflammation and destruction of autologous tissue. SLE is a heterogeneous disease both longitudinally and between affected individuals, and is characterised by periods of exacerbation, known as flares, and periods of remission. The ubiquitous human herpes viruses, cytomegalovirus (HCMV), and Epstein-Barr virus (EBV) have been associated with disease by a variety of mechanisms. Data compiled here suggests SLE patients have elevated IgG responses to HCMV and EBV, but unlike healthy controls these responses do not accumulate with age. No association has been found between the carriage of these viruses, or the magnitude of response against these viruses, and any clinical measurements of disease activity. EBV load is 5.4 times higher in SLE patients than controls. Azathioprine treatment is associated with a 4.4 fold rise in EBV load, no other drugs show associations with EBV load. Among SLE patients EBV load is inversely correlated with CD8+ T-cell IFN\(\gamma\) responses, suggesting impaired T-cell responses are the cause of elevated load. HCMV seropositivity is associated with a 7-year delay in development of disease among SLE patients, and a reduction in plasma IFN\(\alpha\) concentration.

γδ T cells play a central role in the detection of epithelial stress as a component of the lymphoid stress surveillance response. Despite their implication in a range of conditions, including several cancers, little is known about how they interact with their antigenic targets, particularly the interaction of γδ TCRs with their ligands. In this thesis I used molecular and structural modelling techniques to characterise recognition of an epithelial stress ligand, EphA2, by a Vδ1+ γδ T cell, MAU. This resulted in a tripartite model of recognition, involving coordinated interaction of EphA2 with both the TCR and its cognate A-ephrin ligands on the T cell, and the identification of a surface patch on the ligand binding domain of EphA2 that potentially represents a TCR binding site. I also performed sequence-level TCR repertoire analysis to assess γδ T cell populations in human colon and liver, and explored, the effect of chronic cytomegalovirus infection on the Vδ1+ γδ T cell repertoire, the first such analysis of its kind. These studies suggested the Vδ2negative repertoire in humans is diverse and largely private, but also highlighted a Vγ5Vδ1 population that was selectively detected in cytomegalovirus-seropositive individuals, and may be involved in cytomegalovirus immunity.

Glioblastoma is a highly malignant and aggressive high grade glioma with a poor prognosis. The low survival rates stem from tumour progression, late intervention, ineffective therapies and drug resistance, requiring new therapeutic and diagnostic approaches. Lipid droplets are dynamic organelles suggested to be influential facets of cancer metabolism and biology in many tumours. In glioblastoma lipid droplets have been associated with hypoxia higher clinical grades and poor survival however the cellular pathways underlying lipid droplet metabolism remain unclear. Using a publically available database of grade 2 ta 4 glioma gene expression, we observed that genes associated with lipid droplet metabolism were important prognostic survival and tumour progression indicators. Moreover, through confocal microscopy, flow cytometry and NMR-based methods, we observed that uptake of exogenous lipids and adipose triglyceride lipase-mediated lipid shuttling produced lipid droplets whilst autophagy was vital to lipid droplet breakdown. ATGL-mediated lipid shuttling was further observed to prevent unsaturated fatty acid oxidative damage. Finally, we investigated the effect of pharmacological lipid droplet manipulation and observed that autophagy inhibition can improve temozolomide and irradiation cytotoxicity. Taken together our data suggests that understanding lipid droplet metabolic pathways may generate prognostic bio-markers of survival and progression and improve current therapies.