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Despite improvements in tumour response and survival following platinum based therapy in ovarian cancer, both intrinsic and acquired drug resistance remain a significant problem. Mechanisms of acquired drug resistance have been studied extensively in ovarian cancer cells in culture following repeated drug exposure however the relevance of these mechanisms to the clinical situation still needs to be clearly defined. Studies in clinical material have been hampered by the unavailability of sensitive methods to detect the critical drug-induced effects in individual cells. Recently, a modification of the single cell gel electrophoresis (comet) assay has been developed which allows for the first time the sensitive detection of DNA interstrand crosslinks in both tumour and normal cells derived directly from clinical material. In this study the role of DNA crosslink repair as a potential mechanism of clinical resistance to platinum drugs has been examined. Methods for isolating ovarian tumour cells and mesothelial (normal) cells from ascitic fluid, and to prepare a single cell suspension from primary ovarian tumour tissue from surgery have been established. Cells treated ex vivo with cisplatin were examined for crosslink formation and repair (unhooking) using the comet assay. No significant difference in the peak level of crosslinking was observed between tumour and mesothelial cells from an individual patient, or between patients either untreated or previously treated with platinum-based therapy. In the majority of tumours from nine untreated patients little or no repair of crosslinks was evident at 24 hours (mean of 13.6% repair) with seven patients showing less than 10% repair. In contrast, in the majority of ten treated patients a high level of repair was observed in tumour cells (mean 46.5%), with greater than 70% repair in four at 24 hours. Increased interstrand crosslink repair was also observed in a cisplatin acquired resistant human ovarian tumour cell line. Differences in gene expression pattern between the sensitive and resistant cell lines were compared using microarray analysis. The expression of a number of genes, including ERCC1, were consistently elevated in the resistant cell line, which was confirmed using RT-PCR.

Ovarian cancer is a disease which is fatal in the majority of cases. The evolution of surgery and chemotherapy over the past 30 years has resulted in improvements in overall and progression-free survival. However, the rate of relapse in ovarian cancer is very high, suggesting that current treatment strategies are ineffective. Therefore, to overcome the poor prognosis of ovarian cancer, immunotherapeutic strategies have been devised such as the use of anti-CTLA-4 antibody therapy in melanoma. The principle that the immune system can effect either cancer development or clearance has been the subject of debate for over a century. Clinical results of novel immunotherapeutic approaches that aim to exploit and enhance this immunogenicity have had mixed successes such as IL-2 therapy in renal cell carcinoma. It is clear that whilst many tumours possess antigenic component in their make-up, they do not stimulate durable and effective immune responses in vivo. This may reflect the fact that tumours develop a network of escape mechanisms to circumvent tumour-specific immunity. Due to the ineffectual nature of current treatment options and the complexity of the tumour microenvironment a coherent stratagem needs to be composed. This thesis explores, in principle, a contemporary strategy to propagate an anti-tumour immune response within ovarian cancer by using existing drugs in combination to target three different facets of ovarian cancer immunity; Regulatory T cell (Treg) migration, poor release of the tumour associated antigen, MUC1, and reduced cytotoxic T cell (CTL) proliferation. The migration of Regulatory T cell (Treg) to ovarian cancer is principally mediated by the CCR4-CCL22 chemokine receptor-chemokine axis. AZ1, a specific antagonist for the chemokine receptor CCR4, which is highly expressed on Treg, abrogated the migration of these cells to the chemokine. This compound did not alter Treg function suggesting that its activity was specifically against Treg migration. In order to induce an adequate T cell response, sufficient antigen needs to be provided. Camptothecin, a classical topoisomerase inhibitor, demonstrated effective tumour cell death and release of the tumour-associated antigen, MUC1. The increase in tumour antigen release and decrease in tumour load was offset by significant immune toxicity. The incorporation of Camptothecin into a synthetic drug delivery system led to a decrease in immune toxicity while retaining the drug’s anti-tumour activity. Finally, in order to take advantage of tumour antigen release, it would be desirable to stimulate CTL. Imiquimod, the toll-like receptor 7 agonist, widely used in basal-cell carcinoma and melanoma was able to demonstrate a potential enhancement of an anti-tumour response in three ways. Firstly, the drug enhanced the activation and antigen uptake capacity of plasmacytoid dendritic cells. It also had a direct effect on CTL themselves whilst also reducing the suppressive effect of Treg. This thesis illustrates, in principle, the possibility that a poly-pharmaceutical approach can be taken to target ovarian cancer. It indicates that readily available compounds, when used in the correct combination, could be key in developing effective anti-cancer therapy. Future work in this area should focus on using existing chemotherapeutic and immunotherapeutic drugs in combination to illicit enhanced anti-tumour cytotoxicity. Critically, the next step in developing this strategy is to acquire suitable in vivo models. This is key as there is conflicting evidence regarding the efficacy of certain drugs in mice compared to man.

Ovarian cancer is one of the five most common causes of cancer death in women in the USA and UK. It is usually diagnosed when it is well established beyond the ovary in the peritoneum. Intravenous injection of cisplatin is a common palliative therapy for ovarian cancer patients. Intraperitoneal therapy has been shown to improve survival for patients. Poly(lactide-co-glycolide) (PLGA) is a biodegradable polyester which has been proven safe for medical implantation. PLGA microspheres or fibres have been considered in this work as depots for delivering intraperitoneal cisplatin directly to the tumour site. The aims of this work were (1) to develop microsphere depot formulations with improved drug release profiles compared to previous work; (2) Novel cisplatin containing solid and hollow fibres were to be developed and investigated as alternative structures for depot devices; (3) The drug release profiles were to be examined using mathematical models to allow rational comparison of the devices. It was found that cisplatin containing PLGA 65:35 solid and hollow fibres represent a novel, reproducible formulation for encapsulating higher amounts of cisplatin for an equivalent mass of excipient than other polymer formulations. The fibres developed in this study were able to maintain elevated concentrations of unbound cisplatin in the presence of a biological matrix for approximately 100 hours in vitro.