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Vascular endothelial growth factor C (VEGFC) promotes tumor progression in several tumor types, mainly through the stimulation of lymphangiogenesis and lymphatic metastasis. The expression and biological significance of the VEGFCNEGFR3 pathway in ovarian cancer growth and dissemination has been investigated in this thesis using ovarian carcinoma cell lines and tumor animal models. In patient-derived ovarian carcinoma xenografts (HOC), high levels of soluble VEGFC in ascites and serum were detected, in association with disease progression and tumor burden. Peak VEGFC expression preceded para-aortic lymph node infiltration by HOCS neoplastic cells. Histological detection of tumor cells in blood and lymphatic vessels indicated both hematogenous and lymphatic dissemination. VEGFC was over-expressed in the VEGFR3-positive and luciferase-expressing lA9 and IGROVl ovarian cancer cells. In vitro. VEGFC released by the expressing turnor cells stimulated turnor cell migration in an autocrine manner. In vivo, over-expression of VEGFC promoted . IGROVl dissemination after orthotopic intraovarian transplantation in nude mice. VEGFC released in serum of mice correlated with tumor burden and metastasis. Cediranib, a small molecule receptor tyrosine kinase inhibitor of VEGFRl-3 and c-Kit, inhibited in vivo metastasis of VEGFC-overexpressing [GROVI and in vitro autocrine effects on turnor cell migration; cediranib improved the survival of mice bearing the four HOC xenografts analyzed. The therapeutic benefit was proportional to soluble VEGFC levels in serum and ascites and to VEGFR3 expression by tumor cells. Different schedules of cediranib were tested on HOC8 xenograft, The survival benefit of cediranib in maintenance regimen (14 weeks) was superior to 3-weeks treatment. A significant prolongation of mice survival was observed in mice treated with advanced turnor. Treatment benefits were improved combining cediranib with chemotherapy (paclitaxel plus cisplatin). These findings suggest that the VEGFCNEGFR3 pathway acts as an enhancer of ovarian cancer progression through autocrine and paracrine mechanisms, hence offering a potential target for therapy.

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Folates are essential in the de novo nucleotide biosynthetic pathway, folate receptor alpha (FR-α) is a folate transporter which is an attractive target for anticancer drug design due to its limited expression in normal tissues. It has great potential to direct therapies toward pathologic cells whilst minimizing toxicity in normal tissues. The enzyme folylpolyglutamate synthetase (FPGS) polyglutamates folates and folate analogues, trapping them within the cell and increasing their affinity as substrates for subsequent enzymatic reactions. Expression of folate biochemical pathway components, such as FR-α and FPGS, may be indicators of malignancy and also determine response to antifolate chemotherapeutics and other folate pathway targeted therapies currently being evaluated. Knowledge of their expression in tumours may enable optimal use by identifying responsive subgroups of patients. In spite of their importance in the diagnosis and treatment of cancer, the lack of monoclonal anti-FR-α and FPGS antibodies suitable for immunohistochemistry (IHC) analysis of formalin fixed, paraffin embedded biopsy samples or Western blot analysis has limited research in this area. The aim of this project was to generate and fully characterize monoclonal antibodies to detect specific expression of these proteins in formalin fixed, paraffin embedded tissues for use on archival tissue and samples collected prospectively in connection with clinical trials of antifolates. Novel monoclonal antibodies with specificity for sensitive detection of FR-α and FPGS in paraffin-embedded tissues were developed. ELISA and Western blot analysis confirmed specific reactivity of both antibodies to the recombinant protein, a single 40kD protein in whole cell lysates from cell lines known to express FR-α, and a single 60kD protein from cells expressing FPGS. Epitope mapping experiments confirmed specificity restricted to a linear epitope present in the protein target sequences. IHC analysis of FR-α in a panel of normal tissues demonstrated predominantly membrane with cytoplasmic staining limited to some ovarian epithelia (inclusion cysts), placental trophoblasts and proximal kidney tubules; FPGS demonstrated a wider pattern of expression. FR-α analysis of a panel of malignant and benign tissues demonstrated limited expression with variable intensities of staining and patterns of membrane and cytoplasmic Development and Evaluation of Novel Monoclonal Antibodies to FR-α and FPGS VI reactivity between cases. In the majority of malignant ovarian tumours, including an archival tissue microarray (TMA) of 167 tumour samples, membrane staining was observed in 89% of cases. FPGS analysis on a panel of benign and malignant tissues demonstrated frequent and high cytoplasmic expression in a range of tumours compared to normal adjacent tissue. The archival ovarian cancer TMA analysis showed a significant association between high expression of FR-α and poor patient survival (p=0.012, Cox regression) indicating a role for FR-α as a prognostic marker and potential therapeutic target for ovarian cancer and other cancers with expression of FR-α, detectable via the use of our antibody on FFPE tumour samples.

Epithelial ovarian cancer (EOC) has the highest mortality rate in the western world among gynecological malignancies. Approximately 70% of patients achieve complete remission after first-line platinum-based therapy, but, unfortunately, almost all patients relapse with resistant disease. Progress has been made in identifying "hallmarks" of cancer, and the presence of cancer stem cells (CSCs) and the epithelial-mesenchymal transition (EMT) have been associated with aggressiveness and chemoresistant properties. The studies reported here were designed to identify a CSC population, and enhance understanding of the role of EMT in the development of drug resistance in ovarian cancer. The expression of sternness markers, in particular the Side Population phenotype, CD133 and ALDH (aldehyde dehydrogenase), were investigated in ovarian cancer cell lines and in human tumors. Unfortunately, in vivo tumorigenic assays demonstrated that none of these markers could be associated with a CSC. On the other hand, I was able to isolate sphere cultures from fresh human turnors, that were highly tumorigenic in nude mice, and more resistant to drugs currently used in therapy (suggesting a role for these cells in drug resistance), and expressed mesenohymal markers, reinforcing the concept of connection between CSCs and EMT. I obtained ovarian cancer xenografts that mimicked their original human tumors ,and characterized them for sensitivity to cisplatin (DDP) by treating mice with two DDP cycles, mimicking the clinic. On the basis of the response to DDP, xenografts were classified as "Responders" and "Non-Responders". Studies performed to clarify the mechanisms at the basis of the different sensitivity showed that some EMT genes were more highly expressed in cisplatin-"Non-Responder" than in "Responder" xenografts. These data have to be validated in a wider panel of xenografis, in human turnors, and could hopefully defme if EMT markers could be considered predictive of the response to therapy in ovarian cancer.