Investigations on the invasiveness of chemo-resistant epithelial ovarian cancer

Abstract

Epithelial ovarian cancer (EOC) is a debilitating gynecological malignancy wherein patients frequently develop resistance to the first-line chemotherapeutic drugs carboplatin and docetaxel, resulting in poor survival rates. In this dissertation, I have investigated how the acquisition of drug resistance molds the invasion of EOC, distinguishing them phenotypically from their sensitive counterparts. Somatic selection experiments involving treatment with pulses of carboplatin were performed to establish isogenic chemo-resistant variants of OVCAR-3 and COV362 lines, which typify high-grade serous ovarian carcinoma (HGSOC), the most aggressive type of EOC. Resistant cells invaded singly and collectively faster within collagen- and laminin-rich extracellular matrices (ECM) than sensitive controls. Experiments involving time-lapse imaging also showed enhanced collective migration of resistant cells. Suspension cultures of drug-sensitive and -resistant cells resulted in distinct spheroidal morphologies, which show differences in adhesivity on extracellular matrices: as sensitive cells went on to form the low-adhesive blastuloid phenotypes, resistant cells continued to exhibit the adhesive moruloid phenotype. Enrichment analyses indicated resistant cells possessed signatures common to epithelial, mesenchymal, and even amoeboid migrative states, which is consistent with the extension of the phenomenon of epithelial to mesenchymal transition to accommodate amoeboid states into the spectrum. Such predictive hybridity was confirmed through 1. an ontologically predicted and subsequently validated alteration in expression of several extracellular matrix (ECM) genes and their interactors, 2. demonstration of elevated mRNA and protein levels of mesenchymal marker Fibronectin and epithelial marker E-cadherin, 3. decreased cell-ECM adhesion, and 4. propulsion-driven amoeboid behavior. When injected intraperitoneally into immunodeficient mice, the resistant cells dispersed to a greater extent than their sensitive counterparts on the parietal peritoneal mucosae. Videographic analysis of cocultures of cancer cells and untransformed human coelomic mesothelia showed faster clearance of the latter by the resistant cancer cells. These results suggest that coevolution of chemotherapeutic resistance and migration in EOC drives its transcoelomic metastasis. In the next part of the study, I have delved into investigating the population dynamics of resistant niches within bulk tumors, wherein drug-resistant cells coexist with sensitive cells despite their relative tardiness in the proliferation rate when cultured separately. Coculture studies using fluorescently labeled drug-sensitive and -resistant OVCAR-3 variant lines reveal that resistant OVCAR-3, in the absence of chemotherapeutic selection pressures, can survive and even out-compete the sensitive cells. This behavior is driven at least in part by the ECM secreted by resistant OVCAR-3 cells, on which sensitive cells slow down their rate of division. These results provide valuable insights into disease progression and the translational consequences of coevolving cancer behaviors. In the last part of the study, gene expression signatures from the comparative transcriptomics of sensitive and resistant cells are used to obtain subline phenotype-specific metabolic models. These models are compared with genome-scale metabolic models to predict crucial reactions whose fluxes establish these distinct phenotypes. In silico gene knockout strategies are then employed to confirm further these flux dynamics' importance to the chemoresistant phenotype. Information on these reactions is used to explore the DrugBank database to identify drugs capable of impairing such metabolic alterations, thereby resensitizing the resistant cancer cells to chemotherapy. I have experimentally validated the effect of one of the predicted drugs, disulfiram, an aldehyde dehydrogenase inhibitor, in resensitizing drug-resistant OVCAR-3 cells to carboplatin. In summary, the thesis provides insight into the specific contributors of aggressiveness and pharmacological vulnerabilities in an evolved experimental model of drug resistance in epithelial ovarian cancer.