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Seventy percent of ovarian cancer patients die due to consecutive episodes of recurrences resulting from the re-growth of ovarian tumor cells resistant to conventional chemotherapies. In an effort to identify chemoresistance mechanisms, we compared the expression of genes in tumor cells isolated from the ascites of advanced-stage serous ovarian cancer patients prior to (chemonaive, CN) and after chemotherapy treatments (chemoresistant/recurrent, CR). A novel, recently published method was used for the isolation of tumor cells from the ascites of CN and CR patients. Illumina HT-12 platform was used to assess the differential expression of genes (DEGs) between the isolated tumor cells from the ascites of CN and CR patients. The identification of DEGs was achieved by comparing the genetic signatures of CN versus CR samples by a mean expression ratio (fold change) of 2 and P < 0.05. Validation of selected genes was performed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The dominant canonical pathways in the CR versus CN tumor cells were determined by Ingenuity Pathway Analysis. Gene expression analysis revealed differential expression of 414 genes, with 179 genes up regulated and 235 down regulated in the CR group. There were significant differences in gene expressions encoding for proteins involved with cancer stem cells, cell-cell adhesion, embryonic development, tumor suppression, immune surveillance, retinoic acid and energy metabolism in tumor cells isolated from CR compared to CN patients. Pathway analysis revealed that changes in cell cycle pathways, prominently those involved with mitosis and polo-like kinase (PLK1), G2/M DNA damage and proteins linked with cell cycle checkpoint regulation associated with chemoresistance. This preliminary molecular profiling, on a small number of patient samples, suggests an important discrimination of genes in the isolated tumor cells derived from the ascites of CN and CR patients. This type of study on a larg

Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.

Epithelial ovarian cancer (EOC) is an aggressive form of cancer diagnosed at an advanced-stage. A manifestation of this disease is the accumulation of ascites fluid in the abdominal cavity. Surgery and systemic administration of platinum/taxol based combination therapy eliminates most tumour cells, however, resistant residual tumour cells eventually emerge. It is hypothesised that cancer stem cells (CSC) together with epithelial-mesenchymal transition- (EMT) and mesenchymal-epithelial transition (MET)-associated mechanisms contribute to drug resistance. The molecular characteristics accompanying these phenotypes have yet to be fully determined. Hence, the aims of this study were to: • separate and characterise the epithelial and mesenchymal ascites cells prior to and following chemotherapy, • apply gene expression microarray technology to chemonaive (CN) and chemoresistant (CR) samples to determine the gene expression signatures of the epithelial tumour and stromal cells, • determine the role of EMT and CSCs marker profiles in facilitating cisplatin resistance, • establish the role of the JAK2/STAT3 pathway in cisplatin resistant EOC cells by utilising a novel inhibitor (CYT387) which inhibits JAK2/STAT3 pathway and sensitises EOC cells to cisplatin by suppressing CSC-like phenotype and • determine the tumourigenicity of cisplatin treated and untreated EOC cells by inoculating nude mice with cisplatin-treated surviving cells exhibiting features of CSC-like cells. Tumourigenic and non-tumourigenic ascites cells were separated by a novel method developed during this study and were characterised by in vitro assays including wound healing, cell growth and chemosensitivity assays. The expression of genetic markers was assessed using immunofluorescence, flow cytometry, Western blot, qPCR and gene expression microarray. Tumourigenicity of EOC cells was assessed by xenograft studies in mouse. Hematoxylin and Eosin staining performed on histology section of mice organs was u

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.