Surgery (St Vincent's) - Theses

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Subject: chemoresistance × Start date: 2013 × End date: 2014 × Author: LATIFI, ARDIAN ×

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    Molecular phenotypes of ascites cells in drug resistant ovarian carcinomas
    LATIFI, ARDIAN ( 2013)
    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 used to determined invasive tumour behaviours. The significant findings were; • Separation of the epithelial and mesenchymal ascites cells isolated from high-grade EOC patients prior to and following chemotherapy. • Molecular and gene expression profiles of epithelial cells, which were tumourigenic and expressed markers such as CA125, EpCAM, STAT3, Oct4 and E-cadherin, while non-tumourigenic stromal cells exhibited a mesenchymal phenotype and were enriched for CD44, MMP2, MMP9, FSP, CD90, CD105, CD73, N-cadherin and vimentin. • Genetic signatures of the epithelial tumours and stromal cells obtained from gene expression microarray technology applied to CN and CR samples. • EMT and CSCs marker profiles were determined to play an important role in facilitating cisplatin resistance. • The JAK2/STAT3 pathway was implicated in cisplatin resistance EOC cells as the CYT387 inhibitor sensitised EOC cells to cisplatin by suppressing CSC-like phenotype. • Tumourigenicity of cisplatin treated and untreated EOC cells in nude mice - surviving cells exhibited features of EMT- and CSC. Unique populations of EOC cells exhibiting characteristics of CSC-like cells are capable of undergoing cellular changes in line with EMT-like (during cisplatin treatment) and enhanced epithelial phenotypes (in in vivo settings). Together these traits contribute to drug resistance and recurrence, and targeting pathways that facilitate these phenotypes may improve treatment.