Establishment of low passage primary cell lines from colorectal cancer and studies on their biological properties / Melanie Arul Arlusamy

Arul Arlusamy, Melanie (2015) Establishment of low passage primary cell lines from colorectal cancer and studies on their biological properties / Melanie Arul Arlusamy. PhD thesis, University of Malaya.

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Abstract

Colorectal cancer comprises of subpopulations of cells with distinct genotypes and phenotypes that harbor diverse genomic alterations even within the same tumour. Tumour heterogeneity has an implication for biomarker discovery and cancer therapeutics, particularly in the era of targeted treatment. An understanding of tumour heterogeneity is likely to increase with the use of primary cell lines directly from colorectal tumour biopsies and a correlation of the chemoresponses from each cell line to the genetic variation exhibited by each patients. Secondary cell lines purchased from foreign cell culture banks consist of homogeneous entities and may not reflect the diverse spectrum of tumour cells that exists in a population or within an individual. A protocol was designed to ensure routine establishment of primary cell lines following failure in attempts of isolating contamination-free cells by using published methods. Cells were directly isolated from tumour biopsies and cultured in optimized culture conditions. These cells were maintained in ultra-low passages to minimize the chances of their transformation from their original growth characteristics and resemble the primary tumours from which it was derived. Cells from each patient was characterized based on their heterogeneous morphologies and variation in tumour marker expressions. Isolated cells were tested for purity of its tumoral nature with cancer markers such as carcinoembryonic antigen (CEA) and C2 antibodies. The main focus of this study was to analyze the events of drug resistance that has long been identified as a major reason for therapy failure in cancer patients and are thought to be the primary cause of cancer relapse. In order to identify the optimum treatment regimen for the established cell lines, a variety of drug doses and drug combination strategies were analyzed by the Chou-Talalay median-effect principles using secondary cell lines. Cells responded differentially to various drug combinations and monotherapy with synergy seen in specific combinations. The designated doses and combination arrangements were then tested with the established primary cells. Interestingly, primary cells from each patient exhibited significant variation in their chemoresponses. The heterogeneity in drug response converged to focus the attention on identifying the genetic factors underlying the drug resistant variants in primary cancers. The mechanisms that promote or enable drug resistance were investigated with molecular techniques in which a panel of 88 genes were analyzed with real time PCR techniques. Critical factors promoting drug resistance were investigated and this mainly includes drug inactivation, drug target alteration, drug transportation, apoptosis, DNA damage repair and cell cycle. Relatively, chemoresistance as observed in this study was mainly enhanced by involvement of genes participating in repair of DNA damage which promotes cell survival. In this study, the heterogeneity observed within tumours and the relationship of this diversity to drug resistance was analyzed extensively. Studies using primary cancer cells established from each patient helped to increase the understanding of the root of the drug resistance complexities more convincingly than using homogeneous secondary clones. An understanding of the basic molecular heterogeneity of colorectal cancer is essential for the diagnosis and redesigning of targeted treatment for patients.

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