Dunne, Philip

Johnston, Patrick

Kalimutho, Murugan

Longley, Daniel

Van Schaeybroeck, Sandra

BackgroundColorectal cancer (CRC) is the third most common cancer in the UK with around 80% of patients undergoing surgery followed by adjuvant chemotherapy treatment. Among patients with clinicopathologically defined poor stage II and stage III CRC, there are subsets of patients who will not benefit from adjuvant 5-FU or 5-FU/oxaliplatin treatment. In fact, there is preclinical evidence to suggest that some of these patients may actually do worse when treated with adjuvant chemotherapy. The aim of this study was to develop in vitro adjuvant colon cancer models with the ultimate goal to identify novel treatment strategies for early stage CRC.MethodProgressively invasive populations from parental HCT116 were generated using invasion chambers. Analysis of migration and invasion was carried out using the Excelligence system, protein activity/expression using receptor tyrosine kinase array (RTK) and Western blotting, and drug sensitivity using MTT assay and Flow cytometry.ResultsSix sublines of HCT116 cells were generated (I1-6) which displayed an increasing invasive/migratory phenotype along with increasing levels of the mesenchymal markers TWIST and TGF-b, compared to the parental cell line. Interestingly, the invasive I1-6 subpopulations showed decreased sensitivity to 5-FU treatment compared to the parental HCT116 cell line. In addition, exposure to sub-lethal 5-FU(IC20) doses resulted in significant increases in migration in these cell line models. At the molecular level, we found increases in basal activity of a number of RTK and kinases in the invasive sublines, such as HER2 (a phenomenon which is characterized with a more aggressive nature in breast cancers) and STAT3. Silencing STAT3 resulted in significant decreases in migration of the invasive subpopulations.ConclusionWe have generated invasive CRC cells with EMT-like phenotype, altered RTK/kinase profile and resistance to 5-FU treatment. Using siRNA and small molecule approaches, we are now using these models to identify novel treatment strategies in this disease setting

Characterisation of invasive and metastatic subpopulations of colon cancer using a preclinical model

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