Characterisation of the interaction between epithelial cancer cells and mesenchymal cells within an artificial stroma

In the current views tumour microenvironment (TME) contributes significantly to tumour progression and promotes metastasis by facilitating epithelial-tomesenchymal transition (EMT) in carcinomas, thought to be a crucial process for the loss of tissue organisation and the gain of motility. Cancer-associated fibroblasts represent the most abundant cell type in the tumour stroma and, together with other cells and extracellular matrix (ECM) molecules, support EMT advancement. This thesis investigated how the tumour cells respond to the qualitative changes in the TME composition and which changes contribute to the EMT development, using three-dimensional (3D) in vitro tissue culture. Modified alginate capsules were used to provide biologically relevant 3D in vitro experimental models for the culture of HCT116 colon carcinoma cell line, using hyaluronan (HA) or fibronectin (FN) as the ECM components and two types of fibroblasts. Modified alginate capsules were analysed using cryogenic scanning electron microscopy and epithelial and mesenchymal cell cultures within capsules were characterised using microscopy and viability assays. EMT-associated gene expression changes upon transition from two dimensional (2D) to 3D in vitro culture and upon co-culture in the presence of fibroblasts were analysed using qRT-PCR and correlated with the expression and localisation of E-cadherin, MMP-2 and SLUG/SNAIL protein. The microporous structure of the alginate-based capsules and differences between capsules of different composition were demonstrated. Biochemical methods for cell analysis were modified for the use in the 3D alginate-based bio-matrices. The analysis of cell growth and viability showed that normal lung fibroblasts maintained viability in capsules containing FN, whilst transformed neuroblastoma fibroblasts grew preferentially in capsules containing HA; colon cancer cell line grew in all types of capsules. qRT-PCR analysis of EMT-related gene expression changes in HCT116 cancer cells following transition from 2D to 3D in vitro culture suggested the importance of cellcell contact in all types of alginate-based 3D cultures, by the increase of E-cadherin gene expression, but also showed EMT-like changes in the HCT116 cell genotype. The gene expression changes relatively to 2D monolayer culture were similar upon culture in unmodified alginate capsules or modified with FN or HA, suggesting greater influence of 3D nature of culture than the presence of the ECM component. EMT-relevant gene expression following the exposure of HCT116 to the presence and the paracrine signalling from, normal or transformed fibroblasts demonstrated dramatic EMT-like changes following co -culture with transformed fibroblasts. Of those, the expression of vimentin, SLUG, ZEB1, MMP-2 and -9 was up-regulated whereas the expression of E-cadherin, and integrin as was decreased. Only small changes were detected following co-culture with normal fibroblasts. 3D culture within alginate capsules containing HA resulted in changes in E-cadherin protein localisation, however no differences in protein localisation were seen between HCT116 cells cultured as monoculture or in co-culture with transformed fibroblasts. This 3D in vitro approach may help to understand how cancer cells interact with both adjacent and distant tissues and can potentially be used for therapeutic target validation, in particular for approaches targeting EMT or the metastatic phenotype, prior to in vivo studies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo