Development of an in vitro cell-sheet cancer model for chemotherapeutic screening

Epithelial cancer grows in vivo in a microenvironment that comprises tumour, stroma, and immune cells. A three-dimensional (3D) culture model might be able to mimic the tumour microenvironment in vivo; therefore, we developed a new 3D epithelial cancer model using in vitro cell-sheet engineering and compared the results of treatment with several chemotherapeutic drugs among the 3D cell-sheet model, spheroid culture, and 2D cell culture. Methods: The cell sheet comprised keratinocytes and a plasma fibrin matrix containing fibroblasts. Cancer spheroids with or without cancer-associated fibroblasts (CAFs) were interposed between the keratinocytes and fibrin layer. Cell growth, viability, and hypoxia were measured using the cell counting kit-8, LIVE/DEAD assay, and propidium iodide and LOX-1 staining. The morphology, invasion, and mRNA and protein expression were compared among the different cell culture models. Results: Enhanced resistance to sorafenib and cisplatin by cancer spheroids and CAFs was more easily observed in the 3D than in the 2D model. Invasion by cancer-CAF spheroids into the fibrin matrix was more clearly observed in the 3D cell sheet. The expansion of viable cancer cells increased in the 3D cell sheet, particularly in those with CAFs, which were significantly inhibited by treatment with 10 mu M sorafenib or 20 mu M cisplatin (P < 0.05). TGF-beta 1, N-cadherin, and vimentin mRNA and protein levels were higher in the 3D cell-sheet model. Conclusions: The 3D cell sheet-based cancer model could be applied to in vitro observation of epithelial cancer growth and invasion and to anticancer drug testing.