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Stromal fibroblasts are suggested to be a key determinant in the malignant progression of breast cancer. To find an in vitro culture model that best mimics the in vivo tumor microenvironment so we can study the effects of stromal fibroblasts on breast cancer progression, we evaluated several three-dimensional (3D) co-culture models in order to identify the most suitable culture model for our study. The purpose of our study is to co-culturing malignant mouse breast cancer 4T1 cells and murine embryonic fibroblasts (MEF) to form spheroids with matrigel. We found the best culture model for forming the 4T1 aggregates/spheroids was, in the absence of fibroblast, by growing 4T1 cells in the culture wells precoated with matrigel and in the overlay medium containing 2% matrigel. We chose this model as our standard 3D culture to co-culture 4T1 and MEF cells at different ratios. We found that the amount of MEF in the 4T1/MEF mixture affects the morphology of 4T1/MEF aggregates/spheroids: the higher the ratio of MEF in the mixture, the more ductal structures formed among the aggregates, and the more polarized-like alveolar structures they tended to become. Fibroblasts produced protection for the breast cancer cells in the 3D culture, as aggregates/spheroids formed by breast-cancer cells alone were more sensitive to cytotoxic chemo-agents than aggregates formed by the breast-cancer/fibroblast mixture. These results indicate that the selection of a suitable 3D culture model for a particular research focus may be critical to collecting clinically relevant information about tumor progression that involves interplay between different cell types. This 3D co-culture model demonstrated that tumor-surrounding fibroblasts play important roles in distributing and connecting epithelial breast cancer cells in a tumor microenvironment, as well as providing protection for breast cancer cells from chemo-agent killing.

Optimizing a 3D Culture System to Study the Interaction between Epithelial Breast Cancer and Its Surrounding Fibroblasts