A 3D co-culture assay to study the interaction of cancer-associated fibroblasts and immune cells in tumor microenvironment

Abstract

Cancer-associated fibroblasts (CAFs) are the most prominent stromal cells of the tumor microenvironment, and their abundance has been linked to poor prognosis in several cancer types. CAFs play multiple roles in tumor progression, including the modulation of anti-tumor immune responses, which makes them a crucial cell type to consider when developing novel immunotherapies. The crosstalk mechanisms between tumor cells, CAFs, and T cells remain poorly elucidated, and the characterization of these interactions requires the incorporation of novel tools into preclinical research. The aim of this thesis project was to develop, optimize and validate a 3D co-culture model tailored for studying the immune cell – CAF crosstalk occurring in tumor microenvironment. In this thesis project, two distinct protocols for 3D spheroid generation were compared and optimized for MRC-5 fibroblasts co-cultured with OV-90 or A375 tumor cells and activated primary T cells. The pellet culture method for single spheroid generation was found to be the most feasible co-culture method and was validated with immunomodulatory treatments (a DGK inhibitor, dasatinib and a PD-1 blocking antibody). Treatment with the DGK inhibitor resulted, as expected, in strong dose-dependent enhancement of T cell function shown by increased IFN-γ production and upregulation of T cell activation markers in these co-cultures. In contrast, an immunosuppressive compound dasatinib caused a reduction in T cell IFN-γ release accompanied by downregulation of activation markers. There was no significant response to PD-1 blockade in the 3D co-cultures. Unexpectedly, MRC-5 cell line appeared to be immune-promoting when co-cultured with OV-90 tumor cells, which was not observed in co-cultures with A375 cell line. The 3D co-culture model developed in this thesis project will allow further studies of the complex immunomodulatory crosstalk of the tumor microenvironment, and could potentially aid in the discovery of novel immuno-oncology targets in the future. The findings suggest that MRC-5 fibroblasts may exhibit a dual immunomodulatory role influenced by their interactions with different tumor cell lines. Additional studies are needed to elucidate the interaction mechanisms occurring in the 3D co-culture.