Simple SummaryIn breast cancer, adipose-derived stromal cells (ASCs) and adipocytes, as components of the mammary fat pad, come into close contact with tumor cells. To adequately mimic direct cell–cell interactions between tumor and adjacent stromal cells, a 3D co-spheroid model was developed consisting of ASCs or adipocytes and breast cancer cells (MDA-MB-231, MCF-7). Direct contact between MDA-MB-231 tumor cells and ASCs or adipocytes in this model promoted the expression of C-C motif chemokine ligand 5 (CCL5) and specifically the corresponding receptor C-C chemokine receptor type 1 (CCR1). This, in turn, enhanced the migration of triple-negative MDA-MB-231 breast cancer cells. Such tumor-specific markers up-regulated upon cell–cell contact with adjacent stromal cells may represent promising targets for the detection and treatment of aggressive breast cancer.AbstractThe tumor microenvironment (TME) in breast cancer is determined by the complex crosstalk of cancer cells with adipose tissue-inherent cells such as adipose-derived stromal cells (ASCs) and adipocytes resulting from the local invasion of tumor cells in the mammary fat pad. This leads to heterotypic cellular contacts between these cell types. To adequately mimic the specific cell-to-cell interaction in an in vivo-like 3D environment, we developed a direct co-culture spheroid model using ASCs or differentiated adipocytes in combination with MDA-MB-231 or MCF-7 breast carcinoma cells. Co-spheroids were generated in a well-defined and reproducible manner in a high-throughput process. We compared the expression of the tumor-promoting chemokine CCL5 and its cognate receptors in these co-spheroids to indirect and direct standard 2D co-cultures. A marked up-regulation of CCL5 and in particular the receptor CCR1 with strict dependence on cell–cell contacts and culture dimensionality was evident. Furthermore, the impact of direct contacts between ASCs and tumor cells and the involvement of CCR1 in promoting tumor cell migration were demonstrated. Overall, these results show the importance of direct 3D co-culture models to better represent the complex tumor–stroma interaction in a tissue-like context. The unveiling of tumor-specific markers that are up-regulated upon direct cell–cell contact with neighboring stromal cells, as demonstrated in the 3D co-culture spheroids, may represent a promising strategy to find new targets for the diagnosis and treatment of invasive breast cancer.