Simple SummaryDesmoplastic tumors are known to be highly aggressive and difficult to eradicate. In these tumors, the interactions between tumor cells and their microenvironment are pivotal in regulating cancer progression. In particular, activated fibroblasts and/or cancer-associated fibroblasts (CAFs) play a key role in cancer-cell invasiveness and in resistance to chemotherapy. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts. As a proof of principle, we investigated the mechanism of action of soluble factors involved in breast cancer progression, such as TGF-β, PDGF, and IL-6. We posit that advanced transwell and spheroid co-cultures provide a pathologically relevant tractable system to study the role of the tissue micro-environment in cancer progression.AbstractThe interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-β) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-β, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-β, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-β and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach.