Aggressive Breast Cancer Cell-Derived Conditioned Media Induce Inflammatory and Metabolic Reprogramming of Stromal Fibroblasts towarda CAF-like State

Background: The reprogramming of stromal fibroblasts into cancer-associated fibroblasts (CAFs) plays a critical role in breast cancer (BC) progression by promoting extracellular matrix (ECM) remodeling, angiogenesis, tumor cell migration, and immune escape through paracrine signaling. However, it remains unclear whether and how the metastatic properties of BC cells affect the transition of fibroblasts from a normal to an activated state. Aim: In this study, we investigated whether conditioned media (CM) derived from two BC cell lines with distinct metastatic potential, MDA-MB-231 and MCF-7, drives the conversion of human fibroblasts toward a pro-tumorigenic phenotype, using molecular and metabolomic analyses to dissect the mechanisms of tumor-stroma crosstalk. Results: The exposure to MDA-MB-231 CM led to increased secretion of inflammatory cytokines and chemokines, including IL-6, IL-8, ICAM-1, and MCP-1, and production of ECM proteins. Consistently, we observed the upregulation of FAP, a well-known marker for BCCAFs. These effects were moderate or absent when fibroblasts were treated with MCF-7CM. In parallel, we conducted 1H-NMR metabolomic profiling of culture media. Fibroblasts grown in MDA-MB-231 CM showed a more pronounced shift from oxidative phosphorylation to aerobic glycolysis than those cultured in MCF-7 CM. We also observed increased secretion of alanine and TCA intermediates that support cancer cell feeding and favor immune escape mechanisms. All these metabolic changes are concomitant with MYC upregulation. Conclusions: Altogether, our findings suggest that soluble factors secreted by highly aggressive BC cells promote a functional reprogramming of normal fibroblasts toward a pro-tumorigenic, inflammatory phenotype, ultimately contributing to immune evasion and therapy resistance. Elucidating the symbiotic crosstalk between BC cells and the tumor microenvironment holds promise to open new avenues for targeted therapeutic interventions.