An Extracellular Matrix–Producing Subset of Cancer-Associated Fibroblasts Drives Chemoresistance in Breast Cancer via SRC Activation and G0S2 Upregulation

Chemotherapy resistance remains a major hurdle for treating patients with triple-negative breast cancer (TNBC). Although cancer-associated fibroblasts (CAF) as an overall population have been shown to modulate treatment response, innovative approaches are required to decipher which and how distinct CAF populations drive chemoresistance. In this study, by combining analysis of data from patients with TNBC with ex vivo modeling using tumor-on-chip technology, we identified a specific CAF population, the extracellular matrix-producing myofibroblasts (ECM-myCAF), that mediated resistance to chemotherapy. The proportion of ECM-myCAFs decreased after chemotherapy in chemosensitive patients but remained unchanged in chemoresistant patients. In tumor-on-chip models, primary ECM-myCAFs promoted TNBC cell survival under chemotherapy treatment. Single-cell RNA sequencing, advanced cell imaging, and functional assays showed that ECM-myCAFs activated SRC kinases in TNBC cells, likely through secreted factors, and upregulated the apoptosis regulator G0-G1 switch 2 (G0S2). SRC inhibition or G0S2 silencing completely abolished TNBC cell chemoresistance driven by ECM-myCAFs. Altogether, this work reveals the unique role of the specific ECM-myCAF population and identifies G0S2 as a key player in chemoresistance in TNBC. SIGNIFICANCE: Integration of patient data with ex vivo tumor-on-chip modeling identifies an extracellular matrix-producing myofibroblast population that contributes to chemoresistance and can be targeted to improve outcomes in triple-negative breast cancer.