Modeling cancer-associated fibroblast plasticity and breast cancer interactions using patient-derived scaffolds.

Breast cancer progression is intricately influenced by the tumor microenvironment (TME), where cancer-associated fibroblasts (CAFs) play a central role in modulating tumor behavior. To model these interactions, we employed patient-derived scaffolds (PDSs) with preserved native extracellular matrix (ECM) architecture to investigate fibroblast activation and tumor-stroma crosstalk. Mass spectrometry of 63 breast cancer PDSs revealed distinct matrisome clusters associated with tumor grade, recurrence, and CAF-like enrichment, including myofibroblast (myCAFs) and inflammatory CAFs. Normal fibroblasts cultured on PDSs exhibited progressive activation, marked by upregulation of CAF markers (FAP, CD10, PDGFRα), secretory factors (IL6, HGF), and ECM-remodeling genes. Fibroblasts also displayed transcriptional changes linked to cytoskeletal remodeling and cellular plasticity. In transwell co-culture, MDA-MB-231 cells enhanced fibroblast IL6 secretion and altered fibroblast phenotypes, while PDS-grown fibroblasts modulated cancer cell proliferation and stemness transcriptional programs in a subtype-dependent manner. Spatial context also influenced fibroblast activation in direct co-culture. These findings underscore the dynamic, bidirectional nature of tumor-stroma interactions and support PDSs as a physiologically relevant platform for dissecting CAF heterogeneity, ECM-driven modulation, and their potential impact on breast cancer progression.