Abstract Background: Extracellular matrix (ECM) remodeling is a hallmark of tumor progression, driven in part by cancer-associated fibroblasts (CAFs). The N-terminal pro-peptide of type III collagen (PRO-C3), released during collagen formation, is a circulating biomarker reflecting active tumor fibrosis (Nissen et al., 2022). Elevated pre-treatment PRO-C3 levels have been associated with poor outcomes in metastatic HER2+ breast cancer treated with Trastuzumab (Lipton et al., 2018). Additionally, in a Phase II trial of tetrathiomolybdate in high-risk triple-negative breast cancer, patients who remained disease-free exhibited lower PRO-C3 levels (Liu et al., 2021), suggesting a link between reduced collagen formation and improved outcomes. Methods: To investigate the cellular origin and regulation of type III collagen production in breast cancer, we quantified PRO-C3 in conditioned medium (by competitive ELISA) using primary breast CAFs and primary benign breast fibroblast cultured in Ficoll-containing media with ascorbic acid, according to the in vitro Scar-in-a-jar fibroblast model system (Chen et al., 2019). Fibroblasts were stimulated with profibrotic (TGF-β1, PDGF-AB) or inflammatory (IL-1α, IL-6) cytokines, and treated with antifibrotic agents (ALK5i, Fresolimumab) to assess potential changes in PRO-C3. Results: PRO-C3 was elevated intrinsically in CAFs compared to benign fibroblast. Breast CAFs exhibited high basal PRO-C3 levels and with minimal response to TGF-β1, while benign fibroblasts showed low basal levels with marked induction upon stimulation with TGF-β1. TGF-β1 was the dominant driver of PRO-C3 compared to the other cytokines. Both antifibrotic treatments effectively suppressed PRO-C3 to baseline in both CAFs and TGF-β1-induced benign fibroblast. Conclusions: PRO-C3 reflects both intrinsic and inducible fibrotic activity in breast CAFs and may serve as a surrogate marker of fibroblast activation in breast tumors. PRO-C3 is a clinically relevant biomarker of tumor fibrosis in breast cancer, with elevated circulating levels associated with poor prognosis. Our data support a fibroblast-derived origin of PRO-C3 and highlight TGF-β1 as a key upstream regulator. The observed findings underscore the need for individualized anti-fibrotic strategies in breast cancer management. Integration of PRO-C3 in clinical workflows could enable non-invasive monitoring of fibroblast activity and fibrotic burden, informing prognosis and therapeutic response. References: Nissen et al., 2022, PMID: 35159087Lipton et al., 2018, PMID: 29923614Liu et al., 2021, PMID: 34426581Chen et al., 2019, PMID: 19785660 Citation Format: M. PannoneA. HettichR. VestermarkM. KarsdalN. Willumsen. Pro-c3 as a circulating biomarker of tumor fibrosis in breast cancer: functional insights from studying cancer associated fibroblasts in vitro abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-05-03.
Pannone et al. (Tue,) studied this question.