Collagen-derived biomarkers reveal distinct fibrotic responses among cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) are key drivers of extracellular matrix (ECM) deposition and remodeling in the tumor microenvironment (TME), processes that facilitate tumor progression and metastasis. However, CAFs exhibit significant heterogeneity and functional complexity, posing challenges for effective therapeutic targeting. In this study, we evaluated the production of three important ECM proteins - type I (PRO-C1), type III (PRO-C3), and type VI (PRO-C6) collagen - by CAFs in vitro. Four distinct CAFs were cultured in Ficoll-containing media supplemented with ascorbic acid for up to 12 days. Cells were stimulated with profibrotic or inflammatory factors (TGF-β1, PDGF-AB, IL-1α, IL-6) and/or treated with antifibrotic compounds (ALK5i, Fresolimumab). Collagen production was quantified in collected cell culture media using competitive ELISA. Our results reveal distinct fibrotic responses among CAFs. Two CAFs displayed high intrinsic fibrotic activity and minimal additional fibrotic responsiveness to profibrotic stimuli, whereas two CAFs exhibited low intrinsic fibrotic activity and significant increases in PRO-C1, PRO-C3, and PRO-C6 upon profibrotic stimulation. Notably, TGF-β1 was the primary driver of PRO-C3, PDGF-AB was the primary driver of PRO-C6, while IL-1α and IL-6 had no effect on PRO-C1, PRO-C3 and PRO-C6 levels. Antifibrotic treatments with ALK5i and Fresolimumab effectively reduced collagen biomarkers elevated by TGF-β1 to baseline levels or below.These results underscore the heterogeneity of CAFs in ECM remodeling, highlighting the need for tailored therapeutic strategies to target tumors exhibiting high fibrotic activity.