Abstract 4274: Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach
Authors: Hettich Annika , Nissen Neel I. , Karsdal Morten A. , Willumsen Nicholas 
Published: March 2024

Abstract
Background: The tumor microenvironment (TME) plays a crucial role in driving tumor development. Among the constituents of the tumor stroma, cancer-associated fibroblasts (CAFs) are a significant component. CAFs are actively involved in tumor progression by modulating the architecture of the TME through increased deposition of various collagens resulting in tumor fibrosis. Several studies have shown that CAFs have heterogeneity within, and between, individual tissues. TGF-β is thought to be the main driver of tumor fibrosis, however, the field lacks a characterization of the specific collagen deposition of CAFs from different tissues. In this study, we investigated the fibrotic activity of CAFs from various tissues by measuring the production of three specific collagen peptides in vitro by use of non-invasive clinically validated biomarkers.
Methods: Primary human CAFs from pancreas (pCAF), colon (cCAF), breast (bCAF) and lung (lCAF) were cultured over a 12-day period in ficoll-based media under both unstimulated conditions and TGF-β stimulation. Additionally, cells were subjected to treatment with the ALK5/TGF-β1 receptor kinase inhibitor (ALK5i). The assessment of collagen I (PRO-C1), collagen III (PRO-C3), and collagen VI (PRO-C6) formation was conducted in the cell supernatant at day 3, day 6, day 9, and day 12 using competitive enzyme-linked immunosorbent assay (ELISA).
Results: For lCAFs, cCAFs, and pCAFs, the addition of TGF-β led to a notable elevation in the levels of PRO-C1, PRO-C3, and PRO-C6 within the supernatant. Conversely, in the case of bCAFs, stimulation with TGF-β did not elicit remarkable alterations in the levels of PRO-C1 and PRO-C3 compared to unstimulated cells. Notably, bCAFs exhibit elevated intrinsic activity (high PRO-C3) even in the absence of TGF-β stimulation whereas lCAFs and cCAFs exhibited relatively low level of PRO-C3. Additionally, across all different CAFs, the introduction of ALK5 inhibitor (ALK5i) to the culture medium resulted in a considerable reduction in PRO-C1, PRO-C3, and PRO-C6 levels, returning them to the baseline of the unstimulated control or even lower. Distinct collagen formation patterns were observed among the various CAFs. CAFs derived from breast cancer exhibited the highest release of PRO-C1 and PRO-C3, while CAFs isolated from colon cancer displayed the highest levels of PRO-C6.
Conclusion: These findings underscore the heterogeneity in collagen production among different CAFs, providing valuable insights into the extracellular matrix dynamics within distinct tumor microenvironments. Moreover, non-invasive biomarkers demonstrate the capability to differentiate between different CAF subtypes. Lastly, our model proves to be a valuable tool for anti-fibrotic drug screening, as evidenced by the consistent suppression of TGF-β1 signaling and reduced levels of PRO-C1, PRO-C3, and PRO-C6 upon treatment with ALK5i across all cases.
Citation Format: Annika Hettich, Neel I. Nissen, Morten A. Karsdal, Nicholas Willumsen. Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4274.