Abstract 3131: In vitro analysis of collagen deposition in lung cancer-associated fibroblasts using clinically validated biomarkers

Abstract Background: Lung cancer remains the leading cause of cancer-related mortality worldwide, affecting both men and women. This high mortality rate is largely due to the frequent ineffectiveness of interventions and the late-stage diagnosis of the disease, which typically limits available treatment options. Within the tumor stroma of lung cancers, cancer-associated fibroblasts (CAFs) play a crucial role in tumor progression and metastasis by depositing extracellular matrix (ECM) components, such as collagens. To further understand lung cancer biology and support drug discovery, novel tools that replicate the tumor microenvironment are essential. In this study, we developed a pseudo-3D in vitro model incorporating healthy normal lung fibroblasts (lNFs) or lung cancer-associated fibroblasts (lCAFs), combined with clinically validated collagen-based biomarkers, as a translational platform for anti-fibrotic drug screening. Methods: Primary human CAFs from lung adenocarcinoma (lCAFs) and normal lung fibroblasts (lNFs) were cultured for 12 days in ficoll-based media supplemented with ascorbic acid under unstimulated conditions and with TGF-β stimulation. Additionally, cells were treated with the ALK5/TGF-β1 receptor kinase inhibitor (ALK5i) and Fresolimumab, a human monoclonal antibody targeting all TGF-β isoforms. Collagen type I (PRO-C1), III (PRO-C3), and VI (PRO-C6) levels were measured in the cell supernatant on days 3, 6, 9, and 12 using competitive enzyme-linked immunosorbent assay. Results: TGF-β1 stimulation increased type I, III, and VI collagen deposition in both lCAFs and lNFs. Notably, lCAFs showed higher type I collagen formation than lNFs, even after treatment. While untreated lCAFs and lNFs exhibited no type III collagen formation, TGF-β1 treatment significantly induced PRO-C3, with higher levels in lNFs than in lCAFs. PRO-C6 levels were initially comparable in untreated lCAFs and lNFs, yet TGF-β1 stimulation led to a significant increase in type VI collagen in lCAFs compared to a smaller increase in lNFs. Treatment with ALK5i and Fresolimumab reduced levels of PRO-C1, PRO-C3, and PRO-C6 in both lCAFs and lNFs. Conclusion: The pseudo-3D in vitro model using lCAFs and lNFs successfully replicates key aspects of the lung tumor microenvironment, particularly the differential collagen deposition patterns. The model demonstrated that lCAFs exhibit distinct collagen formation responses to TGF-β1, especially in type I and VI collagens, compared to lNFs. Additionally, treatment with ALK5i and Fresolimumab effectively reduced collagen formation, underscoring the model’s potential for preclinical anti-fibrotic drug screening. These findings support the model's application in studying CAF-driven ECM remodeling and assessing therapeutic responses in lung cancer. Citation Format: Annika Hettich, Morten Karsdal, Nicholas Willumsen. In vitro analysis of collagen deposition in lung cancer-associated fibroblasts using clinically validated biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3131.