Abstract 5229: From the patient to the culture plate and beyond: Challenges and opportunities in cancer-associated fibroblast research

Abstract Cancer-associated fibroblasts (CAFs) are a key component of the microenvironment of various solid tumor types. Due to their critical role in tumor progression and therapy resistance, a surge in CAF-related publications is observed exploring the functionality, heterogeneity and potential as therapeutic targets. To facilitate community wide data consolidation and sharing, we initiated a crowdsourcing data-repository together with both national and international collaborators. With this initiative we mapped 85 general and CAF-specific experimental, biological and functional parameters from more than 1000 CAF-related publications from the past decade. This unique set of data indicates a general heterogeneity and underreporting of crucial experimental parameters, related to isolation procedures, culture conditions and CAF characterization. Using single-cell RNAseq we were able to show a phenotypic shift in the CAF population going from the in vivo situation to an in vitro culture. Additionally, we empirically investigated the impact of in vitro aging and hTERT immortalization on the stability of this obtained in vitro CAF phenotype. At the molecular level, single cell RNAseq, bulk proteomics, flow cytometry and Luminex cytokine assay revealed a specific gene/protein and pathway rewiring upon passaging of primary CAF cultures, while immortalization resulted in a loss of heterogeneity in the culture but with gain of phenotype stability. Functional assays evaluating collective migration and type I collagen invasion confirmed the increased phenotypic stability in hTERT immortalized CAF cultures. In conclusion, CAF literature mining combined with empirical evaluation will further support interpretable, reproducible and transparent experimental reporting in the CAF research field. Citation Format: Felix De Vuyst, Sam Ernst, Stephanie Vermeulen, Zihan Xia, Sándor Dedeyne, Olivier De Wever. From the patient to the culture plate and beyond: Challenges and opportunities in cancer-associated fibroblast research [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 5229.