Abstract 750: Optimization of cancer-associated fibroblast expansion and co-culture with patient-derived tumoroids.

Abstract Patient-derived 3D cancer models (tumoroids or cancer organoids) maintain key patient-specific mutations and gene expression profiles during in vitro culture. Gibco™ OncoPro™ Tumoroid Culture Medium Kit was developed as an easy-to-use tumoroid culture system for the expansion of tumoroid lines. Consequently, single cell RNA sequencing of established tumoroid models shows decreases in immune, stromal, and endothelial cells compared to the initial tumor samples from which they were derived. The tumor microenvironment (TME), specifically cancer-associated fibroblasts (CAFs), has been shown to contribute to poor prognosis and therapy resistance. Therefore, there is interest in reliably expanding and culturing CAFs from donor cells for integration with tumoroid cultures and in vitro reconstruction of the TME. Here, we optimized culture conditions for CAF generation from donor cancer samples and explored methods for CAF and tumoroid co-culture. We aimed to create a robust workflow to obtain CAFs from dissociated tumor cells procured from Discovery Life Sciences and, when available, fresh tissue resections. Dissociated cells were plated in Gibco™ Human Fibroblast Expansion (HFE) Medium supplemented with 2% Gibco™ Low Serum Growth Supplement in Thermo Scientific™ Nunc™ Multidishes with Nunclon™ Supra Surface. CAFs were passaged when they reached 80-90% confluency and continuously expanded in Thermo Scientific™ Nunc™ EasYFlask™ Flasks with Nunclon™ Supra Surface for up to 8 passages until a bank was established. Flow cytometry was performed on the CAF cultures, and CAFs were identified as being negative for EpCAM, CD45, and CD31. CAFs were considered a line based on morphology, having less than 15% epithelial, endothelial, and immune populations, and obtaining more than 3 cumulative population doublings. We successfully created CAF lines from 5 out of a total of 6 samples, which included lung, breast, and colorectal DTCs or resected tissue. A colorectal tumoroid line was previously engineered with an eGFP lentivirus and used in co-culture studies. CAFs from this same donor tissue were labeled with Invitrogen™ CellTracker™ Red CMTPX and mixed with the engineered tumoroid line. When cultured in suspension in a 50:50 mixture of HFE medium and OncoPro with 2% Gibco™ Geltrex Flex, the stromal cells and tumoroids self-organized with the stromal cells invading around and into the tumoroids, creating large lobular structures after 7 days in culture. In summary, this method can be leveraged for preclinical studies to develop critical new therapies and further explore the role of CAFs in the TME, enabling the investigation of tumor-stroma crosstalk. We anticipate that CAF-integrated models will be valuable for studies exploring mechanisms of drug resistance, metabolic reprogramming, ECM remodeling, and immunomodulation, as well as for evaluating the efficacy of anti-fibrotic or stroma-targeting agents. Citation Format: Shyanne Salen, Colin D. Paul, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Optimization of cancer-associated fibroblast expansion and co-culture with patient-derived tumoroids abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 750.