Abstract 6043: Deactivating liver metastatic colorectal cancer-associated fibroblasts with all-trans retinoic acid

Abstract Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment (TME) and are known to support tumor growth and disease progression. Reprogramming the stroma, particularly targeting the pro-tumorigenic functions of CAFs, has emerged as a promising therapeutic intervention to disrupt the supportive environment tumors rely upon. An in-house drug screen of FDA-approved oncology therapies was conducted on patient-derived colorectal cancer CAFs and tumor organoids (PDTOs) to identify compounds that may be repurposed as stroma targeting agents. This preliminary screen identified all-trans retinoic acid (ATRA) as a potential candidate that selectively inhibited CAF proliferation without affecting PDTO viability. ATRA is an active metabolite of vitamin A and can inhibit canonical transforming growth factor beta (TGF-β) signaling, a primary means of CAF activation and maintenance of the pro-tumorigenic TME. To investigate the mechanism underlying ATRA-mediated CAF deactivation, patient-derived liver metastatic CAFs were treated with ATRA for 5 days and analyzed at the transcriptional, protein, and metabolic level. qPCR was performed to assess the expression of glycolytic enzymes, CAF activation markers, and ATRA signaling genes. Western blots quantified expression of SMAD 2/3 and α smooth muscle actin (αSMA), a biomarker of CAF activation. ATRA-mediated alterations to CAF secretomes were validated with ELISAs probing for the following CAF activating cytokines: TGF-β, stromal derived factor 1 (SDF-1), and IL-6. Finally, alterations to the metabolic activity in response to ATRA was measured using Fluorescence Lifetime Imaging Microscopy (FLIM), providing a functional readout of TME normalization. All three patient-derived CAF lines treated with ATRA showed increased expression of retinoic acid receptor β (RARβ), a documented indicator of active ATRA signaling. Similarly, αSMA and TGF-β signaling were reduced across all lines in response to treatment. Secretome profiling was also consistent across patient lines, with ATRA lowering the quantities of TGF-β, SDF-1 and IL-6 released from CAFs. However, qPCR analysis revealed heterogeneity in transcriptional response: two CAF lines showed broad downregulation of CAF activation and glycolytic markers, but one line showed variable results. Finally, at a single-cell resolution, FLIM analysis revealed an ATRA-driven shift in metabolism away from aerobic glycolysis, a metabolic state associated with activated CAFs. Overall, these results suggest that ATRA effectively reprograms CAFs toward a deactivated phenotype but also highlights the inherent heterogeneity of CAFs and the impact this may have on drug response. Citation Format: Brandon Choi, Ken Huang, Yuyuan Zhou, Seungil Kim, Eileen Fung, Shannon M. Mumenthaler. Deactivating liver metastatic colorectal cancer-associated fibroblasts with all-trans retinoic acid 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 6043.