Abstract Colorectal adenocarcinoma remains a leading cause of cancer mortality, with chemoresistance and metastatic progression severely limiting treatment options. MUC4, a membrane-bound mucin normally confined to the apical epithelial surface, becomes aberrantly overexpressed across the tumor cell membrane in colorectal and other epithelial cancers. This dysregulated expression promotes immune evasion, cancer survival and metastasis. Therefore, MUC4 serves as a promising candidate for the development of targeted CAR-T therapies. We engineered MUC4-specific CAR-T cells and evaluated their activity against human colorectal cancer cell lines representing distinct clinical features: HT29 (parental), HT29-MTX (methotrexate-resistant), and T84 (metastatic). MUC4 expression was quantified by flow cytometry. Tumor cell responsiveness to IFN-γ and CAR-T-mediated cytotoxicity were assessed in vitro. For in vivo studies, HT29-MTX cells were injected intraperitoneally into NSG mice, creating a highly aggressive and lethal tumor model in which tumor-bearing mice succumb within 30-35 days, to determine the therapeutic impact of MUC4 CAR-T cells. All lines expressed high MUC4 levels. Interestingly, MUC4 CAR-T cells efficiently eliminated HT29-MTX and T84 cells but not HT29 cells. IFN-γ treatment upregulated MHC-I and PD-L1 expression across all lines. However, only HT29-MTX and T84 cells underwent robust IFN-γ-induced apoptosis, paralleling CAR-T cell killing, suggesting that IFN-γ secreted by MUC4 CAR-T cells might contribute to tumor clearance. Strikingly, in an intraperitoneal HT29-MTX tumor growth model, MUC4 CAR-T treatment significantly reduced tumor burden, demonstrating therapeutic efficacy in a setting where chemotherapy typically fails. Mouse necropsy analysis revealed no off-target in vivo toxicities associated with MUC4 CAR-T cell therapy. Our findings establish MUC4 as a potent and clinically relevant CAR-T cell target and demonstrate that MUC4 CAR-T cells control tumor progression in an aggressive human colorectal cancer in vivo model. These results highlight the potential of MUC4-targeted CAR-T cell therapy to fill a critical treatment gap for patients with chemoresistant colorectal cancer and may extend to other MUC4-expressing solid tumors. Funding: This work was supported by Swim Across America Grant 23-1579 to LMRF and an MUSC Specialized Center of Research Excellence (SCORE) 5U54DA016511-18 Pilot Project Award to AC. This study was supported in part by the Flow Cytometry and Cell Sorting Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313). Citation Format: Avik Chattopadhyay, Erin O’Connor, Anna Tingler, Kristi L. Helke, Melinda A. Engevik, Leonardo M. Ferreira, . Development of MUC4-directed CAR-T cells against chemoresistant colorectal carcinoma 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 1541.
Chattopadhyay et al. (Fri,) studied this question.