Autophagy inhibition enhances PD-1 blockade efficacy in mismatch repair-proficient colorectal cancer.

Colorectal cancer (CRC) remains one of the most common and lethal malignancies worldwide, with metastatic disease accounting for the majority of CRC-related mortality. Immunotherapy with programmed death (PD)-1/PD-ligand 1 (PD-1/PD-L1) inhibitors has shown remarkable success in treating metastatic colorectal cancers (CRC) with deficient mismatch repair (dMMR) but is less effective in cancers with proficient mismatch repair (pMMR). Recent studies suggest that enhanced autophagy may negatively regulate the effects of PD-1/PD-L1 inhibitors in pMMR tumors. This study aims to investigate the role of autophagy in reducing the effectiveness of PD-1/PD-L1 inhibitors and explore whether combining autophagy inhibitors can improve treatment outcomes in pMMR CRC. We utilized CRISPR/Cas9 to delete the mismatch repair gene Mlh1 in CT26 colorectal cancer cells, generating CT26-dMMR cells. The parental CT26-pMMR cells and CT26-dMMR cells were treated with the autophagy inhibitor 3-Methyladenine (3-MA) and Tumor Necrosis Factor-alpha (TnfA) to assess cell viability. Flow cytometry was used to analyze the effects of 3-MA and TnfA on the cell cycle, apoptosis, and immune cell populations. Protein levels related to cell cycle regulation, apoptosis, and autophagy were measured by immunoblotting. Additionally, in vivo experiments were conducted using a cell line-derived xenograft (CDX) model to evaluate the anti-cancer effects of combining 3-MA with the PD-1/PD-L1 inhibitor BMS-1. CT26-pMMR cells were less sensitive to TnfA compared to CT26-dMMR cells. Combination treatment with 3-MA and TnfA significantly suppressed the cell cycle regulator Cyclin D1 and activated apoptotic signaling via elevated Casp3/Cleaved Casp3 and Casp9/Cleaved Casp9 levels. In vivo, co-treatment with 3-MA and BMS-1 inhibited tumor growth more effectively than either treatment alone. Notably, regulatory T cells (Tregs) were reduced, and CD8+ T cells increased, indicating an enhanced immune response. Our findings demonstrate that inhibiting autophagy improves the immunotherapeutic efficacy of PD-1/PD-L1 inhibitors in pMMR colorectal cancer. This study offers a new strategy to overcome resistance to PD-1/PD-L1 inhibitors in pMMR metastatic CRC by combining autophagy inhibition with immunotherapy.