Gianluca Mauri

Although microsatellite-instable (MSI) colorectal cancers, reflecting mismatch repair (MMR) deficiency, often respond to immune checkpoint inhibitors, microsatellite-stable (MSS) tumors remain largely resistant. This disparity is typically attributed to differences in neoantigen load. However, whether antigen-independent mechanisms contribute to immune evasion in MSS colorectal cancer remains unclear. To address this, we engineered a model in which MSI and MSS colorectal cancer cells express identical levels of a defined antigen recognized by T-cell receptor–engineered T cells. Despite equivalent antigen presentation, MSS tumors exhibited impaired T-cell activation, reduced cytotoxicity, and resistance to killing. We linked this immune evasion to the MSS tumor secretome, which suppressed immune responses even in immunogenic MSI cells by impairing immune synapse formation. Surfaceome profiling by mass spectrometry identified glycosylation-dependent alterations that impair immune recognition. Our findings demonstrate that MSS colorectal cancer evades immune attack via intrinsic secretome-driven mechanisms, independent of antigenicity. Targeting glycosylation-linked suppressive pathways may restore T-cell responsiveness and improve immunotherapy efficacy in MSS colorectal cancer. Significance:This study reveals that MMR-proficient colorectal cancers resist T-cell attack not only due to low neoantigen load but also through glycosylation-dependent, secretome-mediated immunosuppression. These findings uncover a previously unrecognized immune evasion mechanism and identify actionable pathways to restore immunotherapy responsiveness in resistant colorectal tumors.

Immunohistochemical characterization of patient-derived colorectal cancer organoids, including H&E staining and MMR protein expression.

Effect of MSS and MSI organoid-derived conditioned media on T cell viability, proliferation, IFNγ production, and binding strength to tumor organoids, as well as on immunomodulatory molecule expression in MSI organoids.

Pathway enrichment analysis of membrane proteins upregulated in MSI organoids treated with MSS-conditioned media, and overlap with glycosylation-related proteins.

Intracellular IFNγ production in T cells cocultured with glycosylation inhibitor-treated MSI organoids in the presence of MSS-conditioned media.

Flow cytometry analysis of MHC class I, MHC class II, and HLA-A2 cell-surface expression in patient-derived organoids.

CD137 expression on T cells cocultured with MSS and MSI tumor organoids across increasing antigen concentrations.

IFNγ release in supernatants from T cells cocultured with MSS and MSI tumor organoids across increasing antigen concentrations.

Flow cytometry analysis of PD-L1 cell-surface expression in patient-derived organoids.

Flow cytometry characterization of CD8+ T cells retrovirally transduced with a antigen-specific TCR and validation of antigen-specific reactivity.