Intratumoral MHC-I Heterogeneity: Spatiotemporal Insights into the Immune Microenvironment

Abstract

The loss of major histocompatibility complex class I (MHC-I) molecules on tumor cells is a critical mechanism of resistance to cancer immunotherapy. However, the role of tumor MHC-I heterogeneity in shaping the immune landscape and therapeutic response remains poorly understood. To model this heterogeneity commonly found in human cancers, we co-injected two tumor cell lines-one with constitutive MHC-I expression and another with its irreversible loss-into mice. We then treated these heterogeneous tumors with a combination therapy consisting of DNA immunization and systemic administration of synthetic oligonucleotides. The tumors were rigorously analyzed using a combination of methods: flow cytometry to quantify cell subpopulations, immunohistochemistry for high-resolution imaging, single-cell transcriptomics for in-depth immune cell profiling, and spatial transcriptomics to map cellular interactions. Our model successfully simulated tumor heterogeneity. The two cell lines formed distinct islets, creating a mosaic of MHC-I-positive and MHC-I-negative regions. After immunotherapy, the MHC-I-positive cells were selectively depleted, which shifted the tumor's composition in favor of the MHC-I-negative cells. The therapy significantly slowed overall tumor growth and promoted robust immune cell infiltration. The most expanded immune subpopulations included CD4+ and CD8+ T cells, NK cells, and γδ T cells. Furthermore, we observed polarization of macrophages toward a pro-inflammatory M1 phenotype. The infiltrating immune cells were spatially concentrated at the boundaries between the tumor islets, forming a structured, responsive layer of immune-to-tumor crosstalk. In conclusion, our work revealed that while immunotherapy effectively targeted only MHC-I-positive cancer cells, it significantly altered the complex immune architecture within MHC-I heterogeneous tumors. The differential recruitment and spatial organization of immune cells may create distinct barriers to therapeutic response. These findings could serve as a guide for the development of targeted combination immunotherapies aimed at overcoming the microenvironmental barriers caused by heterogeneous MHC-I expression.