Glycolysis-Driven Immune Evasion in Microsatellite Instability-High Colorectal Cancer: An Integrated Single-Cell and Spatial Transcriptomics Study.

Purpose: Microsatellite instability-high colorectal cancer is characterized by hypermutated genomes and high neoantigen loads, yet a significant proportion of patients exhibit resistance to immune checkpoint blockade. This study aims to investigate tumor cell functional heterogeneity and its role in immune evasion.

Patients and methods: We integrated single-cell RNA sequencing, spatial transcriptomics, and bulk RNA sequencing from microsatellite instability-high colorectal cancer patients. After quality control, normalization, and clustering, malignant epithelial subpopulations were identified through copy number variation analysis and non-negative matrix factorization. Functional characterization employed gene set enrichment analysis. Spatial transcriptomics clarified immune cell and tumor subpopulation localization, and survival analyses assessed prognostic implications.

Results: We identified a glycolysis-enriched tumor subpopulation (MP2) that co-localized with immunosuppressive niches marked by Treg accumulation, effector T-cell depletion, and FOLR2+ tumor-associated macrophages. MP2-high tumors were associated with immune checkpoint blockade resistance and poor prognosis. Mechanistically, MP2 cells secreted lactate, promoting Treg differentiation and macrophage polarization toward an immunosuppressive phenotype. Spatial transcriptomics revealed the precise organization of these lactate-rich, immune-excluded niches within tumors.

Conclusion: These findings establish tumor cell-intrinsic glycolysis as a key driver of immune evasion in microsatellite instability-high colorectal cancer and propose metabolic targeting as a strategy to overcome immune checkpoint blockade resistance.