Integrative single-cell, spatial, and bulk transcriptomics reveal an FMR1-FTO axis linked to the immune-excluded phenotype in gastric cancer.

[BACKGROUND] Immune exclusion is a major barrier to immune checkpoint inhibitor (ICI) efficacy in gastric cancer, yet the spatial mechanisms by which mA regulators drive this phenotype remain unclear.

[METHODS] We integrated single-cell RNA-seq, spatial transcriptomics, and functional assays to map the gastric cancer microenvironment and derived an mA regulator score to quantify the spatial coupling between mA patterns and immune exclusion.

[RESULTS] CAFs emerged as a central hub that excluded T cells via collagen-integrin interactions and MIF signaling, forming a CAF-defined collagen barrier that impeded CD8 T-cell entry into the tumor core. In immune-excluded samples, mA regulator scores were selectively elevated in tumor nests compared with surrounding stroma. Across five bulk transcriptomic datasets, this phenotype was associated with reduced predicted ICI responsiveness, activation of chemotherapy resistance programs, and poor survival. Mechanistically, FTO was highly expressed in the immune-excluded phenotype and correlated with stromal activation and T-cell exclusion. Serial immunohistochemistry and multiplex immunofluorescence revealed high FMR1 protein expression in immune-excluded tumors, with CD8 T cells largely confined to the stroma. Cycloheximide (CHX) chase and MG132 treatment showed that FMR1 depletion reduced FTO protein abundance and accelerated FTO turnover in an MG132-sensitive manner, consistent with a post-translational regulatory relationship.

[CONCLUSIONS] Collectively, our data support an FMR1-FTO module associated with the immune-excluded phenotype and nominate this axis as a potential vulnerability for disrupting stromal immune barriers. The FMR1-FTO axis may represent a candidate target for strategies aimed at relieving immune exclusion and improving immunotherapy sensitivity.