Spatial transcriptomics and single-cell analysis reveal GSTO2-mediated protective networks in oral squamous cell carcinoma tumor microenvironment

• Single-cell sequencing of 50,667 cells reveals nine cell populations in OSCC; epithelial cells show highest glutamine metabolism. • GSTO2-positive epithelial cells exhibit enhanced metabolic reprogramming in fatty acid degradation and amino acid catabolism. • Spatial transcriptomics reveals territorial organization of GSTO2-positive cells with preferential immune cell communication. • PPI analysis identifies five core hubs (MX1, OAS1, UBE2L6, PSMB8, PSMB9) mediating GSTO2 protective effects in OSCC. Oral squamous cell carcinoma (OSCC) exhibits significant cellular heterogeneity and metabolic reprogramming that influence tumor progression and therapeutic responses. However, the molecular mechanisms underlying these processes remain poorly understood. We performed an integrated analysis of single-cell RNA sequencing and spatial transcriptomics analysis on OSCC samples to characterize cellular heterogeneity and identify key regulatory factors. Summary-data-based Mendelian randomization (SMR) analysis was conducted to establish causal links between gene expression and OSCC risk. Functional enrichment, protein–protein interaction (PPI) network analysis, molecular docking, and co-immunoprecipitation (Co-IP) were employed to elucidate molecular mechanisms. Single-cell analysis of 50,667 cells identified nine distinct cell populations with epithelial cells showing the highest glutamine metabolism activity. SMR analysis revealed glutathione S-transferase omega 2 (GSTO2) as a genetically validated protective factor for OSCC risk. GSTO2-positive epithelial cells exhibited enhanced metabolic reprogramming, particularly in fatty acid degradation and amino acid catabolism pathways. Spatial transcriptomics demonstrated that GSTO2-positive cells were spatially organized and showed enhanced communication with immune cells. Protein-protein interaction analysis identified interferon-stimulated genes (MX1, OAS1, UBE2L6) and immunoproteasome subunits (PSMB8, PSMB9) as core regulatory hubs. Molecular docking confirmed direct binding interactions between GSTO2 and these hub proteins, with MX1-GSTO2 showing the strongest binding affinity (−10.7 kcal/mol). Co-immunoprecipitation experiments validated the physical interaction between GSTO2 and MX1. This study indicates that GSTO2 defines an epithelial subset characterized by enhanced metabolic activity and immune interactions, highlighting its potential as a biomarker and therapeutic target in OSCC.