IFN-?-STAT1 Axis Drives MHC-II Antigen Presentation Potentiation in Tumor Associated Macrophages

While the IFN-?-STAT1 signaling pathway is well-characterized in promoting MHC class II (MHC-II)-dependent antigen presentation within pro-inflammatory macrophages during acute infections, its functional dynamics in tumor-associated macrophages (TAMs) remain poorly understood. Here, we systematically investigated the immunomodulatory role of IFN-?-STAT1 axis in TAMs through integrative bioinformatics and experimental validation. Transcriptomic analysis of tumor-infiltrating myeloid cells across multiple cancer cohorts revealed a strong correlation between STAT1 activation and MHC-II pathway enrichment, particularly in IFN-?-high TAM subsets. To mechanistically dissect this relationship, we employed bone marrow-derived macrophages (BMDMs) polarized under tumor-conditioned media and subjected them to IFN-? stimulation. Single-cell RNA sequencing demonstrated that IFN-? triggered STAT1 nuclear translocation, upregulating MHC-II genes. Our findings establish IFN-?-STAT1 as a master regulator of TAM immunogenicity, proposing targeted STAT1 activation as a strategy to overcome myeloid-driven immunosuppression in cancer. Overall design: Collect BMDMs by detaching them from the dish using warm 0.05% trypsin, followed by washing the cells twice with PBS containing 10% FBS. Resuspend these cells with fresh complete medium (DMEM+10%FBS+100 U/ml penicillin 100 mg/ml streptomycin (Coolaber)), and distribute them into 12-well plates (depending on the experimental design). For the M1 activation assay (TH1 cytokines assay), add a final concentration of 100 ng/ml LPS or 100 ng/ml LPS with 50 ng/ml IFN?. For the M2 activation assay (TH2 cytokines assay), add a final concentration of 10 ng/ml IL-4 and 10 ng/ml IL-13 or 10 ng/ml IL-4 and 10 ng/ml IL-13 with 50 ng/ml IFN?. Collect the stimulated BMDMs by detaching them from the dish using 5mM EDTA in PBS, followed by washing the cells twice with PBS containing 10% FBS.