Integration of Bulk and Single-Cell Transcriptomics Reveals Prognostic and Immunological Roles of MTHFD2 in Clear Cell Renal Cell Carcinoma

Tumor-associated macrophages (TAMs) are pivotal in the clear cell renal cell carcinoma (ccRCC) microenvironment. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a central enzyme in one-carbon metabolism, is increasingly recognized for its oncogenic roles in both cancer cells and immune compartments. We integrated bulk and single-cell transcriptomic datasets to interrogate the expression, prognostic impact, and immunomodulatory landscape of MTHFD2 in ccRCC. Robust differential expression, meta-analysis, Cox regression, and cell type deconvolution were performed. MTHFD2 expression and its association with prognosis were validated using tissue microarrays (TMAs), multiplex IHC, and in vitro macrophage polarization assays. MTHFD2 was upregulated in ccRCC tumors and associated with poor prognosis across multiple cohorts. High MTHFD2 expression remained an independent prognostic marker after adjustment for clinical stage. Single-cell analyses identified macrophages as the principal immune subpopulation expressing MTHFD2, with MTHFD2+ macrophages displaying a transcriptional signature of immunosuppression and metabolic adaptation. In vitro, MTHFD2-induced M2 macrophage polarization was reversed by DS18561882, promoting M1 polarization. MTHFD2 is a robust biomarker for poor prognosis in ccRCC, influencing tumor–immune interactions and macrophage polarization. Targeting MTHFD2 may represent a dual-action strategy to suppress tumor growth and reprogram the tumor immune microenvironment.