Immune microenvironment biomarkers linked to ferroptosis in clear cell renal cell carcinoma

Background Clear cell renal cell carcinoma (ccRCC) is characterized by marked metabolic reprogramming and a highly heterogeneous immune microenvironment. Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and has been implicated in ccRCC biology. This study aimed to identify immune-microenvironment-associated hub genes in ccRCC, examine their relationship with ferroptosis-related transcriptional changes, and evaluate their diagnostic and cellular-expression characteristics. Methods Public multi-omics data were analyzed using GSE53757 (72 ccRCC and 72 matched normal kidney tissues), GSE159115 single-cell RNA-sequencing data, and TCGA-KIRC (542 primary tumors and 72 normal kidney tissues). Differential expression, hierarchical co-expression clustering, ferroptosis-gene enrichment, protein-protein interaction analysis, MCP-counter immune scoring, single-cell localization, ROC analysis, and exploratory survival analysis were performed. In addition, in vitro qRT-PCR experiments were performed for SLC7A11, GPX4, ACSL4, and HMOX1 in RPTEC and 786-O cells and for CCR5, CSF1R, and CD86 in macrophage cultures, with three biological replicates per group, using the complete ΔCt/ΔΔCt/2 −ΔΔCt validation workflow. Results GSE53757 identified 8,650 differentially expressed genes, including significant enrichment of detected FerrDb ferroptosis genes (hypergeometric p = 2.3 × 10 −3 ). CCR5, CSF1R, CD86, CD8A, and LCP2 were significantly upregulated and clustered within the ccRCC-associated M03 immune co-expression module. MCP-counter and single-cell analyses localized these signals predominantly to macrophage and T/NK-cell compartments. TCGA-KIRC independently confirmed significant upregulation and diagnostic discrimination (AUC 0.862–0.948; five-gene panel AUC = 0.932), whereas the panel showed no independent overall-survival association. In vitro qRT-PCR measurements confirmed higher relative expression of SLC7A11, GPX4, ACSL4, and HMOX1 in 786-O than in RPTEC cells, with fold changes of 2.55, 1.58, 1.70, and 2.30, respectively; CCR5, CSF1R, and CD86 were also higher in macrophages exposed to 786-O-conditioned medium than in M0 macrophages. Conclusion CCR5, CSF1R, CD86, CD8A, and LCP2 are best interpreted as immune-microenvironment-associated hub genes in ccRCC rather than canonical ferroptosis drivers. Their association with ferroptosis is supported at the pathway and co-expression levels, while direct regulation of iron metabolism, lipid peroxidation, or the GPX4/SLC7A11 axis is not established by the present analyses. The multi-dataset evidence supports their potential diagnostic and immune-stratification value in ccRCC.