M2 macrophage infiltration drives tumor progression and identifies a multigene prognostic signature in esophageal cancer.

OBJECTIVE: This study aimed to elucidate the mechanistic role of M2 tumor-associated macrophages in esophageal cancer (EC) progression and to construct an M2 macrophage-related gene signature for prognostic prediction. METHODS: Integrated analyses of single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data of EC were performed. scRNA-seq data were processed with Seurat and annotated using SingleR. Immune infiltration was evaluated through ssGSEA and CIBERSORT. Weighted gene coexpression network analysis (WGCNA) was used to identify M2 macrophage-associated modules, and candidate genes were intersected with differentially expressed genes (DEGs). Functional enrichment analyses were performed, and a prognostic risk model was established through multivariate Cox regression analysis. The functional roles of key genes were validated through in vitro and in vivo experiments. RESULTS: Twelve cell types were identified by scRNA-seq, with macrophages representing the predominant immune population. M2 macrophages formed the major immunosuppressive subtype and were negatively associated with patient survival. WGCNA and DEG analysis identified 25 M2-related genes, from which a four-gene prognostic signature (SPINK5, A2ML1, IL1RN, IL36G) was constructed. The model effectively stratified EC patients into distinct risk groups with significantly different survival outcomes. Further in vitro experiments demonstrated that silencing IL1RN and IL36G in macrophages markedly suppressed the malignant phenotypes of esophageal cancer cells. Complementary in vivo experiments provided additional evidence, further indicating that IL1RN and IL36G play important functional roles in overall tumor progression. CONCLUSION: A four-gene M2 macrophage-related prognostic model provides reliable prediction of clinical outcomes in EC. Among these genes, IL1RN and IL36G function as key regulators whose silencing inhibits M2 polarization and attenuates tumor proliferation, invasion, migration, and epithelial-mesenchymal transition.