Stromal CD163-positive Macrophage Enrichment Is Associated With a FOXP3-skewed Immune Profile and Poor Prognosis in Esophageal Squamous Cell Carcinoma

Abstract Background/Aim: The prognostic significance of macrophage-centered spatial immune architecture in esophageal squamous cell carcinoma (ESCC) remains incompletely understood. We investigated the spatial distribution and prognostic relevance of CD163-positive macrophages and their association with T-cell subsets in ESCC. Materials and Methods: A total of 139 patients with surgically resected ESCC, including a primary cohort (n=89) and an independent validation cohort (n=50), were analyzed. Double immunofluorescence staining was performed to evaluate CD163-positive macrophages, CD8-positive T cells, and FOXP3-positive T cells in intratumoral and stromal compartments. Associations with clinicopathological features and overall survival were assessed. Public transcriptomic and single-cell RNA-sequencing datasets were analyzed for external biological validation. Results: CD163-positive macrophages, CD8-positive T cells, and FOXP3-positive T cells were predominantly enriched in the stromal compartment. Stromal CD163-positive macrophage infiltration was positively associated with both CD8-positive and FOXP3-positive T-cell infiltration and with an increased stromal FOXP3/CD8 ratio, indicating an inflamed but FOXP3-skewed immune phenotype. Compared to conventional T-cell-based indicators, stromal CD163-related immune features showed stronger and more consistent associations with poor overall survival and remained significantly associated with overall survival after adjustment for age and pathological TNM stage in separate multivariable models. These findings were reproduced in the validation cohort and supported by transcriptomic and single-cell analyses. Conclusion: Stromal CD163-positive macrophage enrichment is associated with a FOXP3-skewed immune architecture and unfavorable prognosis in ESCC. Macrophage-centered spatial immune features may provide complementary prognostic information beyond conventional T-cell-based indicators and warrant further investigation.