Association of intratumoral Fusobacterium nucleatum with stromal remodeling and immune exclusion in esophageal squamous cell carcinoma

Background: Fusobacterium nucleatum ( F. nucleatum ), an oral anaerobe, has been linked to tumor progression and immune suppression in esophageal squamous cell carcinoma (ESCC). However, its relationship with stromal remodeling and immune exclusion within the tumor microenvironment (TME) remains unclear. Methods: We performed integrated analyses combining metagenomic profiling, transcriptomic deconvolution, and histopathologic validation in 93 TCGA-ESCC cases and 126 resected tumors. F. nucleatum status was determined using Centrifuge and quantitative PCR. Immune and stromal cell fractions were estimated by CIBERSORT and EPIC. Immunohistochemistry (IHC) for alpha-smooth muscle actin (alpha-SMA) and NF-κB p65 (RelA) assessed cancer-associated fibroblast (CAF) activation and inflammatory signaling, and fluorescence in situ hybridization (FISH) visualized F. nucleatum within tumors. Clinicopathologic variables, including ECOG performance status, were compared between F. nucleatum -positive and -negative cases. Results: Twenty-three of 93 TCGA-ESCC tumors (25%) were F. nucleatum -positive (≥0.02% relative abundance). Gene set enrichment analysis revealed activation of TNFalpha/NF-κB and epithelial-mesenchymal transition pathways in F. nucleatum -positive tumors. CIBERSORT showed reduced CD8⁺ T-cell infiltration, while EPIC demonstrated a higher CAF fraction (0.72 vs. 0.45, P < 0.01). In the validation cohort, IHC confirmed increased alpha-SMA expression in F. nucleatum -positive tumors, consistent with transcriptomic results. FISH identified F. nucleatum signals within tumor cells adjacent to alpha-SMA-positive fibroblasts, suggesting spatial proximity between infection and stromal activation. Nuclear RelA localization was significantly more frequent in F. nucleatum -positive tumors, indicating NF-κB activation. Cases with both strong alpha-SMA expression and nuclear RelA were markedly enriched in the F. nucleatum -positive group (P < 0.01). These findings support a model in which F. nucleatum activates NF-κB-mediated cytokine signaling that promotes fibroblast recruitment and stromal expansion, contributing to immune exclusion. Clinically, F. nucleatum positivity was associated with poorer performance status despite similar tumor stages, suggesting colonization may reflect systemic vulnerability or impaired oral hygiene. Conclusions: Intratumoral F. nucleatum is associated with an NF-κB-activated, fibroblast-rich, and CD8⁺ T-cell-poor TME in ESCC. By promoting stromal remodeling and immune exclusion, F. nucleatum may enhance tumor aggressiveness. These findings highlight the interplay between microbial colonization, stromal activation, and host condition, offering insight into potential microbiome-stroma-immune interactions in ESCC.