Abstract 4888: Association of intratumoral Fusobacterium nucleatum with stromal remodeling and immune exclusion in esophageal squamous cell carcinoma

Abstract 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 α-smooth muscle actin (α-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 TNFα/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 α-SMA expression in F. nucleatum-positive tumors, consistent with transcriptomic results. FISH identified F. nucleatum signals within tumor cells adjacent to α-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 α-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. Citation Format: Takashi Ofuchi, Qingjiang Hu, Kosuke Kanemitsu, Koshi Mimori, Masaaki Iwatsuki. Association of intratumoral Fusobacterium nucleatum with stromal remodeling and immune exclusion in esophageal squamous cell carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4888.