微生物群（Microbiota）特征提示免疫治疗反应

Spatial Microbiota Signatures Indicate Immunotherapy Responsiveness in ESCC
微生物群（Microbiota）特征提示免疫治疗反应

Objective: Neoadjuvant chemo-immunotherapy (nCIT) improves outcomes in locally advanced esophageal squamous cell carcinoma (ESCC), yet responses vary widely. The esophagus harbors a resident microbiota, but whether tumor-associated microbes modulate immunotherapy efficacy remains unclear.

Methods: Tumor-site esophageal microbiota were sampled from ESCC patients receiving nCIT. Patients were classified as responders (R) or non-responders (NR) by postoperative pathology. We assessed α- and β-diversity, identified differential taxa using Linear Discriminant Analysis Effect Size (LEfSe), and annotated putative functions via KEGG. Genus-level co-occurrence networks were constructed. Public (TCMbio) resources were queried for microbe–neutrophil correlations. Functional assays included co-culture of neutrophils with ESCC cells ± Fusobacterium nucleatum culture supernatant, followed by flow cytometry and transcript analyses. Predictive performance was evaluated with ROC analysis, and an independent validation cohort tested the association between local microbial abundance and neutrophil infiltration.

Results: Microbial diversity (α/β) did not differ significantly between R and NR. Wilcoxon testing and LEfSe consistently showed marked enrichment of Fusobacterium nucleatum(F.n.) in NR at the genus and higher taxonomic ranks, yielding AUC = 0.81 for predicting non-response. Correlation analyses suggested broad co-occurrence of F.n. with multiple taxa. KEGG-based inference indicated enhanced amino-acid transport and polyphenol-processing pathways, features linked to immunosuppressive milieus. TCMbio database indicated a positive association between F.n. and tumor-infiltrating neutrophils(TANs).In vitro, adding F.n. culture supernatant to neutrophil–ESCC co-cultures drove a prototypical immunosuppressive phenotype in neutrophils-upregulating PD-L1, ARG1, and NOS, enhancing degranulation (CD63), increasing CXCR4 expression, and elevating the immunoregulatory cytokines TGF-β and IL-10-as quantified by flow cytometry and RT-qPCR. In an independent cohort, local F.n. positively correlated with TANs.

Conclusions: Tumor-site enrichment of F.n. associates with immunotherapy non-response in ESCC and may act through neutrophil-mediated immunosuppression. These data nominate F.n. and related functional signatures as candidate biomarkers and mechanistic targets for response modulation.

Yuchen Zhang (1), Hongyu Zhang (1), Pingjing Zhou (2), Jie Gu (3), Di Ge (4), (1) Zhongshan Hospital, Fudan University, Shanghai, Shanghai, (2) Fudan University Zhongshan Hospital, shanghai, Shanghai, (3) Zhongshan Hospital Fudan University, Shanghai, Shanghai, (4) Fudan University, Zhongshan Hospital, Shanghai, Shanghai