Single-Cell RNA Sequencing Identifies a Distinct Epithelial Subpopulation Driving Resistance to Neoadjuvant Chemoimmunotherapy in Esophageal Squamous Cell Carcinoma

To investigate the role and mechanism of the transcription factor ETS2 in mediating resistance to neoadjuvant immunotherapy combined with chemotherapy in esophageal squamous cell carcinoma (ESCC). Single-cell RNA sequencing (scRNA-seq) was performed on tumor tissues from ESCC patients who received neoadjuvant therapy. Integrated analyses were conducted using clinical information, genomic and transcriptomic data from public databases (TCGA) and a single-center patient cohort. Weighted gene co-expression network analysis (WGCNA) was employed to identify senescence-associated gene modules. The association between ETS2 expression and cellular senescence or signaling pathway activity was evaluated using gene set enrichment analysis (GSEA) and computational scoring of senescence-related gene sets. ETS2-high epithelial cells were localized at the terminal end of the differentiation trajectory inferred by pseudotime analysis and were enriched in tumors with poor treatment response. ETS2 was highly expressed in ESCC tumor tissues and significantly associated with adverse patient outcomes. Transcriptomic analysis (TCGA) demonstrated significant enrichment of TGF-β in the ETS2-high group. WGCNA identified a senescence-associated “coral” gene module, whose expression was significantly correlated with patient age and induced senescence gene set scores, and exhibited the strongest association with senescence among all modules. High expression of ETS2 in ESCC predicts poor prognosis and resistance to neoadjuvant therapy. Its mechanism may involve inducing a senescent state in tumor cells and promoting aggressive tumor behavior through activation of key signaling pathways such as TGF-β. The “coral” module identified by WGCNA represents a critical senescence-related gene signature in ESCC.