Single-cell transcriptomic analysis reveals distinct cancer-associated fibroblast signatures in treatment-refractory esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) remains a leading cause of cancer-related mortality worldwide. Despite neoadjuvant concurrent chemoradiotherapy (nCCRT), approximately half of patients exhibit incomplete pathological response with persistent lymph node (LN) infiltration. The tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs) and natural killer (NK) cells, plays crucial roles in treatment outcomes, yet specific cellular signatures associated with LN infiltration status remain poorly characterized. We performed single-cell RNA sequencing analysis on tumor and adjacent normal tissues from 10 ESCC patients who underwent nCCRT followed by surgical resection. Patients were stratified into presence (n = 5) and absence (n = 5) groups based on histological draining LN infiltration status according to ypTNM staging. Cell-type identification, subclustering analysis, differential expression analysis, pathway enrichment, and pseudotime trajectory analysis were performed to characterize infiltration-associated signatures. Analysis of 47,907 cells revealed nine cell populations with significant compositional differences between group. Patients with LN infiltration were characterized by significant enrichment of CLEC2BhimyCAF subtype (10.64% vs. 0.58%), highly expressing NFKB1 and associated with immunosuppressive pathways. Conversely, patients without infiltration showed markedly increased abundance of CD56dim NK subset (19.14% vs. 1.73%), linked to enhanced cytotoxic function. Pseudotime trajectory analysis revealed that cells from different groups occupied opposite ends of trajectories. Treatment-associated cellular signatures extended beyond tumor regions to adjacent normal tissues within the radiation field, indicating regional tissue reprogramming associated with therapeutic exposure. This single-cell analysis reveals that LN infiltration status in ESCC is characterized by distinct TME signatures involving CLEC2BhimyCAF in patients with infiltration and F11RhiCD56dim NK in patients without infiltration. These findings provide new insights into cellular mechanisms underlying different pathological outcomes following nCCRT and offer potential biomarkers for patient stratification and therapeutic targets in ESCC management.