Background <p>Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with poor prognosis, where immune checkpoint inhibitors (ICIs), such as anti-PD-1/PD-L1 antibodies, show limited efficacy with heterogeneous patient responses. The tumor microenvironment (TME) and tumor subclonal heterogeneity significantly influence immune evasion and therapeutic resistance. This study aimed to identify distinct tumor subpopulations in ESCC, characterize their roles in immunosuppression, and determine their association with ICI responsiveness.</p> Methods <p>We analyzed single-cell multi-omics data from tumor specimens and peripheral blood mononuclear cells (PBMCs) of 12 ESCC patients receiving neoadjuvant therapy. Using bioinformatics tools (Seurat, CellChat), we identified tumor subpopulations, inferred cell-cell communication, and validated functional mechanisms via in vitro assays (cell culture, flow cytometry, western blotting), patient-derived organoid (PDO) co-culture systems, and humanized mouse models. Clinical correlations were assessed using bulk transcriptome data, survival analysis, and multi-cohort validation (GEO datasets, IMvigor210 cohort).</p> Results <p>A distinct HCAR2⁺ epithelial subclone was identified, characterized by high PD-L1 expression, immunosuppressive cell interactions (e.g., PD-L1/PD-1 axis with exhausted NK-like T cells), and enrichment in pathways driving immune evasion (STAT3, IL-6, PI3K-AKT-mTOR). HCAR2⁺ cells upregulated PD-L1 via STAT3 activation, impairing CD8⁺ T cell cytotoxicity and antigen presentation. Clinically, HCAR2⁺ infiltration correlated with poor prognosis, increased copy number variations (CNVs), high tumor mutational burden (TMB), and enhanced sensitivity to neoadjuvant anti-PD-1 therapy in vivo. Multi-cohort analysis confirmed HCAR2 expression as a predictive biomarker for favorable ICI response, associated with reduced immune exclusion and TIDE scores.</p> Conclusions <p>HCAR2⁺ tumor subclones orchestrate an immunosuppressive TME via PD-L1-mediated CD8⁺ T cell exhaustion while paradoxically conferring sensitivity to PD-L1 blockade. HCAR2 expression serves as a dual biomarker for poor prognosis and ICI responsiveness, offering a novel molecular target for stratifying ESCC patients who may benefit from neoadjuvant immune checkpoint therapy. Targeting the HCAR2-STAT3-PD-L1 axis could enhance immunotherapy efficacy in this aggressive cancer.</p>