Abstract 6115: Deciphering tumor microenvironment dynamics in tumorigenesis and lymph node metastasis of esophageal squamous cell carcinoma using single-cell and spatial transcriptomics

Abstract Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with late diagnosis and high propensity for lymph node (LN) metastasis. Given the major prognostic and therapeutic impact of LN involvement, defining the cellular and molecular features distinguishing metastatic LNs from primary tumors is imperative. We aimed to delineate tumor microenvironment (TME) dynamics between primary tumors and metastatic LNs integrating single-cell RNA/TCR sequencing (scRNA/TCR-seq) with high-resolution spatial transcriptomic validation.We performed scRNA-seq on 344,790 cells from 58 upfront surgical specimens of 18 stage T1-2N0-1M0 ESCC patients. Samples included paired tumor mucosa (TM), normal mucosa (NM), tumor-associated LNs (TLN), and normal LNs (NLN). scTCR-seq in 8 patients delineated clonal trajectories and functional T-cell states. Additionally, spatial transcriptomics using the CosMx 6K platform profiled 3,493,957 cells across 34 specimens (NLN, TLN, and TM) from 13 patients, enabling in situ validation of compartment-specific cellular interactions.Our analysis revealed marked divergence between TM and TLN immune landscapes. TM showed an immunosuppressive milieu dominated by regulatory T cells (TREGs) and sharp loss of cytotoxicity in CD8 exhausted T cells (TEXHs). In contrast, TLN retained partially functional TEXHs following two trajectories: a pre/intermediate-exhausted path with sustained GZMB and PRF1 expression, and a terminally-exhausted path showing steep functional decline—prominent in TM but attenuated in TLN. TCR clonotype analysis supported this, as shared TM-TLN clones preserved cytotoxicity whereas TLN-restricted clones displayed weaker effector programs, suggesting antigen-driven trafficking from primary tumor. Myeloid profiling showed TREM2high macrophages (M) enrichment in TLN, while TM featured TREG-dendritic cell (DC) interactions. Spatial transcriptomics validated these compartmentalized suppressive circuits, demonstrating colocalization of TREM2high Ms with TREGs or TEXHs in TLN. SPP1-mediated signaling between these populations appeared exclusively in TLN, highlighting niche-specific immunoregulatory interactions within metastatic sites.This integrated single-cell and spatial transcriptomic study highlights distinct immunoregulatory programs driving ESCC progression and LN metastasis. While TLNs contained TEXHs with preserved cytotoxicity and reinvigoration potential, TMs exhibited abrupt functional collapse. We identified compartment-specific suppressive networks—TREM2high M-mediated axes in TLN and DC-mediated axes in TM—validated by spatial analysis. These findings offer mechanistic insight into LN metastasis and may inform biomarker development, neoadjuvant treatment optimization, and tailored immunotherapy in ESCC. Citation Format: Tae Hee Hong, Hansoll Na, Chung Lee, Young Ho Yang, Ha Eun Kim, Byung Jo Park, Min Hee Hong, Hye Ryun Kim, Hyun Ki Kim, Dae Joon Kim. Deciphering tumor microenvironment dynamics in tumorigenesis and lymph node metastasis of esophageal squamous cell carcinoma using single-cell and spatial transcriptomics [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 6115.