Purpose: Residual ESCC after neoadjuvant CCRT reflects incomplete treatment response and carries a high risk of recurrence. We sought to define the spatial tumor microenvironmental features of CCRT-resistant residual ESCC (using tumor regression grade (TRG) 2 or 3 tumors as a model of poor pathologic response) with mechanistic refinement at single-cell resolution and outcome assessment in internal and external cohorts. Experimental Design: We profiled post CCRT ESCC tissue microarray (TMA) cores by Visium FFPE whole-transcriptome spatial transcriptomics and orthogonal Xenium single-cell-resolution in situ profiling on serial sections from the same TMA blocks. Spot-level cell-type composition was inferred by CellDART deconvolution against a published ESCC single-cell RNA sequencing (scRNAseq). Local malignant-cell-enriched regions were defined by CancerFinder. We additionally re-analyzed the scRNAseq dataset to characterize SPP1+ and CXCL5+ macrophage states and their ligand-receptor signaling using CellChat. Outcome associations were evaluated in the SNUH cohort and externally tested in the TCGA ESCC cohort. Results: TRG 2-3 residual tumors exhibited effector immune-cell exclusion from malignant-cell-enriched regions, SPP1+/CXCL5+ macrophage accumulation, microvascular rarefaction, and proliferative-metabolic reprogramming. Single-cell re-analysis confirmed that CXCL5+ macrophages constitute a transcriptional subset of the broader SPP1+ macrophage population. CellChat showed that SPP1+ macrophages dominantly signal through SPP1-CD44 and SPP1-integrin axes to stromal and epithelial targets, and additionally engage immunosuppressive NECTIN2-TIGIT, CD86-CTLA4, and LGALS9-HAVCR2 programs with CD8 T cells, providing a mechanistic context for local immune exclusion. Visium LIANA and Xenium distance-gradient profiling localized SPP1-associated signaling preferentially to tumor cells and CAFs. Higher SPP1 expression and lower endothelial abundance were associated with shorter disease-free survival in the SNUH cohort. Higher SPP1 expression was also associated with shorter disease-free survival in the independent TCGA ESCC cohort. Conclusions: CCRT-resistant residual ESCC is characterized by a spatially organized tumor microenvironmental niche centered on SPP1-associated macrophage programs and microvascular rarefaction. These spatially resolved findings identify candidate macrophage- and vasculature-targeted axes for overcoming treatment resistance.
Bae, S., Ohn, J., Choi, H., Lee, H. S., Kim, B., Kang, C. H., Kim, H. J., Na, K. J., Kim, B. H.
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