Abstract 4129: Spatial single-cell transcriptomic profiling reveals distinct immune and stromal landscapes across TIL/PD-L1-defined TNBC subtypes

Abstract Purpose: Triple-negative breast cancer (TNBC) displays marked heterogeneity in immune contexture, influencing response to immune checkpoint blockade. We aimed to characterize spatial and cellular differences among TNBC subtypes stratified by tumor-infiltrating lymphocytes (TILs) and PD-L1 expression using single-cell spatial transcriptomics. Experimental Procedures: We analyzed 36 TNBC cores using the Xenium platform (∼5,000 genes per cell) and annotated major cell populations, including epithelial, fibroblast, and immune cells. Each core was classified into four TIL/PD-L1 subtypes (TIL+PD-L1+, TIL+PD-L1-, TIL-PD-L1+, and TIL-PD-L1-) based on histologic TIL density (≥30%) and PD-L1 CPS (≥10). Pairwise intercellular distances between tumor and stromal/immune cells were computed within a 50-μm radius to quantify spatial immune exclusion and stromal remodeling. Results: Distinct spatial and transcriptional profiles were observed among subtypes. TIL+PD-L1+ tumors exhibited dense immune infiltration and upregulation of antigen-presentation and T-cell activation genes in both CD4+ and CD8+ subsets. In contrast, TIL+PD-L1- tumors showed prominent desmoplastic stroma and reduced immune-cell proximity, accompanied by fibroblast enrichment of POSTN, FBLN1, and SFRP2, suggesting extracellular-matrix remodeling that may hinder immune infiltration. Epithelial cells in TIL+PD-L1- cores displayed higher expression of XBP1, FOXA1, and MLPH, indicating epithelial plasticity and potential immune evasion. Spatial mapping further revealed that tumor margins in TIL+PD-L1- cores contained more aggressive epithelial phenotypes than corresponding cores in TIL+PD-L1+ tumors. Conclusions: Single-cell spatial transcriptomics delineated distinct immune and stromal architectures across TIL/PD-L1-based TNBC subtypes. The TIL+PD-L1+ subtype represents an immune-active microenvironment, whereas the TIL+PD-L1- subtype demonstrates stromal-driven immune exclusion. These findings provide spatial evidence that combined assessment of TILs and PD-L1 can stratify TNBC patients by their potential responsiveness to immunotherapy. Citation Format: Kyungsoo Kim, Soong June Bae, Yoonjin Cha, Yoonwon Kook, Ah Yoon Kim, Jee Hung Kim, Sung Gwe Ahn, Joon Jeong. Spatial single-cell transcriptomic profiling reveals distinct immune and stromal landscapes across TIL/PD-L1-defined TNBC subtypes 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 4129.