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

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.