Keywords
T-cell exhaustion
Breast cancer stem cells
Spatial proteomics
Immunosuppressive niche
Submitted : 2026-05-19
Accepted : 2026-06-03
Published : 2026-06-18
Abstract
Objective: This study aimed to characterize the spatial distribution of CD8A+PD-1+TIGIT+ exhausted T cells in HER2-positive breast cancer and to investigate their spatial relationship with CD44+CD24⁻ breast cancer stem cells. Method: Formalin-fixed paraffin-embedded HER2-positive breast cancer specimens were analyzed using multiplex immunofluorescence, pathological region annotation, and spatial proteomic analysis. Regions of interest were annotated based on histological features, including invasive carcinoma-associated areas, adjacent stroma, peritumoral fibrous tissue, and adipose tissue. CD8A+PD-1+TIGIT+ T cells were identified at the single-cell level, and their regional distribution was quantitatively compared. Spatial mapping, distance-based analysis, and interaction network analysis were performed to evaluate the spatial association between exhausted T cells and CD44+CD24⁻ BCSCs. Pairwise comparisons were conducted using two-tailed Welch’s t-test. Result: CD8A+PD-1+TIGIT+ T cells were preferentially enriched in invasive carcinoma-associated regions and adjacent stromal areas, with significantly lower abundance in peritumoral fibrous and adipose tissues. Within the CD8A+ T-cell compartment, the proportion of triple-positive exhausted T cells was highest in invasive lesions and decreased toward peripheral non-invasive regions. Spatial analyses showed that CD8A+PD-1+TIGIT+ T cells and CD44+CD24⁻ BCSCs were co-enriched in invasive carcinoma, tumor–stromal interface zones, and lymphoid tissue-associated microregions. Distance-based heatmaps and interaction network analyses further confirmed recurrent spatial proximity between these two cell populations. Conclusion: CD8A+PD-1+TIGIT+ exhausted T cells are spatially enriched in invasive pathological microregions of HER2-positive breast cancer and show close spatial association with CD44+CD24⁻ BCSCs. The observed spatial overlap between tumor stemness and T-cell exhaustion defines a localized suppressive niche. This interplay likely underpins the biological mechanisms through which HER2-positive breast cancer evades immune surveillance and develops resistance to therapy.
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