Integrated spatial and single-cell profiling reveals prognostic stromal-immune niches within tertiary lymphoid structures of HER2-low breast cancer

To systematically analyze the molecular and cellular landscape of tertiary lymphoid structures (TLS) in HER2-low breast cancer by integrating public spatial transcriptomics (ST) and single-cell RNA sequencing (scRNA-seq) data, and to validate the prognostic value of key cellular components in an independent clinical cohort. A research strategy of “integrating public data mining with independent clinical validation” was adopted. Public spatial transcriptomic (ST) data (GSE176078, CID4535, HER2-low sample) and single-cell RNA sequencing (scRNA-seq) data (GSE176078, n = 8 HER2-low samples) were integrated to identify TLS-associated differentially expressed genes (DEGs) and trace their cellular origins. An independent retrospective clinical cohort ( n = 27 HER2-low invasive breast cancer patients) was used for immunohistochemical (IHC) validation of key cellular components (FDCs, HEVs, lymphocytes) and prognostic correlation analysis. Statistical analyses included Kaplan–Meier survival analysis with log-rank tests after stratifying patients according to the presence or absence of each TLS subtype. A total of 57 TLS-associated DEGs were identified by ST analysis, among which 15 genes were successfully assigned to specific cell clusters by scRNA-seq, including B-cell markers ( IGLV3-19 , IRF8 , CCR7 , MS4A1 , BLK ), T-cell markers ( SELL , JAK3 ), NK-cell markers ( AKNA , CORO1A , TBC1D10C ), the macrophage-associated gene ( CCL20 ), and tumor-cell-related genes ( SPRR3 , SLPI , TUBA4A , S100A8 ). IHC validation showed that the TLS grade showed a potential association with favorable disease-free survival (DFS) ( P = 0.048). The presence of HEVs, which paralleled the presence of TLS, was associated with longer DFS ( P = 0.028). Within TLS-positive tumors, total HEV density showed a nonsignificant positive correlation with the CD21+ FDC network score (rho = 0.381, P = 0.146), whereas peritumoral TLS-associated HEV density was significantly correlated with it (rho = 0.721, P = 0.002). The presence of peritumoral TLS was associated with longer DFS ( P = 0.028), whereas intratumoral ( P = 0.061) and stromal ( P = 0.248) TLS showed no significant association with DFS. Cell–cell communication analysis revealed that fibroblasts were the core signal senders, while endothelial cells served as major signal receivers, and chemokine signaling and extracellular matrix signaling dominated the communication network. Integration of multi‑omics data enabled systematic mapping of the TLS molecular and cellular landscape in HER2‑low breast cancer. Independent validation suggested that FDC integrity, B‑cell activation, and HEV density may serve as potential prognostic markers, with combined TLS assessment potentially outperforming single indicators, supporting TLS‑based precision pathology and immunotherapy development. Trial registration : The study was retrospectively registered in the Chinese Clinical Trial Registry (ChiCTR2400085188, registered June 2024, http://www.chictr.org.cn/)