Single-cell RNA sequencing reveals age-related heterogeneity in the tumor microenvironment of breast cancer patients

Breast cancer (BC) is the most common malignancy among women, with its progression and prognosis significantly influenced by the tumor microenvironment (TME). Age-related differences in TME composition lead to distinct tumor behaviors: young patients (≤ 40 years) exhibit aggressive tumors, while elderly patients (> 70 years) experience immunosenescence and reduced therapy responses. We performed single-cell RNA sequencing (scRNA-seq) analysis on tumors from 10 breast cancer patients (5 ≤ 40 years, 5 ≥ 70 years), encompassing 33,664 high-quality cells. After cell annotation and batch correction, malignant epithelial cells were identified using inferCNV. We applied pseudotime trajectory analysis, pathway enrichment, and cell-cell communication profiling to investigate age-specific TME dynamics. Survival relevance was assessed using a GEO cohort (GSE20685) of young breast cancer patients, and immunohistochemical staining was performed on clinical tumor and fibroadenoma tissues to validate protein-level expression of key ISGs. In young patients, malignant epithelial cells showed gradual upregulation of interferon-stimulated genes (ISGs) such as IFI44, IFI44L, IFIT1, and IFIT3 along the pseudotime trajectory, suggesting their involvement in early tumorigenesis. High expression of these ISGs was significantly associated with poor overall survival in a young BC cohort (GSE20685). Immunohistochemical validation further confirmed elevated IFIT3 protein levels in young tumor tissues. In contrast, elderly patients had a TME enriched in macrophages and fibroblasts, with activation of immunosuppressive pathways (e.g., SPP1, COMPLEMENT). Our integrative analysis identifies ISGs as key transcriptional drivers of tumorigenesis in young breast cancer, with potential prognostic and therapeutic value. Despite limited sample size, the combination of single-cell transcriptomics, clinical survival data, and protein-level validation provides robust evidence of age-specific TME remodeling. These findings support the development of age-tailored immunotherapy strategies targeting interferon signaling in young patients and immune checkpoint pathways (e.g., LAG3, CTLA4) in elderly individuals.