Ginsenoside Suppresses Triple‐Negative Breast Cancer Growth and Synergizes With Programmed Cell Death Protein 1 ( PD ‐1) Immunotherapy Through Gut Microbiota and Metabolic Regulation

Breast cancer accounts for 30% of all malignancies and ranks second in cancer-related mortality. Triple-negative breast cancer (TNBC) lacks estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) and presents unique therapeutic challenges. Although no TNBC-specific medicines exist, programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) immunotherapy shows promise. Gut microbiota has emerged as a regulator of breast cancer growth, and Traditional Chinese Medicine (TCM) is gaining attention in tumor therapy. This study aimed to evaluate the therapeutic potential of ginsenoside Rg3 and PD-1 immunotherapy in a TNBC mouse model and to assess the impact of gut microbiota and metabolites on treatment outcomes. Breast cancer cell lines (4 T1, MDA-MB-231) were treated with Rg3 and evaluated for viability, apoptosis, migration, and invasion assays. In vivo, 4 T1 tumor-bearing mice were randomized to receive Rg3, anti-PD-1, or combination therapy; tumor growth and biocompatibility were assessed. Fecal samples underwent 16S rRNA gene sequencing and untargeted LC-MS metabolomics with multivariate and statistical analyses. Ginsenoside Rg3 reshaped the tumor microenvironment by promoting M1 macrophage polarization, increasing CD8⁺ and memory T cells, and upregulating Th1 and M1 cytokines while reducing immunosuppressive CD4⁺ T cells, Treg cells, and M2 macrophages. Anti-PD-1 monoclonal antibody (PD-1 mAb)/Rg3 inhibited tumor growth, increased apoptosis, and enhanced M1 polarization and CD8⁺ T-cell responses, with combined treatment further amplifying these effects. Gut microbiota composition and abundance differed across groups, with specific metabolites influencing treatment success. Bacteroides vulgatus showed a positive correlation with sulforaphane-cysteine, suggesting a role for gut microbiota metabolomics in modulating therapeutic benefits. Overall, this study highlights the potential of combining PD-1 immunotherapy with ginsenoside Rg3, underscoring the role of the gut microbiota and its metabolites in TNBC treatment.