Synergistic antitumor and immunomodulatory effects of Bifidobacterium animalis subsp. lactis V9 combined with anti–PD-1 therapy

Background Immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have revolutionized cancer immunotherapy but remain limited by low response rates, immune-related adverse events (irAEs), and reduced efficacy following antibiotic exposure. The gut microbiota critically influences ICI responsiveness, and Bifidobacterium species have emerged as potent immunomodulatory commensals. However, the mechanistic contribution of specific live biotherapeutic strains remains unclear. Methods We systematically characterized Bifidobacterium animalis subsp. lactis V9 ( B. lactis V9), through in-vitro cytokine assays and multiple syngeneic tumor models (CT26, MC38, 4T1). Immunophenotyping, microbiota colonization, and toxicological studies were conducted to evaluate efficacy, immune modulation, colonization stability, and safety. Results B. lactis V9 dose-dependently induced TNF-α, IL-6, and IL-10 secretion in THP-1 macrophages, exhibiting a balanced cytokine profile distinct from LPS stimulation. In vivo, B. lactis V9 alone moderately inhibited tumor growth but synergized with αPD-1 to achieve a 50% tumor growth inhibition and extend survival in CT26 models, accompanied by increased IFN-γ + CD8 + T cells and activated CD86 + CD11c + dendritic cells. The synergy persisted despite antibiotic pretreatment, indicating colonization stability (8×10 8 –10 9 copies/g) and a metabolite-driven mechanism. In 4T1 models, B. lactis V9 co-therapy mitigated αPD-1–induced uterine inflammation and pulmonary hemorrhage by downregulating IL-1α, IL-1β, and IL-17A while maintaining effector cytokines. Toxicology assessments revealed no adverse findings up to 3.52×10 12 CFU/kg (acute) or 5.00×10 11 CFU/kg (90-day repeated dose), with all genotoxicity tests negative. Conclusions B. lactis V9 harmonizes pro- and anti-inflammatory responses through TLR2/TLR4–NF-κB/MAPK signaling, remodels the tumor microenvironment, and enhances αPD-1 efficacy without increasing toxicity. Its colonization resilience and favorable safety profile support clinical translation as a microbiome-based adjunct to immunotherapy. These results provide a mechanistic foundation for combining live biotherapeutic products with ICIs to optimize antitumor immunity.