Metabolite-Driven Immunosuppression in High-Risk NSCLC — with Anti-Tumor Immunity Shaped by Probiotic Strategy, Donor Origin, and Conserved Microbial Mediators

Abstract Elevated serum cytokeratin 19 fragment (CYFRA 21-1) in non-small cell lung cancer (NSCLC) links to immunosuppression, but whether live gut bacteria or their metabolites mediate this effect remains unclear. Using stratified fecal microbiota transplantation in a Lewis lung carcinoma model, we demonstrate that cell-free supernatants, not bacterial pellets, from high CYFRA 21-1 NSCLC donors impair anti-tumor immunity by depleting systemic T cells. Intervention with Bifidobacterium animalis subsp. lactis V9 restored tumor control via two distinct pathways: direct supplementation expanded CD8⁺ T cells and promoted Th1 immunity, while preconditioned microbiota transplantation acted through Th17/regulatory T cell modulation. Donor origin further shaped outcomes: mouse-derived microbiota enabled stable engraftment and robust cytotoxic responses, whereas human-derived transplants induced greater taxonomic shifts but carried opportunistic pathogens and favored Th17 polarization. Integrated metagenomic and untargeted metabolomic profiling revealed that carboxylic acid derivatives, particularly creatine, L-serine, and L-carnitinamide, were consistently associated with therapeutic response across all interventions and donor types. These metabolites represent conserved mediators of microbiota-immune crosstalk. Our findings establish microbial metabolites as central effectors of immunosuppression in high-risk NSCLC and highlight delivery strategy and donor compatibility as critical determinants of microbiome-based therapeutic efficacy.