Synergistic probiotic–chemotherapy remodeling of the intratumoral microbiome as a driver of mature tertiary lymphoid structure formation and anti-tumor immunity in colorectal cancer.

e15672 Background: Neoadjuvant chemo-immunotherapy shows promise for advanced colorectal cancer (CRC), but its efficacy is limited by immune suppression in the tumor microenvironment (TME), potentially modulated by mature tertiary lymphoid structures (mTLSs). Recent studies suggest that the intratumoral microbiome regulates tumor growth and immune function through the TME, but its contribution to mTLS formation in CRC remains unclear. We selected the composite probiotic JC-106 for its immunomodulatory effects and its reported upregulation in chemotherapy-sensitive patients. Methods: We performed multi-omics analysis on 24 non-NAC and 12 NAC CRC patients to investigate chemotherapy's impact on intratumoral microbiota and TLS formation. Using subcutaneous and orthotopic CRC models, we evaluated the anti-tumor effect of the probiotic JC-106 combined with chemotherapy. Comprehensive profiling and in vivo validation revealed the regimen's effects on metabolism and the tumor immune microenvironment. Finally, the efficacy and safety of a triple-combination therapy (JC-106, FOX, and anti-PD-1) were assessed. Results: In subcutaneous syngeneic and AOM-DSS-induced orthotopic CRC mouse models, JC-106 combined with chemotherapy reshaped the intratumoral microbiome and promoted mTLS formation. Mechanistically, the combination therapy increased the intratumoral colonization of the gut-specific symbiont Parabacteroides distasonis and promoted Th17 cell differentiation, both of which were positively correlated with TLS formation. Intratumoral injection of Parabacteroides distasonis enhanced Th17 cell proportions in a linoleic acid depletion-dependent manner, thereby initiating mTLS development. Clinically, JC-106 alleviates chemotherapy-induced PD-1 upregulation to suppress immune escape, and synergizes with chemotherapy to improve immunotherapy efficacy. Conclusions: Our findings support the potential of composite probiotics as a promising strategy to enhance TLS-dependent neoadjuvant chemo-immunotherapy in CRC.