Background: Clostridium butyricum is a known probiotic bacterium with anti-inflammatory activity. We found that it is depleted in patients with colorectal cancer (CRC) from multiple cohorts. Here, we examined the immunomodulatory effect of C. butyricumand its role in anti-PD-1 therapy against CRC.
Methods: The effect of C. butyricum was assessed in two syngeneic mouse models of CT26 (MSS) and MC38 (MSI), transgenic Apc^min/+ mice, and azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC tumorigenesis model. The change of immune landscape was identified by single-cell RNA sequencing and flow cytometry on mouse tumor tissues, and verified using CD34⁺ humanized mice and a co-culture system with patient-derived organoids and lymphocytes. The host-microbes crosstalk was evaluated by direct tissue culture, fluorescence in situ hybridization (FISH), and electron microscopy. Immunomodulatory protein of C. butyricum and the corresponding receptor in CRC cells were identified by mass spectrometry with Biotin pull-down, far western blotting and GST pull-down assays. Functional metabolites generated by C. butyricum were assessed by untargeted and targeted metabolomic profiling.
Results: Oral gavage of C. butyricum significantly improved anti-PD-1 efficacy in two syngeneic mouse models of CT26 (MSS) and MC38 (MSI). The synergistic effect of C. butyricum on anti-PD-1 therapy was validated in Apc^min/+ mice and AOM/DSS-induced tumorigenesis mouse model. Immune analysis on mouse tumor tissues revealed that C. butyricum blunted the recruitment of immunosuppressive tumor-associated macrophages (TAMs), and activated TNFa⁺, IFNg⁺ and Granzyme B⁺ cytotoxic CD8⁺T lymphocytes. C. butyricum was recognized as a tumor-resident bacteria, as demonstrated by direct tissue culture and FISH assay. Mechanistically, C. butyricum colonized in tumors via the binding of its secD protein to the surface receptor glucose-regulated protein (GRP)-78 on CRC cells. Such interplay inactivated GRP78 and reduced the secretion of CCL5 to restrain TAMs and promote intratumoral infiltration of CD8⁺ T cells. Moreover, butyrate is the key metabolite produced by C. butyricum by metabolomic analyses. For validation, CT26 or MC38 syngeneic mice treated with bacterial secD recombinant protein or butyrate synergized with anti-PD-1, improved antitumor immune response and delayed tumor growth. In addition, C. butyricum was capable of enhancing tumor-killing activity of CD8⁺ lymphocytes in patient-derived organoids and improving anti-PD-1 efficacy in CD34⁺ humanized CRC mouse model.
Conclusion: Intratumoral C. butyricum improved anti-PD-1 efficacy in CRC by directly interacting with tumor cells and producing butyrate to enhance antitumor immunity in mice. C. butyrium may be leveraged as a potential adjuvant to augment anti-PD-1 therapy against CRC.

Ruyi Zhang

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