<i>PEPTOSTREPTOCOCCUS ANEROBIUS </i>DIVES IMMUNE THERAPY RESISTANCE THROUGH MODULATING MDSCS IN COLORECTAL CANCER

Background and Aims: Peptostreptococcus stomatis (P. stomatis) is an enriched bacterium in colorectal cancer (CRC) patients as compared to healthy subjects in multiple cohorts. However, the potential role of P. stomatis in colorectal tumorigenesis remains obscure. In this study, we investigate the function of P. stomatis in CRC, and its impact on CRC therapy response.

Methods: Pro-tumorigenic effect of P. stomatis on colorectal carcinogenesis was determined in Apc^Min/+ mice and azoxymethane/Dextran sodium sulfate (AOM-DSS)-treated mice models. In vitro function of P. stomatis was analysed by cell proliferation, apoptosis, and cell cycle assays. Interplay between P. stomatis and CRC cells was evaluated by fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), transmission electron microscopy (TEM), adhesion and invasion assays. The interaction between P. stomatis surface protein and the receptor of CRC cells was investigated by far-western, biotin pulldown, and glutathione S-transferase (GST) pulldown assays.

Results: Oral gavage of P. stomatis significantly promoted CRC tumorigenesis in Apc^Min/+ mice with increased colon tumor number and tumor load, which accompanied with enhanced cell proliferation, reduced apoptosis and impaired gut barrier function. The pro-tumorigenic effect of P. stomatis was validated in another CRC tumorigenesis model induced by AOM-DSS and in CRC cell line Caco2 formed xenograft models. Consistently, P. stomatis co-culture with CRC cells accelerated cell growth, induced G_1-S cell cycle progression, and suppressed apoptosis. FISH, TEM and in vitro adhesion/invasion assays showed that P. stomatis colonized on CRC cells and colonic epithelium cells of mice, with preferential adherence in tumor cells compared to normal colon cells. Mechanistically, P. stomatis surface protein fructose-1,6-bisphosphate aldolase (FBA) binds to integrin α6/β4 receptor of CRC cells and activates ERBB2 phosphorylation, leading to the consequential activation of MEK-ERK-p90 oncogenic signaling. Integrin α6/β4 knockdown abolished P. stomatis adherence and P. stomatis-induced ERBB2-MEK-ERK-p90 activation in vitro, implying that the FBA-integrin α6/β4 interaction is essential for P. stomatis-mediated tumorigenesis. Finally, we demonstrated that P. stomatis co-culture with colon cancer cell lines Caco-2 and HT-29 significantly abolished the therapeutic effects of Cetuximab and Vemurafenib, implying P. stomatis as a novel factor driving tyrosine kinase (RTK) inhibitor resistance through alternative activation of ERBB2 in CRC.

Conclusion: P. stomatis is a novel oncogenic bacterium in promoting colorectal carcinogenesis. P. stomatis surface protein FBA engages an integrin α6/β4-ERBB2 complex leading to aberrant activation of host oncogenic MEK-ERK-p90 signaling to drive CRC development and cause resistance to RTK inhibitors.

BACKGROUND & AIMS: Peptostreptococcus anaerobius is a pathogenic bacterium enriched in colorectal cancer (CRC) and it promotes colorectal tumorigenesis in mice. Here, we investigated the functional role of P. anaerobius in the tumor immune microenvironment (TIME) of CRC, the molecular basis of its action, and the consequential impact on the efficacy of immune checkpoint blockade (ICB) therapy.
METHODS: Immunomodulatory effects of P. anaerobius in Apc^Min/+ , azoxymethane (AOM)- or AOM/ Dextran Sodium Sulfate (DSS)-treated CRC mouse models were investigated by single cell RNA-sequencing (scRNA-seq), flow cytometry and immunofluorescence staining. The effect of P. anaerobius on anti-PD-1 efficacy was evaluated in syngeneic mouse CRC cell line MC38 allograft and in AOM/DSS-induced CRC tumorigenesis in mice. Cytokine array was performed to identify chemokines produced by CRC cells upon P. anaerobius treatment. Immunomodulatory protein secreted by P. anaerobius was identified by mass spectrometry. Microscale thermophoresis and GST pull-down assay were used to identify corresponding receptor on MDSCs.
RESULTS: P. anaerobius significantly induced MDSCs accumulation, whilst suppressed IFNγ⁺CD8⁺ T cells in colonic tumors of Apc^Min/+, which was confirmed in AOM-induced CRC and AOM/DSS-induced CRC mouse models by scRNA-seq, flow cytometry and immunofluorescence staining analyses. By fostering an immunosuppressive TIME, P. anaerobius administration abolished the efficacy of anti-PD-1 therapy both in MC38 allografts and in AOM/DSS-induced CRC tumorigenesis in mice. Mechanistically, P. anaerobius simultaneously promotes MDSCs recruitment and activation through a two-step pathway. Firstly, P. anaerobius co-culture promoted CXCL1 secretion by CRC cells via the integrin α₂/β₁-NF-κB axis. P. anaerobius-induced CXCL1 up-regulation in turn stimulated the migration of CXCR2⁺ MDSCs. Knockdown of integrin α₂/β₁ or NF-κB inhibitor reversed P. anaerobius-induced CXCL1 secretion by CRC cells, accompanied by attenuated MDSCs chemotaxis. Secondly, P. anaerobius culture supernatant directly activated immunosuppressive function of intratumoral MDSCs. Mass spectrometry revealed a P. anaerobius secreted protein, LytC_22, which binds to SLAMF4 receptor on MDSCs to promote ARG1 and iNOS expression, thus enhancing the ability of MDSCs to inhibit cytotoxic T cell proliferation and function. Blockade of integrin α₂/β₁ or Slamf4 receptor abrogated P. anaerobius-induced MDSCs trafficking and activation, and restored anti-PD-1 efficacy in CRC tumor models.
CONCLUSION: P. anaerobius provokes resistance to anti-PD-1 treatment in CRC by promoting MDSCs chemotaxis and activation, which in turn suppresses T cell activation. Targeting integrin α2/β1 or SLAMF4 receptor is a promising strategy to overcome P. anaerobius-induced anti-PD-1 resistance in CRC patients.