ACETATE-PRODUCING BIFIDOBACTERIUM CATENULATUM BOOSTS ANTI-PD1 EFFICACY IN COLORECTAL CANCER VIA ACTIVATION OF CYTOTOXIC CD8+ CELLS

Background: Bifidobacterium catenulatum was reported to attenuate inflammation through modulating gut microbiota. Here, we investigated the roles of B. catenulatum as a potential probiotic in suppressing tumorigenesis and improving the efficacy of immune checkpoint blockade (ICB) therapy in colorectal cancer (CRC).

Methods: B. catenulatum abundance was evaluated in CRC patients (n=308) and healthy subjects (n=288) based on shotgun metagenomic sequencing from multiple cohorts. The effect of B. catenulatum in CRC tumorigenesis was determined in mice induced by azoxymethane (AOM) /dextran sulfate sodium (DSS) injection. The effect of B. catenulatum on anti-PD1 therapeutic efficacy was evaluated in CT26 (MSI-low) and MC38 (MSI-high) syngeneic mouse models. The changes in immune landscapes were identified by multicolor flow cytometry and immunohistochemistry staining. Functional metabolites were identified by non-targeted and targeted metabolomics assays (GC-MS) and verified in human CRC cell lines (HCT116, SW480, HT29). The effect of B. catenulatum and its metabolite on anti-PD1 efficacy were assessed in Apc^min/+ mice and AOM/DSS-induced tumorigenesis mice.

Results: B. catenulatum was significantly depleted in stool samples of CRC patients compared to healthy controls (p<0.05). Oral administration of B. catenulatum significantly inhibited AOM/DSS-induced CRC tumorigenesis in mice. B. catenulatum boosted anti-PD-1 therapy in two syngeneic models of CT26 and MC38, with increased intratumoral infiltration of TNF-α⁺, IFN-γ⁺ and granzyme B⁺ cytotoxic CD8⁺ T cells. Moreover, B. catenulatum enhanced the gut barrier integrity as evidenced by decreased gut leaky of FITC-dextran and increased expressions of tight junction proteins Claudin-1, Occludin and adherent protein E-cadherin in mice. The anti-tumor metabolite in B. catenulatum conditional medium was characterized as <3kD fraction which suppressed CRC cells viability. Acetate was identified as an enriched metabolite of B. catenulatum by non-targeted and targeted metabolomic profiling of bacteria culture supernatants and stool samples from B. catenulatum-treated mice. We confirmed that B. catenulatum and its derived acetate significantly augmented anti-PD1 efficacy and increased the intratumoral infiltration of cytotoxic CD8⁺ T cells in Apc^min/+ mice and CT26 allografts. Mechanistically, acetate directly binds to monocarboxy-late transporter (MCT)-4 in CD8⁺ T cells and activates its downstream Peroxisome proliferator-activated receptors (PPAR) signaling. Pharmacological blockade of MCT-4 by VB124 significantly abolished the activation of CD8⁺ T cells by acetate in vitro.

Conclusions: B. catenulatum protects against CRC tumorigenesis. B. catenulatum generated acetate boosts anti-PD1 efficacy in MSI-low CRC. Live B. catenulatum is a potential probiotic for CRC prevention and adjuvant immunotherapy.