Bifidobacterium breve inhibits colorectal cancer via extracellular vesicles containing formate acetyltransferase

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. The gut microbiota exerts unique therapeutic advantages against CRC, and the probiotic Bifidobacterium breve (B. breve) has been extensively documented to suppress CRC initiation in murine models. Although the role of B. breve in CRC has been established, whether its extracellular vesicles (EVs), as key mediators of bacteria-host crosstalk, exert a functional impact remains undefined. Here, we aim to explore the therapeutic potential of B. breve-derived EVs (B.breEVs) and their active cargo, formate acetyltransferase (pflB), in CRC. Integrative analysis of the curated database of human gut metagenomes cohort (GMrepo) database and an MC38 subcutaneous tumor model revealed a significant reduction of B. breve abundance in faecal samples from CRC patients and tumor-bearing mice. Administration of live B. breve or its cell-free supernatant markedly inhibited tumor growth, whereas pasteurized bacteria or GW4869-mediated EVs blockade abolished this effect, indicating that EVs are the critical effector entities. Isolated B.breEVs selectively accumulated within tumor tissue, directly triggered apoptosis of colorectal cancer cells, and elevated the proportion of IFN-γ⁺ CD8⁺ cytotoxic T lymphocytes (CTLs) in tumor while concurrently ameliorating gut microbial structure and function. Mass-spectrometric profiling identified the pflB as an important active protein within B.breEVs. Recombinant pflB selectively inhibited MC38 cell viability in vitro and significantly reduced CRC burden in vivo. RNA sequencing of tumor issue demonstrated that pflB up-regulated granzyme B, perforin1 and CTL/NK-associated transcripts, and activated the intrinsic apoptotic pathway. Immuno-combination studies further revealed that pflB plus anti-PD1 therapy markedly increased the infiltration of CD8⁺ CTL and NK cells, and enhanced their cytotoxicity compared to either monotherapy. B. breve secretes pflB-loaded EVs that reshape the intestinal micro-ecology, activate CD8⁺ CTL/NK anti-tumor immunity, directly induce mitochondrial apoptosis in malignant cells, and enhance the effects of immune checkpoint blockers to overcome drug resistance, offering a precision “probiotic-EVs-active protein” triadic intervention strategy for CRC.