Bifidobacterium breve inhibits colorectal cancer via extracellular vesicles containing formate acetyltransferase.

B. breve abundance was significantly reduced in fecal samples from CRC patients and tumor-bearing mice.

• Analysis was performed using the curated database of human gut metagenomes cohort (GMrepo) database.
• An MC38 subcutaneous tumor model was used to confirm reduced B. breve abundance in tumor-bearing mice.
• This reduction was identified through integrative analysis combining human metagenomics data and murine tumor models.

Extracellular vesicles (EVs) are the critical effector entities mediating B. breve's anti-tumor activity.

• Administration of live B. breve or its cell-free supernatant markedly inhibited tumor growth.
• Pasteurized bacteria failed to inhibit tumor growth, implicating secreted factors rather than live bacteria alone.
• GW4869-mediated EVs blockade abolished the tumor-inhibitory effect, directly demonstrating the necessity of EVs.
• These results indicate that EVs, not the bacteria themselves, are the critical effector entities.

Isolated B. breve-derived EVs (B.breEVs) selectively accumulated within tumor tissue and directly triggered apoptosis of colorectal cancer cells.

• B.breEVs showed selective accumulation within tumor tissue following administration.
• B.breEVs directly triggered apoptosis of colorectal cancer cells.
• B.breEVs elevated the proportion of IFN-γ+ CD8+ cytotoxic T lymphocytes (CTLs) within the tumor microenvironment.
• B.breEVs concurrently ameliorated gut microbial structure and function.

Mass-spectrometric profiling identified formate acetyltransferase (pflB) as an important active protein within B.breEVs.

• Proteomic profiling of B.breEVs was conducted using mass spectrometry.
• pflB was identified as a key active protein cargo within the EVs.
• Recombinant pflB selectively inhibited MC38 cell viability in vitro.
• Recombinant pflB significantly reduced CRC burden in vivo.

RNA sequencing of tumor tissue demonstrated that pflB upregulated granzyme B, perforin1, and CTL/NK-associated transcripts, and activated the intrinsic apoptotic pathway.

• RNA sequencing was performed on tumor tissue from pflB-treated animals.
• pflB upregulated granzyme B and perforin1, which are key cytotoxic effector molecules.
• CTL/NK-associated transcripts were upregulated by pflB treatment.
• pflB activated the intrinsic (mitochondrial) apoptotic pathway in tumor cells.

Combination of pflB with anti-PD1 therapy markedly increased infiltration of CD8+ CTL and NK cells and enhanced their cytotoxicity compared to either monotherapy.

• Immuno-combination studies were conducted using pflB plus anti-PD1 therapy.
• The combination markedly increased CD8+ CTL infiltration into tumors.
• The combination markedly increased NK cell infiltration into tumors.
• The cytotoxicity of CD8+ CTL and NK cells was enhanced by the combination compared to either monotherapy alone.
• The combination was found to enhance effects of immune checkpoint blockers and overcome drug resistance.