Enterococcus faecalis KU-EF-004 enhances efficacy of anti-CTLA-4 immunotherapy for colorectal cancer via dendritic cell activation and gut microbiota modulation

Immune checkpoint inhibitors (ICI) have improved outcomes in mismatch repair–deficient/microsatellite instability–high colorectal cancer (CRC), yet only a subset of patients achieve durable benefit. The gut microbiota, particularly Enterococcus species, can modulate antitumor immunity and influence ICI efficacy. We investigated the probiotic potential of Enterococcus faecalis KU-EF-004, a non-antibiotic-resistant urinary isolate, as an adjunct to enhance ICI therapy in CRC. MC38 tumor-bearing C57BL/6J mice received combination therapy with KU-EF-004 and anti-PD-1 or anti-CTLA-4 antibodies, and the therapeutic efficacy was compared with E. faecalis ATCC700802 strain and PBS-treated groups. Additionally, combination therapy with KU-EF-004 and either anti-PD-1 or anti-CTLA-4 antibodies was performed to evaluate which immune checkpoint inhibitor was associated with enhanced therapeutic effects. Dendritic cell activation in Peyer’s patches was assessed by flow cytometry, and gut microbiota changes were analyzed using 16S rRNA gene sequencing. KU-EF-004 markedly enhanced anti–CTLA-4 efficacy, reducing tumor growth and prolonging survival, whereas no benefit was observed with anti–PD-1. KU-EF-004 promoted dendritic cell activation and upregulated immune markers (CD80, CD86, IFN-γ, MHC II, IL-6, CD8a) in Peyer’s patches. 16S rRNA sequencing revealed increased microbial diversity and enrichment of Lactobacillus species following combined KU-EF-004 and anti–CTLA-4 treatment. These findings identify E. faecalis KU-EF-004 as a promising probiotic candidate to augment immune checkpoint blockade efficacy in colorectal cancer.