Combination Therapy of Bifidobacterium longum RAPO With Anti-PD-1 Treatment Enhances Anti-tumor Immune Response in Association With Gut Microbiota Modulation

OBJECTIVES: Triple negative breast cancer (TNBC) has a poor prognosis with a high risk of metastasis and relapse, and accounts for approximately 35% of breast cancer death. Immune checkpoint inhibitors (ICI) have been in the spotlight recently as a novel treatment of TNBC due to the higher expressions of programmed cell death ligand 1 (PD-L1) and tumor-infiltrating lymphocytes in TNBC. Considering that gut microbiome is associated with immune response and ICI efficacy, we investigated whether Bifidobacterium longum RAPO supplementation would affect the efficacy of anti-PD-1 therapy against TNBC in vivo. METHODS: Female BALB/c mice bearing 4T1 breast cancer cells were randomly divided into four groups; tumor control (TC), Anti-PD-1, B. longum RAPO (RAPO), or Anti-PD-1 + RAPO (Combi). Anti-PD-1 antibody was injected i.p. 5 times at 3-day intervals and B. longum RAPO was orally administered daily from 2 days before the first injection of anti-PD-1. Spleen and tumor tissues were analyzed by flow cytometry, IHC, and qRT-PCR. Fecal samples were analyzed by 16S rRNA gene sequencing. RESULTS: Tumor volume was reduced in the Combi group than TC and Anti-PD-1 groups on day 12. PD-L1 IHC score and PD-L1 mRNA expression were significantly increased in tumors of the Combi group. The levels of the spleen CD8/CD4 ratio and tumor NK cells were also increased in the Combi group. Compared with the Anti-PD-1 group, the pro-tumor M2 macrophages and related cytokines (IL10, Arg1) were significantly decreased, while anti-tumor M1 cytokines (IFNγ, TNFα) were elevated in tumors of the Combi group. Consistently, immunogenic cell death-associated markers were significantly higher in the Combi group. We found that the relative proportion of the genus Bifidobacterium, Lachnoclostridium, Lachnospiraceae NK4A136 group, and Clostridium sensu stricto 1, which are known to be associated with the better response to ICI therapy, were significantly increased, while the genus Mucispirillum and Dubosiella were decreased in the Combi group than the Anti-PD-1 group. CONCLUSIONS: Our data indicates that combination therapy of B. longum RAPO with anti-PD-1 enhances anti-tumor immune response in association with modulation of gut microbiota, suggesting that B. longum RAPO might be a potential candidate for pharmabiotics in ICI treatment. FUNDING SOURCES: BIFIDO CO, the National Research Foundation of Korea grant.