Bifidobacterium longum RAPO Ameliorates Anti-tumor Efficacy and Immune-Related Adverse Events of Anti-PD-1 Therapy in a Mouse Model of Triple-Negative Breast Cancer

OBJECTIVES: Immune checkpoint inhibitors (ICIs) have been considered as a novel treatment of triple negative breast cancer (TNBC) representing higher risk of early metastasis and relapse. ICIs induce immune-related adverse events (irAEs) including myocarditis, pneumonitis, hepatitis, nephritis, and colitis, which can cause ICIs discontinuation. Thus, finding treatment strategies to enhance efficacy while reducing irAEs of ICIs is important to improve prognosis and quality of life of cancer patients. We investigated whether combination therapy of anti-PD-1 with Bifidobacterium longum RAPO would affect efficacy and irAEs against TNBC. METHODS: 4T1 tumor-bearing mice were assigned to tumor control, B. longum RAPO (RAPO), Anti-PD-1 (Mono), or Anti-PD-1 + RAPO (Combi) group. Tumor tissues were analyzed by IHC, qRT-PCR, and Affymetrix Mouse Gene 2.0 ST Array followed by functional analysis using IPA®. Heart, lung, liver, kidney, and colon tissues were analyzed by qRT-PCR and Western blot. RESULTS: B. longum RAPO supplementation extended survival of tumor-bearing mice compared with other groups. In the tumor tissue, Combi group showed the increased levels of PD-L1 score, TUNEL positive cells, and anti-tumor M1 cytokines such as IFNγ and CXCL9 compared with Mono group, while pro-tumor M2 cytokines were decreased in Combi group. Functional analysis predicted reduction of cardiac lesion and lung inflammation in Combi group. Consistently, expression of CCL3 and CC5, chemokines associated with cardiac lesion, was reduced in the heart tissue of Combi group. Pro-inflammatory cytokines including IL1β and TNFα were significantly decreased, whereas anti-inflammatory cytokine IL10 was increased in the heart, lung, liver, and kidney tissues of Combi group than Mono group. Myeloperoxidase, a pro-inflammatory enzyme released by activated neutrophils, was also significantly reduced in the liver tissue of Combi group. Colonic expression of IL10 and tight junction proteins such as ZO-1 and occludin was significantly increased in Combi group than Mono group. CONCLUSIONS: B. longum RAPO supplementation not only enhances anti-tumor efficacy also improves the risk of irAEs in mice with TNBC, suggesting that combination therapy with B. longum RAPO might be a promising therapeutic strategy for Anti-PD-1 immunotherapy. FUNDING SOURCES: BIFIDO CO., NRF (2020R1C1C1007553 to S-EK).