Dietary supplementation with beetroot modulates gut microbiota-derived diacylglycerol biosynthesis to enhance anti-tumor immunity in MMTV-PyMT (FVB) mice†

Recent studies have established a strong connection between gut microbiota and the tumor immune microenvironment. Beetroot (BRT) has emerged as a promising functional food for cancer prevention and treatment, although its specific effects on breast cancer remain unclear. This study investigates the impact of BRT on polyomavirus middle T antigen (PyMT)-induced breast cancer by examining the gut microbiome, metabolites, and immune responses. Dietary supplementation of 80 g BRT powder per 1000 g of standard diet (8% w/w, 80 g per kg) significantly inhibited tumor growth and metastasis in mouse mammary tumor virus (MMTV)-PyMT (FVB) mice, while simultaneously enhancing anti-tumor immunity. 16S rRNA sequencing revealed that BRT supplementation altered the gut microbiota, notably increasing the abundance of Lachnospiraceae and Ruminococcaceae. An antibiotic (ABX) experiment confirmed that BRT restores gut microbiota balance, thereby enhancing anti-tumor immunity. Metabolomics and ELISA assays demonstrated that BRT regulates diacylglycerol (DAG) biosynthesis through microbiota modulation. Notably, diacylglycerol kinase ζ (DGKζ) inhibitors enhance CD8+ T cell responses by increasing DAG levels, thereby activating Mitogen-Activated Protein Kinase (MAPK) and Protein Kinase C (PKC) signaling pathways. Collectively, our findings suggest that microbiota-derived DAG plays a key role in BRT-mediated enhancement of anti-tumor immunity.