Methylmalonic acid modulates neutrophil function to promote tumor progression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with its progression driven by metabolic reprogramming and an immunosuppressive microenvironment. Methylmalonic acid (MMA), a metabolic byproduct accumulated in CRC, participates in modulating neutrophil function through mechanisms that remain poorly defined. METHODS: We integrated metabolomics, bioinformatics, and multi-omics analyses with in vitro and in vivo experiments. The effects of MMA on neutrophil polarization, proliferation, and invasion were assessed using HL-60-induced and human-derived neutrophils, along with colorectal cancer cell lines (HCT116 and HT29). Functional assays included ELISA, immunofluorescence, EdU proliferation, and invasion assays, while xenograft models assessed tumor growth. Multi-omics interrogation of TCGA, GEO, and CPTAC datasets identified MMA-associated genes and pathways, with hub gene analysis pinpointing key regulators. Plasma MMA levels were quantified in CRC patients via LC–MS/MS, and tumor CXCL8 expression was examined by immunofluorescence. Serum vitamin B12 levels were measured by chemiluminescence immunoassay. RESULTS: MMA induced dose-dependent N2 neutrophil polarization, evidenced by elevated IL-10, TGF-β, MPO secretion, and CD206 expression, alongside reduced iNOS. MMA-conditioned neutrophils enhanced CRC proliferation and invasion in vitro and promoted tumor growth in xenograft models. Multi-omics analysis revealed 50 MMA-associated differentially expressed genes enriched in SMAD signaling, fatty acid metabolism, and mitochondrial pathways, with CXCL8 emerging as a central hub. Mechanistically, MMA upregulated CXCL8 while suppressing p53 and activating TLR4, thereby driving N2 polarization; CXCL8 silencing abrogated these effects. Clinically, CRC patients exhibited elevated plasma MMA and tumor-specific CXCL8 overexpression, without significant alterations in vitamin B12 status. CONCLUSIONS: MMA functions as a metabolic driver of CRC progression, likely by promoting CXCL8-mediated N2 neutrophil polarization through the p53/TLR4 axis. Elevated plasma MMA and tumor CXCL8 expression from CRC patients highlight their potential as biomarkers and therapeutic targets, providing important insights for clinical diagnosis and treatment.