Gut microbiota induces immune-related alterations in gene expression, RNA methylation, and metabolism in glioblastoma revealed by single-cell and spatial multi-omics.

Glioblastoma (GBM) is a highly malignant tumor with poor prognosis and limited effective treatment options. Emerging studies have suggested that gut microbiota may influence glioma progression through the gut-brain axis, though the precise mechanisms remain largely unclear. In this study, we employed a comprehensive multi-omics approach-encompassing single-cell transcriptomics, spatial transcriptomics, metagenomics, metabolomics, and m6A-seq-to investigate how antibiotic-induced gut microbiota disruption impacts glioma progression in a mouse model. Gene expression analysis revealed significant alterations in antibiotics-treated mice (ABX-treated mice), including reduced expression of Epha6 and upregulated expression of Tead1, key genes associated with glioma progression and immune modulation. Spatial transcriptomics and metabolomic profiling identified reduced methionine levels in gliomas of ABX-treated mice, linking gut-derived metabolite changes to epigenetic regulation via m6A methylation. Single-cell RNA sequencing further demonstrated an increased proportion of AC-like cells, disrupted intercellular communication, and aberrations in the EPHA and NRXN signaling pathways. These findings highlight the interplay between gut microbiota, immune signaling, and epigenetic modifications in shaping the glioma microenvironment. This study advances our understanding of the gut-brain axis in glioma biology and proposes the EPHA pathway as a promising biomarker for the immune-mediated modulation of tumor progression, thereby providing new insights into the role of the gut-brain axis in glioma regulation.