Background Despite significant advances in understanding the role of microbiome in cancer and tumor immunity, the impact of microbiome in primary and metastatic brain tumors remains largely unknown. Elucidating the dynamic interaction between microbial entities and the brain tumor microenvironment can provide novel microbiome-based opportunities to enhance care for brain tumor patients.

Methods Brain tumor samples were collected prospectively from glioma and brain metastasis (BrM) patients at the MD Anderson Cancer Center and the University of Texas Health McGovern Medical School. Archived frozen non-cancerous brain samples from epilepsy patients were included as controls. Intra-tumoral bacterial signal was assessed and characterized through fluorescent in situ hybridization, 16S rRNA gene sequencing and metagenomic shotgun sequencing, and bacterial culture. Spatial Molecular Imaging (SMI, CosMx® Platform, NanoString Technologies) was used to determine the spatial distribution of intra-tumoral bacterial signal within the brain tumor microenvironment. Correlation of the bacterial signal with transcriptional or protein profile in carrier cells, their surrounding neighborhoods, and tumor regions was evaluated through SMI and digital spatial profiling (DSP, GeoMx® Platform, NanoString).

Results Bacterial 16S signals were detected in both glioma and BrM tumors, exhibited an intra-cellular localization, and were distributed across various cell types including tumor, stromal, and immune cells. Through sequencing, we identified a total of 16 bacterial taxa in glioma and BrM tumors with association with human commensal microbiota. However, cultivation of freshly resected tumor tissue did not yield bacterial colonies. This finding suggests that the brain tumor microenvironment lacks a viable or cultivable microbiota and the identified intra-cellular bacterial signal in brain tumors may arise from bacterial gene fragments. Lastly, SMI and DSP analyses demonstrated an association between intra-tumoral bacterial signal and anti-microbial, immune, lipid metabolism, and stress response signatures at the level of individual cells, neighborhoods, and tumor regions.

Conclusions Our studies introduce intracellular bacterial signal as a novel component of the brain tumor microenvironment and demonstrate its correlation with distinct tumor and immune transcriptional profiles. These findings offer a new perspective on the brain tumor microenvironment and can guide future mechanistic and translational studies to elucidate the functional role of the intra-tumoral bacterial signal in brain tumor biology.

Ethics Approval Clinical studies were conducted under the institutional approved IRB protocol (2012-4041). Informed consent was obtained from all patients before participation in the study.