Microbial signals in primary and metastatic brain tumors

Gliomas and brain metastases (BrM) are associated with poor prognosis, necessitating a deeper understanding of brain tumor biology and the development of effective therapeutic strategies. While our group and others have demonstrated microbial presence in various tumors, recent controversies regarding cancer-type-specific intra-tumoral microbiota emphasize the importance of rigorous, orthogonal validation. This prospective, multi-institutional study included a total of 243 samples from 221 patients, comprising 168 glioma and BrM samples and 75 non-cancerous or tumor-adjacent tissues. Using stringent fluorescent in situ hybridization, immunohistochemistry, and high-resolution spatial imaging, we detected intracellular bacterial 16S rRNA and lipopolysaccharides in both glioma and BrM samples, localized to tumor, immune, and stromal cells. Custom 16S and metagenomic sequencing workflows identified taxa associated with intra-tumoral bacterial signals in the tumor microenvironment; however, standard culture methods did not yield readily cultivable microbiota. Spatial analyses revealed significant correlations between bacterial 16S signals and anti-microbial and immunometabolic signatures at regional, neighborhood, and cellular levels. Furthermore, intra-tumoral 16S bacterial signals showed sequence overlap with matched oral and gut microbiota, suggesting a possible connection with distant communities. Together, these findings introduce microbial elements as a component of the brain tumor microenvironment and lay the foundation for future mechanistic and translational studies. Two glioma and brain metastasis tissue microarray slides (FFPE, 5 µm) were profiled using the NanoString GeoMx® Digital Spatial Profiler with the Immuno-Oncology Protein Atlas (>570 proteins) and Human Whole Transcriptome Atlas (~18,000 genes), supplemented with custom pan-microbial 16S rRNA probes. Morphology markers were Alexa Fluor 488–SYTO 13 (nuclei) and Alexa Fluor 594–anti-CD3 (TILs). ROIs (300 µm) from high- and low-TIL tumor regions (92 glioma, 90 brain metastases) were UV-cleaved and processed for RNA and protein libraries, pooled at a 2:1 molar ratio, and sequenced on an Illumina NovaSeq 6000 (2 × 27 bp, paired-end). Raw FASTQ files are provided for RNA (WTA + 16S) and protein; processed ROI-level count matrices include both raw and normalized counts.