Abstract

Abstract Metastatic brain tumors are associated with significant morbidity and mortality. Recent trials have demonstrated the potential efficacy of immunotherapy for the management of brain metastasis (BrM), albeit in only a subset of patients. Understanding the BrM microenvironment and identifying the factors that shape this complex milieu can facilitate optimizing treatment strategies for this disease. The microbiome has been recognized as a hallmark of cancer with a major role in shaping response to immunotherapy. However, the role of the microbiome in BrM is largely unknown. We have previously reported that bacterial signals can be detected in BrM tumors via metagenomic analyses and staining. Here we present the spatial correlation between the intra-tumoral bacterial signal and the transcriptional changes in the tumor microenvironment. Tissue digital spatial profiling (DSP) and single-cell DSP were conducted on BrM tissue microarrays using the NanoString GeoMx® and CosMx® platforms, respectively. These assays incorporated a custom panel of pan-bacterial 16S rRNA probes, enabling a comparison between tumor regions and cells that showed a positive signal for bacterial 16S rRNA versus those that did not. DSP of BrM tumors revealed anti-bacterial responses and enrichment of innate immune activity in areas with 16S signal, suggesting an active host response to intra-tumoral 16S signals. Furthermore, single-cell DSP demonstrated a heterogenous distribution of cell types harboring the 16S signal in the tumor microenvironment. In tumor cells, intracellular 16S signal was associated with an upregulation of transcripts related to neuroinflammation, chemokine, and interleukin signaling. Furthermore, immune transcriptional changes were identified in the immediate neighborhood of 16S-positive cells. Overall, this study introduces bacterial signals as a novel element within metastatic brain tumors and demonstrates their association with anti-bacterial responses and immune activity. These findings set the foundation for future mechanistic and translational studies to evaluate microbiome modulation strategies in enhancing care for brain metastasis.