TMOD-30. Microglia and macrophages at the brain-tumor border have enriched expression of interferon gamma and complement signaling in naturally occurring canine brain metastatic melanoma: insight into the mechanism(s) of melanoma brain metastasis from man’s best friend

Abstract Brain metastasis is the leading cause of death for melanoma patients. While approximately 50% of advanced melanoma patients are affected, the mechanisms of melanoma brain metastasis (MBM) are incompletely understood, and treatment options are often ineffective. Pet dogs with naturally occurring melanoma may provide a complementary model to investigate novel therapeutics for MBM. We have demonstrated that MBM develops in approximately 40% of dogs, independent of systemic metastatic burden. Thus, we hypothesized that the primary tumor and brain microenvironment transcriptomic signature is distinct in dogs with MBM compared to melanoma-bearing dogs without MBM. We performed spatial transcriptomics on formalin-fixed paraffin-embedded tissue from primary tumors (n=6) and patient-matched brain with MBM (n=3) or without MBM (n=3) utilizing the Nanostring GeoMX Digital Spatial Profiler (DSP) platform in conjunction with the Canine Cancer Atlas panel. DSP RNA probes were hybridized to tissue sections and stained with fluorescent morphology markers to delineate malignant melanocytes (PNL2) and microglia/macrophages (IBA1). Three regions of interest (ROIs) were selected per tissue location and segmented into areas of illumination (AOIs) based on PNL2+ or IBA1+ immunoreactivity for molecular profiling. Illumina sequencing was performed on the NovaSeqX platform and differential expression analyses conducted. The results demonstrated that MBM-associated microglia/macrophages were indeed molecularly distinct from those in brain tissue without metastases. We further identified distinct gene expression profiles in IBA1+ cells across the tumor core, tumor border, and peritumoral normal brain locations. Specifically, tumor border IBA1+ cells had increased expression of interferon-inducible genes (BST2, GLUL, LRP1) and complement genes (C3, C1QA, C1QB) compared to tumor core IBA1+ cells (p<0.0001). As interdependent pathways, interferon and complement signaling may cooperate to promote metastasis through development of the pre-metastatic niche and immune regulation in MBM. Our continued analysis will inform future studies using naturally occurring canine melanoma as a preclinical model for human MBM.