Abstract 5076: Unravelling melanoma brain metastasis tumor microenvironment characteristics using spatial transcriptomics

Background: Advanced melanoma is an aggressive cancer with a high metastatic potential often resulting in melanoma brain metastases (MBM). The complexity of the MBM tumor microenvironment (TME) impacts tumor progression and therapy response, leading to poor outcomes. Our study integrates spatial transcriptomics profiles with tissue morphology annotations of MBM and clinical data to investigate the TME landscape in relation to treatment and patient outcomes. Methods: We performed spatial transcriptomics on 21 MBM samples collected after various treatment strategies. All samples were preserved in formalin-fixed paraffin-embedded (FFPE) blocks and profiled using the Visium Spatial Gene Expression kit (10X Genomics) with a 6.5x6.5mm Visium Capture slide. All sequencing was performed on a NovaSeq6000 platform (Illumina) with an average of 71,391 reads per spots. H&E slides were annotated by our study pathologist (DL) to enable pathology-guided cluster analysis. Spatial transcriptomics data analysis was conducted using Seurat R pipeline which involved data quality check, normalization, integration, cell type annotation and deconvolution, and clustering. Further differential gene expression analyses were performed between different identified tumor clusters or between clinical groups, leading to the identification of enriched pathways and cell-cell interaction patterns. Results: Our phenotyping of 25,208 spots revealed the presence of diverse cell populations, including neurons, melanoma, plasma, endothelial, stromal, and myeloid cells. We identified 8 distinct functional clusters characterized by specific gene expression and activated pathways. These include 5 tumor clusters with different immune infiltration or expression profiles, identifying differences in cell metabolism and heterogeneity in the TME. Importantly, we found MBM from patients exposed to radiation were significantly enriched for epithelial-to-mesenchymal transition pathway and genes related to cell adhesion. We further showed a reduced immune response in patients received radiation compared those who did not, and we noted that an active immune response was generally positively correlated with survival. Conclusions: Our study is the largest spatial analysis of MBM reported to date. Advances in spatial technologies provide novel insights into the structure and composition of MBM TME. Our findings highlight the importance of spatial context in understanding MBM biology and the potential of spatial transcriptomics in advancing precision oncology. Citation Format: Clemence J. Belle, Sandra Brosda, Vanessa F. Bonazzi, Victor Bulteau, Thomas Stuart, Zherui Xiong, Duncan Lambie, Peter A. Johansson, Lauren G. Aoude, Kalpana Patel, Samantha J. Stehbens, Mitchell S. Stark, Nikolas K. Haass, Wen Xu, Mark B. Pinkham, Matthew C. Foote, Sarah Olson, Victoria Atkinson, Arutha Kulasinghe, Quan H. Nguyen, Andrew Barbour. Unravelling melanoma brain metastasis tumor microenvironment characteristics using spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5076.