Samples & cell type composition in sc/snRNA-seq.

Melanoma brain metastasis (MBM) remains lethal with limited treatment efficacy. Meanwhile, the cellular origins and drivers of brain metastasis from melanoma have yet to be defined. Through integrated single-cell/single-nucleus RNA sequencing of 26 melanoma samples, we identified a pre-brain-metastatic tumor subpopulation (MBMATCs, MBM-associated tumor cells) within a conserved malignant cell trajectory (Mela3). MBMATCs exhibited activated pro-metastatic pathways and upregulated neural/adhesion genes (NRG3, NCAM1), suggesting a cellular origin for brain tropism. MBM ecosystems showed T cell exhaustion (elevated PD-1, HAVCR2, LAG3) and Treg enrichment. An MBM-Index derived from bulk RNA-seq accurately quantifies MBMATCs abundance, independently predicting poor overall survival in both TCGA-SKCM and validation cohorts. Furthermore, we assessed the clinical relevance of the MBM-Index and uncovered five candidate drugs with potential activity against MBMATCs. This study identifies MBMATCs as brain metastasis associated tumor cells and positions the MBM-Index as a biomarker for early stratification of melanoma patients at high risk of brain metastasis.