In-Depth Patient-specific Analysis of Tumor Heterogeneity in Melanoma Brain Metastasis: Insights from Spatial Transcriptomics and Multi-Region Bulk Sequencing

Melanoma brain metastasis (MBM) response to treatment varies greatly among patients, affecting overall prognosis and survival. This study aims to provide a comprehensive multi-omics overview of MBM by investigating tumor tissue composition at spatial, proteomic and transcriptomic levels, elucidating the spatial tumor landscape, characterizing tumor tissue cell populations and identifying enriched signaling pathways in the MBM tumor tissue. We found higher inter-tumoral heterogeneity than intra-tumoral heterogeneity in MBM at the protein and transcriptional levels. The treatment-naive patient exhibited high intra-tumoral heterogeneity (ITH) compared to patient who received treatment, with ITH levels varying across neighboring regions in all patient tumors. Significant global protein and cell type enrichment associated with malignant cells, macrophages, CAFs, and immune cells was observed in MBM using cellular deconvolution and tumor microenvironment (TME) analysis. The presence of MBM cell-type specific gene signatures, and enriched pathways identified using ST and bulk-seq provide an essential framework for understanding tumor composition and potential treatment-related effects in each MBM patient tumor. Taken together, our results provide a comprehensive spatial and molecular view of intra-tumoral and inter-tumoral heterogeneity in MBM, potentially guiding personalized treatment strategies in MBM therapy. Overall design: We obtained fresh frozen tumor tissues from four patients with MBM. The freshly frozen (FF) tissue blocks were OCT-fixed and sectioned using a cryotome. Briefly for multi-region bulk sequencing, 20 sequential sections of 20 µm were collected for four regions, starting with regions (R) 1- 4. We analyzed transcriptome of multiple regions (R1-4) in each patient tumor. Here, multiple region data for patient 1 (P1) is labeled as P1-1 (for R1), P1-2 (for R2), P1-3 (R3) and P1-4 (R4). Likewise, samples for other patients (P2, P3, P4) are also labeled in a similar way.