Single-cell and bulk RNA-seq unveils the immune infiltration landscape associated with cuproptosis in cerebral cavernous malformations

Abstract Background Cerebral cavernous malformations (CCMs) are vascular abnormalities associated with deregulated angiogenesis. Their pathogenesis and optimal treatment remain unclear. This study aims to investigate the molecular signatures of cuproptosis, a newly identified type of cell death, associated with CCMs development. Methods Bulk RNA sequencing (RNA-seq) from 15 CCM and 6 control samples were performed with consensus clustering and clustered to two subtypes based on expression levels of cuproptosis-related genes (CRGs). Differentially expressed genes and immune infiltration between subtypes were then identified. Machine learning algorithms including the least absolute shrinkage and selection operator and random forest were employed to screen for hub genes for CCMs associated with cuproptosis. Furthermore, Pathway enrichment and correlation analysis were used to explore the functions of hub genes and their association with immune phenotypes in CCMs. An external dataset was then employed for validation. Finally, employing the Cellchat algorithm on a single-cell RNA-seq dataset, we explored potential mechanisms underlying the participation of these hub genes in cell-cell communication in CCMs. Results Our study revealed two distinct CCM subtypes with differential pattern of CRG expression and immune infiltration. Three hub genes (BTBD10, PFDN4, and CEMIP) were identified and validated, which may significantly associate with CCM pathogenesis. These genes were found to be significantly upregulated in CCM endothelial cells (ECs) and were validated through immunofluorescence and western blot analysis. Single-cell RNA-seq analysis revealed the cellular co-expression patterns of these hub genes, particularly highlighting the high expression of BTBD10 and PFDN4 in ECs. Additionally, a significant co-localization was also observed between BTBD10 and the pivotal cuproptosis gene FDX1 in Mki67+ tip cells, indicating the crucial role of cuproptosis for angiogenesis in CCMs. The study also explored the cell-cell communication between subcluster of ECs expressing these hub genes and immune cells, particularly M2 macrophages, suggesting a role for these interactions in CCM pathogenesis. Conclusion This study identifies molecular signatures linking cuproptosis to CCMs pathogenesis. Three hub genes—PFDN4, CEMIP, and BTBD10—may influence disease progression by modulating immunity. Further research is needed to understand their precise disease mechanisms and evaluate their potential as biomarkers or therapeutic targets for CCMs.