Single-cell profiling and machine learning identify cuproptosis-related fibroblast subpopulations and fibrogenesis...

Endometriosis is characterized by progressive fibrosis and limited therapeutic options. Cuproptosis, a copper-dependent form of regulated cell death, has been implicated in multiple pathological conditions, but its relevance to fibroblast-mediated fibrotic progression in endometriosis remains unclear. Single-cell RNA sequencing data from normal, eutopic, and ectopic endometrial tissues were analyzed to assess cuproptosis-related gene (CRG) activity and fibroblast heterogeneity. Pseudotime analysis, cell-cell communication analysis and high-dimensional weighted gene co-expression network analysis were performed to identify disease-associated fibroblast states and candidate fibrosis-related genes. Machine learning approaches were applied to prioritize candidate hub genes. Functional validation was conducted in endometrial stromal cells, and a mouse model of endometriosis was used to assess the effects of tetrathiomolybdate (TTM), a copper chelator. Elevated CRG activity was enriched in a distinct fibroblast subpopulation with profibrotic transcriptional features. Network and machine learning analyses consistently prioritized AEBP1 as a candidate fibroblast-associated hub gene linked to cuproptosis-related signatures. In vitro, CuCl₂ plus elesclomol treatment was associated with increased AEBP1 and fibrosis-related marker expression, accompanied by changes in β-catenin pathway-related proteins, whereas FDX1 or AEBP1 knockdown attenuated these effects. In vivo, TTM treatment reduced lesion burden, fibrotic marker expression and collagen deposition in ectopic lesions. Cuproptosis-related molecular alterations are associated with fibroblast activation and fibrotic progression in endometriosis. Targeting copper metabolism may have therapeutic potential in limiting lesion fibrosis.