Single-Cell Transcriptomic Analysis Reveals Ferroptosis-Related Signatures and Tumor Microenvironment Heterogeneity...

Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy. Ferroptosis, an iron-dependent lipid peroxidation-associated cell death mode, provides a new target for tumor therapy. Nevertheless, its impact on EOC tumorigenesis is not well defined. The quality control, dimensionality reduction, and EOC clustering for the gene expression data were performed through the Scanpy pipeline for single-cell RNA sequence from GEO (GSE165897). Ferroptosis pathway scoring, computation of transcription factor activity, and analyzes for cell-cell communication were conducted. An independent validation cohort (GSE300897, n = 19,002 cells) was analyzed to confirm ferroptosis signature robustness. Expression of ferroptosis-related genes in SKOV3 and OVCAR3 cells was confirmed using RT-qPCR and immunoassays after treatment with erastin, RSL3, and ferrostatin-1 rescue. NRF2 siRNA knockdown and co-culture experiments were performed for functional validation. A total of 51,702 cells from 11 patients were analyzed and clustered into 18 clusters that included EOC cells (16.9%), immune cells (67.6%), and stromal cells (15.6%), encompassing 27 fine-grained cell subtypes. KEGG enrichment revealed that ferroptosis, fatty acid metabolism, and glutathione metabolism were the major pathways. Transcription factor profiling indicated increased NRF2 activation in EOC cells, which implied an augmented ferroptosis-resistance capacity. Signaling between cells also revealed three key ligand-receptor interactions, SPP1-CD44, VEGFA-KDR, and CCL2-CCR2, which are involved in the regulation of ferroptosis and were functionally validated in a triple co-culture model. NRF2 siRNA knockdown synergistically enhanced erastin-induced ferroptosis sensitization (C11-BODIPY: 4.52-fold), rescued by ferrostatin-1. Platinum-refractory patients in GSE300897 showed significantly altered ferroptosis scores (Mann-Whitney p = 3.22e-24), linking ferroptosis to clinical platinum sensitivity. Our work uncovers ferroptosis dysregulation as a key driver of EOC pathogenesis, with NRF2 being a master regulator of resistance to ferroptosis and suggesting new therapeutic approaches for ovarian cancer.