Single-cell-derived ferroptosis signature predicts prognosis and therapy response in esophageal squamous cell carcinoma
Background Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with poor prognosis and limited therapeutic options. Ferroptosis, a regulated form of cell death characterized by iron-dependent lipid peroxidation, plays a crucial role in tumor progression and immune regulation. Methods We integrated single-cell RNA sequencing (scRNA-seq) and bulk transcriptomic data to identify ferroptosis-active cellular subpopulations within the ESCC tumor microenvironment. A ferroptosis-related prognostic model was constructed using LASSO-Cox regression and validated across independent cohorts from TCGA and GEO. Associations with immune infiltration, tumor mutation burden, therapeutic response, and drug sensitivity were explored. Furthermore, functional experiments were conducted in vitro using the ESCC cell lines, and the four prognostic core genes were revalidated using an independent single-cell dataset, which was also fully confirmed in clinical ESCC tissue samples. In addition, Western blot analysis was performed to examine the expression levels of ferroptosis-related proteins following CDCA3 knockdown, and to further investigate the impact of CDCA3 depletion on the cellular response to the ferroptosis inducer RSL3. Results Four ferroptosis-related genes (CBS, CDCA3, GALNT14, and IDO1) were identified used to construct a robust risk model, effectively stratifying patients into high- and low-risk groups with significant differences in survival, immune infiltration, and predicted treatment response. In vitro experiments confirmed that CDCA3 knockdown significantly inhibited the proliferation and migration of ESCC cells and induced ferroptosis. GSE188900 single-cell sequencing data further confirmed that the aforementioned genes were significantly upregulated at single-cell resolution in tumor cells, with consistent validation in clinical ESCC tissue samples, Moreover,experimental results showed that knockdown of CDCA3 lead to the downregulation of ferroptosis inhibitor-related genes and upregulation of ferroptosis-promoting genes, thereby enhancing the sensitivity to RSL3-induced ferroptosis. Conclusions Our study presents a single-cell-resolved ferroptosis gene signature with strong prognostic and therapeutic implications for ESCC. The signature was validated in clinical tissue samples, and this model lays the foundation for ferroptosis-targeted therapeutic strategies.