Distinct ROS-adaptive and OXPHOS-dependent cellular programs underlie Ewing sarcoma heterogeneity

Ewing sarcoma (ES) is an aggressive pediatric malignancy driven by the EWSR1-FLI1 fusion oncogene and characterized by substantial cellular heterogeneity. Here, we characterized phenotypically distinct CD44^High and CD99^High ES cell populations using integrated transcriptomic, proteomic, metabolomic, and functional analyses. CD44^High cells exhibited a spindle-shaped morphology and enhanced migratory capacity, whereas CD99^High cells displayed increased proliferation and clonogenicity. Proteomic profiling revealed elevated expression of inflammatory and tumor progression-associated proteins, including IL6, CXCL8, CCL2, SERPINE1, SPARC, and AXL, in CD44^High cells. RNA sequencing demonstrated clear transcriptional segregation between the two populations. Gene set enrichment analysis identified enrichment of epithelial-mesenchymal transition, TNFα/NF-κB signaling, inflammatory response, hypoxia, angiogenesis, and reactive oxygen species (ROS)-related pathways in CD44^High cells. In contrast, CD99^High cells were enriched for E2F targets, G2/M checkpoint, mTORC1 signaling, DNA repair, and oxidative phosphorylation (OXPHOS) pathways. Metabolomic analyses revealed alterations in cystine and kynurenine levels, while integrated multi-omics analyses identified enrichment of glutathione metabolism- and ferroptosis-related pathways in CD44^High cells. Functionally, CD99^High cells exhibited greater sensitivity to tigecycline and doxorubicin. Collectively, these findings identify distinct invasive, stress-associated and proliferative/OXPHOS-associated cellular states underlying ES heterogeneity and highlight biological programs and potential therapeutic vulnerabilities warranting further functional investigation. SIGNIFICANCE: This study reveals distinct proteomic and molecular programs underlying phenotypic heterogeneity in Ewing sarcoma. Integrated multi-omics analyses identified an invasive, stress-adaptive CD44^High state characterized by inflammatory signaling and glutathione-associated metabolic rewiring, and a proliferative CD99^High state associated with oxidative phosphorylation. The findings link proteomic alterations with cellular plasticity and therapeutic response, providing potential biomarkers and therapeutic vulnerabilities that may be exploited to improve treatment outcomes in Ewing sarcoma.