Tissue-Specific Expression of the EWSR1::FLI1 Fusion Protein Identifies col2a1a-Positive Cells as a Source of Ewing Sarcoma-like Tumors in Zebrafish

Ewing sarcoma (ES) is the second most common primary bone malignancy in children and adolescents and remains one of the most lethal pediatric cancers. Found in more than 85% of patients with ES, EWSR1::FLI1 results from the t(11;22)(q24;q12) chromosomal translocation. This fusion encodes an aberrant transcription factor that dysregulates gene expression and drives oncogenic transformation. Although this oncogene was identified over three decades ago, therapeutic progress has been limited, in part due to the lack of robust and permissive animal models. Prior efforts to generate transgenic mouse models have been unsuccessful, and while zebrafish have emerged as a promising system, a tissue context capable of supporting EWSR1::FLI1-driven tumorigenesis has not been defined. Here, we report that tissue-specific expression of EWSR1::FLI1 in zebrafish induces tumor formation that recapitulates the histologic and molecular hallmarks of human ES, including small round blue cell morphology and characteristic biomarker expression. Tumors were driven by the col2a1a promoter and resulted in ~70% incidence of notochord tumors within the first 72–96 h. Of the surviving fish, ~5% developed CD99-positive small round blue cell tumors at ~9 months post-fertilization. This work establishes a stable tissue-specific transgenic model of ES, providing a powerful in vivo platform to investigate disease pathogenesis and evaluate novel therapeutic strategies.