Automated compound testing in zebrafish xenografts identifies combined MCL-1 and BCL-XL inhibition to be effective against Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue cancer for which new therapies to improve disease outcome and to reduce adverse effects of current standard treatments are urgently needed. To identify new and effective drugs, phenotypic drug screening has proven to be a powerful method and a cancer model ideally suited for this approach is the larval zebrafish xenograft system. Complementing mouse xenografts, zebrafish offer high-througput screening possibilities in an intact complex vertebrate organism. Here, we generated Ewing sarcoma xenografts in zebrafish larvae and established a workflow for automated imaging of xenografts, tumor cell recognition within transplanted zebrafish and quantitative tumor size analysis over consecutive days by high-content imaging. The increased throughput of our in vivo screening setup allowed us to identify combination therapies effective against Ewing sarcoma cells. Especially, combined inhibition of MCL-1 and BCL-XL, two anti-apoptotic proteins, was highly efficient at eradicating tumor cells in our zebrafish xenograft assays with two Ewing sarcoma cell lines and with patient-derived cells. Transcriptional analysis across Ewing sarcoma cell lines and tumors revealed that MCL-1 and BCL2L1, coding for BCL-XL, are the most abundantly expressed anti-apoptotic genes, suggesting that combined MCL-1/BCL-XL inhibition might be a broadly applicable strategy for Ewing sarcoma treatment.