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Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-X

Engineered chimeric antigen receptor T cells (CAR T cells) have emerged as a promising immunotherapy for cancer and have proven to be effective for B cell malignancies. Currently, great efforts are undertaken to expand the application of CAR T cells to other cancer entities, to increase the efficacy of CAR T cell-mediated killing of cancer cells and to reduce possible side effects of CAR T cell therapy. This creates a need for preclinical models to test the many emerging novel CAR designs. Traditionally, mouse xenograft models are applied to investigate the efficacy of CAR T cells in vivo. Here, we describe a complementing xenograft protocol for testing CAR T cells against human leukemia cells in zebrafish embryos. The embryonic zebrafish xenograft promises to be a fast and cost-efficient model and particularly offers live imaging opportunities of CAR T cell distribution and killing of cancer cells in vivo.

Models that more accurately reflect Ewing sarcoma (ES) will enable the prioritization of novel targeted agents from bench to clinic. To date, development of an ES mouse model have been unsuccessful1 and engraftment of human ES in mice is variable2. Coupled with a moral obligation to minimize the use of mammals, we have established and characterized models using patient-derived ES cultures in vitro and in larval zebrafish. The transcriptomes of ES cell lines and patient-derived ES cultures3 were analyzed and compared using total RNAseq followed by DESeq2 and Gene Set Enrichment analysis. Cells in 2D, as 3D spheroids, in combination with cells of the tumor microenvironment and in larval zebrafish (Danio rerio) were characterized. Six cell lines clustered independently from patient-derived ES cultures (n=20), reflecting differential expression of 15,709 RNAs (p10μM, p0.05), although there was heterogeneity in spheroid weight (range 205-3639ng, p 1Minas et al. 2017.Oncotarget,8(21):34141-34163.2Nanni et al. 2019.Sci Rep,9(1):12174 3Roundhill et al. 2021.Cell Oncol,44(5):1065-1085. Citation Format: Elizabeth A. Roundhill, Elton J. Vasconcelos, David R. Westhead, Sarah Grissenberger, Martin Distel, Susan A. Burchill. Developing human Ewing sarcoma in vitro models to prioritise new treatments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4683.

Tumor models allowing for the in vivo investigation of molecular mechanisms driving tumor progression and metastasis are important to develop novel strategies for cancer treatment. Unfortunately, for Ewing sarcoma no adequate genetic animal models are currently available. Mouse xenograft models are the state of the art to model Ewing sarcoma in vivo. Here, we describe an alternative Ewing sarcoma xenograft model in embryonic and larval zebrafish. This xenograft model offers live imaging and easy compound testing opportunities hereby complementing mouse xenograft models. In this chapter, we provide a detailed protocol how to xenograft Ewing sarcoma cells (shSK-E17T) into 2-day-old zebrafish and how xenografted zebrafish can be imaged and analyzed over consecutive days to study tumor proliferation.

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.

Tumor models allowing for the in vivo investigation of molecular mechanisms driving tumor progression and metastasis are important to develop novel strategies for cancer treatment. Unfortunately, for Ewing sarcoma no adequate genetic animal models are currently available. Mouse xenograft models are the state of the art to model Ewing sarcoma in vivo. Here, we describe an alternative Ewing sarcoma xenograft model in embryonic and larval zebrafish. This xenograft model offers live imaging and easy compound testing opportunities hereby complementing mouse xenograft models. In this chapter, we provide a detailed protocol how to xenograft Ewing sarcoma cells (shSK-E17T) into 2-day-old zebrafish and how xenografted zebrafish can be imaged and analyzed over consecutive days to study tumor proliferation.

The aim of this study was to develop effective and specific anti-cancer drugs based on membrane active peptides. In previous studies we showed that human lactoferricin (hLFcin) derived peptides facilitate specific killing of cancer cells. These antitumor peptides were found by conventional melanoma two-dimensional (2D) cell cultures to induce apoptosis of cancer cells and to specifically target lipid phosphatidylserine located on the outside of cancer cell membranes. In order to have a more relevant in vitro model able to mimic the natural microenvironments of tumor tissues we established three-dimensional (3D) multicellular tumor spheroids (MCTS). We used a set of (retro) di-peptides derived from LF11, an 11 amino acid long fragment of hLFcin, which differed in peptide length, positive net charge and hydrophobicity and determined antitumor activity and non-specific toxicity on non-neoplastic cells using 2D and 3D model systems. 2D studies unveiled a correlation between length, positive net charge and hydrophobicity of peptides and their specific antitumor activity. (Retro) di-peptides as R-DIM-P-LF11-215 and DIM-LF11-322 with a net charge of +9 and moderate hydrophobicity exhibited the highest specific antitumor activity. Further evaluation of the peptides anticancer activity by 3D in vitro studies confirmed their higher activity and cancer specificity compared to their parent R-DIM-P-LF11, with the exception of DIM-LF11-339. This highly hydrophobic peptide caused cell death mainly at the border of tumor spheroids indicating that too high hydrophobicity may prevent peptides from reaching the center of the spheroids.

Ewing sarcoma is a malignant bone and soft tissue tumor in children and adolescents. In 85% of all cases the formation of Ewing sarcoma is caused by a chromosomal translocation, leading to the expression of the oncogene EWS-FLI1. This aberrant transcription factor is the main driver of the disease and leads to massive transcriptional dysregulation. Although the genetic mechanism driving Ewing sarcoma is well understood, an animal model adequately mimicking the disease is still lacking. One reason why modeling attempts have remained difficult is the elusive cell of origin in Ewing sarcoma. Several cell types including neural crest progenitor cells and mesenchymal stem cells have been proposed, but none of them has yet been confirmed as cell of origin of Ewing sarcoma. Towards identifying this cell of origin, we are currently following a novel cross-species approach by exploring the use of enhancers, which are specifically active in Ewing sarcoma cells, but were not created de novo by EWS-FLI1. Injection of reporter constructs into zebrafish embryos allows for visualization of cell types with activity of the selected enhancers and provides us with cell-of-origin candidate cell types. To investigate which of these candidate cells are permissive for development of Ewing-like tumors, we will also express EWS-FLI1 in these cells. If successful, this approach will not only reveal the Ewing sarcoma cell of origin but also result in a zebrafish Ewing sarcoma model, which will help to understand tumor initiation and progression and, furthermore, will be a valuable tool to develop novel therapeutic strategies. Citation Format: Sarah Grissenberger, Anna Poetsch, Monika Heinzl, Luisa Morelli, Nathan Sheffield, Niko Popitsch, Heinrich Kovar, Martin Distel. A cross-species enhancer activity analysis approach to identify the Ewing sarcoma cell of origin [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B28.

Precise regulation of signaling pathways in single cells underlies tissue development, maintenance and repair in multicellular organisms, but our ability to monitor signaling dynamics in living vertebrates is currently limited. We implemented kinase translocation reporter (KTR) technology to create DREKA ("dynamic reporter of Erk activity") zebrafish, which allow one to observe Erk activity in vivo at single cell level with high temporal resolution. DREKA zebrafish faithfully reported Erk activity after muscle cell wounding and revealed the kinetics of small compound uptake. Our results promise that kinase translocation reporters can be adapted for further applications in developmental biology, disease modeling, and in vivo pharmacology in zebrafish.