Abstract 4683: Developing human Ewing sarcoma in vitro models to prioritise new treatments

Abstract

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 (p<0.01). Interrogation of the differentially expressed genes using Reactome, KEGG and Gene Ontology identified up regulation of genes associated with mitotic spindle and G2/M G1/S DNA damage spindle checkpoints, homologous recombination and the cell cycle in the cell lines compared to patient-derived cultures (p<0.01). These data are consistent with the quicker doubling time (DT) and increased sensitivity of ES cell lines to chemotherapy (actinomycin D, doxorubicin, vincristine, etoposide; DT range=19-33h, EC50 range=4-34nM) compared to patient-derived cultures (DT range=55-197h, EC50>10μM, p<0.05). All patient-derived ES cultures formed spheroids of similar diameter (range 105-258µm) at 96h (p>0.05), although there was heterogeneity in spheroid weight (range 205-3639ng, p<0.05) and migration index (MI; range 30-170, p<0.05). Treatment with actinomycin D (1-10 µM) significantly increased spheroid diameter (p<0.05), whereas 4-hyperoxyifosfamide (active metabolite of ifosfamide, 7-30µM) decreased spheroid diameter (p<0.05). Doxorubicin did not significantly change the diameter of spheroids. When propagated in combination with mesenchymal stem cells there was a 2-fold increase in viable cell number (p<0.0001) compared to ES cultures alone. Patient-derived ES cultures in zebrafish were observed at the injection site (perivitelline space). Some cells were detected in circulation 3 days post injection. This is consistent with heterogeneity and the high MI of patient-derived ES cultures. The transcriptome of ES cell lines is significantly different from that of ES patient-derived cultures and is associated with increased doubling times and response to chemotherapies. This highlights the importance of developing preclinical models using patient-derived ES cultures. Patient-derived ES cultures in 2D and 3D models in vitro and in zebrafish may provide a reliable cost-effective preclinical pipeline 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.