In vitro and in ovo characterization of Ewing sarcoma cell lines: Comparison with neuroblastoma cell lines and lymphatic endothelial cells.

Ewing sarcoma (EwS) is characterized by a balanced chromosomal translocation in which a member of the FET gene family is fused with an E26 transformation specific (ETS) transcription factor: the most common fusion being EWSR1-FLI1. Traditionally, EwS includes Ewing-like tumors, Askin tumors, and peripheral primitive neuroectodermal tumors (PNET), indicative of a neuroectodermal relationship. Previously, in the absence of genetic diagnostics, extraosseous EwS could be mistaken for neuroblastoma (NB), which has become particularly clear in the history of the CHP100 cell line. In previous studies we characterized the behavior of NB cell lines in the chick chorioallantoic membrane (CAM) assay. Here we focused on four EWSR1-FLI1 gene-fusion-transcript-containing EwS cell lines (CHP100, TC71, RH1, SK-N-MC) and compared them to NB cell lines. We show that EwS cells form highly aggressive CD99- and vimentin-positive tumors in CAM, with blood-filled cysts, very similar to NB tumors in CAM. We observed newly formed blood vessels in the tumors, but not lymphangiogenesis. However, the main characteristics were arrosion of blood vessels and hemorrhage. At RNA and protein level we found important differences between EwS and NB cell lines (23 NB cell lines were used for qPCR studies): Adrenergic receptor ß1 (a potential therapeutic target) was exclusively found in EwS. In contrast, VANGL2, LRP5, PROX1, HAND2, PHOX2A and PHOX2B were found in NB cells, with few exceptions only. EWSR1, FLI1, ERG, CD99 and vimentin are characteristically expressed in lymphatic endothelial cells (LECs). Previously, EWSR1-FLI1-silencing studies revealed critically regulated genes in a mixture of cells containing mesenchymal stem cells. By whole genome RNASeq, we found 32 of 38 of these genes in LECs. With gene set enrichment analysis of publicly available data sets, we found increased similarity of EWS-FLI/ERG-silenced EwS cell lines with LECs and blood vascular ECs. In sum, EwS tumors in CAM recapitulate the high aggressiveness of the malignancy. EwS and NB cell lines show characteristic molecular differences despite similar behavior in CAM. EwS and ECs show a certain degree of molecular similarity, and the original description of EwS as an endothelioma of bone should still be considered as a possibility. We discuss our data in the light of the little work that exists on the innervation and lymphatic supply of bone.