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

IF 3.7 2区 生物学 Q2 CELL BIOLOGY
Jürgen Becker, Cherim Jeon, Jörg Wilting
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Abstract

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.
尤因肉瘤细胞系的体外和卵内特性:与神经母细胞瘤细胞系和淋巴内皮细胞的比较
Ewing肉瘤(EwS)以平衡染色体易位为特征,其中FET基因家族成员与E26转化特异性(ETS)转录因子融合:最常见的融合是EWSR1-FLI1。传统上,EwS包括ewing样肿瘤、Askin肿瘤和周围原始神经外胚层肿瘤(PNET),表明神经外胚层之间存在关系。以前,在缺乏遗传诊断的情况下,骨外EwS可能被误认为是神经母细胞瘤(NB),这在CHP100细胞系的历史中已经变得特别清楚。在以前的研究中,我们在鸡绒毛膜尿囊膜(CAM)试验中表征了NB细胞系的行为。本研究以四种含有EWSR1-FLI1基因融合转录的EwS细胞系(CHP100、TC71、RH1、SK-N-MC)为研究对象,并将其与NB细胞系进行比较。我们发现EwS细胞在CAM中形成高度侵袭性的CD99和vimentin阳性肿瘤,具有充满血液的囊肿,与CAM中的NB肿瘤非常相似。我们在肿瘤中观察到新形成的血管,但没有淋巴管生成。但主要表现为血管侵蚀和出血。在RNA和蛋白质水平上,我们发现EwS和NB细胞系之间存在重要差异(23个NB细胞系用于qPCR研究):肾上腺素能受体ß1(一种潜在的治疗靶点)仅在EwS中发现。相反,在NB细胞中发现了VANGL2、LRP5、PROX1、HAND2、PHOX2A和PHOX2B,只有少数例外。EWSR1、FLI1、ERG、CD99和vimentin在淋巴内皮细胞(LECs)中特征性表达。此前,ewsr1 - fli1沉默研究揭示了含有间充质干细胞的细胞混合物中的关键调控基因。通过全基因组RNASeq,我们在lec中发现了38个这些基因中的32个。通过对公开数据集的基因集富集分析,我们发现EwS - fli / ergg沉默的EwS细胞系与LECs和血管ECs的相似性增加。总之,CAM中的EwS肿瘤再现了恶性肿瘤的高侵袭性。EwS和NB细胞系在CAM中表现出相似的分子差异。EwS与ECs在分子上有一定的相似性,将EwS描述为骨内皮瘤仍有可能。我们讨论我们的数据在少量的工作,存在的神经支配和淋巴供应骨。
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来源期刊
Cellular signalling
Cellular signalling 生物-细胞生物学
CiteScore
8.40
自引率
0.00%
发文量
250
审稿时长
27 days
期刊介绍: Cellular Signalling publishes original research describing fundamental and clinical findings on the mechanisms, actions and structural components of cellular signalling systems in vitro and in vivo. Cellular Signalling aims at full length research papers defining signalling systems ranging from microorganisms to cells, tissues and higher organisms.
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