Purpose

The development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets.

Methods

Thirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts.

Results

ES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation.

Conclusions

Genes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

中文翻译：

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患者来源细胞的 RNA 测序和功能研究表明，neurexin-1 和该通路的调节剂与尤文肉瘤的不良预后有关

目的

迫切需要为尤文肉瘤 (ES) 患者开发生物标志物和分子靶向疗法，以最大限度地减少发病率并改善结果。在这里，我们着手分离和表征患者来源的 ES 原代细胞培养物和子代癌症干细胞样细胞 (CSC)，以确定高风险疾病的生物标志物和候选治疗靶点。

方法

使用功能性方法从未治疗的肿瘤和从这些培养物中分离的原代 ES-CSC 建立了 32 种源自患者的原代培养物。通过 RNA 测序，我们分析了 ES 患者来源细胞 ( n  = 24) 和 ES-CSCs ( n  = 11)的转录组，以确定最丰富和差异表达的基因 (DEG)。与 ES 细胞相比，ES-CSC 中顶部 DEG 的表达在 RNA 和蛋白质水平上得到验证。使用敲低研究和两个独立肿瘤队列的免疫组织化学研究了最重要基因（neurexin-1）的功能和预后潜力。

结果

ES-CSC 从所有原代细胞培养物中分离出来，这与 ES 是 CSC 驱动的癌症的前提一致。转录分析证实这些细胞是间充质来源的，揭示了用于调节细胞-细胞外基质相互作用的新细胞表面治疗靶点，并确定了进展和复发的候选驱动因素。neurexin-1 的高表达和其活性调节剂 APBA1 和 NLGN4X 的低水平与较差的无事件生存率和总生存率有关。neurexin-1 的组合式减少了活细胞数量和球体形成。

结论

调节细胞外相互作用的基因，包括 neurexin-1，是 ES 的候选治疗靶点。诊断时高水平的 neurexin-1 与较差的结果相关，并识别出局部疾病会复发的患者。这些患者可以从更强化或新颖的治疗方式中受益。应该独立验证 neurexin-1 的预后意义。