Children's Cancer Research Group, Leeds Institute of Medical Research, St. James's University Hospital, Leeds, LS9 7TF, UK.
Royal Orthopaedic Hospital NHS Foundation Trust, Bristol Road South, Northfield, Birmingham, B31 2AP, UK.
Cell Oncol (Dordr). 2021 Oct;44(5):1065-1085. doi: 10.1007/s13402-021-00619-8. Epub 2021 Aug 17.
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.
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.
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.
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.
为了降低发病率并改善结果，迫切需要为尤文肉瘤（ES）患者开发生物标志物和分子靶向治疗。在这里，我们着手分离和鉴定患者来源的 ES 原代细胞培养物和衍生的癌症干样细胞（CSC），以鉴定高危疾病的生物标志物和候选治疗靶点。
从治疗初治肿瘤中建立了 32 个患者来源的原代培养物，并使用功能方法从这些培养物中分离出 ES-CSC。通过 RNA 测序，我们分析了 ES 患者来源细胞（n=24）和 ES-CSC（n=11）的转录组，以鉴定最丰富和差异表达的基因（DEG）。在 RNA 和蛋白质水平上验证 ES-CSC 中与 ES 细胞相比表达最高的 DEG。通过敲低研究和两个独立肿瘤队列的免疫组织化学，研究了最显著基因（神经连接蛋白-1）的功能和预后潜力。
从所有原代细胞培养物中分离出 ES-CSC，这与 ES 是由 CSC 驱动的癌症这一前提一致。转录谱分析证实这些细胞来源于间充质，为治疗提供了新的细胞表面靶点，这些靶点调节细胞-细胞外基质相互作用，并鉴定了进展和复发的候选驱动因素。神经连接蛋白-1 的高表达和其活性调节剂 APBA1 和 NLGN4X 的低水平与不良无事件和总生存率相关。敲低神经连接蛋白-1可减少活细胞数量和球体形成。
调节细胞外相互作用的基因，包括神经连接蛋白-1，是 ES 的候选治疗靶点。诊断时神经连接蛋白-1水平高与不良预后相关，并鉴定出局部复发的疾病患者。这些患者可能受益于更密集或新型的治疗方式。神经连接蛋白-1的预后意义应独立验证。
