Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Via Sergio Pansini 5, 80131 Naples, Italy.
CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Naples, Italy.
Int J Mol Sci. 2019 Feb 19;20(4):893. doi: 10.3390/ijms20040893.
Tumors often show intra-tumor heterogeneity because of genotypic differences between all the cells that compose it and that derive from it. Recent studies have shown significant aspects of neuroblastoma heterogeneity that may affect the diagnostic-therapeutic strategy. Therefore, we developed a laboratory protocol, based on the combination of the advanced dielectrophoresis-based array technology and next-generation sequencing to identify and sort single cells individually and carry out their copy number variants analysis. The aim was to evaluate the cellular heterogeneity, avoiding overestimation or underestimation errors, due to a bulk analysis of the sample. We tested the above-mentioned protocol on two neuroblastoma cell lines, SK-N-BE(2)-C and IMR-32. The presence of several gain or loss chromosomal regions, in both cell lines, shows a high heterogeneity of the copy number variants status of the single tumor cells, even if they belong to an immortalized cell line. This finding confirms that each cell can potentially accumulate different alterations that can modulate its behavior. The laboratory protocol proposed herein provides a tool able to identify prevalent behaviors, and at the same time highlights the presence of particular clusters that deviate from them. Finally, it could be applicable to many other types of cancer.
肿瘤通常表现出肿瘤内异质性，因为构成肿瘤的所有细胞以及源自肿瘤的细胞之间存在基因型差异。最近的研究表明神经母细胞瘤异质性的重要方面可能会影响诊断-治疗策略。因此，我们开发了一种实验室方案，该方案基于基于先进的介电泳阵列技术和下一代测序的组合，以单独识别和分选单细胞并进行其拷贝数变异分析。目的是评估细胞异质性，避免由于对样本进行批量分析而导致的高估或低估错误。我们在两种神经母细胞瘤细胞系 SK-N-BE(2)-C 和 IMR-32 上测试了上述方案。在这两种细胞系中，都存在多个增益或缺失的染色体区域，这表明单个肿瘤细胞的拷贝数变异状态具有很高的异质性，即使它们属于永生化细胞系。这一发现证实，每个细胞都可能积累不同的改变，从而改变其行为。本文提出的实验室方案提供了一种能够识别常见行为的工具，同时突出了存在偏离这些行为的特定聚类。最后，它可以应用于许多其他类型的癌症。
