Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells.
Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells.
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DOI：
10.3389/fonc.2014.00201
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发表时间：
2014
影响因子：
4.7
通讯作者：
Mosse YP
中科院分区：
文献类型：
--
作者：
Carpenter EL;Rader J;Ruden J;Rappaport EF;Hunter KN;Hallberg PL;Krytska K;O'Dwyer PJ;Mosse YP
我们对逃脱原发肿瘤和种子微转移的细胞多样性的了解仍然很初级，研究循环和播散性肿瘤细胞的方法一直受到以下因素的限制：吞吐量和灵敏度低，依赖单参数分选，注重计数而不是表型和基因特征。在这里，我们利用一种高度敏感的微流控和介电泳法来分离和分析单个肿瘤细胞。我们使用荧光标记技术从11个细胞系(包括8个神经母细胞瘤细胞系)的刺激性实验中分离出208个单个细胞，并获得了每106个白细胞(WBC)中1个肿瘤细胞的捕获灵敏度。样品固定或冷冻对细胞捕获没有可检测到的影响。在捕获的单个肿瘤细胞的全基因组扩增产物中准确检测到点突变，而在阴性对照WBC中未检测到点突变。我们应用这种方法从10例神经母细胞瘤患者的骨髓样本中捕获144个单个肿瘤细胞。在这种儿科恶性肿瘤中，高危患者通常表现为广泛的血行转移，但很难或不可能获得原发肿瘤。在这里，我们使用基于流动的分选来预富集肿瘤受累程度低于0.02%的样本。对于所有已经在其原发肿瘤中检测到间变性淋巴瘤激酶基因突变的患者，在他们的骨髓单个细胞中也检测到了相同的突变。这些发现证明了一种新的、非侵入性的、适应性强的方法来捕获和分析癌症患者的单个肿瘤细胞。
Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.