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Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip
Nano-Micro Letters ( IF 31.6 ) Pub Date : 2017-11-14 , DOI: 10.1007/s40820-017-0168-y
Ren Li , Mingxing Zhou , Jine Li , Zihua Wang , Weikai Zhang , Chunyan Yue , Yan Ma , Hailin Peng , Zewen Wei , Zhiyuan Hu

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger’s sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger’s sequencing to be a routine test before performing targeted cancer therapy.
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中文翻译:

通过微流控芯片鉴定EGFR表达的细胞并检测单细胞水平的EGFR多突变

EGFR突变伴随诊断已被证明对于酪氨酸激酶抑制剂靶向癌症治疗的疗效至关重要。为了发现少数EGFR突变细胞中发生的多种突变,可能被大多数未突变细胞的噪声所掩盖,目前正成为一项迫切的临床需求。在这里,我们介绍了一种基于微流控芯片的方法在单细胞水平检测EGFR多突变的验证。通过在专门设计的硅微孔中捕获单个细胞并对其进行免疫荧光成像,可以轻松鉴定表达EGFR的细胞。通过原位裂解单细胞,无需交叉污染即可回收表达EGFR的细胞的细胞裂解液。得益于排除了没有EGFR表达的细胞产生的噪音,Sanger测序简单而经济高效,而不是使用整个细胞群体的昂贵的深度测序来发现多重突变。我们通过从样本中发现了三个最重要的与EGFR药物相关的突变来验证新方法,在该样本中,EGFR突变的细胞仅占整个细胞群体的一小部分。微流控芯片不仅能够发现特定的EGFR多重突变,而且还能发现其他有价值的单细胞水平信息:突变发生在哪些特定细胞上,或者同一细胞是否共存不同的突变。这种微流控芯片构成了一种有前途的方法,可将简单且经济高效的Sanger测序提升为进行靶向癌症治疗之前的常规测试。我们通过从样本中发现了三个最重要的与EGFR药物相关的突变来验证新方法,在该样本中,EGFR突变的细胞仅占整个细胞群体的一小部分。微流控芯片不仅能够发现特定的EGFR多重突变,而且还能发现其他有价值的单细胞水平信息:突变发生在哪些特定细胞上,或者同一细胞是否共存不同的突变。这种微流控芯片构成了一种有前途的方法,可将简单且经济高效的Sanger测序提升为进行靶向癌症治疗之前的常规测试。我们通过从样本中发现了三个最重要的与EGFR药物相关的突变来验证新方法,在该样本中,EGFR突变的细胞仅占整个细胞群体的一小部分。微流控芯片不仅能够发现特定的EGFR多重突变,而且还能发现其他有价值的单细胞水平信息:突变发生在哪些特定细胞上,或者同一细胞是否共存不同的突变。这种微流控芯片构成了一种有前途的方法,可将简单且经济高效的Sanger测序提升为进行靶向癌症治疗之前的常规测试。微流控芯片不仅能够发现特定的EGFR多重突变,而且还能发现其他有价值的单细胞水平信息:突变发生在哪些特定细胞上,或者同一细胞是否共存不同的突变。这种微流控芯片构成了一种有前途的方法,可将简单且经济高效的Sanger测序提升为进行靶向癌症治疗之前的常规测试。微流控芯片不仅能够发现特定的EGFR多重突变,而且还能发现其他有价值的单细胞水平信息:突变发生在哪些特定细胞上,或者同一细胞是否共存不同的突变。这种微流控芯片构成了一种有前途的方法,可将简单且经济高效的Sanger测序提升为进行靶向癌症治疗之前的常规测试。
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更新日期:2017-11-14
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