Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip.
Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip.
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DOI：
10.1007/s40820-017-0168-y
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发表时间：
2018
影响因子：
26.6
通讯作者：
Hu Z
中科院分区：
文献类型：
--
作者：
Li R;Zhou M;Li J;Wang Z;Zhang W;Yue C;Ma Y;Peng H;Wei Z;Hu Z
EGFR突变伴随诊断已被证明对酪氨酸激酶抑制剂靶向癌症治疗的疗效至关重要。发现少数EGFR突变细胞中发生的多个突变，这些突变可能被大多数未突变细胞的噪声所掩盖，目前已成为临床迫切的需求。在这里，我们提出了一种基于微流控芯片的方法，用于检测EGFR多突变在单细胞水平的验证。通过在专门设计的硅微孔中捕获和免疫荧光成像单细胞，EGFR表达的细胞很容易识别。通过原位裂解单细胞，EGFR表达细胞的细胞裂解物被回收而没有交叉污染。受益于排除来自没有EGFR表达的细胞的噪声，使用简单且具有成本效益的桑格测序，而不是昂贵的整个细胞群体的深度测序来发现多突变。我们通过从EGFR突变细胞仅占整个细胞群的一小部分的样本中精确发现三种最重要的EGFR药物相关突变来验证新方法。微流控芯片不仅能够发现特定EGFR多突变的存在，还能够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，或者不同突变是否共存于同一细胞上。这种微流控芯片构成了一种有前途的方法，以促进简单和具有成本效益的桑格的测序是一个常规的测试之前，进行靶向癌症治疗。 本文的在线版本（10.1007/s40820-017-0168-y）包含补充材料，可供授权用户使用。
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. The online version of this article (10.1007/s40820-017-0168-y) contains supplementary material, which is available to authorized users.
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影响因子：
1.6
作者：
BONNER, WA;SWEET, RG;HERZENBERG, LA
通讯作者：
HERZENBERG, LA
影响因子：
6.1
作者：
Yang, Yoonsun;Rho, Hoon Suk;Terstappen, Leon W. M. M.
通讯作者：
Terstappen, Leon W. M. M.
影响因子：
8
作者：
Gazdar, A. F.
通讯作者：
Gazdar, A. F.
影响因子：
15.8
作者：
Greulich H;Chen TH;Feng W;Jänne PA;Alvarez JV;Zappaterra M;Bulmer SE;Frank DA;Hahn WC;Sellers WR;Meyerson M
通讯作者：
Meyerson M
影响因子：
11.2
作者：
Tang, XN;Shigematsu, H;Wistuba, II
通讯作者：
Wistuba, II