Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

作     者：Ren Li Mingxing Zhou Jine Li Zihua Wang Weikai Zhang Chunyan Yue Yan Ma Hailin Peng Zewen Wei Zhiyuan Hu 

作者机构：CAS Key Laboratory of Standardization and Measurement for Nanotechnology CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety CAS Center for Excellence in Nanoscience National Center for Nanoscience and Technology of China Academy for Advanced Interdisciplinary Studies Peking University University of Chinese Academy of Sciences Sino-Danish College University of Chinese Academy of Sciences Yangtze River Delta Academy of Nanotechnology and Industry Development Research 

出 版 物：《Nano-Micro Letters》 (纳微快报（英文版）)

年 卷 期：2018年第10卷第1期

页      面：148-157页

核心收录：

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

基　　金：supported by the National HighTech R&D Program of China(No.2015AA020408) National Natural Science Foundation of China(No.61204118,81500900 and21503054) Beijing Municipal Science and Technology Project(No.Z171100002017013) Key Research Program of the Chinese Academy of Sciences,Grant NO.KFZD-SW-210 

主　　题：EGFR mutation Single-cell analysis Microfluidic chip Tyrosine kinase inhibitor 

摘      要：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 unmutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multimutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cellswere 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 drugrelated 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.