Supplementary MaterialsSupplementary materials 1 (PDF 859?kb) 40820_2017_168_MOESM1_ESM. sequencing of the whole cell population, was Raf265 derivative 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 Sangers sequencing to be a routine test before performing targeted cancer therapy. Electronic supplementary material The online version of this article (10.1007/s40820-017-0168-y) contains supplementary material, which is available to authorized users. strong class=”kwd-title” Keywords: EGFR mutation, Single-cell analysis, Microfluidic chip, Tyrosine kinase inhibitor Highlights Discovering Raf265 derivative not only the existence of specific EGFR multi-mutations occurred in minority of EGFR-mutated cells which may be covered by the noises from majority of un-mutated cells, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. Trapping and identifying EGFR-expressed single cells to exclude interferences from EGFR-unexpressed cells. Introduction Epidermal growth factor receptor (EGFR) has been proved to be related with the pathogenesis and progression of multiple carcinoma types, including lung cancer [1], breast cancer [2], prostatic cancer [3] and pancreatic cancer [4]. Previous clinical trials demonstrated that inhibitors of EGFR tyrosine kinase (TK) effectively retarded disease progression of non-small cell lung cancer (NSCLC) patients [5, 6]. Evidences suggest that mutated EGFR proteins are inhibited by small-molecule tyrosine kinase inhibitors (TKIs) which compete with ATP binding to the TK domain of the receptor and block signal transduction [6]. Mutations mediate oncogenic results by changing downstream anti-apoptotic and signaling systems [1, 7]. For example, L858R in exon 21 and Del E749-A750 in exon 19 mutations raise the TKIs level of sensitivity [8], while T790M in exon 20 can be a drug-resistant mutation, abrogating inhibitors binding with EGFR [9, 10]. Since these Raf265 derivative mutations influence Raf265 derivative the potency of targeted medication considerably, EGFR analysis is now increasingly more a regular test before choosing targeted therapy for related malignancies, such as for example NSCLC [11C13]. Immunohistochemistry of tumor cells may be the most medically utilized solution to detect EGFR at protein level [14, 15]. Also, directly sequencing cells extracted from tumor tissue has also been clinically accepted to detect EGFR mutation sequences [16, 17]. However, either the protein analysis or the gene sequencing of tumor tissue provides only averaged information of the whole cell population. Since the tumor cells are heterogeneous [18, 19], the mutations occurred on a small amount of cells could be covered Raf265 derivative by the other normal cells [20]. To reveal EGFR mutation on individual cells, fluorescence-activated cell sorting (FACS) was previously introduced [21] to sort single cells from a large cell amount, usually larger than 105 cells [22]. For cell samples fewer than 105 cells, the emerging microfabrication technologies have advanced the examinations of protein expression or gene mutation at single-cell level by preciously controlling single cells and their surrounding environments. At protein level, by employing immunofluorescence identification, microfluidic chips are capable of identifying [23, 24] or enumerating [25] EGFR-expressed cells. However, the application of protein level analyses is limited by the diverse specificity of different antibodies and Rabbit Polyclonal to HES6 the lack of detailed mutation information. At gene level, on-chip single-cell isolation, lysis and gene amplification have been realized using microchambers [26] or droplets [27], enabling the sequencing of the disease-related gene fragments [28, 29] or even the whole genome [30]. However, the lack of on-chip identification of EGFR expression and corresponding sorting of EGFR-expressed cells compromises the feasibility of selectively sequencing EGFR-expressed cells which possibly make up a small portion of all cells extracted from tumor tissue. Clinically, before performing targeted therapy, it is crucial to understand not merely if EGFR appearance occurs but also just how many types of disease-related mutation can be found and the actual mutated sequences specifically are [31]. This immediate demand is however to be satisfied with a precise, cost-effective and simple method, regardless of the advancements which were attained on EGFR mutation perseverance currently, with or without the help of microfluidic chips. To handle this necessity, we developed.