Shigeto Hajime, Yamada Eriko, Kitamatsu Mizuki, Ohtsuki Takashi, Iizuka Akira, Akiyama Yasuto, Yamamura Shohei
Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan.
Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan.
Micromachines (Basel). 2020 Jun 27;11(7):628. doi: 10.3390/mi11070628.
Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0-20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.
在单细胞水平上对携带与抗癌药物耐药性相关基因突变的癌细胞进行研究，重点在于癌症的诊断或治疗。已经报道了几种在大量未突变细胞中检测基因突变细胞的方法；然而，在大量细胞样本（如癌组织）中靶向单个核苷酸突变细胞仍然难以分析。在本研究中，开发了一种新系统，通过结合单细胞微阵列芯片和肽核酸（PNA）-DNA探针，从多个未突变癌细胞中检测和分离表达T790M突变型表皮生长因子受体（EGFR）mRNA的单癌细胞。单细胞微阵列芯片由聚苯乙烯制成，有62410个微腔（直径31-40μm）。T790M突变的肺癌细胞系NCI-H1975和未突变的肺癌细胞系A549成功地在芯片上的每个微腔中分离成单个细胞。仅用异硫氰酸荧光素（FITC）偶联的PNA探针在芯片上对NCI-H1975细胞进行染色，以特异性检测T790M突变。根据掺入浓度，在1小时内对掺入A549细胞中的NCI-H1975细胞的0-20%进行了定量分析。因此，我们的系统可能有助于分析含有少量抗癌耐药细胞的癌组织。
