Analysis of single nucleotide-mutated single-cancer cells using the combined technologies of single-cell microarray chips and peptide nucleic acid-DNA probes

Hajime Shigeto, Eriko Yamada, Mizuki Kitamatsu, Takashi Ohtsuki, Akira Iizuka, Yasuto Akiyama, Shohei Yamamura

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Article number628
JournalMicromachines
Volume11
Issue number7
DOIs
Publication statusPublished - Jul 1 2020

Keywords

  • Cell microarray
  • Epidermal growth factor receptor (EGFR)
  • Lung cancer
  • Peptide nucleic acid (PNA) probe
  • Single nucleotide mutation
  • Single-cell analysis
  • T790M mutation

ASJC Scopus subject areas

  • Control and Systems Engineering
  • Mechanical Engineering
  • Electrical and Electronic Engineering

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