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

doi:10.3390/mi11070628

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.

Highlights

Single-cell analysis offers great potential for understanding the complex biology of various diseases and can assist with diagnosis
The developed peptide nucleic acid (PNA)-DNA probes showed increases in fluorescence intensity against dose-dependent, in vitro target epidermal growth factor receptor (EGFR)-mutated sequences, which indicates that fluorescein isothiocyanate (FITC)-PNA probes are separated from quencher-conjugated DNA (Q-DNA) probes, and bind to the target sequence of mutated EGFR mRNA (Figure 1b)
Various cancer cells can be separated into single cells, regardless of their type, suggesting that single-cell microarray chip technology could be used for the analysis and diagnosis of a wide range of cancers

Summary

Introduction

Single-cell analysis offers great potential for understanding the complex biology of various diseases and can assist with diagnosis. The fluorescent labeled antibodies [14,15,16,17,18] or fluorescent labeled DNA-based probes [19,20,21,22,23,24,25,26] are commonly used to screen for and analyze target cells. These probes have high sensitivity and specificity, it is difficult to detect slightly expressed proteins or the few nucleotide-mutated genes. The screening and detection of anticancer drug-resistant cancer cells harboring single nucleotide-mutated genes has focused on cancer diagnosis [27,28,29]; we aimed to detect and isolate the single cells expressing the single nucleotide-mutated mRNA from multiple non-mutated cancer cells using our original cell chip technology and peptide nucleic acid (PNA)-based probes with high specificity

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Conclusion