TY - JOUR
T1 - Analysis of single nucleotide-mutated single-cancer cells using the combined technologies of single-cell microarray chips and peptide nucleic acid-DNA probes
AU - Shigeto, Hajime
AU - Yamada, Eriko
AU - Kitamatsu, Mizuki
AU - Ohtsuki, Takashi
AU - Iizuka, Akira
AU - Akiyama, Yasuto
AU - Yamamura, Shohei
N1 - Funding Information:
This work was supported by the JSPS KAKENHI Grant Numbers 18K15299 and the JST CREST Grant Number JPMJCR15G4. We appreciate the help of SEIKO GIKEN Co., Ltd., Chibe, Japan in fabricating single-cell microarray chips.
Funding Information:
Funding: This work was supported by the JSPS KAKENHI Grant Numbers 18K15299 and the JST CREST Grant Number JPMJCR15G4.
Publisher Copyright:
© 2020 by the authors.
PY - 2020/7/1
Y1 - 2020/7/1
N2 - 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.
AB - 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.
KW - Cell microarray
KW - Epidermal growth factor receptor (EGFR)
KW - Lung cancer
KW - Peptide nucleic acid (PNA) probe
KW - Single nucleotide mutation
KW - Single-cell analysis
KW - T790M mutation
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U2 - 10.3390/MI11070628
DO - 10.3390/MI11070628
M3 - Article
AN - SCOPUS:85088369447
VL - 11
JO - Micromachines
JF - Micromachines
SN - 2072-666X
IS - 7
M1 - 628
ER -