Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes
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, ta...
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| Format: | Article |
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MDPI AG
2020-06-01
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| Series: | Micromachines |
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| Online Access: | https://www.mdpi.com/2072-666X/11/7/628 |
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| author | Hajime Shigeto Eriko Yamada Mizuki Kitamatsu Takashi Ohtsuki Akira Iizuka Yasuto Akiyama Shohei Yamamura |
| author_facet | Hajime Shigeto Eriko Yamada Mizuki Kitamatsu Takashi Ohtsuki Akira Iizuka Yasuto Akiyama Shohei Yamamura |
| author_sort | Hajime Shigeto |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T18:51:21Z |
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| id | doaj.art-66ea72feeabb45078f2d29be6bc06530 |
| institution | Directory Open Access Journal |
| issn | 2072-666X |
| language | English |
| last_indexed | 2024-03-10T18:51:21Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Micromachines |
| spelling | doaj.art-66ea72feeabb45078f2d29be6bc065302023-11-20T05:08:43ZengMDPI AGMicromachines2072-666X2020-06-0111762810.3390/mi11070628Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA ProbesHajime Shigeto0Eriko Yamada1Mizuki Kitamatsu2Takashi Ohtsuki3Akira Iizuka4Yasuto Akiyama5Shohei Yamamura6Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, JapanHealth and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, JapanDepartment of Applied Chemistry, Faculty of Science and Engineering, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, JapanDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, JapanImmunotherapy Division, Shizuoka Cancer Center Research Institute, Shizuoka 411-8777, JapanImmunotherapy Division, Shizuoka Cancer Center Research Institute, Shizuoka 411-8777, JapanHealth and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, JapanResearch 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.https://www.mdpi.com/2072-666X/11/7/628single-cell analysispeptide nucleic acid (PNA) probecell microarraysingle nucleotide mutationT790M mutationlung cancer |
| spellingShingle | Hajime Shigeto Eriko Yamada Mizuki Kitamatsu Takashi Ohtsuki Akira Iizuka Yasuto Akiyama Shohei Yamamura Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes Micromachines single-cell analysis peptide nucleic acid (PNA) probe cell microarray single nucleotide mutation T790M mutation lung cancer |
| title | Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes |
| title_full | Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes |
| title_fullStr | Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes |
| title_full_unstemmed | Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes |
| title_short | Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes |
| title_sort | analysis of single nucleotide mutated single cancer cells using the combined technologies of single cell microarray chips and peptide nucleic acid dna probes |
| topic | single-cell analysis peptide nucleic acid (PNA) probe cell microarray single nucleotide mutation T790M mutation lung cancer |
| url | https://www.mdpi.com/2072-666X/11/7/628 |
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