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Updated: Dec 16, 2025
Counting Proteins in Single Cells with Addressable Droplet Microarrays
Published on: July 6, 2018
Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray
Hajime Shigeto1, Eriko Yamada1, Mizuki Kitamatsu2
1Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan.
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.
Insights
This study introduces a novel system for detecting single cancer cells with specific gene mutations, like T790M-mutated epidermal growth factor receptor (EGFR), within larger cell populations. This advancement aids in analyzing drug-resistant cancer cells more effectively.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Detecting single cancer cells with specific gene mutations, particularly those conferring drug resistance, remains a challenge in cancer research.
- Existing methods struggle to analyze target single nucleotide-mutated cells within complex samples like cancer tissue.
Purpose of the Study:
- To develop a new system for detecting and isolating single cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA.
- To enable the analysis of rare drug-resistant cancer cells within a heterogeneous population.
Main Methods:
- Utilized a single-cell microarray chip with 62,410 microchambers for cell separation.
- Employed peptide nucleic acid (PNA)-DNA probes conjugated with fluorescein isothiocyanate (FITC) for specific T790M mutation detection.
- Successfully separated T790M-mutated (NCI-H1975) and non-mutated (A549) lung cancer cell lines into individual microchambers.
Main Results:
- The developed system successfully separated lung cancer cell lines into single cells within microchambers.
- Fluorescein isothiocyanate (FITC)-conjugated PNA probes specifically stained T790M-mutated NCI-H1975 cells.
- Quantitative analysis of T790M-mutated cells spiked into non-mutated cells was achieved within 1 hour, with detection rates varying from 0-20% based on concentration.
Conclusions:
- The combined use of single-cell microarray chips and PNA-DNA probes provides an effective system for detecting and isolating rare, drug-resistant cancer cells.
- This technology holds significant potential for analyzing cancer tissue containing a low percentage of anticancer drug-resistant cells.
- The system facilitates a deeper understanding of cancer cell heterogeneity and drug resistance mechanisms at the single-cell level.
