Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes

Abstract

<jats:p>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.</jats:p>

FRET probe for detecting two mutations in one EGFR mRNA

“…Although the intracellular concentration of EGFR mRNA is unknown, it could be detected with this probe set as the mRNA was detected intracellularly by another probe with a detection limit of around 5 nM. 40,41…”

Selective cell retrieval method using light-responsive gas-generating polymer-based microarrays

“…We have also attempted to retrieve CTC using micromanipulators for further functional analysis (e.g., EGFR-mutated genes in drug resistant cancers). 33,34 Although the LGP microarray system need for further integration and improvement in future, by using the proposed LGP microarray method, it is expected that CTC can be easily retrieved with low damage.…”

Single-Cell Microarray Chip with Inverse-Tapered Wells to Maintain High Ratio of Cell Trapping

“…In passive SCM, the important factors to consider when determining the well geometry are the well dimensions and shape. The two-dimensional geometry of a conventional well fabricated using photolithography is flexible in design [ 29 , 30 , 31 ]. In contrast, we propose an SCM with inverse-tapered three-dimensional (3D) wells (fabricated via 3D photolithography) to achieve single-cell immobilization and prevent cell dropout during the liquid exchange [ 32 ].…”