Isolation and molecular analysis of circulating tumor cells from lung cancer patients using a microfluidic chip type cell sorter

Abstract

Circulating tumor cells (CTCs) are a tumor-derived material utilized for liquid-based biopsy; however, capturing rare CTCs for further molecular analysis remains technically challenging, especially in non-small-cell lung cancer. Here, we report the results of a clinical evaluation of On-chip Sort, a disposable microfluidic chip-based cell sorter, for capture and molecular analysis of CTCs from patients with lung adenocarcinoma. Peripheral blood was collected from 30 metastatic lung adenocarcinoma patients to enumerate CTCs using both On-chip Sort and CellSearch in a blind manner. Captured cells by On-chip Sort were subjected to further molecular analysis. Peripheral blood samples were also used for detection of EGFR mutations in plasma using droplet digital PCR. Significantly more CTCs were detected by On-chip Sort (22/30; median 5; range, 0-18 cells/5 mL blood) than by CellSearch (9/30; median, 0; range, 0-12 cells/7.5 mL) (P < 0.01). Thirteen of 30 patients who had a negative CTC count by CellSearch had a positive CTC count by On-chip Sort. EGFR mutations in CTCs captured by On-chip Sort were observed in 40.0% (8/20) of patients with EGFR-mutated primary tumor. EGFR mutations were often observed in 53.3% (8/15) of patients detected in plasma DNA. Expressions of EGFR and vimentin protein on CTCs were also successfully assessed using On-chip Sort. These results suggest that On-chip Sort is an efficient method to detect and capture rare CTCs from patients with lung adenocarcinoma that are undetectable with CellSearch. Mutation detection using isolated CTCs remains to be further tackled (UMIN000012488).

Keywords: EGFR; cell sorter; circulating tumor cell; liquid biopsy; lung cancer.

Figure 1

Circulating tumor cell (CTC) count using the On‐chip Sort system compared with the CellSearch system. (A) CTC count/7.5 mL blood is shown for 10 healthy donors and 30 patients with non‐small‐cell lung carcinoma (NSCLC). Paired blood samples were analyzed by CellSearch according to the manufacturer's protocol and On‐chip Sort by immunolabeling analysis. (B) Direct comparison of CTC counts between CellSearch and On‐chip Sort. Gray line represents the theoretical perfect correlation. The cut‐off levels (3 CTCs for On‐chip Sort and 2 CTCs for CellSearch per sample) are indicated by the dashed lines

Figure 2

Gallery of cells captured by On‐chip Sort from patients with non‐small‐cell lung carcinoma. Cells were stained with FITC‐labeled anti‐cytokeratin antibody, phycoerythrin‐labeled anti‐vimentin, and Alexa700‐labeled anti‐CD45 antibody. CTC, circulating tumor cell

Figure 3

Assessment of protein expression in circulating tumor cells (CTCs). Vimentin (A) and epidermal growth factor receptor (EGFR) (B) expressions were assessed by On‐chip Sort during sorting. Intensity of fluorescent‐labeled protein expression on CTCs was analyzed by FlowJo software 7.6.5. Cell images of EGFR expression on CTCs are embedded in (B). PE, phycoerythrin