Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices

Hei-Jen Jou; Hsin-Cheng Ho; Kuan-Yeh Huang; Chen-Yang Chen; Sheng-Wen Chen; Pei-Hsuan Lo; Pin-Wen Huang; Chung-Er Huang; Ming Chen

doi:10.3390/ijms232315139

Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices

Int J Mol Sci. 2022 Dec 1;23(23):15139. doi: 10.3390/ijms232315139.

Authors

Hei-Jen Jou^{1

2

3}, Hsin-Cheng Ho⁴, Kuan-Yeh Huang⁴, Chen-Yang Chen⁴, Sheng-Wen Chen^{4

5}, Pei-Hsuan Lo¹, Pin-Wen Huang⁶, Chung-Er Huang⁴, Ming Chen^{7

8

9

10

11}

Affiliations

¹ Departments of Obstetrics and Gynecology, Taiwan Adventist Hospital, Taipei 105404, Taiwan.
² Departments of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei 100225, Taiwan.
³ School of Nursing, National Taipei University of Nursing and Health Science, Taipei 112303, Taiwan.
⁴ Cytoaurora Biotechnologies Inc., Hsinchu Science Park, Hsinchu 302058, Taiwan.
⁵ Department of Electrical Engineering, National Chung Cheng University, Chiayi 621301, Taiwan.
⁶ Department of Emergency Medicine, Show Chwan Memorial Hospital, Changhua 500009, Taiwan.
⁷ Department of Genomic Medicine, Changhua Christian Hospital, Changhua 500209, Taiwan.
⁸ Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua 500209, Taiwan.
⁹ Department of Molecular Biotechnology, Da-Yeh University, Changhua 515006, Taiwan.
¹⁰ Department of Obstetrics and Gynecology, College of Medicine, National Taiwan University, Taipei 106319, Taiwan.
¹¹ Department of Medical Genetics, National Taiwan University Hospital, Taipei 100225, Taiwan.

Abstract

Single-cell sequencing provides promising information in tumor evolution and heterogeneity. Even with the recent advances in circulating tumor cell (CTC) technologies, it remains a big challenge to precisely and effectively isolate CTCs for downstream analysis. The Cell Reveal^TM system integrates an automatic CTC enrichment and staining machine, an AI-assisted automatic CTC scanning and identification system, and an automatic cell picking machine for CTC isolation. H1975 cell line was used for the spiking test. The identification of CTCs and the isolation of target CTCs for genetic sequencing were performed from the peripheral blood of three cancer patients, including two with lung cancer and one with both lung cancer and thyroid cancer. The spiking test revealed a mean recovery rate of 81.81% even with extremely low spiking cell counts with a linear relationship between the spiked cell counts and the recovered cell counts (Y = 0.7241 × X + 19.76, R² = 0.9984). The three cancer patients had significantly higher TTF-1⁺ CTCs than healthy volunteers. All target CTCs were successfully isolated by the Cell Picker machine for a subsequent genetic analysis. Six tumor-associated mutations in four genes were detected. The present study reveals the Cell Reveal^TM platform can precisely identify and isolate target CTCs and then successfully perform single-cell sequencing by using commercially available genetic devices.

Keywords: EGFR; ERBB2; KRAS; STK 11; TTF-1+ CTCs; automatic cell picker; circulating tumor cells; lung cancer; nanostructured microfluidic chip; single-cell sequencing.

MeSH terms

Cell Line, Tumor
Cell Separation
Humans
Lab-On-A-Chip Devices
Lung Neoplasms* / genetics
Neoplastic Cells, Circulating* / pathology

Grants and funding

This research received no external funding.