Establishment and characterization of a patient-derived circulating lung tumor cell line in vitro and in vivo

Zujun Que; Bin Luo; Zhiyi Zhou; Changsheng Dong; Yi Jiang; Lin Wang; Qihui Shi; Jianhui Tian

doi:10.1186/s12935-019-0735-z

Establishment and characterization of a patient-derived circulating lung tumor cell line in vitro and in vivo

Cancer Cell Int. 2019 Jan 29:19:21. doi: 10.1186/s12935-019-0735-z. eCollection 2019.

Authors

Zujun Que^#^{1

2}, Bin Luo^#², Zhiyi Zhou², Changsheng Dong¹, Yi Jiang², Lin Wang³, Qihui Shi⁴, Jianhui Tian^{1

2}

Affiliations

¹ Institute of Traditional Chinese Medicine Oncology, Shanghai Institute of Traditional Chinese Medicine, Shanghai, 200032 People's Republic of China.
² 2Department of Oncology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032 People's Republic of China.
³ 3Department of Nephrology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032 People's Republic of China.
⁴ 4Key Laboratory of Medical Epigenetics and Metabolism, Minhang Branch, Zhongshan Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433 People's Republic of China.

^# Contributed equally.

Abstract

Background: Circulating tumor cells (CTCs) have been described as a population of cells that may seed metastasis, which is a reliable target for the prevention of metastases in lung cancer patients at the early stage. The culturing of CTCs in vitro can be used to study the mechanism of lung cancer metastasis and to screen antimetastasis drugs. This study aims to establish CTC cell line in vitro and explore the potential mechanism of its metastasis.

Methods: A mixture of EpCAM- and EGFR-coated immunomagnetic microbeads in microfluidic Herringbone-Chip was used to capture CTCs. The CTCs, 95-D and A549 cells was evaluated by cell proliferation assays, clonal formation assays, migration assays and drug resistance. Flow cytometry and cytokine protein chip were used to detect the difference in phenotype and cytokine secretion between CTCs, 95-D and A549 cells. The NOD/SCID mice were used to study tumorigenicity, lung organ colonization and metastasis of CTCs. The H&E staining, immunohistochemistry and immunofluorescence assay were used to detect the pathological status of CTCs.

Results: The number of EpCAM(+)/EGFR(+)/CK(+)/CD45(-) lung CTCs showed a weak negative correlation with clinical stages in patients with non-small cell lung cancer (NSCLC). In a phase IIa lung cancer patient, we successfully establish a permanent CTC cell line, named CTC-TJH-01. In vitro studies showed the CTC-TJH-01 cells were in the intermediate stage of epithelial to mesenchymal transition (EMT), had stem cell characteristics and were drug resistant. In vivo studies showed that CTC-TJH-01 cells can induce tumorigenesis, lung organ colonization and metastasis after xenografting in immunodeficient mice. In addition, the low expression level of CX3CL1 and high expression level of CXCL5 in the CTC-TJH-01 cells may be an important mechanism for their metastasis.

Conclusions: We successfully established a permanent CTC cell line with metastatic ability, which can be used to screen antimetastatic drugs and study the mechanism of lung cancer metastasis.

Keywords: Circulating tumor cell; Metastasis; Non-small cell lung cancer; Organ colonization; Prevention.