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. 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. 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. 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.

Lung cancer remains the leading cause of cancer-related death globally, with metastasis driven by circulating tumor cells (CTCs)—particularly clusters—being a major treatment challenge. Despite their critical role, the biological differences between single CTCs and CTC clusters remain unclear. Here, we comprehensively compared their behavioral, transcriptomic, and proteomic profiles in lung cancer models. Compared with single cells, CTC clusters present enhanced metastatic potential, greater survival in the bloodstream and increased resistance to microenvironment. Mechanistically, the Src/FN1 pathway is centrally activated in clusters, promoting intercellular cohesion and protecting against immune clearance and stress in circulation. Pharmacological inhibition of Src with the clinical inhibitor KX2-391 disrupted clustering, impaired CTC survival, and reduced metastasis in preclinical models. Our findings identify the Src/FN1 pathway as a key vulnerability in CTC cluster–driven metastasis, suggesting that Src inhibitors are promising therapeutic strategies to disrupt clustering and improve outcomes in patients with metastatic lung cancer.