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Aim: Circulating tumor cells (CTCs) are crucial to tumor metastasis and valuable for prediction of clinical outcome in patients with solid tumors. Here, the authors aimed to establish a method for enumeration and characterization of CTCs from liquid biopsies. Methods: Peripheral blood mononuclear cells (PBMCs) were separated from blood samples from patients with metastatic cancer using Ficoll-Hypaque gradients and cultured to isolate and enumerate CTCs. Cultured CTCs were morphologically characterized by light and phase contrast microscopy. The tumorigenicity of Ficoll-Hypaque-separated PBMCs was examined, in addition to their expression of mRNA metastasis markers. Results: CTCs were isolated in culture and enumerated by counting under phase contrast microscopy, demonstrating that 0.01-0.04% of total PBMCs were CTCs. CTCs were dormant, with large, oval-shaped, spiky morphology. PBMCs obtained from liquid biopsies exhibited anchorage-independent growth, forming numerous colonies in soft agar assays. Molecular profiling demonstrated expression of several metastatic genes, but not of cadherin 1 (encoding the adhesion protein), in all patients. Conclusion: The authors successfully isolated, enumerated, and characterized CTCs from liquid biopsies of metastatic cancer patients. This study has potential to facilitate the development of new diagnostic and therapeutic methods using liquid biopsies, for application in metastatic cancers. Key words:Circulating tumor cells, liquids biopsies, molecular markers, soft agar assay, metastasis, metastatic genes, epithelial mesenchymal transition, tumorigenic ABSTRACTArticle history: