Objective:Circulating tumor cells (CTCs) play a critical role in cancer metastasis,but their prevalence and significance remain unclear.This study attempted to track the epithelial-mesenchymal transition (EMT) status of CTCs in breast cancer patients and investigate their clinical relevance.Methods:In this study,the established negFACS-IF:E/M platform was applied to isolate rare CTCs and characterize their EMT status in breast cancer.A total of 89 breast cancer patients were recruited,including stage 0-Ⅲ (n =60) and late stage (n =29) cases.Results:Using the negFACS-IF:E/M platform,it was found that in human epidermal growth factor receptor 2 (HER2)+ patients,mesenchymal CTCs usually exhibited a high percentage of HER2+ cells.Stage Ⅳ breast cancer patients had considerably more CTCs than stage 0-Ⅲ patients.Among stage 0-Ⅲ breast cancers,the HER2 subtype included a significantly higher percentage of mesenchymal and biphenotypic (epithelial and mesenchymal) CTCs than the luminal A or B subtypes.Among stage Ⅳ patients,CTCs were predominantly epithelial in cases with local recurrence and were more mesenchymal in cases with distant metastasis.By applying a support vector machine (SVM) algorithm,the EMT status of CTCs could distinguish between breast cancer cases with metastasis/local recurrence and those without recurrence.Conclusions:The negFACS-IF:E/M plafform provides a flexible and generally acceptable method for the highly sensitive and specific detection of CTCs and their EMT traits in breast cancer.This study demonstrated that the EMT status of CTCs had high clinical relevance in breast cancer,especially in predicting the distant metastasis or local recurrence of breast cancer.