Epithelial-mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterized by up-regulation of Vimentin, Twist, snail, slug and SIP-1 among others. Metastasis is also associated with the presence of circulating (CTCs) and disseminated (DTCs) tumor cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far.

The expression of Twist and Vimentin in CTCs of 25 metastatic and 25 early breast cancer patients was investigated using double immunofluorescence experiments in isolated peripheral blood mononuclear cells (PBMCs)' cytospins using anti-cytokeratin (CK) anti-mouse (A45-B/B3) and anti-Twist or anti-Vimentin anti-rabbit antibodies.

Vimentin- and Twist-expressing CK(+) CTCs were identified in 77% and 73%, respectively, of early breast cancer patients and in 100% for both markers of metastatic patients (P=0.004 and P=0.037, respectively). Among patients with early disease, 56% and 53% of the CK (+) CTCs were double stained with Vimentin and Twist, whereas the corresponding values for metastatic patients were 74% and 97%, respectively (P=0.005 and P=0.0001, respectively). The median expression of CK+Vimentin+ and CK+Twist+ cells per patient in metastatic setting was 98% and 100%, whereas in adjuvant setting the corresponding numbers were 56% and 40.6%, respectively. Triple-staining experiments revealed that all CK+Twist+ or CK+Vimentin+ cells were also CD45-negative, confirming their epithelial origin. Immunomagnetic separation of CTCs and triple immunofluorescence with anti-CK/Twist/Vimentin antibodies demonstrated that both mesenchymal markers could be co-expressed in the same CK(+) cell since 64% of the total identified CTCs were triple stained. There was a significant correlation (P=0.005) between the number of CTCs expressing Twist and Vimentin within the same setting.

CTCs expressing Twist and Vimentin, suggestive of an epithelial-mesenchymal transition, are identified in patients with breast cancer. The high incidence of these cells in patients with metastatic disease compared to early stage breast cancer strongly supports the notion that EMT is involved in the metastatic potential of CTCs.

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