Abstract
BackgroundCTCs expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported. However, no information exists for keratin expression levels of CTCs in association with disease status, whereas assays for the characterization of transitional EMT phenotypes of CTCs in breast cancer are rather lacking. We investigated the correlation between keratin expression of CTCs and patients’ outcome and characterized the EMT status of CTCs via the establishment of a numerical “ratio” value of keratin and vimentin expression levels on a single cell basis.MethodsKeratin expression was evaluated in 1262 CTCs from 61 CTC-positive patients with metastatic breast cancer, using analysis of images obtained through the CellSearch System. For the determination of vimentin/keratin (vim/K) ratios, expression levels of keratin and vimentin were measured in cytospin preparations of luminal (MCF-7 and T47D) and basal (MDA.MB231 and Hs578T) breast cancer cell lines and 110 CTCs from 5 CTC-positive patients using triple immunofluorescence laser scanning microscopy and image analysis.ResultsMCF-7 and T47D displayed lower vim/K ratios compared to MDA.MB231 and Hs578T cells, while MCF-7 cells that had experimentally undergone EMT were characterized by varying intermediate vim/K ratios. CTCs were consisted of an heterogeneous population presenting variable vim/K values with 46% of them being in the range of luminal breast cancer cell lines. Keratin expression levels of CTCs detected by the CellSearch System correlated with triple negative (p = 0.039) and ER-negative (p = 0.025) breast cancer, and overall survival (p = 0.038).ConclusionsKeratin expression levels of CTCs correlate with tumor characteristics and clinical outcome. Moreover, CTCs display significant heterogeneity in terms of the degree of EMT phenotype that probably reflects differential invasive potential. The assessment of the vim/K ratios as a surrogate marker for the EMT status of CTCs merits further investigation as a prognostic tool in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1386-7) contains supplementary material, which is available to authorized users.
Highlights
Circulating tumor cells (CTCs) expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported
We propose a new approach for the designation of Epithelial-to-Mesenchymal Transition (EMT) status of CTCs, based on the quantification of fluorescence intensity of keratin and vimentin on a single cell basis and the generation of a numerical ‘ratio’ value corresponding to their relative expression
When the expression values were presented as a vimentin to keratin ratio, which we introduce as an EMT index, it was shown that the “epithelial” MCF-7 and T47D cell lines are characterized by low vim/K ratios (0.19 ± 0.05 for MCF-7 and 0.20 ± 0.07 for T47D cells), while “mesenchymal” MDA.MB231 and Hs578T cells display high vim/K (4.44 ± 1.98 and 13.14 ± 5.08, respectively) ratios (Table 1 and Figure 3)
Summary
CTCs expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported. No information exists for keratin expression levels of CTCs in association with disease status, whereas assays for the characterization of transitional EMT phenotypes of CTCs in breast cancer are rather lacking. It has been suggested that modulation of keratins due to Epithelial-to-Mesenchymal Transition (EMT) occurs frequently in CTCs of breast cancer patients and may be associated with an unfavorable outcome [1]. During EMT, epithelial cells display decreased expression of epithelial markers (loss of epithelial keratins, including 8, 18 and 19, and downregulation of E-cadherin, occludins, claudins and desmoplakin) and acquire mesenchymal traits (up-regulation of vimentin, N-cadherin, fibronectin, alpha-smooth muscle actin). Human cancer cells induced to undergo EMT have been shown to exhibit stem cell–like properties and increased metastatic potential [8]
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