Variable expression levels of keratin and vimentin reveal differential EMT status of circulating tumor cells and correlation with clinical characteristics and outcome of patients with metastatic breast cancer

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.

The Landscape of Circulating Tumor Cell Research in the Context of Epithelial-Mesenchymal Transition

“…Presence of mesenchymal CTCs was associated with shorter survival in MBC [4] , which was also confirmed using a single CTC analysis approach [44] . MBC patients Whereas epithelial cells seem to be more capable of establishing tumor colonies at distant sites (proliferative phenotype -Ki-67+), mesenchymal cells are more resilient to changing conditions, thus are better equipped to survive during the metastatic cascade.…”

“…Recently, plastin-3, a specific, novel marker useful for the detection of CTCs undergoing EMT, has been described [42,43] . Also, although with a slight difference, some investigators used a ratio of epithelial and mesenchymal markers to describe CTC phenotypes [19,44] .…”

TTF-1- and/or CD56-positive Circulating Tumor Cells in patients with small cell lung cancer (SCLC)

“…This observation should be attributed to the finding that, in these particular patients, IF staining could not reveal the presence of either TTF-1 + /EpCAM + or CK + /EpCAM + CTCs since CS recognizes CK + CTCs which are, previously, captured by an anti-EpCAM antibody. It is well known that epithelial tumor cells migrating through the bloodstream undergo EMT which is characterized, among others, by a loss of expression of epithelial markers325455; therefore, it is reasonable to hypothesize that, in this small subgroup of patients without detectable CTCs by CS, the TTF-1 + /EpCAM − CTCs probably represent CTCs undergoing EMT. This is in accordance with studies reporting that the CTC detection rate was higher using EpCAM-independent enrichment methods56.…”

Cancer metastasis through the prism of epithelial‐to‐mesenchymal transition in circulating tumor cells

“…Markers of EMT in CTCs have the potential to provide both prognostic and predictive information. As discussed above, in breast cancer, the number of mesenchymal CTCs has been linked to disease progression and decreased overall survival (Polioudaki et al, 2015;Yu et al, 2013). In colorectal cancer, a study of CTCs in over a thousand patients found that biphenotypic and mesenchymal CTCs correlated with clinical stage, lymph node, and distant metastases (Zhao et al, 2017).…”