title: 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: BACKGROUND
  CTCs 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.
  
  
  METHODS
  Keratin 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.
  
  
  RESULTS
  MCF-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).
  
  
  CONCLUSIONS
  Keratin 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.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, Heraklion, Greece. poloudaki@med.uoc.gr.
authors.forename: Hara
authors.initials: H
authors.lastname: Polioudaki
authors.affiliation: Laboratory of Τumor Cell Βiology, School of Medicine, University of Crete, Heraklion, Greece. agelaki@med.uoc.gr.|Department of Medical Oncology, University General Hospital of Heraklion, Heraklion, Greece. agelaki@med.uoc.gr.
authors.forename: Sofia
authors.initials: S
authors.lastname: Agelaki
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, Heraklion, Greece. renachiotaki@gmail.com.
authors.forename: Rena
authors.initials: R
authors.lastname: Chiotaki
authors.affiliation: Laboratory of Τumor Cell Βiology, School of Medicine, University of Crete, Heraklion, Greece. lenapolitaki@med.uoc.gr.
authors.forename: Eleni
authors.initials: E
authors.lastname: Politaki
authors.affiliation: Laboratory of Τumor Cell Βiology, School of Medicine, University of Crete, Heraklion, Greece. mavrudis@med.uoc.gr.|Department of Medical Oncology, University General Hospital of Heraklion, Heraklion, Greece. mavrudis@med.uoc.gr.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Mavroudis
authors.affiliation: Department of Medical Oncology, University General Hospital of Heraklion, Heraklion, Greece. almatikas@gmail.com.
authors.forename: Alexios
authors.initials: A
authors.lastname: Matikas
authors.affiliation: Laboratory of Τumor Cell Βiology, School of Medicine, University of Crete, Heraklion, Greece. georgoul@med.uoc.gr.|Department of Medical Oncology, University General Hospital of Heraklion, Heraklion, Greece. georgoul@med.uoc.gr.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Georgoulias
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, Heraklion, Greece. takis@med.uoc.gr.
authors.forename: Panayiotis A
authors.initials: PA
authors.lastname: Theodoropoulos
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006801:Humans; D007633:Keratins / Q000378:metabolism*; D061986:MCF-7 Cells; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D016019:Survival Analysis; D014746:Vimentin / Q000378:metabolism*; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D014746:Vimentin; D007633:Keratins