title: Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients.
abstract: BACKGROUND
  Epithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour 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.
  
  
  METHODS
  The expression of Twist and vimentin in CTCs of 25 metastatic and 25 early breast cancer patients was investigated by using double-immunofluorescence experiments in isolated peripheral blood mononuclear cell cytospins using anti-cytokeratin (anti-CK) anti-mouse (A45-B/B3) and anti-Twist or anti-vimentin anti-rabbit antibodies.
  
  
  RESULTS
  Among early breast cancer patients, vimentin-and Twist-expressing CK(+) CTCs were identified in 77% and 73% of the patients, respectively, and in 100% of the patients with metastatic breast cancer for both markers (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, and 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 patients was 98% and 100%, and in an adjuvant chemotherapy 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(-), confirming their epithelial origin. Immunomagnetic separation of CTCs and triple-immunofluorescence with anti-CK/anti-Twist/anti-vimentin antibodies demonstrated that both mesenchymal markers could be coexpressed 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.
  
  
  CONCLUSIONS
  CTCs expressing Twist and vimentin, suggestive of EMT, 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.
authors.affiliation: Laboratory of Τumor Cell Βiology, University of Crete, Heraklion, Greece. kalergi@med.uoc.gr
authors.forename: Galatea
authors.initials: G
authors.lastname: Kallergi
authors.forename: Maria A
authors.initials: MA
authors.lastname: Papadaki
authors.forename: Eleni
authors.initials: E
authors.lastname: Politaki
authors.forename: Dimitris
authors.initials: D
authors.lastname: Mavroudis
authors.forename: Vassilis
authors.initials: V
authors.lastname: Georgoulias
authors.forename: Sophia
authors.initials: S
authors.lastname: Agelaki
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000276:immunology; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000097:blood / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D017024:Chemotherapy, Adjuvant; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006367:HeLa Cells; D006801:Humans; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D051879:Twist-Related Protein 1 / Q000276:immunology / Q000378:metabolism*; D014746:Vimentin / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1; D014746:Vimentin; D017493:Leukocyte Common Antigens