title: Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines.
abstract: BACKGROUND
  Tumour heterogeneity and resistance to systemic treatment in urothelial carcinoma (UC) may arise from cancer stem cells (CSC). A recent model describes cellular differentiation states within UC based on corresponding expression of surface markers (CD) and cytokeratins (CK) with CD90 and CK14 positive cells representing the least differentiated and most tumourigenic population. Based on the fact that this population is postulated to constitute CSCs and the origin of cisplatin resistance, we enriched urothelial carcinoma cell lines (UCCs) for CD90 and studied the tumour-initiating potential of these separated cells in vitro.
  
  
  METHODS
  Magnetic- and fluorescence-activated- cell sorting were used for separation of CD90(+) and CD90(-) UCCs. Distribution of cell surface markers CD90, CD44, and CD49f and cytokeratins CK14, CK5, and CK20 as well as the effects of short- and long-term treatment with cisplatin were assessed in vitro and measured by qRT-PCR, immunocytochemistry, reporter assay and flow cytometry in 11 UCCs.
  
  
  RESULTS
  We observed cell populations with surface markers according to those reported in tumour xenografts. However, expression of cytokeratins did not concord regularly with that of the surface markers. In particular, expression of CD90 and CK14 diverged during enrichment of CD90(+) cells by immunomagnetic sorting or following cisplatin treatment. Enriched CD90(+) cells did not exhibit CSC-like characteristics like enhanced clonogenicity and cisplatin resistance. Moreover, selection of cisplatin-resistant sublines by long-term drug treatment did not result in enrichment of CD90(+) cells. Rather, these sublines displayed significant phenotypic plasticity expressing EMT markers, an altered pattern of CKs, and WNT-pathway target genes.
  
  
  CONCLUSIONS
  Our findings indicate that the correspondence between CD surface markers and cytokeratins reported in xenografts is not maintained in commonly used UCCs and that CD90 may not be a stable marker of CSC in UC. Moreover, UCCs cells are capable of substantial phenotypic plasticity that may significantly contribute to the emergence of cisplatin resistance.
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Universitaetsstrasse 1, 40225, Düsseldorf, Germany. Margaretha.Skowron@uni-duesseldorf.de.
authors.forename: Margaretha A
authors.initials: MA
authors.lastname: Skowron
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Universitaetsstrasse 1, 40225, Düsseldorf, Germany. Guenter.Niegisch@med.uni-duesseldorf.de.
authors.forename: Günter
authors.initials: G
authors.lastname: Niegisch
authors.affiliation: Institute of Toxicology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Universitaetsstrasse 1, 40225, Düsseldorf, Germany. fritz@uni-duesseldorf.de.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Fritz
authors.affiliation: Department of Forensic Medicine, Medical Faculty, Heinrich-Heine-University Duesseldorf, Moorenstr. 5, 40225, Düsseldorf, Germany. tanja.arent@med.uni-duesseldorf.de.
authors.forename: Tanja
authors.initials: T
authors.lastname: Arent
authors.affiliation: Department of Urology, Maastricht University Medical Centre, P. Debyelaan 25, 6202, AZ, Maastricht, The Netherlands. joep.van.roermund@mumc.nl.
authors.forename: Joep G H
authors.initials: JG
authors.lastname: van Roermund
authors.affiliation: Department of Obstetrics and Gynaecology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Centre, P. Debyelaan 25, 6202, AZ, Maastricht, The Netherlands. a.romano@maastrichtuniversity.nl.
authors.forename: Andrea
authors.initials: A
authors.lastname: Romano
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Universitaetsstrasse 1, 40225, Düsseldorf, Germany. Peter.Albers@med.uni-duesseldorf.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Albers
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Universitaetsstrasse 1, 40225, Düsseldorf, Germany. Wolfgang.Schulz@hhu.de.
authors.forename: Wolfgang A
authors.initials: WA
authors.lastname: Schulz
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Universitaetsstrasse 1, 40225, Düsseldorf, Germany. Michele.Hoffmann@uni-duesseldorf.de.
authors.forename: Michèle J
authors.initials: MJ
authors.lastname: Hoffmann
mesh_terms: D015415:Biomarkers; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D006801:Humans; D016130:Immunophenotyping; D053547:Keratin-14 / Q000378:metabolism*; D060787:Neoplasm Grading; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D018800:Thy-1 Antigens / Q000378:metabolism*; D014523:Urethral Neoplasms / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000187:drug effects
chemical_list: D015415:Biomarkers; D053547:Keratin-14; D018800:Thy-1 Antigens; D002945:Cisplatin