title: Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model.
abstract: OBJECTIVE
  To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells.
  
  
  METHODS
  HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids.
  
  
  RESULTS
  The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression.
  
  
  CONCLUSIONS
  We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids.
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Gagliano
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Celesti
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Lorenza
authors.initials: L
authors.lastname: Tacchini
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Pluchino
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Chiarella
authors.initials: C
authors.lastname: Sforza
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Rasile
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Valerio
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Laghi
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Conte
authors.affiliation: Nicoletta Gagliano, Lorenza Tacchini, Vincenza Valerio, Chiarella Sforza, Vincenzo Conte, Patrizia Procacci, Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Procacci
mesh_terms: D015703:Antigens, CD; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D045744:Cell Line, Tumor; D048430:Cell Shape; D002470:Cell Survival; D058750:Epithelial-Mesenchymal Transition; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism; D000098853:Podoplanin
chemical_list: D015703:Antigens, CD; D014408:Biomarkers, Tumor; D015820:Cadherins; D008562:Membrane Glycoproteins; D051176:beta Catenin; C513384:CDH1 protein, human; C495270:CTNNB1 protein, human; C117468:PDPN protein, human; D000098853:Podoplanin
keywords: 3D-spheroids; E-cadherin; Epithelial-to-mesenchymal transition; Pancreatic ductal adenocarcinoma; Podoplanin