Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 14, 2016; 22(18): 4466-4483
Published online May 14, 2016. doi: 10.3748/wjg.v22.i18.4466
Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model
Nicoletta Gagliano, Giuseppe Celesti, Lorenza Tacchini, Stefano Pluchino, Chiarella Sforza, Marco Rasile, Vincenza Valerio, Luigi Laghi, Vincenzo Conte, Patrizia Procacci
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
Giuseppe Celesti, Luigi Laghi, Laboratory of Molecular Gastroenterology, Humanitas Clinical and Research Center, 20089 Rozzano, Milan, Italy
Stefano Pluchino, Department of Clinical Neurosciences, Wellcome Trust-Medical Research Council Stem Cell Institute and NIHR Biomedical Research Centre, University of Cambridge, Cambridgeshire CB2 0PY, United Kingdom
Marco Rasile, Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 20089 Rozzano, Milan, Italy
Author contributions: Gagliano N and Procacci P were responsible for the study concept and design, data acquisition, analysis, interpretation, and drafting the article; Celesti G, Rasile M, Valerio V, and Conte V performed data acquisition and analysis, as well as assisting with drafting the article; Tacchini L, Pluchino S, Sforza C, and Laghi L drafted and critically revised the manuscript; all authors gave their final approval for the version of the manuscript to be published.
Supported by the University of Milan (Project B grant) and core support grant from the Wellcome Trust and MRC to the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute.
Institutional review board statement: In this study, only commercial cell lines were used, therefore no approval by an Institutional Review Board is needed.
Institutional animal care and use committee statement: In this study, only commercial cell lines were used, therefore no Institutional Animal Care and Use Committee statement is needed.
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists.
Data sharing statement: Technical appendix and dataset are available from the corresponding author at nicoletta.gagliano@unimi.it.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Nicoletta Gagliano, PhD, Associate Professor of Histology, Department of Biomedical Sciences for Health, Università degli Studi di Milano, via Mangiagalli 31, 20133 Milan, Italy. nicoletta.gagliano@unimi.it
Telephone: +39-2-50315374 Fax: +39-2-50315387
Received: January 20, 2016
Peer-review started: January 21, 2016
First decision: February 18, 2016
Revised: March 2, 2016
Accepted: March 14, 2016
Article in press: March 14, 2016
Published online: May 14, 2016
Abstract

AIM: 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.

CONCLUSION: 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.

Keywords: Epithelial-to-mesenchymal transition, E-cadherin, 3D-spheroids, Podoplanin, Pancreatic ductal adenocarcinoma

Core tip: The functions of living tissue can be mimicked by three-dimensional (3D) cell cultures, thereby providing a method of decoding the information encoded in the tissue architecture. We aimed to analyze the effect of 3D-arrangement on the expression of some key markers of epithelial-to-mesenchymal transition in pancreatic adenocarcinoma (PDAC) cells cultured in either 2D-monolayers or 3D-spheroids. Our results 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.