Abstract Epithelial-to-Mesenchymal Transition (EMT) is a stepwise and complex biological mechanism which plays an important role in tumor progression. During EMT, epithelial cells undergo a phenotypical switch to mesenchymal cells, acquiring motility and invasion capability. We aimed at clarifying the role of this complex mechanism in the Pancreatic Ductal Adenocarcinoma (PDAC) focusing on the expression of EMT markers in different cell culture conditions, such as monolayer and spheroids. For this study we used immunofluorescence and confocal microscopy, zymography, differential proteomics, real-time pcr and western blot to analyse and characterize three commercial PDAC cell lines ( HPAF-II, HPAC and PL45) cultured as monolayer or 3D- spheroids. Our analyses revealed that PDAC cells cultured in monolayer show different epithelial characteristics, such as retention of the E-cadherin/β-catenin complex – and, consequently, keep zonula adherens junctions – as well as some mesenchymal and EMT markers, such as high levels of matrix metalloproteinases (MMPs). The analysis of 3D-spheroids revealed that the E-cadherin/ β-catenin complex was similarly expressed at the cell boundaries on the plasma membrane in 2D-monolayers as well as in the 3D-spheroids, but cleavage fragments were detectable in lysates obtained from monolayers. By contrast, N-cadherin expression was observed in few HPAC cells grown in monolayers but increased in 3D-spheroids, and some cells expressing collagen type I were observed in 3D-spheroids. Podoplanin and α-smooth muscle actin were similarly expressed in both experimental conditions, as well as Snail, Slug and Zeb1. Our results confirm that EMT plays a role in PDAC. In particular, the concomitant retention of an epithelial phenotype based on the retention of zonula adherens junctions together with the expression of mesenchymal markers and proteins involved in actin cytoskeleton reorganization, strongly suggest a collective migration mechanism for PDAC cells. Moreover, the overall information provided by this study support the use of 3D cultures in biomedical research to bridge the gap between traditional cell cultures and in vivo settings. 3D cultures seem provide powerful information on PDAC cells phenotype possibly better reflecting the in vivo organization than monolayer cell culture systems, and could therefore represent a pre-clinical model to identify and validate tumor markers and to study new molecular tools to inhibit signalling pathways, or to target EMT transcription factors.

TRANSIZIONE EPITELIO-MESENCHIMALE IN CELLULE DI CARCINOMA DUTTALE DEL PANCREAS: STUDIO MORFOLOGICO E MOLECOLARE / M. Clerici ; tutor: N. Gagliano ; co-tutor: I. Dalle Donne ; coordinator: C. Sforza. - : . DIPARTIMENTO DI SCIENZE BIOMEDICHE PER LA SALUTE, 2015 Jan 16. ((27. ciclo, Anno Accademico 2014. [10.13130/clerici-marco_phd2015-01-16].

TRANSIZIONE EPITELIO-MESENCHIMALE IN CELLULE DI CARCINOMA DUTTALE DEL PANCREAS: STUDIO MORFOLOGICO E MOLECOLARE

M. Clerici
2015

Abstract

Abstract Epithelial-to-Mesenchymal Transition (EMT) is a stepwise and complex biological mechanism which plays an important role in tumor progression. During EMT, epithelial cells undergo a phenotypical switch to mesenchymal cells, acquiring motility and invasion capability. We aimed at clarifying the role of this complex mechanism in the Pancreatic Ductal Adenocarcinoma (PDAC) focusing on the expression of EMT markers in different cell culture conditions, such as monolayer and spheroids. For this study we used immunofluorescence and confocal microscopy, zymography, differential proteomics, real-time pcr and western blot to analyse and characterize three commercial PDAC cell lines ( HPAF-II, HPAC and PL45) cultured as monolayer or 3D- spheroids. Our analyses revealed that PDAC cells cultured in monolayer show different epithelial characteristics, such as retention of the E-cadherin/β-catenin complex – and, consequently, keep zonula adherens junctions – as well as some mesenchymal and EMT markers, such as high levels of matrix metalloproteinases (MMPs). The analysis of 3D-spheroids revealed that the E-cadherin/ β-catenin complex was similarly expressed at the cell boundaries on the plasma membrane in 2D-monolayers as well as in the 3D-spheroids, but cleavage fragments were detectable in lysates obtained from monolayers. By contrast, N-cadherin expression was observed in few HPAC cells grown in monolayers but increased in 3D-spheroids, and some cells expressing collagen type I were observed in 3D-spheroids. Podoplanin and α-smooth muscle actin were similarly expressed in both experimental conditions, as well as Snail, Slug and Zeb1. Our results confirm that EMT plays a role in PDAC. In particular, the concomitant retention of an epithelial phenotype based on the retention of zonula adherens junctions together with the expression of mesenchymal markers and proteins involved in actin cytoskeleton reorganization, strongly suggest a collective migration mechanism for PDAC cells. Moreover, the overall information provided by this study support the use of 3D cultures in biomedical research to bridge the gap between traditional cell cultures and in vivo settings. 3D cultures seem provide powerful information on PDAC cells phenotype possibly better reflecting the in vivo organization than monolayer cell culture systems, and could therefore represent a pre-clinical model to identify and validate tumor markers and to study new molecular tools to inhibit signalling pathways, or to target EMT transcription factors.
GAGLIANO, NICOLETTA
SFORZA, CHIARELLA
EMT; PDAC; adenocarcinoma; epithelial; mesenchymal
Settore BIO/06 - Anatomia Comparata e Citologia
Settore BIO/16 - Anatomia Umana
TRANSIZIONE EPITELIO-MESENCHIMALE IN CELLULE DI CARCINOMA DUTTALE DEL PANCREAS: STUDIO MORFOLOGICO E MOLECOLARE / M. Clerici ; tutor: N. Gagliano ; co-tutor: I. Dalle Donne ; coordinator: C. Sforza. - : . DIPARTIMENTO DI SCIENZE BIOMEDICHE PER LA SALUTE, 2015 Jan 16. ((27. ciclo, Anno Accademico 2014. [10.13130/clerici-marco_phd2015-01-16].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/252716
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