Epithelial-To-Mesenchymal Transition Markers and CD44 Isoforms Are Differently Expressed in 2D and 3D Cell Cultures of Prostate Cancer Cells.

Fabrizio Fontana,Monica Marzagalli,Patrizia Limonta,Michele Sommariva,Michela Raimondi,Nicoletta Gagliano

doi:10.3390/cells8020143

Abstract

Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues and provide key information encoded in tissue architecture. Considered the pivotal role of epithelial-to-mesenchymal transition (EMT) in carcinoma progression, including prostate cancer (PCa), we aimed at investigating the effect of the 3D arrangement on the expression of some key markers of EMT in cultured human prostate cancer (PCa) cells, to better understand PCa cell behavior. PC3 and DU145 PCa cells were cultured in RPMI cell culture medium either in 2D-monolayers or in 3D-spheroids. The main EMT markers E-cadherin, N-cadherin, α-smooth muscle actin (αSMA), vimentin, Snail, Slug, Twist and Zeb1 were evaluated by confocal microscopy, real-time PCR and Western blot. Confocal microscopy revealed that E-cadherin was similarly expressed at the cell boundaries on the plasma membrane of PCa cells grown in 2D-monolayers, as well as in 3D-spheroids, but resulted up-regulated in 3D-spheroids, compared to 2D-monolayers, at the mRNA and protein level. Moreover, markers of the mesenchymal phenotype were expressed at very low levels in 3D-spheroids, suggesting important differences in the phenotype of PCa cells grown in 3D-spheroids or in 2D-monolayers. Considered as a whole, our findings contribute to a clarification of the role of EMT in PCa and confirm that a 3D cell culture model could provide deeper insight into the understanding of the biology of PCa.

Highlights

Prostate cancer (PCa) is a urological disease associated with significant morbidity and mortality, representing the second most common cause of cancer-related deaths in male population [1].There are considerable data suggesting that epithelial-to-mesenchymal transition (EMT) plays a pivotal role in prostate cancer (PCa) progression [2]
Some heterogeneity in E-cadherin expression has been previously described in pancreatic adenocarcinoma [31,39] as well as in PCa, showing variable E-cadherin expression in metastatic tissues compared to primary tumor tissues [15,16,17,18,19,20], increasing the relevance of studies aimed at characterizing the phenotype of PCa cells in relation to the expression of EMT markers, and especially of E-cadherin, in order to better understand PCa development and progression
CD44 standard splice isoform (CD44s) and CD44 variant splice isoforms (CD44v) are related, respectively, to a more mesenchymal or epithelial phenotype, our results suggest that DU145 cells have a more epithelial phenotype, compared to PC3 cells, consistent with the stronger cell adhesion observed under the microscope

Summary

Introduction

There are considerable data suggesting that epithelial-to-mesenchymal transition (EMT) plays a pivotal role in PCa progression [2]. An EMT program is required by cancer cells to acquire functional characteristics for metastasis, and involves multiple steps including the acquisition of invasiveness, intravasation in systemic blood or lymphatic systems, subsequent extravasation, and growth at distant organs [3]. During EMT, cancer cells shed their epithelial features, detach from epithelial sheets, loose their polarity, and undergo cytoskeletal modifications towards a mesenchymal phenotype, acquiring a high degree of motility and invasive potential [4]. Repression of E-cadherin expression by EMT transcription factors was described in vivo and in various cancer cell lines, including lung, breast, colorectal and ovarian cancer, inducing tumor malignancy [8,9,10]

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