Enhanced morphological and functional differences of pancreatic cancer with epithelial or mesenchymal characteristics in 3D culture

Yuuki Shichi,Junko Aida,Masashi Toyoda,Toshiyuki Ishiwata,Kaiyo Takubo,Fumio Hasegawa,Norihiko Sasaki,Yoko Matsuda,Masaki Michishita,Kimimasa Takahashi,Tomio Arai,Hisashi Yoshimura,Fujiya Gomi

doi:10.1038/s41598-019-47416-w

Abstract

Pancreatic cancer, composed of heterogeneous cancer cells, alters epithelial to mesenchymal features during growth and metastasis. In this study, we aimed to characterize pancreatic ductal adenocarcinoma (PDAC) cells showing epithelial or mesenchymal features in 3D culture. In 3D culture, PK-1 cells had high E-cadherin and low vimentin expression and exhibited a round-like appearance encircled by flat cells. PANC-1 cells had high vimentin and low E-cadherin expression and formed grape-like spheres. PK-1 cells had secretary granules and many microvilli, desmosomes, and adherens junctions, while PANC-1 cells had few microvilli, adherens junction, and no desmosomes. Cytokeratin 7, trypsin, CA19-9, and E-cadherin were highly expressed in PK-1 cells but not in PANC-1 cells. Ki-67 was diffusely expressed in PANC-1 spheres but was restricted to the peripheral flat cells of PK-1 spheres. PANC-1 and PK-1 cells were positive for transforming growth factor (TGF) β receptor II and phosphorylated smad2/3, but PK-1 cells were smad4 negative. Taken together, 3D culture enhanced morphofunctional differences of PDAC cells showing epithelial or mesenchymal characteristics, and epithelial phenotype maintenance may be due to the ineffectiveness of the TGF- β pathway. Clarification of heterogeneity using 3D culture may be useful for development of individualized diagnostic and therapeutic methods in patients with PDAC.

Highlights

Due to the development of early detection methods and new treatments including surgery, chemotherapy, radiotherapy, and immunotherapy for cancers, the average survival rates of major cancers are over 50%1
To clarify the EMT features of pancreatic ductal adenocarcinoma (PDAC) cells, we examined mRNA expression levels of typical EMT markers, E-cadherin and vimentin, in four PDAC cell lines
MIA PaCa-2 showed high vimentin mRNA levels, but PK-1 cells showed low vimentin mRNA levels (Fig. 1B). These results suggest that MIA PaCa-2 and PANC-1 cells have mesenchymal phenotypes, while T3M-4 and PK-1 cells have epithelial phenotypes

Summary

Introduction

Due to the development of early detection methods and new treatments including surgery, chemotherapy, radiotherapy, and immunotherapy for cancers, the average survival rates of major cancers are over 50%1. Many of 3D culture systems have been reported These include spheroid culture using an ultra-low attachment plate, 3D matrix scaffolds and microsquare patterns on the bottom of the plates, ex vivo culture, multilayered postconfluent cell culture, and growing of the cells in extracellular matrix gel[15,16,17]. We report that 3D culture enhances the morphological and functional differences of PDAC cells showing epithelial or mesenchymal features, and that the epithelial phenotype may be partly maintained due to the ineffectiveness of TGF-β1 induction of the EMT pathway

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Conclusion