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

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.

SUBMITTER: Shichi Y

PROVIDER: S-EPMC6659675 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Shichi Yuuki Y Sasaki Norihiko N Michishita Masaki M Hasegawa Fumio F Matsuda Yoko Y Arai Tomio T Gomi Fujiya F Aida Junko J Takubo Kaiyo K Toyoda Masashi M Yoshimura Hisashi H Takahashi Kimimasa K Ishiwata Toshiyuki T

Scientific reports 20190726 1

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 spher ...[more]

PMID: 31350453

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Project description:The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-β), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for in vitro research using 3D high-density culture systems.

Project description:BackgroundGeneration of insulin-producing cells from human pluripotent stem cells (hPSCs) in vitro would be useful for drug discovery and cell therapy in diabetes. Three-dimensional (3D) culture is important for the acquisition of mature insulin-producing cells from hPSCs, but the mechanism by which it promotes β cell maturation is poorly understood.MethodsWe established a stepwise method to induce high-efficiency differentiation of human embryonic stem cells (hESCs) into mature monohormonal pancreatic endocrine cells (PECs), with the last maturation stage in 3D culture. To comprehensively compare two-dimensional (2D) and 3D cultures, we examined gene expression, pancreas-specific markers, and functional characteristics in 2D culture-induced PECs and 3D culture-induced PECs. The mechanisms were considered from the perspectives of cell-cell and cell-extracellular matrix interactions which are fundamentally different between 2D and 3D cultures.ResultsThe expression of the pancreatic endocrine-specific transcription factors PDX1, NKX6.1, NGN3, ISL1, and PAX6 and the hormones INS, GCG, and SST was significantly increased in 3D culture-induced PECs. 3D culture yielded monohormonal endocrine cells, while 2D culture-induced PECs co-expressed INS and GCG or INS and SST or even expressed all three hormones. We found that focal adhesion kinase (FAK) phosphorylation was significantly downregulated in 3D culture-induced PECs, and treatment with the selective FAK inhibitor PF-228 improved the expression of β cell-specific transcription factors in 2D culture-induced PECs. We further demonstrated that 3D culture may promote endocrine commitment by limiting FAK-dependent activation of the SMAD2/3 pathway. Moreover, the expression of the gap junction protein Connexin 36 was much higher in 3D culture-induced PECs than in 2D culture-induced PECs, and inhibition of the FAK pathway in 2D culture increased Connexin 36 expression.ConclusionWe developed a strategy to induce differentiation of monohormonal mature PECs from hPSCs and found limited FAK-dependent activation of the SMAD2/3 pathway and unregulated expression of Connexin 36 in 3D culture-induced PECs. This study has important implications for the generation of mature, functional β cells for drug discovery and cell transplantation therapy for diabetes and sheds new light on the signaling events that regulate endocrine specification.

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

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

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