Hollow-spheres: a new model for analyses of differentiation of pancreatic duct epithelial cells.
We discovered a unique feature of a subclone of the pancreatic carcinoma cell line A818. A818-1-derived hollow-spheres developed under three-dimensional growth conditions. Hollow-spheres consist of a single layer of 50-200 epithelial cells surrounding an inner lumen. In contrast to A818-1, the subclone A818-4 and all other pancreatic tumor cell lines tested (n = 5), formed spheroids as the only three-dimensional phenotype. A dramatically reduced proliferation rate compared to the corresponding monolayer was observed in hollow-spheres when bromodeoxyuridine (BrdU) incorporation was measured. This finding was confirmed by immunostaining using the MIB-1 antibody. Mechanically disrupted hollow-spheres not only attached but also grew as monolayer with the same doubling time as the founder cells. Hollow-spheres developed in fetal calf serum (FCS) containing RPMI 1640 medium without additionally added cytokines. A818-1 hollow-sphere formation and integrity was influenced by interferon-gamma. Tumor necrosis factor-alpha (TNF-alpha) led to cell death. Exogenously added hepatocyte growth factor (HGF) showed no effect neither on hollow-sphere formation nor on the integrity of completely developed hollow-spheres. Moreover, no changes were observed when cells were treated with a neutralizing antibody for HGF. Interestingly, hollow-spheres showed intensive immunoreactivity for the HGF-receptor (c-met) and its ligand (HGF). Immunostaining for the biliary glycoprotein (BGP), the non-specific cross-reacting antigen 95 (NCA95) and beta-catenin revealed a polar organization of hollow-spheres. Immunhistochemically, hollow-spheres were negative for the carcinoembryonic antigen (CEA). When hollow-spheres were embedded into matrigel, duct-like tubes grew out. Taken together, A818-1 hollow-spheres resemble normally differentiated duct-like structures and will serve as an excellent model to study differentiation of human pancreatic epithelial cells.
Department of Molecular Oncology, Christian Albrechts University of Kiel, Germany.
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Lehnert
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Trost
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Schmiegel
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Röder
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Kalthoff
D000368:Aged; D002454:Cell Differentiation; D002455:Cell Division; D016764:Cell Polarity; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008954:Models, Biological; D010183:Pancreatic Ducts / Q000166:cytology*; D010190:Pancreatic Neoplasms / Q000473:pathology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma