A pancreatic adenocarcinoma cell line differentiates into polarized epithelial hollow-spheres: A in vitro model to study pre-lesions of the pancreatic duct

Background: We discovered a subclone of the pancreatic carcinoma cell line A818 called A818 -1,which developed hollow-spheres under three-dimensional growth conditions. Methods and results: Hollow-spheres consist of a single layer of 50-200 epi thelial cells surrounding an inner lumen. In contrast, to A818-1, a subclon e called A818-4 formed spheroids as the only three-dimensional phenotype. A dramatically reduced proliferation rate compared to the corresponding mono layer was observed in hollow-spheres when BrdU-incorporation was measured. Hollow-spheres developed in FCS-containing RPMI-1640-Medium without additio nally added cytokines. A818-1 hollow-sphere formation and intigrity was inf luenced by Interferon-gamma TNF-alpha lead to cell death. Exogenously added HGF neither showed an effect on hollow-sphere formation nor on the integri ty of completely developed hollow-spheres. Moreover, no changes were observ ed when cells were treated with a neutralizing antibody for HGF. Interestin gly, hollow-spheres showed intensive immunoreactivity for (both,) the HGF-r eceptor (c-met) (and its ligand HGF). Immunostaining for BGP, NCA95 and bet a-catenin revealed a polar organisation of hollow-spheres. Immunhistochemic ally, hollow-spheres were negative for CEA-180 Interestingly, CEA-180 mRNA was detected by RT-PCR and released protein was found in the supernatant. T his resembles the normal situation in gastrointestinal epithelial tissues. Mechanically disrupted hollow-spheres not only attached but also grew as mo nolayer with the same doubling time as the founder cells. When embedded int o matrigel, duct-like tubes grew out. Conclusion: Taken together, A818-1 hollow-spheres resemble normally differe ntiated duct-like structures and will serve as an excellent model to study differentiation of pancreatic epithelial cells.