Establishment and characterization of two Merkel cell tumor cultures.

Two Merkel cell tumor cultures (MC-MA1, MC-MA2) have been established from metastases of typical Merkel cell tumors. The mestastases in vivo were characterized by co-expression of cytokeratins 8, 18, 19, 20 and neurofilaments, presence of intermediate filament whirls, expression of synaptophysin, neuron-specific enolase, and chromogranin A, rare and weak immunostaining for plakoglobin but absence of cadherins and desmoplakins. Both cultures grow, using supplemented RPMI medium on human irradiated fibroblast feeder layers, as loosely arranged floating small aggregates. Their karyotypes are mostly hyperdiploid. The mean doubling times were about 84 h in the first 8 months and later increased. Ultrastructural and immunoelectron microscopic studies of the Merkel cell tumor cells in vitro (MC-MA1, MC-MA2) revealed sparse membrane-bound neuroendocrine granules and typical IFs that were partly arranged in paranuclear whirls and were labeled by antibodies against cytokeratins and neurofilaments. In immunocytochemical studies using antibodies to cytokeratins 8, 18, 19, and 20 and neurofilament protein NF-L, Merkel cell tumor cells in vitro showed a uniform staining appearing as paranuclear whirls and cytoplasmic fibrils as well. Double-labeling experiments showed a co-localization of both intermediate filament types in most cells. Biochemically we found cytokeratins 8, 18, 19, and 20, and NF-L in tumor cells in vitro. Immunocytochemical staining was negative for desmoplakins, various cadherins, and cell adhesion molecules, whereas plakoglobin was only rarely detectable in some Merkel cell tumor cells in vitro. By immunoluminometric assay chromogranin A was detected in cell homogenates and culture supernatants as well. Immunocytochemically, synaptophysin and neuron-specific enolase were detectable additionally in some of the cells. These established cell cultures will allow further studies devoted to the biology, differentiation, and hormone secretion of Merkel cell tumors that may also increase our knowledge about normal Merkel cells.