Establishment and Characterization of Two Merkel Cell Tumor Cultures I. Moll, E. Bohnert, C. Herbst, W. Fbrster,* R. Moll;, and W. W. Franke:!: Department of Dermatology, Mannheim Medical School, University of Heidelberg, Mannheim; "Institute of Human Genetics, University of Giessen, Giessen; tInstitute of Pathology, University of Mainz, Main:z;; and tDivision of Cell Biology, German Cancer Research Center, Heidelberg, Germany Two Merkel cell tumor cultures (MC-MA 1, MC-MA2) have neurofilament protein NF-L, Merkel cell tumor cells in vitro been established from metastases of typical Merkel cell showed a uniform staining appearing as paranuclear whirls tumors. The mestastases in vivo were characterized by co-ex­ and cytoplasmic fibrils as well. Double-labeling experiments pression of cytokeratins 8, 18, 19, 20 and neurofilaments, showed a co-localization of both intermediate filament types presence of intermediate filament whirls, expression of syn­ in most cells. Biochemically we found cytokeratins 8,18,19, aptophysin, neuron-specific enolase, and chromogranin A, and 20, and NF-L in tumor cells in vitro. Immunocytochemi­ rare and weak immunostaining for plakoglobin but absence cal staining was negative for desmoplakins, various cadher­ of cadherins and desmoplakins. Both cultures grow, using ins, and cell adhesion molecules, whereas plakoglobin was supplemented RPM I medium on human irradiated fibroblast only rarely detectable in some Merkel cell tumor cells i,.,. vitro. feeder layers, as loosely arranged floating small aggregates. By immunolurninometric assay chromogranin A was de­ Their karyotypes are mostly hyperdiploid. The mean dou­ tected in cell homogenates and culture supernatants as well. bling times were about 84 h in the first 8 months and later Immunocytochemically, synaptophysin and neuron-specific increased. Ultrastructural and immunoelectron microscopic enolase were detectable additionally in some of the cells. studies of the Merkel cell tumor cells in vitro (MC-MA1, These established cell cultures will allow further studies de­ MC-MA2) revealed sparse membrane-bound neuroendo­ voted to the biology, differentiation, and hormone secretion crine granules and typical IFs that were partly arranged in of Merkel cell tumors that may also increase our knowledge paranuclear whirls and were labeled by antibodies against about normal Merkel cells.] Invest DermatoI102:346-353, cytokeratins and neurofilaments. In immunocytochemical 1994 studies using antibodies to cytokeratins 8, 18, 19, and 20 and hen first described by Toker [1], neuroendo­ CK 20 turned out to represent a selective marker of Merkel cells in crine carcinoma of skin (Merkel cell carci­ skin [4] . Because CK 20 is also detectable in Merkel cell tumors, it noma) was referred to as trabecular carci­ would seem very likely that Merkel cell tumors are derived from noma. Ultrastructural examination of this epidermal Merkel cells [4,6] although the former, in addition, ex­ tumor revealed the presence of neurosecre­ press neurofilaments (NF) [7] . Wtory granules within the tumor cells. As in the skin, these features The hormone content of these granules is, at present, incom­ are highly characteristic of Merkel cells, so the term Merkel cell pletely understood. Monoamines have not been detected [2], but tumor was considered to be appropriate. Merkel cells are neuroen­ vasoactive intestinal polypeptide (VIP), bombesin, and calcitonin­ docrine cells of the epidermal basal layer that are characterized by gene-related protein (CGRP) have so far been demonstrable in such the presence of neuroendocrine granules (80 -100 nm in diameter granules in humans ([8-11]; for other species, see [12]). Thus, the [2]) . function of Merkel cells is not entirely clear [2] . They may function In addition, Merkel cells ultrastructurally exhibit loosely as slowly adapting mechanoreceptors [12,13] or may be involved in arranged bundles of intermediate filaments (IF) , these comprising the processes of cell growth and differentiation, as suggested by exclusively cytokeratins (CKs) 8, 18, 19, and 20 [2-5]. Especially recent results concerning Merkel cell distribution in fetal tissues [3] and the secretion of nerve growth factor (NGF) by rat Merkel cells [1 4] or of substance P by pig Merkel cells [10]. As both normal human Merkel cells and Merkel cell tumor tissues are difficult to obtain, especially in the amounts of material required for thorough studies, we established and characterized two cell Manuscript received June 2, 1993; accepted for publication October 27, cultures, MC-MAl and MC-MA2, derived from metastasizing 1993. Merkel cell tumors growing on human irradiated fibroblast feeder Reprint requests to: PD Dr. Ingrid Moll, Hautklinik, Klinikum der Stadt layers. Mannheim, Theodor-Kutzer-Ufer, D-68167 Mannheim, Germany. Abbreviations: CgA, chromogranin A; CGRP, calcitonin gene-related MATERIALS AND METHODS protein; CK, cytokeratin; NE, neuroendocrine; NEPHG, non-equilibrium pH gradient; NF, neurofilament; NSE, Neuron-specific enolase; VIP, vaso­ Materials The first Merkel cell tumor (case 1; MC-MA1) cultivated was active intestinal polypeptide. derived from an inguinal lymph-node mass (61-year-old female patient). 0022-202X/94/S07.00 Copyright © 1994 by The Society for Investigative Dermatology, Inc. 346 VOL. 102. NO. 3 MARCH 1994 TWO MERKEL CELL TUMOR CULTURES 347 Table I. List of Antibodies Used Antibody Specificity Source MoAb 6B10 CK 4' Euro-Diagnostics, Apeldoorn, The Netherlands MoAb A.E14 CK5 Kindly provided by Dr. T.-T. Sun, New York, USA MoAb CK-7 CK 7 Boehringer, Mannheim, Ger- many MoAbCAM CK8 Becton Dickinson, Heidelberg, 5.2 Germany MoAbM20 CK8 Euro-Diagnostics, 1\peldoorn, The Netherlands MoAb 1C7 CK 13 Euro-Diagnostics, Apeldoorn, The Netherlands MoAb Ks8.12 CKs 13, 15, 16 Bio-Makor, Rehovot, Israel MoAb E3 CK 17 Kindly provided by Dr. S.M. Troyanovsky, Moscow, Russia MoAb CK 18 Progen Biotcchnics, Heidelberg, Ks18.174 Germany MoAb CK 19, Hax Progen Biotechnics, Heidelberg, Ks19.2- Germany Z10S MoAb IT- CK20 Progen Biotechnics, Heidelberg, Ks20.3,6,8 Germany MoAb NR4 NF-L Boehringer, Mannheim, Ger- many MoAb 2F11 NF-L, NF-H Biochrom, Berlin, Germany MoAb VIM- Vimentin Progen Biotechnics, Heidelberg, 3B4 Germany MoAb a-sm-1 Smooth muscle-type Progen Biotechnics, Heidelberg, actin Germany MoAb Desmoplakins I and II Boehringer, Mannheim, Ger- Figure 1. Routine hematoxylin and eosin staining (a) and ABC-peroxidase DP1&2- many staining (paraffin sections) of Merkel cell tumor i" vivo (case 1) using anti­ 215 bodies against CK 8 (b; CAM 5.2) against CK 20 (c; IT-Ks20.8) and antibody MoAb Plakoglobin [19] Kindly provided by Dr. P. 2Fll (d) aga inst neurofilament proteins NF-L and NF-H. Note the decora­ PGS.l72 Cowin, New York, USA tion of both paranuclear plaques (fibrous bodies) and cytoplasmic fi laments MoAb l1/S Vinculin [20] Kindly provided by Dr. B. with CK antibodies, but exclusive (and focal) plaque staining with the neur­ Geiger, Rchovot, Israel ofilament antibody. Bar, SO 11m . MoAb DC Desmocollin [21] Kindly provided by Dr. P. Koch, 227 Heidelberg, Germany MoAb DG Desmoglein [22] Kindly provided by Dr. M. 133 Schmelz, Heidelberg, Ger- Establishment of the Cell Cultures and In Vitro Morphology Imme­ many diately after their excision, samples of fresh tumor tissue were transferred MoAb SH9 E-Cadherin [23] Kindly provided by Prof. W . into RPMI culture medium supplemented with lS% heat-inactivated fetal Birchmeier, Essen, Germany calf serum, penicillin, and streptomycin. The tumor tissue was finely MoAb SY38 Synaptophysin Progen Biotechnics, Heidelberg, minced, and this mechanically dissociated tumor material, consisting of Germany small cell clumps, was seeded into culture flasks containing a feeder layer of Rabbit anti- NSE Dako, Hamburg, Germany lethally irradiated adult human dermal fibroblasts (primary cultures between bodies passages 3 and 18, radiation doses 2S00 cGy) and maintained at 37.0'C in an N-CAM Immunotech S.A., Marseille, MoAb atmosphere containing 5% CO2 , The culture flasks were left undisturbed H28.123 France for approximately 2 weeks, during which time only some RPMI medium MoAb CgA Boehringer, Mannheim, Ger- was added, the amount and frequency depending on the growth of the LK2H10 many cultures. When sufficient multiplication of the tumor colonies had been MoAb 8211 NGF-receptor Boehringer, Mannheim, Ger- obtained, the cells were centrifuged and resuspended in culture medium in many flasks containing human feeder layers. This was repeated every 1- 3 weeks MoAbEGFRI EGF-receptor Amersham-Buchler, Braunschw- depending on the cell growth rate. Floating cell clumps were now cautiously eig, Germany disaggregated by tiny pipettes. At varying intervals, aliquots of the cells were MoAb CD29 lntegrin VLA PI Dianova GmbH, Hamburg, G(;r- cryopreserved in RPMI supplemented with glycerol (10%) or with dimeth­ many ylsulfoxide (10%) and then stored in liquid nitrogen. After the establishment MoAb Integrin a lP2 Dianova GmbH, Hamburg, G(;r- of the cell lines (i.e., after 5-7 months), insulin (5 I1g/ml) and transferrin (5 Cdw49b many Ilg/ml) were added in case 1 (MC-MA1), whereas in case 2 (MC-MA2) both MoAb CDS7 Carbohydrate antigen Dianova GmbH, Hamburg, Geor- were added from the beginning of cultivation. The cells used in the present of neuroectoder- many study had been in culture for more than 12 months. To estimate the doubling mal and other cells time, tumor cells were seeded into four flasks containing freshly prepared feeder layers. Samples from