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 each flask were counted separately over 4 d • Additional references for the antibodies used arc given in (18]. using a Neubauer chamber. Tumor cells of early passages were injected into nude mice (four experi­ Histologic examination demonstrated the presence of a large metastaric ments). Merkel cell tumor that had largely destroyed the lymph nodes and \Vas For light microscopy, cytocentrifuged cells (Cytospin 3, Shandon, USA) infiltrating the surrounding fatty tissue and adjacent skin. The primary were stained using the Pappenheim method. In addition, samples of the tumor was located at distal upper leg. cultured cells were centrifuged in Eppendorf tubes, and the pellets were The second case (MC-MA2) was established from an abdominal cutane­ processed for electron microscopy by exposure to 2.S% sodium-cacodylate­ ous metastasis (73-year-old male patient; primary tumor at the back) show­ buffered glutaraldehyde solution and postfixed in sodium-cacodylate-buf­ ing similar histologic features . Moreover, frozen tissue samples from three fered osmium tetroxide solution (1 %) prior to Epon embedding and section­ more cases (two female, 80 and 73 years old and one male, 70 years old, cases ing as described elsewhere [15]. Electron photomicrographs were taken with 3 - S) were available. a Philips 301 electron microscope. In addition, immunoelectronmlcroscopy 348 MOLL ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2. Li ght and electron micrographs of Merkel cell tumor in culture (MC-MA 1). The li ght microscopy (a; Pappenheim stain) shows ce ll clusters containing cell s with large nuclei and littl e cytoplasm. Electron microscopy (b , e) reveals the typical NE granules at the cell borders, indented nuclei, prominent endoplasmic reticulum, and the typical fibrous body (e) consisting of IF-whirls. Bars, 10 11m (a); 0.5 11m (b); 0.5 11m (e) . VOL. 102, NO.3 MARCH 1994 TWO MERKEL CELL TUMOR CULTURES 349 was done using anti-CK 20 and anti-NF antibodies and gold-labeled second­ crine markers, were poslt!ve in a heterogenous pattern (not ary antibodies (for methods, see [7,15]). shown). NGF-receptor and EGF-receptor were not detectable as well as the CDS7 antigen. Various junction proteins and cell adhe­ Immunohistochemistry sion molecules were checked by a battery of antibodies. Desmopla­ Merkel Cell Til tnO rs Itl Vivo: Frozen samples of the inguinal-node metastasis kin I and II (antibody DP 2.15) as well as various other constitutive and the adjacent infiltrated dermis (MC-MA1), of the cutaneous metastasis desmosomal proteins (see Table I) could be localized weakly and (MC-MA2), and primary tumors (cases 3-5) were available. In all cases very heterogenously at the cell surface in cases 3 and 5, but not in S-,um - thick cryostat sections were cut, fixed in acetone, and processed for indirect immunoperoxidase staining as previously described [16]. In case 1, cases 1, 2, and 4. E-cadherin was not detected. The antibody PG IF proteins, CgA, and NSE were, in addition, tested on paraffin sections 5.172 against plakoglobin, a protein typical for intermediate junc­ using the avidin-biotin-complex peroxidase method (see [6]). tions and desmosomes as well, was heterogeneously and very weakly positive at the cell surface of tumor cells in cases 2, 3, and 5 Merkel Cell Tllmors In Vitro: Floating aggregates of the Merkel cell tumors and homogenously positive in epidermal keratinocytes of all cases in I/itro were spun down onto polylysine-coated microscope slides using a (not shown). cytocentrifuge (Cytospin 3, Shandon, USA) and then immediately fixed first in methanol (-20·C, 5 min) and subsequently in acetone (-20·C, 10 seconds). For NSE and CgA-staining, buffered 4% paraformaldehyde was Growth Properties and Morphology In Vitro We established used as fixative too. Immunofluorescence microscopy was performed as cell cultures derived from metastatic, subcutaneously infiltrating described earlier [17]. tumor masses (case 1; MC-MA1) and from cutaneous metastasis The primary antibodies applied are listed in Table I (see also [18-23)). As (case 2; MC-MA2). On human fibroblast feeder layers these cells secondary antibodies, we used Texas red- or fluorescein isothiocyanate­ grew in suspension in the form of isolated cells or of floating cell labeled goat antibodies to mouse or rabbit immunoglobulins (Oianova, clumps, lacking central necrosis. The size of the clumps was vari­ Hamburg, Germany) for immunofluorescence microscopy [17] and peroxi­ able, about 0.1-0.2 mm, containing roughly 10 - 80 cells (Fig 2a). dase-labeled goat antibodies to mouse or rabbit immunoglobulins (Oianova, These cell cultures were rather homogenous, containing mostly Hamburg) for immunoperoxidase microscopy [16]. neuroendocrine (NE) Merkel cell tumor cells resembling those of Biochemical Analysis of Cytoskeletal Components Two-dimen­ the in lIillo Merkel cell tumors. The individual NE cells were round sional gel electrophoresis of cytoskeletal preparations obtained from the or polygonal, exhibiting very large, moderately pleomorphic nuclei cultured Merkel cell tumor cells was performed using non-equilibrium pH­ with one or several rather prominent nucleoli and relatively little gradient (NEPHG) electrophoresis in the first dimension and sodium dode­ cytoplasm. The mean doubling time was about 84 h for about 8 cylsulfate (SOS) polyacrylami?e gel electroRhoresis .(P.AGE) in the second months, and later it increased. The MC-MAl and MC-MA2 cells dimension. The gels were stamed usmg a silver stammg method [24]. In have been in continuous culture for 12 months, i.e., about 28 pas­ other experiments, electrophoretically (SOS-PAGE) separated proteins sages. were transferred to nitrocellulose membranes, and specific polypeptides were identified by immunoblotting [6,24] using antibodies to various IF proteins.

Immunoluminometric Assay These assays using defined s~ernatants and cell homogenates were performed according to Bender et aI125]. Brief, acridinium ester-labeled antibodies to CgA were used and the bound lumi­ nescence was measured. Cytogenetic Analysis The standard Giemsa banding technique was used. The cultured cell suspension was exposed to colcemid (0.05 ,ug/ml for 2 h) and fixed using acetic acid and methanol at a ratio of 1 : 3. The fixative was changed, and air-dried sections were prepared. Chromosome abnorn1al­ ities were recorded according to the International System of Human Cyto­ genetic Nomenclature (1978).§

RESULTS Immunohistochemistry of the Merkel Cell Tumors in vivo Routine hematoxylin and eosin staining revealed in all cases exten­ sive solid epithelial tumor growing in sheets and trabecules. The uniform tumor cells contained relatively large, round-to-oval, ve­ sicular nuclei with relatively loosely textured chromatin, as well as small to medium-sized nucleoli and little cytoplasm (Fig la). Mi­ toses were very frequent. To obtain a detailed characterization as well as to confirm the diagnosis, immunohistochemistry was per­ formed on frozen and/or paraffin-embedded material by applying antibodies against IF proteins as well as antibodies against NSE, egA, and synaptophysin. Antibodies specific for CK 8, CK 18, CK 19, and CK 20 stained the cytoplasm of all tumor cells, this staining often being concentrated in a small, dense paranuclear plaque (Fig lb,c). Neurofilament antibodies stained many, but not all, tumor cells, producing a positive reaction only in such plaques (Fig ld), indicating the co-expression of both IF types at these sites. This pattern is highly typical and characteristic of Merkel cell tumors l6,26,27]. Vimentin could not be detected in the tumor cells at all, whereas the staining patterns for actin and the micro-filament­ associated vinculin were weak and heterogenous in cases 2 and 3 (not shown). CgA and synaptophysin, major neuroendo- Figure 3. Immunoelectron microscopy of MC-MAl cells using gold-la­ beled CK 20- (a; IT-Ks20.8) and NF-antibodies (b; NR4). a shows the fibrous body and b a thin ncurofilamcnt bundle within the cytoplasm. Bar, § For references sec Cytogetlet Cell Cellet 21:309, 1978. 0.25,um. 350 MOLL ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

By electron microscopy, the cells of MC-MAl and MC-MA2 The diameter of these aggregates ranged from 1 to 5 fJ.m, and some cultures were rather uniform in appearance in early and in late contained typical dense-core granules. Slender microvilli were de­ passages. The large nuclei contained relatively loosely textured tectable on the cell surface. chromatin and prominent nucleoli. In the cytoplasm, a variable, By electron microscopical immunogold labeling the presence of usually moderate number of typical "dense-core" NE granules were abundant CK filaments and fewer neurofilaments constituting the present, ranging in diameter from 0.070 to 0.12 fJ.m (Fig 2b). Most paranuclear whirls or running throughout the cytoplasm (Fig 3a,b) of these granules were located at the cell periphery (Fig 2c), and only could be detected. a few could be detected in central regions of the cytoplasm, often in The tumor cells produced no tumors in nude mice. association with the prominent, active Golgi apparatus. In addition, numerous small, pale or empty vesicles were detectable, these being Immunocytochemical and Biochemical Analysis of Merkel mainly concentrated in the Golgi regions (Fig 2). Mitochondria Cell Tumors In Vitro Immunocytochemistry was performed were frequently encountered. The cells ofMC-MAl and MC-MA2 in early and late passages of both cell cultures. Antibodies against cultures also often exhibited a dense, whirled paranuclear aggregate CK. 8 (not shown) and CK. 18 (Fig 4a) produced a prominent cyto­ of IFs (Fig 2c), a typical feature of Merkel cell tumors ([44]; Fig 2c). plasmic immunofluorescent staining of essentially all MC-MA 1

Figure 4. Immunofluorescence staining of MC-MA2 culture cells with CK (a-d) and NF antibodies (e-f). MoAbs Ks18.174 (a) and Ks19.2.Z.10S (b) decorate in most cells the fibrou s bodies and cytoplasmic fibrils. MoAb IT-K s20.3 (c; d, phase contrast) decorates all cel ls brightly in a si milar pattern. MoAb NR4 (against NF-L; e;J, phase contrast) stains predominantly the fibrous bodies an.d only rare cytoplasmic fibrils. Bar, 10 11m. VOL. 102, NO.3 MARCH 1994 TWO MERKEL CELL TUMOR CULTURES 351

and MC-MA2 culture cells; this staining took the form of either NEPHG~ a variably extensive, irregular fibrillary network or a dense S plaque. D The CK-19 antibody also stained most cells albeit to a markedly • S more restricted degree, in that only a few fibrils or a dense plaque were stained in each cell (Fig 4b). CK 20, a cytokeratin characteris­ tic of Merkel cells and Merkel cell tumor ill vivo [7 ,27,28]' was widely distributed within MC-MAl and MC-MA2 culture cells in the form of branching and often bizarrely curved fibrils and as a plaque in all cells, including mitotic ones (Fig 4c, d). CK 4 (antibody 6 B 10), CK 5 (antibody AE 14), CK 7 (anti­ 18- body CK-7), CK 13 (antibody 1 C 7), CKs 13/ 15/16 (antibody K, r: -19 8.12), and CK 17 (antibody E 3) could not be demonstrated in the cells of MC-MAI and MC-MA2 cultures of early and late pas­ sages, and the staining for vimentin was also negative (not shown). Figure 5. Two-dimensional gel electrophoresis of MC-MA 1 culture cel ls However, the neurofilament protein NF-L was detectable in (cytoskeleton) using NEPHG electrophoresis in the first dime nsion most cells, being predominently concentrated in a dense paranu­ (NEPHG; from rig h~ to left) and 50S-PAGE in the second dimension (50S; clear plaque, with only a sparse fibrillar distribution (Fig 4e{). from top to bottom) using sil ver staini ng. The numbers denote CKs 8, 18, Antibodies to desmoplakms, desmoglems, and desmocoll ll1s 19, and 20 present according to the Moll catalog [5J; V, vimentin; A, actin. were negative, whereas plakoglobin revealed a weak if any reaction, indicating the presence of few proteins of ad herin g junctions of both the desmosome and the fasc ia adherens type. The same was (lp+), two copies of the rearranged chromosome 5 (+i5p), and a true for antibodies to vinculin and sm-actin (see T ables I and II; not translocatiQn from chromosome 1 to chromosome 12 (p 13; q13). shown). Four additional abnormalities were detectable in parts of the meta­ Two-dimensional gel electrophoresis of the cytoskeletal proteins phase cells investigated: deletion of the short arm (p) at chromo­ from MC-MAl cells revealed the presence of CKs 8,18,19, and 20 so me 1, deletion at chromosome 2 (q33), and additional long arms (Fig 5). At imm~noblotti.ng, t~e identity of CK ~O could be con­ at chromoso mes 6 and 12. However, the second culture MC-MA2 firmed by its specific reaction with monoclonal antibody IT-K,20.6; showed some different chromosomal abnormalities in 2S0 meta­ in addition, the presence of the neurofilament protein, NF-L phases studied. Trisomy 6 and 20 as well as a deletion 11 (q21) and a (68 kD), could b~ demonstrated by immunoblotting (not shown). translocation from chromosome 5 to 17 (pIS; p13) were present in Various integnns and CD 57-reactivity were not detectable as 111 all cells. In addition, mosaics with YY and a translocation (1 ; 7) M erkel cell tumors irl vivo (see T ables I and II). But in MC-MA 1 and (q 32; pIS) were detected. MC-MA2 culture cell s, the immunoreactivity for synaptophysin appeared as a finely granular pattern in the cytoplasm of so me, but Investigation of the Supernatants T o gain insight into th.e se­ not all, cells when antibody SY -38 was applied (Fig 6a). The CgA cretion activity of the cell lines, defined supernatants and homoge­ and NSE antibodies also produced weak granular cytoplasmic stain­ nized cells were studied for CgA by immunoluminometric assays. ing in some cells (Fig 6b) . Again antibodies to NGF-R and EGF-R The cells of MC-MAI cultures secreted at day 619 Jlg/l (superna­ showed no significant staining (not shown). tant) CgA, w hereas homogenized tumor cell s contained 10 Jlg/l (homogenate). Cytogenetic Analysis Both MC-MA cultures exhibited various chromosomal abnormalities. The examination of more than 300 metaphases of cells of MC-MAI cultures revealed 45 - 50 chromo­ DISCUSSION somes with different abnormalities (Fig 7). Each of the metaphase Two ce ll cultures (MC-MAl; MC-MA2) have been established cells analyzed showed an additional short ann of chromosome 1 from metastases of Merkel cell tumors, w hich have now been grow­ ing for about 12 months in vitro in the form of floating aggregates on human irrad iated fibroblasts serving as a feeder layer. Table II. Intermediate Filaments, Junction Proteins, and In the cells of MC-MAI and MC-MA2 cultures, the electron­ Neuronal Markers of Merkel Cell Tumors III Vivo and In Vitro microscopic features of typical neurosecretory granules as well as of In Vivo MA-M Cl, MA-MC2 paranuclear plaq ues (fibrous bodies) composed of IF whirls closely correspond to those seen in Merkel cell tumor cells ill villa . More­ CK 8,18,19 ++ ++ over, the unique molecular composition of the IFs of Merkel cell CK20 ++ ++ tumors is perfectly preserved in both MC-MAI and MC-MA2 cul­ CK4, 5, 7,13,15,16,17 tures, i.e., the simultaneous presence of NFs and simpl e-epithelial NF-L + + CKs that characteristically include CK 20 [4,28,29]. The presence Vimentin Actin a-sm-1 Desmoplakin I-II (+)/- Plakoglobin (+)/- (+) Vinculin Desmocollin Desmoglein E-cadherin Synaptophysin + + NSE + (+) CgA + + Neuronal cel l adhesion molecule C057-antigcn Integrin VLA PI Integrin a1p2 Figure 6. Immunoflu orescence stai ning using antibody SY-38 (against NGF-rcceptor synaptophysin; a) and LK2H10 (against CgA; b) . Both antibodies decorate EGF-receptor some cell s in a gran ul ar pattern (arrows). Bar, 10 tim. 352 MOLL ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

A 1 2 3 B 5 -.

c 6 7 8 9 10 11 12

I' I D 13 14 15 E 16 17 18

F 19 20 G 21 22 xv

Figure 7. Karyotype of a banded metaphase cell (trypsin-Giemsa) from MC-MA2 culture cells. Trisomy 6 and 20, the deletion of chromosome 11 and a translocation from chromosome 5 to 17 are present in all cells.

of CK 20 distinguishes cells of MA-MC1 and MA-MC2 cultur~s Concerning specific NE markers, the presence of both synapto­ from PC 12 cells derived from a neuroblastoma, whereas the latt~r physin (an integral membrane protein of presynaptic vesicles [33] also co-express CKs and neurofilaments [30]. Also the contributi(.)p and CgA (a specific marker of neuroendocrine cells in general; see of both CKs (including CK 20) and neurofilaments to the form3-­ review [33]) could be demonstrated in MC-MAl and MC-MA2 tion of the fibrous bodies, as detected by both immunofluorescence cells, but the amounts of such substances do not appear to be partic­ and immunoe1ectron microsco).'y, is identical to the situation of ularly abundant, as suggested by the rather discrete immunostaining Merkel cell tumor cells in vivo l7,28,29]. Normal human fetal al).d and secretion, respectively. In normal human Merkel celis, such adult Merkel cells exhibit the same CK pattern but lack neurofil;,J.­ immunostaining is similarly discrete [10,34]. By electron micros­ ments [3 ,4,28]. Thus, during tumorigenesis, Merkel cell tumo~s copy in our two cell cultures (and in that of Rosen e/ al [35]), rela­ acquire additional neuronal/NE features: neurofilaments and fi­ tively low numbers of granules were present in most cultured cells, brous bodies, although these characteristics are somewhat variab1e but on the other hand the prominent Golgi apparatus indicates high among individual tumors [7,27]. There was even an increase of levels of sy nthesis activity in these cells [35,36]. Thus, it is possible neurofilament expression in the cell cultures as compared to the that secretory products, such as peptide hormones, might be se­ tumors i" vivo . Like in human Merkel cell tumors in vivo [3] strati­ creted rapidly into the culture medium rather than accumulating in fied-epithelial CKs were not detectable. The presence of all these the cells. Further analyses of conditioned medium for neuropeptides specific features of Merkel cell tumors in the NE cells of MC-M1\.l are currently in progress. and MC-MA2 cultures can be taken as definite proof of their derivl'l­ Karyotyping of the cells of MC-MAl and MC-MA2 cultures tion from such tumors. Their continued presence also demonstrates revealed a chromosome number ranging between hypo- and hyper­ a surprisingly high degree of differentiation maintenance in cultute, diploid. Chromosomes 1, 5, and 6 were involved in both cases at without any apparent manifestation of the dedifferentiation that is least in mosaic. The Merkel cell tumor cultures described by frequently observed when cell lines are established from sohd Rosen et at [35] also revealed the involvement of chromosomes 1 tumors [31]. and 5. It is worthy of note that isochromosome 5 p was uniformly In contrast to most other types of epithelial tumors [7,27,31], present in two lines; however, this marker has not been detected Merkel cell tumors showed only a poor and inconsistent reaction for in cells of our MC-MA2 cultures and in any primary Merkel cell desmoplakins, the major plaque proteins of true desmosomal junc­ tumor studied so far [37 ,38], although it has been observed in tions [7,32]' and other adhering junction markers. In MC-MAl at).d various other carcinomas, such as breast and bladder tumors [40]. MC-MA2 cell cultures, these proteins were also detected in minor Abnormalities in chromosome 1 similar to ours have been observed amounts, if at all. Whether this reduction of junctions reflects tlJ.e in four Merkel cell tumors in vivo [37], but also in various other tumor state or results from growth as individual cells or clusters in malignant tumors, such as neuroblastoma and melanoma [37]. An suspension remains to be seen. Other epithelial cell lines that gr(.)"W involvement of chromosome 7, which was found in various Merkel similarly in floating cultures, such as PC 12 cells, are also poor in cell carcinomas [37,38], could not have been observed in cells of our junction proteins [30]. Me-MAl culture. In conclusion, the present and previous data VOL. 102, NO.3 MARCH 1994 TWO MERKEL CELL TUMOR CULTURES 353

suggest that, although there is no cytogenetic marker that is specific 16. Franke WW, Moll R: Cyroskeletal components oflymphoid organs. I. Synthesis for Merkel cell tumors, alterations especially in chromosomes 1 and of cytokeratins 8 and 18 and des min in subpopulations of extrafollicular reticu­ lum cells of human lymph nodes, tonsils, and spleen. Differelltiation 36:145- 5 might be involved in the transformation process of these carci­ 163, 1987 nomas. 17. Franke WW, Schmid E, Osborn M, Weber K: Different intermediate-sized fila­ Vos et at have described the production of NGF in primary cul­ ments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci tures of rat Merkel cells, and they have suggested that Merkel cells USA 75:5034 - 5038, 1978 18. Moll I, Troyanovsky M, Moll R: Special program of differentiation expressed in play an important role during nerve development in rodents keratinocytes of human Haarscheiben: an analysis of individual cytokeratlll [14,41]. Whether human Merkel cells and their tumors also pro­ polypeptides.] Invest Derrnatol100:69-76, 1993 . duce NGF remains to be clarified. 19. Cowin P, Kapprell H-P, Franke WW, Tamkun], Hynes RO: Plak~globll1: a In conclusion, the two cell lines characterized in the present study protein common to different kinds of intercellular adhering junctlons. Cell 146:1063-1073,1986 provide a valuable m?del.for analyses o~NGF and ~ther ~eurotra.ns­ 20. Geiger B, Avnur Z, Volberg T, Volk T: Molecular domains of adherens junctions. mitters aimed at castmg hght on the stlll rather elllgmatlC secretmg In: Edelman GM, Thie.ry ]P (eds.). The Cell in Contaet. john Wiley and Sons, products and probable function(s) of Merkel cells. New York, 1985, pp 427-460. 21. Koch PJ, Goldschmidt MD, Walsh MJ, Zimbelmann R, Schmelz M, Franke Note Added in Proof Recently in vitro cell cultures of human Mer­ WW: Amino acid sequence of bovine muzzle epithelial desmocollin derived kel cell tumors have been described by Baudoin et al: Cancer Res from cloned eDNA: A novel subtype of desmosomal cadherins. Differentiation 47:29-36, 1991 53:1175-1181,1993. 22. Schmelz M, Duden R, Cowin P, Franke WW: A constitutive transmembrane glycoprotein of M., 165000 (desmoglein) in epidermal and non-epidermal des­ mosomes. II. Immunolocalization and micro injection studies. Ellr] Cell Bioi We thank Mrs. C/'. Grund (D ivision oj Cell Biology, German Cancer Research 42:184-199,1986 23. Moll R, Mitze M, Frixen UH, Birchmeier W: Differential loss of E-cadherin Center, Heidelberg, Germat'Y) , Mrs. Ch. Nohl (It,stilllte oj Human Get,eties, expression in infiltrating ductal and lobular breast carcinomas. Am] Pathol (in Utliversity oj Giessen, Giessen, Gem,at'Y), and Mrs. U. 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