[CANCER RESEARCH 45, 6024-6033, December 1985] Characterization of Three New Variant Type Cell Lines Derived from Small Cell Carcinoma of the Lung Lou de Leij,1 Pieter E. Postmus, Charles H.C.M. Buys, Job D. Elema, Frans Ramaekers, Sibrand Poppema, Marjolein Brouwer, Anneke Y. van der Veen, Geert Mesander, and T. Hauw The Departmentsof Clinical Immunology[L. de L, G. M., T. H. T.], PulmonaryDiseases[P. E. P.], Human Genetics [C. H. C. M. B., A. Y. v. d. V.¡, and Pathology [J. D. E., S. P.], University ölGroningen, Groningen; DepartmentoÃ-Pathology, University of Nijmegen, Nijmegen ¡F.R.]; and the Netherlands Cancer Institute, Amsterdam [M. B.¡,TheNetherlands ABSTRACT Three new, well growing cell lines (GLC-1, GLC-2, and GLC- 3) have been established from small cell lung carcinoma (SCLC) and characterized. A subclone (GLC-1-M13) markedly different from its parent line GLC-1 was also isolated and characterized. Cytogenetic analysis of the cell lines revealed deletions in the short arm of chromosome 3 as a most consistent chromosomal aberration. The deleted region was not identical in all meta- phases, 3p(21-23) being the shortest region of overlap. Despite their SCLC origin GLC-1, GLC-2, and GLC-3 do not show pronounced SCLC differentiation features. Neurosecretory gran ula were very rare (GLC-1) or completely absent (GLC-2 and GLC-3), whereas the SCLC-related enzyme and hormone mark ers L-3,4-dihydroxyphenylalanine decarboxylase, neuron-specific enolase, creatine kinase BB, and bombesin-like immunoreactivity were variably expressed. Although the subclone GLC-1-M13 was derived from the poorly differentiated GLC-1, it behaved according to the above criteria as a differentiated "classic" SCLC cell line. When assessed with specific monoclonal antibodies the different cell lines appeared to express different subsets of intermediate filament proteins, indicative for different stages and directions of differentiation: "undifferentiated" (GLC-1 and GLC- 2); "neural tissue related" (GLC-2); "simple epithelium" related (GLC-1-M 13); and a combination of simple and squamous epi thelium related (GLC-3). We conclude that GLC-1, GLC-2, and GLC-3 represent dedif- ferentiated forms of SCLC, related to the recently described "variant" type of SCLC, whereas the donai derivate GLC-1-M13 behaves like a differentiated "classic" SCLC cell line. INTRODUCTION According to the latest WHO classification five major forms of lung cancer can be recognized on histological grounds, epider- moid (squamous), small cell, large cell, adeno-, and adenosqua- mous cell carcinoma (1). Such a classification might not be fully satisfying however, since mixing of different forms and also transitions from one form to another have been reported (2-4). In vitro similar conversions have been documented (5-7). There fore it has been proposed that lung cancer comprises in fact a continuous spectrum of cancer types, possibly originating from one common malignantly transformed "stem cell" population, in which different histological appearances reflect preferentially ex pressed (normal) differentiation pathways (6). The establishment Received 12/11/84; revised 7/19/85; accepted 7/23/85. 1To whom requests for reprints should be addressed, at Department of Clinical Immunology, University Hospital, Oostersingel 59, 9713 EZ Groningen, The Neth erlands. of a growing number of lung tumor-derived cell lines has been reported in the past years (6,8,9). The availability of continuously growing cell lines facilitates the study of the biology of lung cancer and it can be anticipated that such a study will eventually elucidate the relations between the different forms of this dis ease. Here we describe the isolation and characterization of three new, SCLC2-derived cell lines which, although partly undif ferentiated still show combinations of features characteristic for different forms of differentiated lung cancer. MATERIALS AND METHODS Morphological Methods. For light microscopy the tumor biopsy ma terial was fixed in 8% formaldehyde, embedded in Epon, and stained with toluidine blue. For transmission electron microscopy the tumor biopsy or, in the case of cell lines, the isolated cell pellets were incubated in 2% glutaraldehyde in phosphate-buffered saline. Postfixation was performed in 2% osmium tetroxide in phosphate-buffered saline. The material was embedded in Epon. Further preparations were according to routine procedures. Growth Conditions and Media. Cell lines were kept at 37°C in a humidified atmosphere in a CO2 incubator (5% CO2) and subcultured once or twice weekly by dilution in fresh growth medium. The growth medium was RPM11640 (Gibco, Paisley, United Kingdom) supplemented with 5x 10~5 M 0-mercaptoethanol, 2 mw glutamine, 1 mw pyruvate, and gentamicin (50 ¿/g/ml). Serum-containing medium was supplemented with 15% fetal calf serum, whereas in serum-free medium hydrocortisone (10~8 M), bovine insulin (5 Mg/ml), human transferrin (10 /¿g/ml),17/3- estradiol (10~e M), sodium selenite (3 x 10~8 M), bovine serum albumin (1%, w/v), arginine vasopressin (10 ng/ml), bombesin (10~7 M), and ethanolaminephosphorylethanolamine (10"5 M) was added according to the method of Minna ef a/. (10). Growth in semisolid medium was assessed after triturating the cell clumps and filtering the obtained suspension (Nylar filter, 20 ^m). In this way a single cell suspension was obtained. The cells were harvested by centrifugation and subsequently suspended in serum-containing medium to which 2% (w/v) low melting agarose (Seaprep 15/45; FMC, Rockland, ME) was added. Chromosome Analysis. Chromosome studies were carried out on cell cultures that had been in continuous serial passage for 8-14 months after explantation. The cells received fresh medium 1 day before harvest. Vinblastine sulfate (Lilly, Indianapolis, IN) was added to a final concentra tion of 20 ng/ml for the last 2.5 h in order to arrest in metaphase those cells that entered mitosis. Cell were collected, treated with a hypotonie solution, and fixed as described by Yu ef al. (11). Air-dried metaphases were banded by staining with 4,6-diamidino-2-phenylindole after a pre treatment with actinomycin D (12). A total of 32 metaphases were karyotyped: 10 from line GLC-1 ; 8 from GLC-1-M13; 5 from GLC-2; and 9 from GLC-3. 2The abbreviations used are: SCLC, small cell lung carcinoma; dopa, u-3,4- dihydroxyphenylalanine;RGE 53, monoclonalantibody directed against cytokeratin 18, and specific for glandular epithelium; RKSE 60, monoclonal antibody specific for keratins in keratinizing cells. CANCER RESEARCH VOL. 45 DECEMBER 1985 6024 Research. on November 24, 2021. © 1985 American Association for Cancer cancerres.aacrjournals.org Downloaded from