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[CANCER RESEARCH 40. 2500-2506. 0008-5472/80/0040-OOOOS02.00 July 1980] Cell Line Derived from a Metastasis of a Human Testicular Germ Cell Tumor1 David L. Bronson,2 Peter W. Andrews, Davor Solter, Jaroslav Cervenka, Paul H. Lange, and Elwin E. Fraley Departments of Urologie Surgery [D. L. B.. P. H. L., E. E. F.] and Oral Pathology ¡J.C.¡,University of Minnesota Minnesota 55455. and the Wistar Institute. Philadelphia, Pennsylvania 19104 [P. W. A.. D. S.J College of Health Sciences, Minneapolis. ABSTRACT sue, which suggests the formation by teratoma cells of fully A cell line, designated 833K-E, has been established from a differentiated elements. Much of the information about these tumors was obtained metastasis of a human testicular germ cell tumor that consisted from studies of mouse testicular teratomas, which were first of four histological types of tumor cells. The 833K-E cells have observed by Stevens and Little (28) and were described in morphological and ultrastructural characteristics of epithelial detail by Stevens (25) and by Pierce (22). Testicular teratomas cells and a hyperdiploid karyotype indicative of their human occur spontaneously at high frequency in strain 129 mice and male origin. The cells grow in agar cultures and produce in can be induced by grafting genital ridges of 12-day embryos nude mice tumors which have the histological features of or whole embryos into the testes of syngeneic males (26, 27). embryonal carcinoma without differentiated elements. Many of These teratomas consist of a variety of tissues representing the cells express a stage-specific mouse embryonic antigen, derivatives of all 3 germ layers. and low levels of the major histocompatibility antigens and ß2Some of the induced and spontaneous mouse teratomas can microglobulin also were detected on a large percentage of the be serially transplanted in syngeneic adult animals. The transcells. A lymphoblastoid cell line (833K-LC) established from plantable tumors also contain EC cells and thus are classified the same tumor specimen expresses major histocompatibility as teratocarcinomas. Several EC cell lines have been estab antigens and /?2-microglobulin but does not express the embry lished in vitro from these tumors. Some are nullipotential lines, onic antigen. which exhibit little or no tendency to differentiate, whereas others are multipotential lines, which differentiate readily in INTRODUCTION vitro and in vivo. These EC cells express cell surface (embry onic) antigens that also are detected on undifferentiated cells Carcinoma of the testis causes 11 to 13% of the deaths from from other species, including humans, but are not expressed cancer in North American men between the ages of 15 and 34 by differentiated cells (reviewed in Rets. 10, 13, and 19). years, and more than 90% of the testicular tumors in this age We are aware of reports of only 4 cell lines, Tera-1, Tera-2, group are classified as germ cell tumors (18). Current evidence SuSa, and NEC-8, that were derived from human testicular suggests that these tumors arise from the primordial germ germ cell tumors (9, 11, 30). In a previous communication (4), cells, which line the seminiferous tubules and are the direct we described ultrastructural observations of the production, precursors of sperm. although at low frequency, of particles morphologically identi In the histological classification system proposed by the cal to the human placental retrovirus (6, 16, 29) by cells World Health Organization, testicular germ cell tumors are classified further as EC,3 teratoma, teratocarcinoma (EC with (designated 833K-E cells) established in vitro from an abdom inal metastasis of a human testicular germ cell tumor. This teratoma), choriocarcinoma, yolk sac tumor, and seminoma report describes some additional properties of the 833K-E cell (20). EC consists of anaplastic, undifferentiated cells that re line and compares these properties with those of other human semble undifferentiated normal cells of an embryo in the earli est stages of development. Approximately 40% of testicular and mouse cell lines of similar classification. germ cell tumors contain elements of more than one (e.g., teratocarcinoma), or even all, of the histological types of these MATERIALS AND METHODS tumor cells, and it is thought that the other nonseminomatous Clinical History. A right radical orchiectomy was performed germ cell tumors are formed by the differentiation of EC cells in September 1975 on a 19-year-old Caucasian male. Histo(14, 18, 20). Thus, malignant transformation of the germ cells leads to the development of seminoma or, by a separate pathological examination of the testis revealed teratoma, EC, seminoma, and foci of choriocarcinoma. Chemotherapy was pathway, of EC, and the EC cells differentiate to form chorio initiated with methotrexate, cyclophosphamide, and actinomycarcinoma, yolk sac tumor, or teratoma. In addition, some teratomas contain cartilage, epithelium, and neural tis- cin D, but EC was discovered in a left periaortic lymph node in February 1976, and the patient died 2 months later with wide ' This investigation was supported in part by Grants CA 15551. CA 10815, spread métastases. and CA 21069 from the National Cancer Institute; T32-GM 07511-03 from the Tissue Culture. Tissue from an abdominal metastasis, con National Institute of General Medical Sciences; HD 12487 from the National Institute of Child Health and Human Development; and 1-301 from the National sisting of choriocarcinoma with elements of EC, teratoma, and Foundation-March of Dimes. seminoma, was obtained at autopsy. The specimen was placed 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: EC. embryonal carcinoma; HLA, human histo in culture by the coverslip method (8), and cultures were compatibility leukocyte antigens: SSEA-1, stage-specific embryonic antigen; incubated at 35° in Roswell Park Memorial Institute Medium PBS, phosphate-buffered saline (0.9 ITIM CaCI2, 2.7 mM KCI, 1.5 RIM KH2PO4. 138 mM NaCI. and 9.6 mM Na2HPO«.pH 7.4). Received August 21. 1979; accepted April 10. 1980. 2500 1640 containing 10% tryptose phosphate broth, 15% heatinactivated (56°,30 min) fetal bovine serum, 2 mM L-glutamine, CANCER RESEARCH VOL. 40 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. Cell Line from Germ Cell Testicular Cancer 100 units of penicillin per ml, and 100 /ig of streptomycin per ml. Cells were detached from culture vessels by incubation for 7 min at 37°with trypsin:citrate diluted with an equal volume of Grand Island Biological Co. Salt Solution A. Trypsinxitrate contains 0.25% trypsin (Difco Laboratories, Detroit, Mich.; 1: 250) in Salt Solution A and, per liter, 10 g of sodium citrate, 5.5 g of sodium chloride, and 0.02 g of phenol red (pH 7.4; formula provided by H. T. Holden, National Cancer Institute). After several passages by this method, cells were subcultured with undiluted trypsin:citrate. Assays for Mycoplasma were done by in vitro cultivation techniques (Flow Laboratories, Inc., Rockville, Md.) and by ultrastructural examination of the cells. No Mycoplasma were detected. Tumorigenicity. The methods for seeding cells in agar cul tures have been described (7). Athymic (nude) mice were given s.c. inoculations of 5 to 10 x 106 cells, and tumors were removed for histological examination when approximately 5 mm in diameter. Cytogenetics. Chromosome analyses were done by conven tional staining methods as well as by Q- and G-banding tech niques (7). Antisera. Monoclonal antibody W6/32, specific for a deter minant common to the 43,000-dalton chains of HLA-A, -B, and -C (2), and monoclonal Antibody B.BM1, reactive with human /?2-microglobulin (3), were kindly provided by Colin Barnstable, University of Oxford. The production and specificity of a mono clonal antibody, anti-SSEA-1, which recognizes a stage-spe cific mouse embryonic antigen and which reacts with an anti gen expressed by human teratocarcinoma cells and sperm, has been described (24). Two monospecific HLA-typing sera with specificities for HLAA2 (Pinquette, No. FD 89 2-50-6-03-15-03) and HLA B12 (O'Driscoll, No. P3672B) were supplied by NIH and by Julia Bodmer, University of Oxford. Serology. Cells in monolayer cultures were harvested with trypsin, which does not affect the antigens studied." The mono clonal antibodies were assayed by indirect radioimmunobinding as described previously (24). Briefly, diluted antibody was incubated for 60 min at 4°with 2 x 10s target cells in a total volume of 100 /J of PBS containing 2.5% fetal bovine serum. After being pelleted and washed 3 times with PBS containing 2.5% fetal bovine serum and 0.1% sodium azide, the cells were suspended in 50 /il of the same washing solution containing 50,000 cpm of 125l-labeled rabbit anti-mouse immunoglobulin (it-specific, or heavy- and light-chain-specific, as appropriate). The cells were incubated at 4°for 60 min, pelleted, and washed 3 times as above, and the 125Ibound to the cell pellet was determined as cpm in a gamma counter. F9 cells served as targets for anti-SSEA-1, and 833K-LC cells served as targets for W6/32 and B.BM1 antibodies. Determinations were made in triplicate and the variation between the triplicate samples did not exceed 5%. Quantitative absorptions were performed by incubating increasing numbers of cells at 4°for 60 min with 200 fil of monoclonal antibody that was diluted (anti-SSEA-1, 1:16,000; W6/32, 1:2,000; B.BM1, 1:10,000) to give 90% of maximum binding. After the cells were pelleted, residual anti body in the supernatant was assayed by the indirect radioim' P. W. Andrews and D. Seller, unpublished data. JULY munobinding assay described above. The negative controls for these absorptions of W6/32 and B.BM 1 antibodies were Daudi cells, which express neither HLA nor /J2-microglobulin (23). The lymphoid cells were typed for HLA by standard tech niques in the Tissue Typing Laboratory, University of Pennsyl vania. Expression of HLA alloantigens by 833K-E cells was determined by incubating the cells with 12 jul of alloantiserum (Pinquette or O'Driscoll) on ice for 60 min. The cells were then pelleted in a Beckman microfuge, and the residual activity of the serum was titered on peripheral blood lymphocytes from donors of established HLA type [A1, A2, B8; and A3, A11, B12 (B5, B18, BW35)] by the microcytotoxicity assay. Another human testicular tumor cell line, designated 1156Q-E,5 of known HLA type (A1, A3, B7, B15) was used as a negative control. For indirect immunofluorescence assays, cells were incu bated with antibody and then with rabbit anti-mouse immuno globulin of appropriate specificity (Cappell Laboratories, Cochranville, Pa.) conjugated with fluorescein isothiocyanate. Incu bations were at 4°in the presence of 0.1 % sodium azide. After the cells had been washed 3 times, they were suspended in 50% glycerol in PBS and examined under UV epiillumination with a BP 390-490 violet-blue exciting filter and a No. 515 suppression filter. RESULTS Tissue Culture. Numerous small islands of epithelial cells were present in plates and on coverslips a few days after the tissue fragments were placed in culture. Secondary cultures were established with free-floating cells and by transferring coverslips with areas of epithelial cell growth to 35-mm plates. Subsequent passages were made with trypsin:citrate. The cells exhibited scant cytoplasm, large nuclei, and prominent nucleoli (Fig. 1). Ultrastructural examination revealed microvilli, desmosomes, and cytoplasmic tonofibrils (Fig. 2), which are char acteristics of epithelial cells. Dome formation, another charac teristic of epithelial cells (21), is observed frequently in aged 833K-E cultures. The cells have been subcultured more than 100 times and thus constitute an established cell line. In addition, lymphocytes persisted in a few of the primary cultures, and proliferation of these cells was noted within 5 to 6 weeks after the tissue was explanted. Each time the medium was replaced ¡nthese plates, floating cells were pelleted and seeded in a separate vessel. The continued growth of these cells resulted in establishment of a lymphoblastoid (833K-LC) cell line. The 833K-LC cells grow in suspension (i.e., they do not attach to the vessel growth surface) as single cells or small clusters, are highly pleomorphic, and exhibit uropods. A herpes-type virus was detected by ultrastructural examinations of these cells in early culture, and all cells examined expressed Epstein-Barr virus antigens.6 Of the lymphoid cells, the EpsteinBarr virus selectively infects and transforms B-lymphocytes; thus, the expression of these viral antigens suggests that the 833K-LC are derivatives of polyclonal B-lymphocytes (15). Cytogenetics. Chromosomal analyses were performed with 833K-E cells in passages 5 and 59 with virtually identical results. The cells have a hyperdiploid number of chromosomes, 5 D. L Bronson, unpublished data. 6 G. R. Dreesman, personal communication. 1980 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. 2501 D. L. Branson et al. with a modal number of 56 to 61 in 38 of 58 mitotic spreads counted. Five karyotypes were constructed by G-banding, and 20 mitotic spreads were analyzed. In each of the karyotypes, the A-group chromosomes were overrepresented, whereas the number of chromosomes of the other groups did not deviate consistently from diploid constitution. In all metaphases, 2 or 3 isochromosomes of F-group size were observed, as were 3 to 8 chromosomes of abnormal morphology that could not be identified with certainty. No marker chromosome was found that would identify this cell line specifically. The Y chromosome was detected by Q-banding in all mitotic figures (Fig. 3). Tumorigenicity. 833K-E cells in passage 85 were inoculated into 5 nude mice. Tumors at least 5 mm in diameter were produced at the site of inoculation in 4 of the animals between 35 and 76 days after inoculation. No métastaseswere noted. The tumor-free animal died on Day 59 after inoculation. Sec tions of the tumors from all 4 animals showed cells arranged in tightly packed, convoluted sheets. The tumor cells exhibited a uniform morphology consistent with EC without differentiated elements, although the convoluted areas suggested poorly defined glandular structures (Fig. 4). No tumors formed in 9 nude mice within 44 to 141 days after inoculation (;'.e., at the time of death of the animal) with 833K-LC cells. The 833K-E cells also formed colonies in agar cultures with an efficiency of 1 to 2%. Forty-four clones have been isolated in 3 separate experiments and grown in mass culture for additional characterization studies. The clones exhibit the cel lular morphology and growth pattern of the parent 833K-E cell line. Antigen Expression. Quantitative absorptions of monoclonal antibodies W6/32, B.BM1, and anti-SSEA-1 indicated that cells of the 833K-E line express HLA, /8?-microglobulin, and the cross-reacting mouse embryonic antigen, SSEA-1. The latter antigen is absent from the 833K-LC cells (Chart 1). Like other lymphoid cell lines, the 833K-LC cells express more HLA and /i?-microglobulin than do peripheral blood lymphocytes. However, the 833K-E cells express approximately 50% less HLA and /i?-microglobulin on a per-cell basis than do peripheral blood lymphocytes even though the 833K-E cells are much larger (roughly 5:1 based on the volume of equivalent numbers of packed cells). Unotsorbed Similarly, in immunofluorescence assays, the 833K-E cells showed only a weak, stippled fluorescence after reaction with W6/32 and B.BM1 antibodies, and some of the cells were scored as negative (Table 1). However, the apparent lack of HLA and /?2-microglobulin on those cells may reflect only the technical difficulties of scoring a weak system. More certain was the observation that some 833K-E cells did not express SSEA-1, because reactive cells fluoresced strongly. Absorption of HLA typing sera confirmed that HLA of both the A and B loci are expressed by 833K-E and 833K-LC cells (Chart 2). Similar results were obtained with 833K-E cells in passages 34 and 117. DISCUSSION The 833K-E cell line was established from metastatic tumor tissue consisting of 4 histological types of testicular tumor cells, which raises the question of which type(s) of testicular tumor cell is (are) represented by this cell line. As a general observation, it can be stated that the 833K-E cells exhibit ultrastructural and growth properties of epithelial cells and are morphologically similar (i.e., have scant cytoplasm, large nu clei, and prominent nucleoli) when continuously subcultured at a high cell density. All clones derived from this cell line exhibit Table 1 Indirect immunotluorescence assays Cell suspensions were incubated with monoclonal antibody at 4°for 60 min. pelleted, washed 3 times, and then incubated at 4°for 60 min with rabbit antimouse immunoglobulin conjugated with fluorescein isothiocyanate. The cells were pelleted, washed 3 times, and resuspended in 50% glycerol in PBS for examination under UV. % of positive cells Anti-SSEA-1 W6/32 (anti-HLA) Cell line )"0087(+ (+ + + 833K-E833K-LCOaudiF9LNSV667 f100(+ (+ +)0010080< + )100(++ +)00NTC + +)092+ + + + , strong fluorescence; + , weak, stippled fluorescence. 1Human skin fibroblasts transformed with SV40 (5). NT. not tested. Serum Unorjsorbed o o o. x 15 30 Serum O_ 2 « 2 E 7.5 B.BMI (anti-ft-microglobulin) o. O 3.8 7.5 15 30 3.8 75 15 30 Cells x 100,000 Cells x 100,000 Cells 100,000 Chart 1. A. absorption analysis of monoclonal antibody (anti-SSEA-1) reactivity. After absorption of antibody by 833K-E (A), 833K-LC (•). or F9 (CDcells, residual antibody in the clarified serum was tested by radioimmunoassay on F9 cells as described previously (24) Each point is the average of the results of triplicate assays. B, absorption analysis of monoclonal antibody W6/32 (anti-HLA) reactivity on 833K-E (A). 833K-LC (•). Daudi (O) cells, or peripheral blood lymphocytes O by methods described in A. Residual antibody in the clarified serum was tested on 833K-LC cells. C. absorption analysis of monoclonal antibody B BM1 (anti-/i2microglobulin) reactivity on 833K-E (A). 833K-LC (•). or Daudi (O) cells or peripheral blood lymphocytes (O) by methods described in A. Residual antibody in the clarified serum was tested on 833K-LC cells. 2502 CANCER RESEARCH VOL. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. 40 Cell Line from Germ Cell Testicular Cancer 12 14 Serum Dilution II 12 14 Serum Dilution Chart 2. Cytotoxicity assays of unabsorbed alloantisera (O) and of sera after incubation with 833K-E (A), 833K-LC (O). or 1156Q-E (•)cells. C'C (A), complement control. The activities of unabsorbed alloantisera. anti-HLA-A2 (A) or anti-HLA-B12 (6) and of absorbed alloantisera were titered on peripheral blood lymphocytes of known HLA type. The scores, representing the percentage of lymphocytes that were lysed by antibody, are: 1 - 0 to 10%; 2 = 11 to 25%; 4 = 26 to 50%; 6 = 51 to 80%; 8 = 81 to 100%. parison, SuSa cells also have an aneuploid karyotype (11 ), as do the murine somatic cell hybrids (1). Thus, in some aspects (origin, morphology, and expression of SSEA-1), the 833K-E cells resemble other teratocarcinoma stem cells, and they also produce EC in nude mice. In other aspects, such as expression of major histocompatibility antigens, although weakly and in karyotype, they are dissimilar from mouse teratocarcinoma stem cells. It is possible that 833K-E cultures contain more than one type of malignant cell and, perhaps, variable numbers of differ entiated cells (which could lack SSEA-1 but express HLA and /?2-microglobulin), with only the EC cells capable of producing tumors in nude mice. The cultures occasionally contain cells of different morphology, depending in large part on culture con ditions, which might represent in vitro differentiation, other types of testicular tumor cells, or both. The immunofluorescence data also suggest heterogeneity of the cultures. The developmental relationships between these possible subpopulations, and the question of whether the stem cells express HLA and /?2-microglobulin, will become clearer after studies of the clones derived from 833K-E and other human testicular tumor cell lines. these same properties and are morphologically indistinguish able from the parent line. Chromosome analyses demonstrated that the cells are of human male origin, and, although the ACKNOWLEDGMENTS chromosomal constitution is grossly abnormal, it is stable, as The authors thank Peter Waldron and Hannelore Asmussen for excellent is indicated by analyses of cells in widely different passages. The tumors produced by inoculation of 833K-E cells into technical assistance, Donna Ritzi for photomicrography, and Judith Gunn Bronson for editing the manuscript. nude mice were composed of undifferentiated small cells typi cal of the EC cells seen in biopsies of human EC and of the REFERENCES stem cells found in transplantable murine teratocarcinomas. Although human teratocarcinomas have been passaged by 1. Andrews. P. W.. and Goodfellow, P. N. Antigen expression by somatic cell hybrids of embryonal carcinoma cells with thymocytes and L cells Somat others in nude mice and in cheek pouches of cortisone-treated Cell Genet., in press. 1980 hamsters, only human testicular tumor cell line NEC-8 has 2. Barnstable. C. J., Bodmer. W. F., Brown, G . Galfre. G.. Milstein. C.. Williams, A. F . and Ziegler. A Production of monoclonal antibodies to previously been reported to produce tumors in ¡mmunodeficient Group A erythrocytes. HLA and other human cell surface antigens—new hosts (30). tools for genetic analysis. Cell. 14: 9-20. 1978 All murine EC cells examined to date are characterized by 3. Brodsky, F. M.. Bodmer. W. F.. and Parham. P. Characterization of a monoclonal /}2-microglobulin antibody and its use in the genetic and bio the presence of the embryonic cell surface antigen, SSEA-1 chemical analysis of major histocompatibility antigens. Eur. J. Immunol , 9 (24), and by the lack of major histocompatibility antigens. A 536-545. 1979. priori, there is no reason to assume that human EC cells must 4 Bronson, D L , Ritzi. D. M., Fraley, E. E., and Dalton. A. J. Morphologic evidence for retrovirus production by epithelial cells derived from a human show a similar cell surface phenotype. However, in 2 other cell testicular tumor metastasis. J. Nati. Cancer Inst.. 60 1305-1308. 1978. lines (Tera-2 and SuSa) derived from human testicular germ 5. Croce. C. M.. Girardi, A. J.. and Koprowski, H Assignment of the T-antigen cell tumors, a large percentage of the cells lack HLA and ß2gene of Simian Virus 40 to human chromosome C-7. Proc. Nati. Acad. Sci. U. S. A.. 70. 3617-3620. 1973. microglobulin and express a mouse embryonic antigen, F9 (11, 6. Dirksen. E. R . and Levy, J. A. Virus-like particles in placentas from normal 12), that evidently has been conserved during mammalian individuals and patients with systemic lupus erythematosus. J Nati Cancer Inst , 59. 1187-1192, 1977. evolution. The F9 and SSEA-1 antigens show similar patterns 7. Elliott, A. Y., Bronson. D. L., Cervenka. J., Stein. N.. and Fraley. E. E. of reactivities, and SSEA-1, like F9, is detected on Tera-2 (24) Properties of cell lines established from transitional cell cancers of the and SuSa cells.4 human urinary tract. Cancer Res.. 37. 1279-1289. 1977. Therefore, the expression of SSEA-1 by some 833K-E cells is consistent with an EC phenotype. However, this expression is not conclusive evidence that these are EC cells because SSEA-1, like F9 antigen, is also detected on cells of the male germ line and on some cells of the kidney and brain (24). The expression of HLA and /?2-microglobulin on most, if not all, 833K-E cells provides a contrast with the murine teratocarcinoma stem cells. However, the level of expression is low and cannot be taken in isolation as evidence against the EC nature of 833K-E cells. We note that certain murine somatic cell hybrids have properties of pluripotent murine EC cells, although they express low levels of H-2 antigens (1). The aneuploid karyotype of 833K-E cells also contrasts with the near-diploid karyotype of many murine teratocarcinoma stem cells. In com 8 9. 10. 11. 12. 13. 14. 15. Elliott, A. Y.. Bronson, D. L.. Stein. N , and Fraley. E. E. In vitro cultivation of epithelial cells derived from tumors of the human urinary tract Cancer Res., 36. 365-369. 1976 Fogh. J. 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Bronson et al. 16. 17. 18. 19. 20. 21. 22. II. Presence of Epstein-Barr virus receptors on B lymphocytes. J. Exp. Med.. Õ38. 1365-1378, 1973. Kalter, S. S., Helmke, R. J., Heberling, R. L., Panigel, M., Fowler, A. K., Strickland, J. E., and Hellman, A. C-type particles in normal human placen tas. J. Nati. Cancer Inst., 50. 1081-1084, 1973. Miller, R. A., and Ruddle, F. H. Properties of teratocarcinoma-thymus so matic cell hybrids. Somat. Cell Genet.. 3. 247-261, 1977. Mostofi, F. K., and Price, E. B. Tumors of the male genital system. In: Atlas of Tumor Pathology, H. I. Firminger (ed.), 2nd Series, Fase. 8, pp. 1-20. Washington, D. C.: Armed Forces Institute of Pathology, 1973. Nicolas, J. F.. Avner, P.. Gaillard, J., Guenet. J. L., Jakob. H., and Jacob, F. Cell lines derived from teratocarcinomas. Cancer Res., 36. 4224-4231, 1976. Nochomovitz, L. E., DeLa Torre, F., and Rosai, J. Pathology of germ cell tumors of the testis. Urol. Clin. North Am.. 4: 359-378, 1977. Picket!. P. B., Pitelka, D. R., Narriamolo. S. T., and Misfeldt. D. S. Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells. J. Cell Biol.. 66. 316-332, 1975. Pierce, G. B. Teratocarcinoma: model for a developmental concept of cancer. Curr. Top. Dev. Biol., 2. 223-246, 1967. 2504 23. Poulik. M. D.. Ferrone, S., Pellegrino. M. A., Sevier, D. E., Oh, S. K., and Reisfeld, R. A. Association of HL-A antigens and /32-microglobulin: concepts and questions. Transplant. Rev., 21: 106-125, 1974. 24. Solter, D., and Knowles, B. B. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc. Nati. Acad. Sei. U. S. A.. 75. 5565-5569, 1978. 25. Stevens. L. C. The biology of teratomas. Adv. Morphog., 6. 1-31, 1967. 26. Stevens, L. C. Origin of testicular teratomas from primordial germ cells in mice. J. Nati. Cancer Inst., 30. 549-552, 1967. 27. Stevens, L. C. The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos. Dev. Biol., 21: 364-382, 1970. 28. Stevens. L. C., and Little, C. C. Spontaneous testicular teratomas of an inbred strain of mice. Proc. Nati. Acad. Sei. U. S. A., 40:1080-1087, 1954. 29. Vernon, M. L., McMahon. J. M., and Hacke«, J. J. Additional evidence of type-C particles in human placentas. J. Nati. Cancer Inst., 52. 987-989, 1974. 30. Yamamoto, T., Komatsubara. S.. Suzuki, T., and Oboshi, S. In vitro cultiva tion of human testicular embryonal carcinoma and establishment of a new cell line. Gann, 70. 677-680, 1979. CANCER RESEARCH VOL. 40 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. Cell Line from Germ Cell Testicular Cancer Fig. 1. Phase-contrast photomicrograph of 833K-E cells in culture, x 300. Fig. 2. Electron micrograph of 833K-E cells with desmosomes (thick arrows) and tonofibrils (thin arrows), x 60,000 Fig. 3. Trypsin-Giemsa banding of chromosomes from 833K-E cells in passage 5 showing a hyperdiploid karyotype with a Y chromosome. Fig. 4. Photomicrograph of a section of a tumor taken from a nude mouse inoculated with 833K-E cells. The tumor consisted of undifferentiated into convoluted sheets. H & E, x 240. JULY cells organized 1980 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. 2505 CO (M CM 00 IO (D (D lü *• (O 0) } ü o ce O co GO (D IO ÃŽo (M (O 00 o M 0) Ir- o) X X Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. Cell Line Derived from a Metastasis of a Human Testicular Germ Cell Tumor David L. Bronson, Peter W. Andrews, Davor Solter, et al. Cancer Res 1980;40:2500-2506. 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