In Vitro Cell. Dev. Biol. 27A:349-351, May 1991 © 1991 Tissue Culture Association 0883-8364/91 $01.50+0.00
Letter to the Editor NEW MURINE MAMMARY TUMOR CELL LINES
Dear Editor: All inbred strains of mice can transmit mammary tumor virus (MMTV) exogenously by way of salivary glands, seminal fluids or milk, and endogenously via germ cells. The response to MMTV may differ in detail depending on the host, for instance, histological types of induced tumors, virulence, antigenicity and mode of transmission (4). The feral mouse, especially M.m.musculus Sub-Jyg (JYG mouse) transmits exogenous MMTV only; the lack of endogenous integrated MMTV differs from all inbred strains of mice (unpublished data). Several groups reported the establishment of mouse mammary tumor cell lines (6,9,14,16). However, little attention has been devoted to feral mammary tumors. Culture lines of such tumors should prove useful for detailed analysis of viral tumorigenesis. In this paper, we describe the first establishment of murine mammary tumor cell lines (JYG-MC) from feral M.m.musculus Sub-Jyg mice. Their properties are described. Of particular interest is the observation that the inoculation of these cell lines into BALB/enude mice resulted in mammary tumors with 100% metastasis to lung after short latent periods. JYG mouse has been recently inbred by Dr. K. Moriwaki at the National Institute of Genetics in Japan. The incidence of mammary tumors is very high and the latent period is short which is quite different from another feral mouse, Czech II (3). Czech II mice are European wild mice (M.m.musculus) and they also lack endogenous MMTV genomes.
The jYG-MC cell lines were established from spontaneous mammary carcinomas in the left #1 and # 4 fat pads of a 10-month-old female JYG mouse. Histologically, the L1 and L4 mammary tumors were respectively a medullary adenocarcinoma (called JYG-MC Type A) and a papillary adenocarcinoma (called JYG-MC Type B). Metastases were found in the lung, and liver and the spleen. The mammary tumors were cultured using Dulbecco's modified Eagle's minimum essential medium (DMEM) with 10% fetal bovine serum (FBS). As shown in Table 1, doubling times of the JYG-MC cell lines were approximately 26.5 h in cell line A and 26.1 h in cell line B. The cells grew to a saturation density of 7.2 × 105/cm 2 in cell hne A and 4.4 × 105/cm 2 in cell line B, respectively. Chromosomal analysis of JYG-MC cell lines showed a hyperdiploid chromosome number ranging 37 to 42 (average 40). A small number of cells in culture B had chromosome numbers around 80. Microscopically, JYG-MC B cells were mainly cuboidal and JYGMC A cells were spindle shaped during growth (Fig. 1 a,b). Both cell lines, however, were arranged into a pavement-like monolayer at confluence, which is a typical mammary epithelial cell. After confluence, "domes" also developed in both cell lines (Fig. 2), which are characteristic of actively secretory cell cultures (1,11). Therefore both cell lines (JYG-MC A & B) showed the features of typical epithelial mammary tumor cell lines. There were differences between JYG-MC A and B cell lines in morphology and saturation density, but the doubling times of both cell lines were almost the same. These two cell lines have been carried for over 14 passages.
TABLE 1 GROWTH PARAMETERS AND TRANSPLANTABILITY OF JYG-MC CELL LINESa In Vitro
In Vivo
Cell Lines
Saturation Density (No. of Cells)
Population Doubling Time (h)
JYG-MC A
7.2 × 10S/cm2
26.5
JYG-MC B
4.4 X 105/cmz
26.1
Sacrificed Weeks After Transplantation
Transplantability in Nude Mice
Ex. 1 Ex. 2 Ex. 3 Ex. 1 Ex. 2 Ex. 3
100% 100% 100% 100% 100% 100%
4.5 8 6 4.5 7 6
(4/4) (6/6) (5/5) (6/6) (4/4) (4/4)
Metastasis to Lung
50% (2/4) 50% (3/6) 100% (5/5) 17% (1/6) 100% (4/4) 1009"o (4/4)
Mammary tumors were cultured using Dulbecco's modified Eagle's minimum essential medium (DMEM) without serum and centrifuged for 10 min at 1000 rpm. Pellets were resuspended in DMEM with 10% fetal bovine serum (FBS)(Gibco New Zealand LTD) and the resulting cell dispersions were cultured in plastic bottles at 37 ° C in an atmosphere of 7% CO2 in air. Medium was changed once a week. Once a month the piled up cells were dispersed with 0.12% trypsin (Difco Labs., Detroit, MI) and 0.012% EDTA in Ca++, Mg++-free phosphaie buffered salin [PBS(-)], pH 7.2, and subcuhured at a spirit ratio of 1:4.
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FiG. 3. Medullary adenocarcinoma that developed 1.5 months after injecting Jyg-MC B cells into Balb/c-nude mouse. H&E stain, X200.
MMTVgp52 antigen was detected in cell extracts and culture supernatants from both Jyg-MC cell lines by radioimmunoassay (5). MMTVgp52 antigen was also detected in transplanted tumors by immunohistochemistry using monoclonal antibody to MMTVgp52
(10). Electron microscopically, intracytoplasmic A particles and mature B particles were observed in both Jyg-MC cell lines and acinar areas of transplanted tumors (Fig. 4). Therefore, JYG-MC cell lines is a good source from which to obtain exogenous MMTV. 5 - 8 )< 106 Cells were inoculated s.c. into female nude mice (BALB/c-AJcl-nu) to determine whether the Jyg-MC cells (A and B) were tumorigenic. As shown in Table 1, 29 of 29 (100%) of mice inoculated with Jyg-MC cells (A and B) developed mammary tumors at injection sites after 1-2 months. No mammary tumors developed in other strains of mice. The histological type of these tumors was medullary adenocarcinoma (Fig. 3). Macroscopic and microscopic examination revealed extensive metastatic lesions in lungs (Table 1). By 6 - 8 weeks after transplantation most mice had such lesions. The cell line Jyg-MC A and B were highly oncogenic in nude mice with metastases to lungs at 100% by as soon as 6 weeks after transplantation subcutaneously. It has been reported that the metastatic rate of mammary tumors is not so high in mice: for instance, 19.5% (25/128) macroscopically and 39.4% (26/66) microscopically in C3H/HeNSa mice reported by Sato (15), and 1 0 - 6 9 % in several mice strains by Pitelka et al. (12).
FIG. 2. Confluent culture of Jyg-MC B epithelial cells. Note "dome" (upper left). Phase contrast >(200.
FIG. 4. Electron mtcroscopy shows intracytoplastmic A and mature B particles in culture cells; JygMC B. ×20 000.
FIG. 1. a. Growing stage of Jyg-MC A epithelial cells. Note "spindle shape" of non-confluent cells. Phase contrast >(200. b. Growing stage of Jyg-MC B epithelial cells. Note "cuboidal shape". Phase contrast >(200.
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NEW MAMMARY TUMOR CELL LINES Therefore these cell lines will be a unique and effective model for analyzing metastasis mechanisms of mammary tumors. It is unclear what features of these cells lead to their high metastatic rate. Possibilities include the increased synthesis of hyaluronic acid (8), ultrastructural organization (7), basal lamina structures (13), and virulence of the exogenous mammary tumor virus of these cells. Further studies should clarify the mechanism of mammary tumor metastasis using these established cell lines.
REFERENCES 1. Das, N. K.; Hosick, H. L.; Nanndi, S. Influence of seeding density of multicellular organization and nuclear events in cultures of normal and neoplastic mouse mammary epithelium. J. Natl. Cancer Inst. 52:849-861; 1974. 2. Dunn, T. B. Morphology of mammary tumors in mice. In: Homburger, F., ed. Physiopathology of cancer, 2nd ed. New York: Hoeber; 1959:38-84. 3. Gallahan, D.; Callahan, R. Mammary tumorigenesis in feral mice: identification of a new int locus in mouse mammary tumor virus (Czech II) induced mammary tumors. J. Virology 61:66-74: 1987. 4. Hilgers, J.; Bentvelzen, P, Interaction between viral and genetic factors in murine mammary cancer. Adv. Cancer Res. 29:143-195; 1978. 5. Imai, S.; Hilgers, J. Levels of mammary tumor virus proteins (MTVp27 and MTVgp52) in the milk of low and high mammary cancer mouse strains of Japanese origin compared with European and American strains. Int. J. Cancer 24:359-364; 1979. 6. Keydar, J.; Gilead, Z.; Hartman, J. R,, et al. In vitro production of mouse mammary tumor virus in a mouse mammary tumor ascites cell line. Proc. Natl. Acad. Sci. USA 70:2983-2987; 1973. 7. Khaisman, A. E.; Tereshenko, I. P.; Yagubov, A. S. Correlation between the ultrastructural organization of mammary gland cancer in mice and ability to metastasize. Bull. Exp. Biol. Med. 87:340-343; 1979. 8. Kimata, K.; Honma, Y.; Okayama, M., et al. Increased synthesis of hyaluronic acid by mouse mammary carcinoma cell variants with high metastatic potential. Cancer Res. 43:1347-I354; 1983. 9. Lasfargues, E. Y.; Kramarsky, B.; Sarker, N. H., et al. An established RIll mouse mammary tumor virus production. Proc. Soc. Exp. Biol. Med. 139:242-247; 1972. 10. Morimoto, J. Production and properties of monoclonal antibodies against mouse mammary tumor virus (DI)-MMTV)--with special reference to horizontal transmission of MMTV. J. Nara Med. Assoc. 38:501-516; 1987.
11. Morimoto, J.: Imai, S.: Taniguchi, Y., et al. Establishment and characterization of a new murine mammary tumor cell line, BALB/c-MC. In Vitro 23:755-758; 1987. 12. Pitelka, D. R.; Hamamoto, S. T.; Taggart, B. N. Epithehal cell junctions in primary and metastatic mammary tumors of mice. Cancer Res. 40:1588-1599: 1980. 13. Pitelka, D. R.: Hamamoto. S. T.: Taggart, B. N. Basal lamina and tissue recognition in malignm~t mammary tumors. Cancer Res. 40:1600-1611: 1980. 14. Sarker, N. H.: Pomenti, A. A.; Dion, A. S. Replication of mouse mammary tumor virus in tissue culture. 1. Establishment of a mouse mammary tumor cell line, virus characterization, and quantitation of virus production. Virology 77:12-30: 1977. 15. Sato, H.; Kuroki, T.: Narisawa, T., et al. Characteristics of an inbred substrain (C3H/HeNSa) of C3H mouse, with reference to the mammary cancer incidence and metastasis. GANN 56:605-609; 1965. 16. Yagi, M. J. Characteristics of mammary tumor cultures from four mouse strains infected with MTV. Cancer Res. 35:370-373; 1975. Junji Morimoto Shunsuke Imai Satomi Haga Yosbiaki Iwai Mineko lwai
Shingo Hiroishi Nobumoto Miyashita Kazuo Moriwaki Howard L. Hosick
Department of Pathology, Nara Medical University 8 4 0 Shijo-cho, Kashihara-shi, Nara 634, JAPAN (J. M., S. I., S. H., Sh. H.); Research Institute for Advanced Science and Technology, University of Osaka Prefecture, 1 7 4 1 6 Shingacho, Sakaishi, Osaka 593, JAPAN (Y. I., M. I.); National Institute of Genetics, 1111 Tanidacho, Mishimashi, Shizuoka 411, JAPAN (N. M., K. M.); and Department of Zoology, Washington State University, Pullman, Washington 9 9 1 6 4 (H. L. H.) (Received 31 October 1990)