Isolation and characterization of a spontaneously transformed ...

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Carcinogenesis vol.19 no.11 pp.1907–1911, 1998. Isolation and characterization of a spontaneously transformed malignant mouse mammary epithelial cell line ...
Carcinogenesis vol.19 no.11 pp.1907–1911, 1998

Isolation and characterization of a spontaneously transformed malignant mouse mammary epithelial cell line in culture

Abhik Bandyopadhyay, Michael L.Cibull1 and LuZhe Sun2 Department of Pharmacology and 1Department of Pathology, University of Kentucky College of Medicine, 800 Rose Street, MS-311 UKMC, Lexington, KY 40536-0084, USA 2To

whom correspondence should be addressed Email: [email protected]

A method is described that permits the selection of spontaneously transformed mammary epithelial colonies from an untransformed mouse mammary epithelial cell line, NMuMG, and utilizes a long-term anchorageindependent growth of the transformants on soft agarose. These transformed cells (NMuMG-ST) are shown to be distinguishable from the untransformed cells by morphology, growth characteristics, induced carcinomas when transplanted into nude mice and ability to metastasize. This transformed phenotype displayed focal, multilayer growth and higher saturation density in comparison with the untransformed phenotype. Transplanted tumors as well as metastatic lung tumors in nude mice were adenocarcinomas morphologically similar to typical mammary tumors in humans. This selection procedure of mutant mammary cells from an immortalized cell line derived from normal mammary glands could be very useful to identify the genomic biomarkers in the growth regulation and malignant progression of breast cancer. Introduction One of the major objectives of cancer research is the elucidation of the fundamental molecular–biochemical–biological modulations that are responsible for the development of the cancer phenotype and its evolution into increasingly malignant behavior (1). An accumulation of altered gene expression and/ or mutant genes, whose products mediate signal transduction, control cell cycle, maintain genomic stability, and mediate apoptosis and cellular senescence, is central to carcinogenesis. A healthy cell during the process of replication or growth carries the constant hazard of genetic mutation, and random changes that can impair the regulatory profile of a cell (2). Both spontaneous and chemically induced mutations may contribute to tumor progression. To study the pathogenesis and progression of breast cancer, chemical carcinogen-treated tissue culture and animal model systems have been used extensively with limited success (3). Mutations that cannot be attributed to the exogenous causes are considered spontaneous and may arise from endogenous causes such as depurination and depyrimidation of DNA, proofreading and mismatch errors during DNA replication, deamination of 59methylcytocine to generate a C→T transition mutation and damage to DNA by-products of metabolism such as oxygen free radicals. Abbreviations: FBS, fetal bovine serum; TGF-β, transforming growth factor beta. © Oxford University Press

Spontaneous mutation may also be initiated by deficiencies in the cellular defense mechanism. These include defective DNA repair, low levels of antioxidants, antioxidant enzymes and enzymes that conjugate nucleophiles with DNA damaging electrophiles (4,5). Spontaneous mutagenesis may lead to spontaneous carcinogenesis. Since there are hundreds of genes that are required for the control of cell growth and the stability of the genome in somatic cells, mutations in any one of these genes could initiate tumorigenesis and/or genetic instability, which in turn contributes to the multiplicity of gene mutations and modulations observed in tumors (6). A progressive accumulation of genetic damage and epigenetic alterations can apparently cause normal cells to become transformed and even metastatic. Identification of the mutated genes and the genes whose expression is altered is necessary for our understanding of cancer etiology and for development of strategies for chemoprevention and cancer therapy. Currently, many transformation model systems have been developed in vitro for the elucidation of genetic and epigenetic changes during carcinogenesis (4,7,8). The NMuMG mouse mammary epithelial cell line was established through spontaneous immortalization of normal mouse mammary epithelial cells (9). The cells exhibit many normal, untransformed features (9) and were used previously for the establishment of a morphogenic model in culture (10). In this study, we intended to develop a method to select spontaneously transformed cells from this apparently normal cell population in culture. The sustained anchorageindependent growth potential of transformed cells was exploited in a modified soft agarose assay in which a large number of cells were suspended in a culture medium for several weeks. During this period of adverse growth environment, untransformed cells underwent apoptosis, whereas transformed cells proliferated. We report here the successful isolation of a small number of colonies of transformed NMuMG cells and the characterization of this cell line for its malignant behavior. Materials and methods Animals Female athymic nude mice (nu/nu), 5–6 weeks old, were obtained from Harlan Sprague–Dawley (Indianapolis, IN). The animals were housed under specific pathogen-free conditions. Cell culture The NMuMG mouse mammary gland epithelial cell line (9) was obtained from ATCC (Rockville, MD) at passage number 12. The cell line was adopted to McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS), pyruvate, vitamins, amino acids and antibiotics (11). Working cultures were maintained at 37°C in a humidified atmosphere of 6% CO2. Isolation of clonogenic cells under suspension culture on soft agarose NMuMG cells at passage number 18 were suspended in the 10% FBS medium and plated on the top of 1 ml solidified underlayer of 0.8% soft agarose (Life Technologies, MD) in a 6 well culture plate at 63104 cells/well. After 5 weeks of incubation, visible colonies identified by naked eye were selected and plated on plastic in the 10% FBS medium. The viable cells designated NMuMG-ST were passaged several times before being characterized.

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A.Bandyopadhyay, M.L.Cibull and L.Sun Growth assays Growth curves were determined to compare the growth rates and saturation density between NMuMG and NMuMG-ST cells. The cells were plated in 6 well plates at 63104 cells/well in 10% FBS medium. The medium was changed twice a week. Viable cells (trypan blue exclusion) were counted with a hemocytometer from triplicate wells every other day up to 9 days. Soft agarose assays To determine whether the selected cells are transformed, we first performed soft agarose assays to compare the anchorage-independent clonogenicity between the selected cells and the parental cells as described previously (12). Briefly, cells (1.23104) were suspended in 1 ml of 0.4% soft agarose prepared in the 10% FBS medium and plated on top of a 1 ml underlayer of 0.8% soft agarose in 6 well culture plates. The plates were incubated for 2 weeks in a humidified atmosphere of 6% CO2 at 37°C. Cell colonies were visualized by staining with 1 ml of p-iodonitrotetrazolium violet (Sigma, St Louis, MO). Tumorigenicity and lung metastasis studies in nude mice Tumorigenicity studies were performed as described previously (12). Briefly, cells from exponential cultures of NMuMG and NMuMG-ST cells were inoculated subcutaneously in the flanks of 5-week-old female athymic nude mice. The growth of tumors was monitored every other day and tumor sizes were measured with a caliper in two dimensions. Tumor volumes were calculated with the equation V 5 (L3W2)30.5, where L is length and W is width of a tumor. For the lung metastasis study, lungs were removed during autopsy and examined for nodular macroscopic metastasis. These were verified by histological examinations. Histological examinations Subcutaneous tumor and lung specimens for histological examination were fixed in 10% buffered formalin (Fisher Scientific) for at least 24 h. Representative sections of the tumor and lung were embedded in paraffin. Tissue sections (5 µm) were cut, stained with hematoxylin and eosin and examined by light microscopy.

Results Isolation of spontaneously transformed cells on soft agarose To isolate a population of transformed cells from the untransformed NMuMG cells, we took advantage of the fact that transformed cells can often survive and proliferate under an anchorage-independent condition. The sustained anchorageindependent growth of a few colonies of NMuMG cells while floating on the soft agarose and their subsequent passages on plastic led to the isolation of a cell line designated NMuMGST. These cells were found to be remarkably different from untransformed parent NMuMG cells in morphology, growth properties, tumorigenicity and ability to cause lung metastasis.

Fig. 1. Morphology of the untransformed NMuMG and spontaneously transformed NMuMG-ST cell lines in culture. (A) NMuMG, passage 20, showing a single layer of cuboidal epithelial cells (3200); (B) NMuMG-ST, passage 8, multilayers piled up in focal areas and islands (3200).

Altered growth characteristics of NMuMG-ST cells NMuMG cells grew as a single layer of cuboidal epithelial cells on plastic (Figure 1A). In contrast, NMuMG-ST cells piled up in focal areas and grew as islands (Figure 1B) exhibiting a transformed phenotype (13). The multilayer growth was confirmed after we compared the growth rates and saturation density between NMuMG-ST cells and NMuMG cells. As shown in Figure 2, after a 3-day lag phase, NMuMGST cells started to proliferate at a much faster rate than NMuMG cells. On day 7, NMuMG cells reached plateau phase while NMuMG-ST cells were still growing although at a slower rate. The saturation density of NMuMG-ST cells on day 9 was ~2.5-fold higher than that of NMuMG cells because of the multilayer growth of NMuMG-ST cells. Clonogenicity of NMuMG-ST cells in soft agarose As mentioned earlier, being untransformed, NMuMG cells do not proliferate under anchorage-independent growth conditions. Therefore, they did not form any detectable colonies in soft agarose after 2 weeks of incubation (Figure 3). In contrast, NMuMG-ST cells, being selected by suspension culture on 1908

Fig. 2. Growth curves of untransformed NMuMG and spontaneously transformed NMuMG-ST cells. Cells were plated in 6 well tissue culture plates at 63104 cells/well. Cell numbers in each well at each time point were determined twice using a hemocytometer. Values are means 6 SE of triplicate wells with six determinations.

Spontaneously transformed mammary epithelial cells

Fig. 3. Anchorage-independent colony formation inside soft agarose of untransformed NMuMG and spontaneously transformed NMuMG-ST cells. Exponentially growing cells (1.23104 cells) were suspended in 1 ml 0.4% soft agarose then 10% FBS medium and plated on top of a 1 ml solidified underlayer of 0.8% agarose in the same medium in a 6 well tissue culture plate. After 2 weeks of incubation, cell colonies were visualized by staining with 1 ml p-iodonitrotetrazolium violet.

Fig. 4. Tumor formation by spontaneously transformed NMuMG-ST cells. Exponentially growing cells were inoculated subcutaneously in the flanks of athymic nude mice at a low and a high inoculum, 53106 and 103106 cells, respectively. Tumors were measured externally on the indicated days in two dimensions using a caliper. Tumor volume was determined by the equation V 5 (L3W2)30.5, where L 5 the length and W 5 the width of the tumor. Values are the means 6 SE of 8 and 10 tumors for the low and the high inoculum, respectively.

soft agarose, were able to form colonies when embedded in soft agarose, and exhibited another transformation phenotype. Tumorigenicity of NMuMG-ST cells in nude mice Since the ability of anchorage-independent growth in vitro is often associated with tumorigenic potential in vivo, we next studied the tumorigenicity of NMuMG-ST cells in athymic nude mice. NMuMG cells did not form any visible tumors when inoculated subcutaneously in the flanks of athymic nude mice at 107 cells/site and incubated for up to 10 weeks. In contrast, NMuMG-ST cells started to form tumors within 5 days after inoculation at both 53106 and 107 cells/site (Figure 4). The higher inoculum contributed to the higher tumor growth rate (Figure 4). The mice with the higher inoculum started to die 18 days after inoculation. Sixty percent of them died within 28 days of the study, whereas 100% of the mice with the lower inoculum survived. However, the tumor incidence was 100% for both groups. Most of the tumors in both groups also showed ulceration of overlaying skin when the tumors were ~7–9 mm in diameter. The histopathology of the tumors showed poorly differentiated adenocarcinoma morphologically similar to mammary

Fig. 5. Histology of a tumor formed by the spontaneously transformed NMuMG-ST cell. A representative tumor formed by NMuMG-ST cells at passage 8 was cut into 5 µm tissue sections which were stained with hematoxylin and eosin. The morphology of the tumor cells exhibit a poorly differentiated adenocarcinoma.

Fig. 6. Lung metastases formed by the spontaneously transformed NMuMG-ST cells. Lung tissue sections were obtained from a tumor-bearing mouse inoculated with NMuMG-ST cells and stained with hematoxylin and eosin. Multiple nodular metastases were visible at 340 magnification as shown in panel (A). The tumor tissue (M) is readily distinguishable from the lung tissue depicted with (L). Tumors revealed poorly differentiated adenocarcinoma (B) similar to the primary tumors shown in Figure 5 at a magnification 3200.

tumors in humans (Figure 5). The epithelial nature of the tumor cells was confirmed by morphology as well as positive staining for epithelial cytokeratins (data not shown). Metastatic potential of NMuMG-ST cells The pulmonary invasion potential of the NMuMG-ST tumors was evaluated by the examination of nodular tumors on lungs. The macroscopic lung metastases (data not shown) were verified by histological examination of hematoxylin and eosin stained tissue sections. These metastases were poorly differentiated adenocarcinomas similar to the primary tumors (Figure 6A and B). All the animals inoculated with both the low and high cell numbers developed lung metastases by the termination of the experiments. Discussion This study focuses on the selection of spontaneously transformed malignant cells from the spontaneously immortalized NMuMG mouse mammary epithelial cell line. The normal cellular behavior and characteristics of NMuMG cells were 1909

A.Bandyopadhyay, M.L.Cibull and L.Sun

previously utilized for the establishment of a morphogenic model in culture (10), the study of transdifferentiation of mammary epithelial cells to mesenchymal cells (15), and early gene responses to TGF-β1 (16). In the present study, we observed that the growth of NMuMG cells required adhesion to substratum and was inhibited by cell–cell contact. In contrast, the growth of the spontaneously transformed NMuMG-ST cells was independent of anchorage and was not inhibited by cell–cell contact. NMuMG-ST cells grew into foci and showed multilayer growth with a saturation density of 2.5-fold higher than that of NMuMG cells. In addition, whereas NMuMG cells inoculated at 10 million cells/site had not formed any tumors at up to 10 weeks, NMuMG-ST cells, when inoculated into nude mice at 5 million cells/site, were highly tumorigenic and metastatic. These phenotypes of NMuMG-ST cells are all consistent with the characteristics of malignant transformation (13,16). We had initially sought to select spontaneously transformed cells with the conventional soft agarose assay. However, there was no colony formation when the cells were individually separated and embedded in soft agarose after weeks of incubation. The modified soft agarose assay described in this report allowed cells to adhere to one another while still suspended in the medium. Cell aggregation appeared to have assisted the proliferation of a few transformed cells in our assay although the molecular mechanism of the cell–cell adhesion-dependent proliferation remains to be elucidated. Nevertheless, this modified soft agarose method appears to be an effective way by which cells transformed either by exogenous or endogenous factors can be isolated. A mouse rather than a human untransformed cell line was chosen to test the efficacy of the modified soft agarose method because a mouse cell is much more likely to be fully transformed than a human cell (17,18). As a result, the likelihood of isolating transformed cells from a mouse cell line is much higher. On the other hand, it is known that the number of events taking place during tumor progression is similar in mouse and human cells (18). Thus, while cellular transformation can be obtained by treatment of carcinogens or overexpression of oncogenes, the spontaneously transformed NMuMG-ST cells selected in this study may comprise a cell culture model of spontaneous human breast cancer progression, which will facilitate the identification of the progressive changes in key biomarkers that mark the progression of nonmalignant to fully malignant cellular phenotype. Immortalization of somatic cells is characterized by their indefinite growth in vitro. However, the molecular changes that lead a cell to such a phenotype are not yet fully understood. During immortalization, cells escape several crisis periods from senescence. The changes in cellular properties in the act of emerging from the crisis may continue in some susceptible cells during in vitro culture. Thus, the spontaneous mutations leading to the transformation of NMuMG cells may be initiated originally during immortalization and initial passages. Alternatively, the mutations may have been accumulated during the selection by the modified soft agarose method in which the NMuMG cells were left floating for a prolonged period of time with limited proliferation. Goldie and Coldman (19) demonstrated mathematically that higher mutational frequency was favored in slow growing spontaneous human solid tumors. In a recent report, Richards et al. (20) provided evidence that two human cell lines deficient in a key DNA mismatch repair protein accumulated many more mutations when the cells were maintained at high density with greatly diminished proliferation 1910

than when the cells were allowed to proliferate rapidly. In addition, it has been proposed by Strauss (21) that it is possible that mutations in some cells may accumulate in a timedependent manner in the absence of growth. Thus, adverse growth conditions such as denial of adhesion to substratum may promote spontaneous mutations which may result in the malignant transformation as in the case of NMuMG-ST cells. Normal as well as untransformed cells undergo apoptosis when they are detached from extracellular matrix (22,23 and our unpublished observation). On the other hand, metastatic transformation involves degradation of extracellular matrix and translocation of freely mobile cancer cells to a new site (24). The modified soft agarose method described in this report not only promotes apoptosis of the untransformed cells but also selects the malignant cells that can proliferate in suspension. As such, a cell line selected with this method is devoid of untransformed cells. More importantly, the cells are capable of surviving without attachment to a substratum, a prerequisite for metastasis. Indeed, the highly tumorigenic and metastatic potential of the NMuMG-ST cells is probably attributable to this type of selection process. Therefore, spontaneously transformed cells selected with the modified soft agarose method may be uniquely suited for the identification of the genes involved in tumor invasion and metastasis. Acknowledgements This work was supported by NIH grants CA 63480 and CA 75253 from the National Cancer Institute (to L.-Z.S.).

References 1. Farber,E. (1996) The step-by-step development of epithelial cancer: from phenotype to genotype. Adv. Cancer Res., 70, 21–48. 2. Cavanee,W.K. and White,R.L. (1995) The genetic basis of cancer. Sci. Am., 272, 72–79. 3. Ip,C. (1996) Mammary tumorigenesis and chemoprevention studies in carcinogen-treated rats. J. Mamm. Gland Biol. Neoplasia, 1, 37–47. 4. Venitt,S. (1996) Mechanisms of spontaneous human cancers. Environ. Health Perspect., 104, 633–637. 5. Ames,B.N., Gold,L.S. and Willett,W.C. (1995) The causes and prevention of cancer. Proc. Natl Acad. Sci. USA, 92, 5258–5265. 6. Loeb,L.A. and Christians,F.C. (1996) Multiple mutations in human cancers. Mutat. Res., 350, 279–286. 7. Calaf,G. and Russo,J. (1993) Transformation of human breast epithelial cells by chemical carcinogens. Carcinogenesis, 14, 483–492. 8. Band,V. (1995) Preneoplastic transformation of human mammary epithelial cells. Semin. Cancer Biol., 185–192. 9. Owens,R.B., Smith,H.S. and Hackett,A.J. (1974) Epithelial cell cultures from normal glandular tissue of mice. J. Natl Cancer Inst., 53, 261–269. 10. Hall,H.G., Farson,D.A. and Bissell,M.J. (1982) Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture. Proc. Natl Acad. Sci. USA, 79, 4672–4676. 11. Wu,S.P., Theodorescu,D., Kerbel,R.S., Willson,J.K., Mulder,K.M., Humphrey,L.E. and Brattain,M.G. (1992) TGF-beta 1 is an autocrinenegative growth regulator of human colon carcinoma FET cells in vivo as revealed by transfection of an antisense expression vector. J. Cell. Biol., 116, 187–196. 12. Sun,L., Wu,G., Willson,J.K. et al. (1994) Expression of transforming growth factor beta type II receptor leads to reduced malignancy in human breast cancer MCF-7 cells. J. Biol. Chem., 269, 26449–26455. 13. Band,V. and Sager,R. (1989) Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc. Natl Acad. Sci. USA, 86, 1249–1253. 14. Miettinen,P.J., Ebner,R., Lopez,A.R. and Derynck,R. (1994) TGF-beta 5nduced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J. Cell. Biol., 127, 2021–2036. 17. Koskinen,P.J., Sistonen,L., Bravo,R. and Alitalo,K. (1991) Immediate early gene responses of NIH 3T3 fibroblasts and NMuMG epithelial cells to TGF beta-1. Growth Factors, 5, 283–293.

Spontaneously transformed mammary epithelial cells 16. Filmus,J. and Kerbel,R.S. (1993) Development of resistance mechanisms to the growth-inhibitory effects of transforming growth factor-beta during tumor progression. Curr. Opin. Oncol., 5, 123–129. 17. DiPaolo,J.A. (1983) Relative difficulties in transforming human and animal cells in vitro. J. Natl Cancer Inst., 70, 3–8. 18. Holliday,R. (1996) Neoplastic transformation: the contrasting stability of human and mouse cells. Cancer Survey, 28, 103–115. 19. Goldie,J.H. and Coldman,A.J. (1985) A model for tumor response to chemotherapy: an integration of the stem cell and somatic mutation hypothesis. Cancer Invest., 3, 553–564. 20. Richards,B., Zhang,H., Phear,G. and Meuth,M. (1997) Conditional mutator phenotypes in hMSH2 deficient tumor cell lines. Science, 277, 1523–1526. 21. Strauss,B.S. (1992) The origin of point mutations in human tumor cells. Cancer Res., 52, 249–253. 22. Frisch,S.M. and Francis,H. (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol., 124, 619–626. 23. Boudreau,N., Sympson,C.J., Werb,Z. and Bissell,M.J. (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science, 267, 891–893. 24. Jiang,W.G., Puntis,M.C. and Hallett,M.B. (1994) Molecular and cellular basis of cancer invasion and metastasis: implications for treatment. Br. J. Surg., 81, 1576–1590. Received on March 18, 1998; revised on July 2, 1998; accepted on July 21, 1998

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