Two Syngeneic Cell Lines from Human Breast Tissue: The Aneuploid Mammary Epithelial (Hs578T) and the Diploid Myoepithelial (Hs578Bst) Cell Lines 1.2 Adeline J. Hackett, 3.4 Helene S. Smith, 3 E. Louise Springer, 3 Robert B. Owens, 3 Walter A. Nelson-Rees, 3 John L. Riggs,S and Murray B. Gardner 6 • 7 ABSTRACT-We characterized two human cell lines (Hs578T and Hs578Bst), which provide several unique features that should be useful in the study of breast disease. Hs578T, derived from a carcinosarcoma, is epithelial In origin. Hs578Bst, established from normal tissue peripheral to the tumor, Is myoepithelial in origin. This is the first report of companion cell lines, one malignant and one normal, established from the same organ.-J Natl Cancer Inst 58: 1795-1806, 1977.
MATERIALS AND METHODS
Patient history and pathology.-The patient was a 74year-old Caucasian woman with no family history of cancer or any other disease and no known exposure to carcinogens. The breast tissue excised at surgery showed an infiltrating ductal carcinoma in which a prominent fibrous (desmoplastic) stromal reaction with intermingled lymphocytes obscured most of the carcinoma cells. The large, bizarre, hyperchromatic sarcoma-like cells were thought to be spindle-shaped carcinoma cells or truly malignant mesenchymal cells. In the latter instance, this could be considered a carcinosarcoma (fig. 1). The norVOL. 58, NO.6, JUNE 1977
ABBREVIATIONS USED: FCS = fetal calf serum; EM = electron microscopy; SEM = scanning electron microscopy; TEM = transmission electron microscopy; Kd = dissociation constant; CIM = contactinhibited monolayer; ATS = antithymocyte serum; M-PMV = MasonPfizer monkey virus; p = protein; MuMTV = murine mammary tumor virus; MuRLV = murine Rauscher leukemia virus; RER rough endoplasmic reticulum. Received September 14, 1976; accepted December 2, 1976. Supported by Public Health Service contract NOI CP33237, NO! CP53502, and NO! CP43209 from the Virus Cancer Program within the Division of Cancer Cause and Prevention, National Cancer Institute. 3 Cell Culture Laboratory, School of Public Health, University of California, Berkeley, Calif. 94720. 4 Address reprints requests to Dr. Hackett, Naval Biomedical Research Laboratory, Naval Supply Center, Oakland, Calif. 94625. 5 Viral and Rickettsial Diseases Laboratory, State Department of Health, Berkeley, Calif. 94704. 6 Department of Pathology, University of Southern California (USC), School of Medicine, Los Angeles, Calif. 90033. 7We thank A. Hiller,j. Weaver, S. Sylvester, and R. Flandermeyer for technical assistance.
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The highly variable nature of breast cancer suggests that it consists of several biologically different diseases (1, 2). One possible source of this variability resides in the origin or degree of differentiation of the cell that becomes malignant. Developing and characterizing cell lines derived from each type of breast cancer offer a means of determining whether a cellular basis exists for the variation. At present, however, cell lines that have been verified as human mammary epithelium in origin are scarce (3); BT-20, which was developed from ductal cells of the breast, is the best known and completely characterized cell line established from a primary breast carcinoma (4), although another line Bot-2 has been described recently (5). Also, several cell lines have been established from breast tumors metastatic to the lung (6-11), but none derived from other metastatic sites; only one, HBI00, derived from normal human mammary epithelium (12, 13). The best characterized of these metastatic cell lines is MCF-7 (8), which produces tumors in mice (14), is hormone-responsive (15, 16), and is secretory (17). In this communication we describe two cell lines, derived from the same breast, which provide several unique features that should be useful in the study of breast cancer. One line, Hs578T was derived from a carcinosarcoma, which is a rare form of breast cancer. It is epithelial, aneuploid, with distinctive chromosomes, produces casein, and lacks estrogen-receptor protein. The other cell line, Hs578Bst, established from normal tissue peripheral to the tumor, is diploid and possibly of myoepithelial origin.
mal tissue was ta~en from the same breast, but from an area distal to the tumor; no tumor cells were identified in it. Initiation of cultures.-The samples of breast tissue were shipped at melting-ice temperature to this laboratory from Dr. M. B. Gardner (USC School of Medicine) on October 15, 1971. Tissues were minced to fine 1- to 2mm 2 fragments, seeded in Falcon flasks (Falcon Plastics, Oxnard, Calif.), and covered with Leibovitz's L-15 medium (18) with 30% FCS. The medium was changed to Dulbecco's modification of Eagle's minimum essential medium (Gibco, Grand Island, N.Y.) with 10% FCS (Gibco) after outgrowth was obtained. The tumor cells were designated Hs578T (Homo sapiens No. 578, tumor cells), grew well, and were subcultured three times before storage of several am pules in liquid nitrogen. The normal culture, Hs578Bst (H. sapiens No. 578, breast cells), also grew well and was frozen-stored at passage 3 as well. Morphology and growth pattern.-Replicate cultures of each cell line were made in Leighton tubes containing glass cover slips. The cultures were fixed in methanol and stained with May-Griinwald-Giemsa. Cultures on glass cover slips were dehydrated in acetone:xylene and mounted in Coverbond (Harleco, Gibbstown, N.J .). Saturation density.-A confluent culture of each line was subcultured, and eight replicate cultures were seeded in 25-cm2 plastic flasks. These cultures were fed every 2-3 days throughout the test. When confluent, two cultures were harvested and the cells counted in a hemacytometer at 2- or 3-day intervals until no further increase in cell number was observed. Karyology.-For conventional morphology and enumeration of chromosomes, meta phases were stained with Giemsa following hypotonic treatment and air-
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MuRLV p30 was obtained from Dr. T. August (Albert Einstein School of Medicine, New York, N.Y.). The RD 114 virus p30 was prepared by Dr. J. Riggs. The following cell lines were utilized as positive controls in the experiments to detect virus antigens: monkey cell line, CMMT, chronically infected with M-PMV (31); murine mammary tumor cell line, Mm5Mt, releasing MuMTV (32); dog cell line, D17, chronically infected with ATS-124 virus or RD114 virus (33); and murine cell line, JLS-V5, releasing MuRL V (34). Methods for growth and maintenance of cell lines have been described (32). RESULTS Morphology of Tumor Cell Line Hs578T
The Hs578T culture formed a mosaic growth pattern on plastic surfaces. Two morphologic forms were present in the culture at early passage: polygonal, stellate cells and large spindle-shaped cells. The nuclei were large, bizarre, and lobulated with multiple nucleoli. Aberrant mitotic figures, binucleate cells, and multinucleate cells were common. The nuclear-cytoplasmic ratio was high. Both morphologic forms were still present at passage "26. A colony was then picked and passaged. This culture, now at passage 52, consists of a single stellate cell form (fig. 2A). Further passage of the original culture also yielded a homogeneous population of the stellate cell type by passage 30. It appears that the large spindle-shaped cells described in the pathology report and the early passages were a minority component and were lost during passaging. We cannot be certain whether they are malignant myoepithelial or stromal cells or simply reflect a pleomorphism of the carcinoma cells. EM of cells at passages 9 and 11 showed both morphologic forms. The cells contained several ultrastructural features consistent with cancer, such as large, dentated nuclei with clumped marginated heterochromatin and multiple aberrant nucleoli; intracytoplasmic duct-like vacuoles lined with microvilli and containing filamentous material; confronting cisternae; and aberrant mitochondria (figs. 3A-C). The cells also contained ultrastructural features of epithelium, such as tight junctions and desmosomes. The presence of lipid droplets, well-developed Golgi apparatus, vacuoles, dilated RER, vesicularized smooth endoplasmic reticulum, and numerous polyribosomes indicate secretory activity that is a characteristic of mammary epithelial cells (figs. 3D, E). The mammary origin of the cells was confirmed by ultrastructural evidence for the presence of the milk protein, casein. Casein production was most prominent when cells had been held at confluence and dome formation (35) had taken place. Casein was identified by observation of aggregates of protein granules with the periodicity and organization of casein (fig. 3F). Morphologic Features of Normal Cell Line Hs578Bst
The Hs578Bst cell line had a fibroblast-like appearance in the light microscope. At confluence the cells VOL. 58, NO.6, JUNE 1977
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drying. The trypsin-Giemsa method (19, 20) for Gbanding was used for a study of marker chromosomes and Q-banding after the method of Lin et al. (21) revealed the presence or absence of the human Y-chromosome. EM.-Semiconfluent and confluent monolayers of both cell lines were prepared as previously described for SEM (22) and TEM (23). Mycoplasma test5 .-The two lines were repeatedly tested for the presence of mycoplasma and were consistently negative. Tests were done at the Naval BioSciences Laboratory (Oakland, Calif.) according to a modification of the method of M. Barile and by SEM and TEM. Estrogen-specific binding protein.-The techniques described by Wittliff (24) and Wittliff and Savlov (25) for the dextran-coated charcoal assay for estrogen-receptor protein were used. Specific binding curves were calculated from the difference between the noncompetitive and competitive binding reactions. The Kd and number of binding sites were calculated by the method of Scatchard (26). We normalized the results for protein content using the Folin method for protein determination. MCF-7 cells (15) were used as positive controls. Growth in semisolid medium.-Two ml growth medium plus 0.9% agar (Bacto-agar; Difco Laboratories, Detroit, Mich.) was added to 60-mm diameter tissue culture dishes. After the agar hardened, cells were trypsinized and suspended in growth medium containing 1.2% Methocel [4,000 cP, # 60 (high grade); Dow Chemical Co., Midland, Mich.] and various numbers of cells were plated over the agar layer. Plates were incubated at 37° C without subsequent feeding. One day after plating, the dishes were observed with an inverted microscope to determine that no cell clumps were present initially. The number and size of the colonies that subsequently formed were estimated 3 weeks post seeding. Colony Formation CIM.-Confluent monolayers were prepared in tissue culture dishes 60 mm in diameter. Hs578T and Hs578Bst cells were seeded onto a confluent monolayer of an epithelial-like cell line FHs 74 Int (23) and a fibroblast-like line Hs392Sk (27). Cultures to be tested were trypsinized and seeded at 103 and 100 cells/dish; the fluid was changed twice weekly for 21 days, fixed in methanol, and stained with May-Griinwald-Giemsa. Tumorigenicity.-Adult female NAMRU mice (Naval Biomedical Research Laboratory) were inoculated with A TS (Microbiological Associates, Los Angeles, Calif.) as described (28). Cells were trypsinized, centrifuged, and resuspended in medium lacking serum and in 0.1 or 0.2 ml volumes containing 5x 106 cells immediately injected intradermally into the interscapular region. No palpable nodules were observed in the 20 control mice inoculated with medium only. Virus detection.-Supernatants were assayed at various passage levels for RNA-dependent DNA polymerase with the use of the technique described previously (29). Immunofluorescence was done by the indirect methods described by Riggs (30). The M-PMV p27 and MuMTV p27 were obtained from Dr. R. Cardiff (Department of Pathology, University of California, Davis, Calif.).
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Growth Characteristics
Growth characteristics for both cell lines, at two passages, are listed in table 1. The tumor-derived cells grew rapidly to saturation at 22 X 104 cells/cm 2 , forming a mosaic pattern. The normal tissue-derived cells grew rapidly and reached saturation at 5.5X 104 cells/cm 2 • Malignant Characteristics
Both cell lines were tested for properties associated with cancer; they were grown in semisolid medium, immunosuppressed mice, and on CIM. Growth in semisolid medium (Methocel) was assayed at passages 13, 28, and 35 for the tumor-derived line and at passages 8 and 15 for the normal tissue-derived line. VOL. 58, NO.6, JUNE 1977
The latter did not grow in Methocel at either passage, whereas all passages of the tumor line grew well. Colony formation was consistently better in monkey serum than in FCS. Representative data are shown in table 2. Both cell lines were injected intradermally into immunosuppressed mice, at dosages of 3,5, and lOX 106 cells. Neither line produced tumors. Hs578T culture readily formed colonies of equal efficiency when plated on CIM of human epithelial and fibroblast cells. The tumor cells formed flat, smooth colonies sharply defined at the edge on the epithelial cell monolayers (fig. 5, inset). The cells on fibroblastic monolayers formed irregular colonies that followed the pattern of the substrate layer. Dilution plating of the tumor cells on both types of CIM at passage levels 22, 28, 31, and 35 yielded plating efficiences of 50%, which were reproducible at each passage level. The diploid Hs578Bst line did not form colonies on either monolayer. A representative experiment is shown in figure 5, along with an inset of a typical colony of tumor cells on an epithelial monolayer. Karyology and Isoenzymes
The tumor cells Hs578T had a unique, abnormal karyotype. Many rearranged or "marker" chromosomes were present, in addition to at least one normal (haploid) set of human chromosomes per cell. Figure 6 presents the proposed origin of five marker chromosomes presumably arising by rearrangement of normal chromosomes. Marker #1 involved the rearrangement of an X-chromosome and an autosome (#ll). One normal Xchromosome was noted. At early passages, 3 and 4, the TABLE
I.-Growth characteristics ofHs578T and Hs578Bst cell lines
Characteristics Growth rate Saturation density/ cm 2 Growth pattern Cell type
TABLE
Hs578Bst, passages 7,30
Hs578T, passages 10,16 Rapid 22x10 4
Moderate 5.5x10 4
Mosaic, multilayering "dome" formation Pleomorphism, short-spindle, polygonal
Flat, swirling multilayered Spindle
2.-Growth of Hs578T and Hs578Bst cells in semisolid medium (Methocel)
Cell line: Passage level Hs578T: 28 Hs578T: 35 Hs578Bst: 8 Hs578Bst: 15
FCSa
Monkey serum a
No. of cells seeded
Small colonies
Large colonies
Small colonies
Large colonies
104 103 104 103 104 103 104 103
125 14 TMb 6 0 0 0 0
TMb 1 17 0 0 0 1 0
150 6 182
25 0 42 2 0 0 0 0
22
0 0 0 0
a Plates were scored for colonies 3 wk post seeding; small colony=4-100 cells/colony and large colony = >100 cells/colony. b TM=too many to count.
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formed a swirling pattern and multiple layers, whereas at subconfluence the cells were uniformly spindleshaped and oriented in parallel rows. However, these cells also had features not characteristic of fibroblasts; the nuclei seemed more round to ovoid than elongated, as expected for fibroblasts, and they were sometimes displaced rather than centrally located; multiple nucleoli were conspicuous. The cells had features characteristic of nonmalignant cells: The nuclear-cytoplasmic ratio was low, the nuclear margins were regular, the nucleoli were small, and the cytoplasm was eosinophilic and pale (fig. 2B). Certain ultrastructural features of the Hs578Bst cells were more consistent with a myoepithelial rather than fibroblastic origin. One of the most distinctive features of myoepithelial cells is the presence of micro filaments of 60 A diameter, which in localized areas show thickening and electron density analogous to the Z-bands in muscle cells (36). The number and arrangement of the microfilaments vary with the degree of differentiation of the myoepithelial cell. In addition, these cells generally have evidence of secretory activity and contain pinocytotic vesicles clustered between the regional areas of filament thickening (36, 37). Ultrathin sections of Hs578Bst cells showed these typical myoepithelial features. A cross section showed multiple layers of elongated cells containing cells with two different levels of differentiation (fig. 4). Both cell types contained the characteristic myoepithelial microfilaments, but in varying amounts. Areas of increased electron density (Zband-like) could be seen. As is often seen in myoepithelial cells in vivo, the parallel microfilaments were sometimes confined to one side of the cell only (36). Both cells also had pinocytotic vesicles with typical myoepithelial cell localization. Only one of the two cell types showed features suggestive of secretory activity. These cells had extensive dilated RER containing electrondense material typical of cells actively synthesizing protein. These cells also contained fine filamentous threads emanating from the cell surfaces that resembled elastin fibers found in myoepithelial cells in vivo (36, 37). Possibly this amorphous, electron-dense intercellular material is basement membrane (37). Myoepithelial cells in vivo form few desmosomes, and none was observed in vitro. Collagen, characteristic of fibroblasts, was not observed either (fig. 4).
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Hormone Receptors
The estrogen-receptor protein was not detectable in either the tumor line Hs578T at passage 14 or normal Hs578Bst at passage 13; whereas under similar conditions, the MCF-71ine bound 42.5 fmole protein/mg with a Kd of 3.3x 10-9 • These values agreed with data obtained on MCF-7 by Brooks et aJ. (15). In addition, the 4S sedimentatio'n coefficient for MCF-7 reported in (16) was confirmed. Associated Endogenous Virus
Evidence for the presence of endogenous virus was sought by EM, assay for reverse transcriptase, and immunofluorescence methods. Extensive EM surveys of Hs578T and Hs578Bst cells at various passage levels revealed no evidence of particles characteristic of type C, type B, or herpesvirus. Assays for reverse transcriptase in supernatants were consistently negative. Cover slip-grown monolayers were tested by immunofluorescence with both fixed and live cells. Antisera prepared against purified viral proteins of known mammalian oncornaviruses were used. No evidence of viral antigen was detected (table 3). In addition, sera from the donor and 6 other breast cancer patients were tested for the presence of antibodies to the cellular antigens of the two cell lines, and no antibodies were found. TABLE
Cell lines tested a Hs578T HS578Bst D17 infected with RD114 CMMT MmsMt D17 infected with ATS-124 JLS-V5
DISCUSSION
In normal breast tissue, ductal epithelium and myoepithelium are closely associated, and maintenance of an integrated structure depends on this association (38, 39). In carcinosarcomas, which are a rare form of breast disease, a complex histologic pattern involving these two cell types exists (38, 40, 41). Typically, this tumor displays epithelial cells with squamous differentiation composed of spindle-shaped or stellate cells in a mucoid basophilic stroma (40). Hyperplasia of myoepithelial cells has been noted peripheral to the tumor (41). This form of breast disease might be included in the scirrhous carcinoma classification of Murad (38), who suggested a myoepithelial cell origin for this class. In this study both cell types were isolated from a carcinosarcoma, mammary tumor epithelial cell line Hs578T and normal myoepithelial cell line Hs578Bst. The characteristics that identify the Hs578T cell line as epithelial and mammary in origin are listed in table 4. The polygonal shape, dome formation, and the presence of desmosomes identify the cells as epithelial; cytoplasmic organelles for secretion, the presence of lipid droplets, and casein granules identify the cell as mammary. Although several other cell lines derived from metastatic mammary tumors produce domes at confluence (7, 8, 10), casein production detectable by EM has not been previously reported in a human cell line. However, Gaffney (12) and Polanowski (13) report that cells established in culture from human milk produce casein detectable by biochemical techniques. The presence of casein in a tumor from a postmenopausal woman is expected, since elevated serum levels of casein have been found by radioimmunoassay in women with breast cancer (42). The absence of estrogen-receptor protein suggests that estrogen may not be necessary for casein synthesis. This cell line offers a system for the study of hormonal effects on casein synthesis free of the synergistic and antagonistic interplay between hormones encountered in vivo. The malignancy of the Hs578T cell line is suggested by the properties listed in table 5. The growth properties of cultured cells that are correlated with cancer (27, 43) are found in Hs578T with the exception that tumors were not produced in immunosuppressed mice. This property may be due to a need for hormonal stimulation
3.-Attempts to detect evidence for endogenous viruses in Hs578T and Hs578Bst cell lines RTb
ND
EMc
IF A using sera to viral antigen d ATS-124
MuMTV
MuRLV
+
+ +
,:
ND
+ +
±
+
+
±
+
+
M-PMV
RD114
+
+
+ +
a Source
and characteristics of cell lines are given in "Materials and Methods." RT=reverse transcriptase; ND=not determined. c Survey of >300 cells by EM. dlFA=indirect fluorescent antibody; source of antibody and type listed in "Materials and Methods." b
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number of chromosomes ranged from 31 to 59 (a mode of 58), and there was 25% polyploidy for the original aberrant karyotype with cells containing approximately 110 chromosomes. At later passages, 6, 12, 23, 24, and 29, the frequency of this polyploidy increased to 75%. At passage 26, a colony was picked; at passages 40-42 thereof, most cells (75-85%) were polyploid. The Hs578Bst cells had the G-banding pattern of a normal diploid female. Through 7 passages diploidy was consistent, and only 1-5% of the cells were polyploid. For unknown reasons, mitotic arrest with Colcemid was not easily achieved in either line. Isoenzyme mobility patterns for glucose-6-phosphate dehydrogenase were tested by Dr. W. D. Peterson,]r. (Children Research Center of Michigan, Detroit, Mich.) and revealed type B or "slow" movement for both cell lines, conforming to the race of the donor, a Caucasian.
SYNGENEIC MAMMARY EPITHELIAL MYOEPITHELIAL CELL LINES TABLE
4.-Characteristics of mammary epithelial cells displayed by Hs578T and Hs578Bst cell lines
Characteristics Ultrastructure: Desmosomes Tonofibrils Intracytoplasmic ductlike vacuoles Casein production Lipid production 60 A microfilaments Biochemistry: Estrogen-receptor sites
Hs578T
Hs578Bst
+ +
+ + +
+
5.-Characteristics of malignant cells displayed by Hs578T and Hs578Bst cell lines
Characteristics Morphology: High nUclear-cytoplasmic ratio Basophilia Bizarre nuclei Karyotype: Aneuploid Growth: High saturation density Multilayered Growth in Methocel Growth on CIM Growth in immunosuppressed mice
Cell lines Hs578T
Hs578Bst
+ + + + + + + +
+
over and above the levels in normal adult NAMRU mice (14). The aneuploidy, which was present at early passage and which appeared to stabilize at passage 40, is also compatible with cancer. The line is made unique and distinctive by virtue of banding markers in the chromosome complement (fig. 6). Data presented here support the identification of the Hs578Bst cell line as normal (table 5). The cells are diploid and have no unusual features, with the possible exception of the number of nucleoli. Further studies to confirm the origin of these cells are in progress; evidence is being sought for the components of myosin and responsiveness to prolactin and oxytocin. It has been suggested that epithelial and myoepithelial cells act in concert and that myoepithelial cells respond to growth regulatory stimuli received from the blood and regulate information and transport of nutrients to epithelial cells (36, 37). Uncontrolled proliferation of epithelial cells may result in concomitant myoepithelial hyperplasia. Such myoepithelial hyperplasia in the tumor-bearing breast may account for the successful isolation of the Hs578Bst myoepithelial cell line. Some obvious questions concerning mesenchymal and epithelial interactions in mammary tumorigenesis may now be amenable to experimental study with these cell lines. REFERENCES (1) KENNEDY BJ: Hormonal therapies in breast cancer. Semin Oncol 1: 119-130, 1974 VOL. 58, NO.6, JUNE 1977
(2) - - - : Appearance and spread of human breast cancer. Recent
Results Cancer Res 42:31-37, 1973
(3) LASFARGUES EY: New approaches to the cultivation of human
breast carcinomas. In Human Tumor Cells In Vitro (Fogh J, ed). New York: Plenum Press, 1975, p 51 (4) LASFARGUES EY, OZELLO L: Cultivation of human breast carcinoma. J Natl Cancer Inst 21:1131-1147,1958 (5) NORDQUIST RE, ISHMALL DR, LOVIG CA, et al. The tissue culture and morphology of human breast tumor cell line BOT -2. Cancer Res 35:3100-3105, 1975 (6) YOUNG RK, CAILLEAU RM, MACKAY B, et al: Establishment of epithelial cell line MDA-MB-157 from metastatic pleural effusion of human breast carcinoma. In Vitro 9:239-245, 1974 (7) CAILLEAU RM, MACKAY B, YOUNG RK, et al: Tissue culture studies on pleural effusions from breast carcinoma patients. Cancer Res 34:801-809, 1974 (8) SOULE HD, VASQUEZ J, LONG AS, et al: A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 51:1409-1416, 1973 (9) REED MV, GEY GO: Cultivation of normal and malignant human lung tissue. Lab Invest 11 :639-652, 1962 (10) CAILLEAU R, CRUCIGER Q, HOKANSON KM, et al: Morphological, biochemical and chromosomal characteristics of breast tumor lines from pleural effusions. In Vitro 12 :331, 1976 (11) FOGH J, TREMPE G: New human tumor cell lines. In Human Tumor Cells In Vitro (Fogh J. ed). New York: Plenum Press, 1975, pp 115-159 (12) GAFFNEY EV: The hormone response of secreting and nonsecreting human breast cells in culture. In Vitro 12:328, 1976 (13) POLANOWSKI FP, GAFFNEY EV, BURKE RE: HBL100: A cell line established from human breast milk. In Vitro 12:328, 1976 (14) RussoJ, BRENNAN MJ, RICH MA: Induction of tumor growth by inoculation of a human breast cancer cell line MCF-7 into ovary or pituitary grafted nude mice. Proc Am Cancer Soc 19:116, 1976 (15) BROOKS SC, LOCKE ER, SOULE HD: Estrogen receptor in a human cell line (MCF-7) from breast carcinoma.J BioI Chem 248:62516253, 1973 (16) HORWITZ KB, COSTLOW ME, McGUIRE WL: MCF-7: A human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors. Steroids 26:785-795, 1975 (17) ROSE HN, McGRATH CM: a-Lactalbumin production in human mammary carcinoma. Science 190:673-675, 1975 (18) LEIBOVITZ A: The growth and maintenance of tissue cell cultures in free gas exchange with the atmosphere. Am J Hyg 78:173180, 1963 (19) NELSON-REES WA, FLANDERMEYER RR, HAWTHORNE PK: Distinctive banded marker chromosomes of human tumor cell lines. Int J Cancer 16:74-82, 1975 (20) - - - : Banded marker chromosomes as indicators of intraspecies cellular contamination. Science 184:1093-1096, 1974 (21) LIN CC, UCHIDA lA, BYRNES EA: A suggestion for the nomenclature of the fluorescent banding patterns in human metaphase chromosomes. Can J Genet Cytol 13:361-363, 1971 (22) SPRINGER EL, HACKETT AJ, NELSON-REES WA: Alteration of the cell membrane architecture during suspension and monolayer culturing. Int J Cancer 17:407-415, 1976 (23) OWENS RB, SMITH HS, NELSON-REES WA, et al: Epithelial cell cultures from normal and cancerous human tissues. J Natl Cancer Inst 56:843-849, 1976 (24) WITTLIFF JL: Specific estrogen receptors in the lactating mammary gland of the rat. Biochemistry 12:3090-3096, 1973 (25) WITTLIFF JL, SAVLOV ED: Estrogen-binding capacity of cytoplasmic forms of the estrogen receptors in human breast cancer. In Estrogen Receptors in Human Breast Cancer (McGuire WL, Carbone PP, Vollmer EP, eds). New York: Raven Press, 1975, p 72 (26) SCATCHARD G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672, 1949 (27) SMITH HS, OWENS RB, HILLER AJ, et al: The biology of human cells in tissue culture. I. Characterization of cells derived from osteogenic sarcomas. IntJ Cancer 17:219-234, 1976 (28) ARNSTEIN P, TAYLOR DO, NELSON-REES WA, et al: Propagation of human tumors in antithymocyte serum-treated mice. J Natl Cancer Inst 52:71-84, 1974 J NATL CANCER INST
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TABLE
Cell lines
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(36) ELLIS RA: Fine structure of the myoepithelium of the exocrine sweat glands of man. J Cell BioI 27:551-563, 1965 (37) OZZELLO L: Epithelial-stromal junction of normal and dysplastic mammary glands. Cancer 25:586-600, 1970 (38) MURAD TM: A proposed histochemical and electron microscopic classification of human breast cancer according to cell of origin. Cancer 27:288-299, 1971 (39) DUNN TB: Morphology of mammary tumors in mice. In The Physiopathology of Cancer (Hornberger F, ed), 2d ed. New York: Paul B. Hoeber, 1959, pp 238-292 (40) HARRIS M, PERSAUD V: Carcinosarcoma of the breast. J Pathol 112:99-105, 1974 (41) HAMPERL H: The myothelia (myoepithelial cells) normal state, regressive changes, hyperplasia, tumors. Curr Top Pathol 53: 162-220, 1970 (42) HENDRICK LC, FRANCHIMONT P: Rdaioimmunoassay of casein in the serum of normal subjects and of patients with various malignancies. Eur J Cancer 10:725-730, 1974 (43) TOOZE J: The molecular biology of tumor viruses, chapt 2. Cold Spring Harbor Laboratory Press, New York, 1973, pp 74-172
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(29) HACKETT AJ: Monitoring for presence of oncogenic virus in tissue culture. In Tissue Culture Methods and Applications (Kruse PF, Patterson MK, eds). New York: Academic Press, 1973, pp 539-548 (30) RIGGS JL: Application of fluorescent antibody techniques to viral infections. Industr Med Surg 34:269-277, 1965 (31) NELSON-REES WA, HOOSER LE, HACKETT AJ: Chronic poliovirus infection of co-cultivated monkey cells harboring the MasonPfizer monkey virus. J Natl Cancer Inst 49:713-725, 1972 (32) OWENS RB, HACKETT AJ: Tissue culture studies of mouse mammary tumor cells and associated virus. J Natl Cancer Inst 49:1321-1332, 1972 (33) RIGGS JL, McALLISTER RM, LENNETTE EH: Immunofluorescent studies of RD114 virus replication in cell culture. J Gen Virol 25:21-29, 1974 (34) WRIGHT BS, LASFARGUES EY: Multiplication of the Rauscher leukemia virus in a mixed culture of mouse spleen and thymus cells. Proc Am Assoc Cancer Res 5:70, 1964 (35) MCGRATH CM: Replication of mammary tumor virus in tumor cell cultures: Dependence on hormone-induced cellular organization. J Natl Cancer Inst 47:455-467, 1971
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I.-Histopathologic section of tumor of origin (HT860). Infiltrating ductular carcinoma with prominent fibrous stromal reaction. Hyperchromatic sarcoma-like cells could be malignant mesenchymal cells, in which case this could be considered carcinosarcoma.
FIGURE
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FIGURE
1802
2.-Light micrographs of monolayer cultures: A) Hs578T, passage 22; B) Hs578Bst, passage 7.
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FIGURE 3.-EM of Hs578T cells showing ultrastructural markers associated with cancer (A-C) and mammary epithelial cells (D-F). A) Nucleus
with 4 nucleoli with dense nucleonemal network and spotty pars amorpha. X 8,800. B) Microvillus-lined intracytoplasmic duct-like vacuole containing fibers and cell debris. x 11,000. C) Triad and duplex of confronting cisternae. x 40,000. D) Two cells with well-formed desmosomal junctions and tonofibrils. x 30,000. E) Perinuclear vesicular Golgi (g), lipid droplet (I), aberrant mitochondria (m). X 7,000. F) Compact protein granule (casein) in vacuole near cell surface. x 60,000
HACKETT ET AL.
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4.-EM of multilayered Hs578Bst cells. One cell has dilated RER (R); another is filled with 60 A microfilaments with regional electrondense condensation (c) and slender elongated mitochondria (M). Threads, similar to elastin (arrow), contribute to extracellular matrix resembling basement membrane. X 24,000
FIGURE
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~
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5
5.-Colony formation on CIM of epithelial cells (A) and fibroblasts (B). Note colony of Hs578T cells growing on epithelial CIM (top, center). Dishes, top row, mock. Middle row, Hs578T cells seeded at two dilutions, 100 and 1,000 cells/dish. Hs578Bst cells were seeded similarly (bottom row).
FIGURE
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3
3 ,0
6
6.-Karyotype of Hs578T cell, passage 24, with 68 chromosomes. At least one complement of normal human chromosomes, including X, is present, but most chromosomes are in multiple copies. Rearranged "marker" chromosomes (bottom row), including X-autosome translocation, are labeled according to presumed origin.
FIGURE
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HACKETT ET AL.
MATERIALS AND METHODS
Patient history and pathology.-The patient was a 74year-old Caucasian woman with no family history of cancer or any other disease and no known exposure to carcinogens. The breast tissue excised at surgery showed an infiltrating ductal carcinoma in which a prominent fibrous (desmoplastic) stromal reaction with intermingled lymphocytes obscured most of the carcinoma cells. The large, bizarre, hyperchromatic sarcoma-like cells were thought to be spindle-shaped carcinoma cells or truly malignant mesenchymal cells. In the latter instance, this could be considered a carcinosarcoma (fig. 1). The norVOL. 58, NO.6, JUNE 1977
ABBREVIATIONS USED: FCS = fetal calf serum; EM = electron microscopy; SEM = scanning electron microscopy; TEM = transmission electron microscopy; Kd = dissociation constant; CIM = contactinhibited monolayer; ATS = antithymocyte serum; M-PMV = MasonPfizer monkey virus; p = protein; MuMTV = murine mammary tumor virus; MuRLV = murine Rauscher leukemia virus; RER rough endoplasmic reticulum. Received September 14, 1976; accepted December 2, 1976. Supported by Public Health Service contract NOI CP33237, NO! CP53502, and NO! CP43209 from the Virus Cancer Program within the Division of Cancer Cause and Prevention, National Cancer Institute. 3 Cell Culture Laboratory, School of Public Health, University of California, Berkeley, Calif. 94720. 4 Address reprints requests to Dr. Hackett, Naval Biomedical Research Laboratory, Naval Supply Center, Oakland, Calif. 94625. 5 Viral and Rickettsial Diseases Laboratory, State Department of Health, Berkeley, Calif. 94704. 6 Department of Pathology, University of Southern California (USC), School of Medicine, Los Angeles, Calif. 90033. 7We thank A. Hiller,j. Weaver, S. Sylvester, and R. Flandermeyer for technical assistance.
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The highly variable nature of breast cancer suggests that it consists of several biologically different diseases (1, 2). One possible source of this variability resides in the origin or degree of differentiation of the cell that becomes malignant. Developing and characterizing cell lines derived from each type of breast cancer offer a means of determining whether a cellular basis exists for the variation. At present, however, cell lines that have been verified as human mammary epithelium in origin are scarce (3); BT-20, which was developed from ductal cells of the breast, is the best known and completely characterized cell line established from a primary breast carcinoma (4), although another line Bot-2 has been described recently (5). Also, several cell lines have been established from breast tumors metastatic to the lung (6-11), but none derived from other metastatic sites; only one, HBI00, derived from normal human mammary epithelium (12, 13). The best characterized of these metastatic cell lines is MCF-7 (8), which produces tumors in mice (14), is hormone-responsive (15, 16), and is secretory (17). In this communication we describe two cell lines, derived from the same breast, which provide several unique features that should be useful in the study of breast cancer. One line, Hs578T was derived from a carcinosarcoma, which is a rare form of breast cancer. It is epithelial, aneuploid, with distinctive chromosomes, produces casein, and lacks estrogen-receptor protein. The other cell line, Hs578Bst, established from normal tissue peripheral to the tumor, is diploid and possibly of myoepithelial origin.
mal tissue was ta~en from the same breast, but from an area distal to the tumor; no tumor cells were identified in it. Initiation of cultures.-The samples of breast tissue were shipped at melting-ice temperature to this laboratory from Dr. M. B. Gardner (USC School of Medicine) on October 15, 1971. Tissues were minced to fine 1- to 2mm 2 fragments, seeded in Falcon flasks (Falcon Plastics, Oxnard, Calif.), and covered with Leibovitz's L-15 medium (18) with 30% FCS. The medium was changed to Dulbecco's modification of Eagle's minimum essential medium (Gibco, Grand Island, N.Y.) with 10% FCS (Gibco) after outgrowth was obtained. The tumor cells were designated Hs578T (Homo sapiens No. 578, tumor cells), grew well, and were subcultured three times before storage of several am pules in liquid nitrogen. The normal culture, Hs578Bst (H. sapiens No. 578, breast cells), also grew well and was frozen-stored at passage 3 as well. Morphology and growth pattern.-Replicate cultures of each cell line were made in Leighton tubes containing glass cover slips. The cultures were fixed in methanol and stained with May-Griinwald-Giemsa. Cultures on glass cover slips were dehydrated in acetone:xylene and mounted in Coverbond (Harleco, Gibbstown, N.J .). Saturation density.-A confluent culture of each line was subcultured, and eight replicate cultures were seeded in 25-cm2 plastic flasks. These cultures were fed every 2-3 days throughout the test. When confluent, two cultures were harvested and the cells counted in a hemacytometer at 2- or 3-day intervals until no further increase in cell number was observed. Karyology.-For conventional morphology and enumeration of chromosomes, meta phases were stained with Giemsa following hypotonic treatment and air-
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MuRLV p30 was obtained from Dr. T. August (Albert Einstein School of Medicine, New York, N.Y.). The RD 114 virus p30 was prepared by Dr. J. Riggs. The following cell lines were utilized as positive controls in the experiments to detect virus antigens: monkey cell line, CMMT, chronically infected with M-PMV (31); murine mammary tumor cell line, Mm5Mt, releasing MuMTV (32); dog cell line, D17, chronically infected with ATS-124 virus or RD114 virus (33); and murine cell line, JLS-V5, releasing MuRL V (34). Methods for growth and maintenance of cell lines have been described (32). RESULTS Morphology of Tumor Cell Line Hs578T
The Hs578T culture formed a mosaic growth pattern on plastic surfaces. Two morphologic forms were present in the culture at early passage: polygonal, stellate cells and large spindle-shaped cells. The nuclei were large, bizarre, and lobulated with multiple nucleoli. Aberrant mitotic figures, binucleate cells, and multinucleate cells were common. The nuclear-cytoplasmic ratio was high. Both morphologic forms were still present at passage "26. A colony was then picked and passaged. This culture, now at passage 52, consists of a single stellate cell form (fig. 2A). Further passage of the original culture also yielded a homogeneous population of the stellate cell type by passage 30. It appears that the large spindle-shaped cells described in the pathology report and the early passages were a minority component and were lost during passaging. We cannot be certain whether they are malignant myoepithelial or stromal cells or simply reflect a pleomorphism of the carcinoma cells. EM of cells at passages 9 and 11 showed both morphologic forms. The cells contained several ultrastructural features consistent with cancer, such as large, dentated nuclei with clumped marginated heterochromatin and multiple aberrant nucleoli; intracytoplasmic duct-like vacuoles lined with microvilli and containing filamentous material; confronting cisternae; and aberrant mitochondria (figs. 3A-C). The cells also contained ultrastructural features of epithelium, such as tight junctions and desmosomes. The presence of lipid droplets, well-developed Golgi apparatus, vacuoles, dilated RER, vesicularized smooth endoplasmic reticulum, and numerous polyribosomes indicate secretory activity that is a characteristic of mammary epithelial cells (figs. 3D, E). The mammary origin of the cells was confirmed by ultrastructural evidence for the presence of the milk protein, casein. Casein production was most prominent when cells had been held at confluence and dome formation (35) had taken place. Casein was identified by observation of aggregates of protein granules with the periodicity and organization of casein (fig. 3F). Morphologic Features of Normal Cell Line Hs578Bst
The Hs578Bst cell line had a fibroblast-like appearance in the light microscope. At confluence the cells VOL. 58, NO.6, JUNE 1977
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drying. The trypsin-Giemsa method (19, 20) for Gbanding was used for a study of marker chromosomes and Q-banding after the method of Lin et al. (21) revealed the presence or absence of the human Y-chromosome. EM.-Semiconfluent and confluent monolayers of both cell lines were prepared as previously described for SEM (22) and TEM (23). Mycoplasma test5 .-The two lines were repeatedly tested for the presence of mycoplasma and were consistently negative. Tests were done at the Naval BioSciences Laboratory (Oakland, Calif.) according to a modification of the method of M. Barile and by SEM and TEM. Estrogen-specific binding protein.-The techniques described by Wittliff (24) and Wittliff and Savlov (25) for the dextran-coated charcoal assay for estrogen-receptor protein were used. Specific binding curves were calculated from the difference between the noncompetitive and competitive binding reactions. The Kd and number of binding sites were calculated by the method of Scatchard (26). We normalized the results for protein content using the Folin method for protein determination. MCF-7 cells (15) were used as positive controls. Growth in semisolid medium.-Two ml growth medium plus 0.9% agar (Bacto-agar; Difco Laboratories, Detroit, Mich.) was added to 60-mm diameter tissue culture dishes. After the agar hardened, cells were trypsinized and suspended in growth medium containing 1.2% Methocel [4,000 cP, # 60 (high grade); Dow Chemical Co., Midland, Mich.] and various numbers of cells were plated over the agar layer. Plates were incubated at 37° C without subsequent feeding. One day after plating, the dishes were observed with an inverted microscope to determine that no cell clumps were present initially. The number and size of the colonies that subsequently formed were estimated 3 weeks post seeding. Colony Formation CIM.-Confluent monolayers were prepared in tissue culture dishes 60 mm in diameter. Hs578T and Hs578Bst cells were seeded onto a confluent monolayer of an epithelial-like cell line FHs 74 Int (23) and a fibroblast-like line Hs392Sk (27). Cultures to be tested were trypsinized and seeded at 103 and 100 cells/dish; the fluid was changed twice weekly for 21 days, fixed in methanol, and stained with May-Griinwald-Giemsa. Tumorigenicity.-Adult female NAMRU mice (Naval Biomedical Research Laboratory) were inoculated with A TS (Microbiological Associates, Los Angeles, Calif.) as described (28). Cells were trypsinized, centrifuged, and resuspended in medium lacking serum and in 0.1 or 0.2 ml volumes containing 5x 106 cells immediately injected intradermally into the interscapular region. No palpable nodules were observed in the 20 control mice inoculated with medium only. Virus detection.-Supernatants were assayed at various passage levels for RNA-dependent DNA polymerase with the use of the technique described previously (29). Immunofluorescence was done by the indirect methods described by Riggs (30). The M-PMV p27 and MuMTV p27 were obtained from Dr. R. Cardiff (Department of Pathology, University of California, Davis, Calif.).
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Growth Characteristics
Growth characteristics for both cell lines, at two passages, are listed in table 1. The tumor-derived cells grew rapidly to saturation at 22 X 104 cells/cm 2 , forming a mosaic pattern. The normal tissue-derived cells grew rapidly and reached saturation at 5.5X 104 cells/cm 2 • Malignant Characteristics
Both cell lines were tested for properties associated with cancer; they were grown in semisolid medium, immunosuppressed mice, and on CIM. Growth in semisolid medium (Methocel) was assayed at passages 13, 28, and 35 for the tumor-derived line and at passages 8 and 15 for the normal tissue-derived line. VOL. 58, NO.6, JUNE 1977
The latter did not grow in Methocel at either passage, whereas all passages of the tumor line grew well. Colony formation was consistently better in monkey serum than in FCS. Representative data are shown in table 2. Both cell lines were injected intradermally into immunosuppressed mice, at dosages of 3,5, and lOX 106 cells. Neither line produced tumors. Hs578T culture readily formed colonies of equal efficiency when plated on CIM of human epithelial and fibroblast cells. The tumor cells formed flat, smooth colonies sharply defined at the edge on the epithelial cell monolayers (fig. 5, inset). The cells on fibroblastic monolayers formed irregular colonies that followed the pattern of the substrate layer. Dilution plating of the tumor cells on both types of CIM at passage levels 22, 28, 31, and 35 yielded plating efficiences of 50%, which were reproducible at each passage level. The diploid Hs578Bst line did not form colonies on either monolayer. A representative experiment is shown in figure 5, along with an inset of a typical colony of tumor cells on an epithelial monolayer. Karyology and Isoenzymes
The tumor cells Hs578T had a unique, abnormal karyotype. Many rearranged or "marker" chromosomes were present, in addition to at least one normal (haploid) set of human chromosomes per cell. Figure 6 presents the proposed origin of five marker chromosomes presumably arising by rearrangement of normal chromosomes. Marker #1 involved the rearrangement of an X-chromosome and an autosome (#ll). One normal Xchromosome was noted. At early passages, 3 and 4, the TABLE
I.-Growth characteristics ofHs578T and Hs578Bst cell lines
Characteristics Growth rate Saturation density/ cm 2 Growth pattern Cell type
TABLE
Hs578Bst, passages 7,30
Hs578T, passages 10,16 Rapid 22x10 4
Moderate 5.5x10 4
Mosaic, multilayering "dome" formation Pleomorphism, short-spindle, polygonal
Flat, swirling multilayered Spindle
2.-Growth of Hs578T and Hs578Bst cells in semisolid medium (Methocel)
Cell line: Passage level Hs578T: 28 Hs578T: 35 Hs578Bst: 8 Hs578Bst: 15
FCSa
Monkey serum a
No. of cells seeded
Small colonies
Large colonies
Small colonies
Large colonies
104 103 104 103 104 103 104 103
125 14 TMb 6 0 0 0 0
TMb 1 17 0 0 0 1 0
150 6 182
25 0 42 2 0 0 0 0
22
0 0 0 0
a Plates were scored for colonies 3 wk post seeding; small colony=4-100 cells/colony and large colony = >100 cells/colony. b TM=too many to count.
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formed a swirling pattern and multiple layers, whereas at subconfluence the cells were uniformly spindleshaped and oriented in parallel rows. However, these cells also had features not characteristic of fibroblasts; the nuclei seemed more round to ovoid than elongated, as expected for fibroblasts, and they were sometimes displaced rather than centrally located; multiple nucleoli were conspicuous. The cells had features characteristic of nonmalignant cells: The nuclear-cytoplasmic ratio was low, the nuclear margins were regular, the nucleoli were small, and the cytoplasm was eosinophilic and pale (fig. 2B). Certain ultrastructural features of the Hs578Bst cells were more consistent with a myoepithelial rather than fibroblastic origin. One of the most distinctive features of myoepithelial cells is the presence of micro filaments of 60 A diameter, which in localized areas show thickening and electron density analogous to the Z-bands in muscle cells (36). The number and arrangement of the microfilaments vary with the degree of differentiation of the myoepithelial cell. In addition, these cells generally have evidence of secretory activity and contain pinocytotic vesicles clustered between the regional areas of filament thickening (36, 37). Ultrathin sections of Hs578Bst cells showed these typical myoepithelial features. A cross section showed multiple layers of elongated cells containing cells with two different levels of differentiation (fig. 4). Both cell types contained the characteristic myoepithelial microfilaments, but in varying amounts. Areas of increased electron density (Zband-like) could be seen. As is often seen in myoepithelial cells in vivo, the parallel microfilaments were sometimes confined to one side of the cell only (36). Both cells also had pinocytotic vesicles with typical myoepithelial cell localization. Only one of the two cell types showed features suggestive of secretory activity. These cells had extensive dilated RER containing electrondense material typical of cells actively synthesizing protein. These cells also contained fine filamentous threads emanating from the cell surfaces that resembled elastin fibers found in myoepithelial cells in vivo (36, 37). Possibly this amorphous, electron-dense intercellular material is basement membrane (37). Myoepithelial cells in vivo form few desmosomes, and none was observed in vitro. Collagen, characteristic of fibroblasts, was not observed either (fig. 4).
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Hormone Receptors
The estrogen-receptor protein was not detectable in either the tumor line Hs578T at passage 14 or normal Hs578Bst at passage 13; whereas under similar conditions, the MCF-71ine bound 42.5 fmole protein/mg with a Kd of 3.3x 10-9 • These values agreed with data obtained on MCF-7 by Brooks et aJ. (15). In addition, the 4S sedimentatio'n coefficient for MCF-7 reported in (16) was confirmed. Associated Endogenous Virus
Evidence for the presence of endogenous virus was sought by EM, assay for reverse transcriptase, and immunofluorescence methods. Extensive EM surveys of Hs578T and Hs578Bst cells at various passage levels revealed no evidence of particles characteristic of type C, type B, or herpesvirus. Assays for reverse transcriptase in supernatants were consistently negative. Cover slip-grown monolayers were tested by immunofluorescence with both fixed and live cells. Antisera prepared against purified viral proteins of known mammalian oncornaviruses were used. No evidence of viral antigen was detected (table 3). In addition, sera from the donor and 6 other breast cancer patients were tested for the presence of antibodies to the cellular antigens of the two cell lines, and no antibodies were found. TABLE
Cell lines tested a Hs578T HS578Bst D17 infected with RD114 CMMT MmsMt D17 infected with ATS-124 JLS-V5
DISCUSSION
In normal breast tissue, ductal epithelium and myoepithelium are closely associated, and maintenance of an integrated structure depends on this association (38, 39). In carcinosarcomas, which are a rare form of breast disease, a complex histologic pattern involving these two cell types exists (38, 40, 41). Typically, this tumor displays epithelial cells with squamous differentiation composed of spindle-shaped or stellate cells in a mucoid basophilic stroma (40). Hyperplasia of myoepithelial cells has been noted peripheral to the tumor (41). This form of breast disease might be included in the scirrhous carcinoma classification of Murad (38), who suggested a myoepithelial cell origin for this class. In this study both cell types were isolated from a carcinosarcoma, mammary tumor epithelial cell line Hs578T and normal myoepithelial cell line Hs578Bst. The characteristics that identify the Hs578T cell line as epithelial and mammary in origin are listed in table 4. The polygonal shape, dome formation, and the presence of desmosomes identify the cells as epithelial; cytoplasmic organelles for secretion, the presence of lipid droplets, and casein granules identify the cell as mammary. Although several other cell lines derived from metastatic mammary tumors produce domes at confluence (7, 8, 10), casein production detectable by EM has not been previously reported in a human cell line. However, Gaffney (12) and Polanowski (13) report that cells established in culture from human milk produce casein detectable by biochemical techniques. The presence of casein in a tumor from a postmenopausal woman is expected, since elevated serum levels of casein have been found by radioimmunoassay in women with breast cancer (42). The absence of estrogen-receptor protein suggests that estrogen may not be necessary for casein synthesis. This cell line offers a system for the study of hormonal effects on casein synthesis free of the synergistic and antagonistic interplay between hormones encountered in vivo. The malignancy of the Hs578T cell line is suggested by the properties listed in table 5. The growth properties of cultured cells that are correlated with cancer (27, 43) are found in Hs578T with the exception that tumors were not produced in immunosuppressed mice. This property may be due to a need for hormonal stimulation
3.-Attempts to detect evidence for endogenous viruses in Hs578T and Hs578Bst cell lines RTb
ND
EMc
IF A using sera to viral antigen d ATS-124
MuMTV
MuRLV
+
+ +
,:
ND
+ +
±
+
+
±
+
+
M-PMV
RD114
+
+
+ +
a Source
and characteristics of cell lines are given in "Materials and Methods." RT=reverse transcriptase; ND=not determined. c Survey of >300 cells by EM. dlFA=indirect fluorescent antibody; source of antibody and type listed in "Materials and Methods." b
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number of chromosomes ranged from 31 to 59 (a mode of 58), and there was 25% polyploidy for the original aberrant karyotype with cells containing approximately 110 chromosomes. At later passages, 6, 12, 23, 24, and 29, the frequency of this polyploidy increased to 75%. At passage 26, a colony was picked; at passages 40-42 thereof, most cells (75-85%) were polyploid. The Hs578Bst cells had the G-banding pattern of a normal diploid female. Through 7 passages diploidy was consistent, and only 1-5% of the cells were polyploid. For unknown reasons, mitotic arrest with Colcemid was not easily achieved in either line. Isoenzyme mobility patterns for glucose-6-phosphate dehydrogenase were tested by Dr. W. D. Peterson,]r. (Children Research Center of Michigan, Detroit, Mich.) and revealed type B or "slow" movement for both cell lines, conforming to the race of the donor, a Caucasian.
SYNGENEIC MAMMARY EPITHELIAL MYOEPITHELIAL CELL LINES TABLE
4.-Characteristics of mammary epithelial cells displayed by Hs578T and Hs578Bst cell lines
Characteristics Ultrastructure: Desmosomes Tonofibrils Intracytoplasmic ductlike vacuoles Casein production Lipid production 60 A microfilaments Biochemistry: Estrogen-receptor sites
Hs578T
Hs578Bst
+ +
+ + +
+
5.-Characteristics of malignant cells displayed by Hs578T and Hs578Bst cell lines
Characteristics Morphology: High nUclear-cytoplasmic ratio Basophilia Bizarre nuclei Karyotype: Aneuploid Growth: High saturation density Multilayered Growth in Methocel Growth on CIM Growth in immunosuppressed mice
Cell lines Hs578T
Hs578Bst
+ + + + + + + +
+
over and above the levels in normal adult NAMRU mice (14). The aneuploidy, which was present at early passage and which appeared to stabilize at passage 40, is also compatible with cancer. The line is made unique and distinctive by virtue of banding markers in the chromosome complement (fig. 6). Data presented here support the identification of the Hs578Bst cell line as normal (table 5). The cells are diploid and have no unusual features, with the possible exception of the number of nucleoli. Further studies to confirm the origin of these cells are in progress; evidence is being sought for the components of myosin and responsiveness to prolactin and oxytocin. It has been suggested that epithelial and myoepithelial cells act in concert and that myoepithelial cells respond to growth regulatory stimuli received from the blood and regulate information and transport of nutrients to epithelial cells (36, 37). Uncontrolled proliferation of epithelial cells may result in concomitant myoepithelial hyperplasia. Such myoepithelial hyperplasia in the tumor-bearing breast may account for the successful isolation of the Hs578Bst myoepithelial cell line. Some obvious questions concerning mesenchymal and epithelial interactions in mammary tumorigenesis may now be amenable to experimental study with these cell lines. REFERENCES (1) KENNEDY BJ: Hormonal therapies in breast cancer. Semin Oncol 1: 119-130, 1974 VOL. 58, NO.6, JUNE 1977
(2) - - - : Appearance and spread of human breast cancer. Recent
Results Cancer Res 42:31-37, 1973
(3) LASFARGUES EY: New approaches to the cultivation of human
breast carcinomas. In Human Tumor Cells In Vitro (Fogh J, ed). New York: Plenum Press, 1975, p 51 (4) LASFARGUES EY, OZELLO L: Cultivation of human breast carcinoma. J Natl Cancer Inst 21:1131-1147,1958 (5) NORDQUIST RE, ISHMALL DR, LOVIG CA, et al. The tissue culture and morphology of human breast tumor cell line BOT -2. Cancer Res 35:3100-3105, 1975 (6) YOUNG RK, CAILLEAU RM, MACKAY B, et al: Establishment of epithelial cell line MDA-MB-157 from metastatic pleural effusion of human breast carcinoma. In Vitro 9:239-245, 1974 (7) CAILLEAU RM, MACKAY B, YOUNG RK, et al: Tissue culture studies on pleural effusions from breast carcinoma patients. Cancer Res 34:801-809, 1974 (8) SOULE HD, VASQUEZ J, LONG AS, et al: A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 51:1409-1416, 1973 (9) REED MV, GEY GO: Cultivation of normal and malignant human lung tissue. Lab Invest 11 :639-652, 1962 (10) CAILLEAU R, CRUCIGER Q, HOKANSON KM, et al: Morphological, biochemical and chromosomal characteristics of breast tumor lines from pleural effusions. In Vitro 12 :331, 1976 (11) FOGH J, TREMPE G: New human tumor cell lines. In Human Tumor Cells In Vitro (Fogh J. ed). New York: Plenum Press, 1975, pp 115-159 (12) GAFFNEY EV: The hormone response of secreting and nonsecreting human breast cells in culture. In Vitro 12:328, 1976 (13) POLANOWSKI FP, GAFFNEY EV, BURKE RE: HBL100: A cell line established from human breast milk. In Vitro 12:328, 1976 (14) RussoJ, BRENNAN MJ, RICH MA: Induction of tumor growth by inoculation of a human breast cancer cell line MCF-7 into ovary or pituitary grafted nude mice. Proc Am Cancer Soc 19:116, 1976 (15) BROOKS SC, LOCKE ER, SOULE HD: Estrogen receptor in a human cell line (MCF-7) from breast carcinoma.J BioI Chem 248:62516253, 1973 (16) HORWITZ KB, COSTLOW ME, McGUIRE WL: MCF-7: A human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors. Steroids 26:785-795, 1975 (17) ROSE HN, McGRATH CM: a-Lactalbumin production in human mammary carcinoma. Science 190:673-675, 1975 (18) LEIBOVITZ A: The growth and maintenance of tissue cell cultures in free gas exchange with the atmosphere. Am J Hyg 78:173180, 1963 (19) NELSON-REES WA, FLANDERMEYER RR, HAWTHORNE PK: Distinctive banded marker chromosomes of human tumor cell lines. Int J Cancer 16:74-82, 1975 (20) - - - : Banded marker chromosomes as indicators of intraspecies cellular contamination. Science 184:1093-1096, 1974 (21) LIN CC, UCHIDA lA, BYRNES EA: A suggestion for the nomenclature of the fluorescent banding patterns in human metaphase chromosomes. Can J Genet Cytol 13:361-363, 1971 (22) SPRINGER EL, HACKETT AJ, NELSON-REES WA: Alteration of the cell membrane architecture during suspension and monolayer culturing. Int J Cancer 17:407-415, 1976 (23) OWENS RB, SMITH HS, NELSON-REES WA, et al: Epithelial cell cultures from normal and cancerous human tissues. J Natl Cancer Inst 56:843-849, 1976 (24) WITTLIFF JL: Specific estrogen receptors in the lactating mammary gland of the rat. Biochemistry 12:3090-3096, 1973 (25) WITTLIFF JL, SAVLOV ED: Estrogen-binding capacity of cytoplasmic forms of the estrogen receptors in human breast cancer. In Estrogen Receptors in Human Breast Cancer (McGuire WL, Carbone PP, Vollmer EP, eds). New York: Raven Press, 1975, p 72 (26) SCATCHARD G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672, 1949 (27) SMITH HS, OWENS RB, HILLER AJ, et al: The biology of human cells in tissue culture. I. Characterization of cells derived from osteogenic sarcomas. IntJ Cancer 17:219-234, 1976 (28) ARNSTEIN P, TAYLOR DO, NELSON-REES WA, et al: Propagation of human tumors in antithymocyte serum-treated mice. J Natl Cancer Inst 52:71-84, 1974 J NATL CANCER INST
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TABLE
Cell lines
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(36) ELLIS RA: Fine structure of the myoepithelium of the exocrine sweat glands of man. J Cell BioI 27:551-563, 1965 (37) OZZELLO L: Epithelial-stromal junction of normal and dysplastic mammary glands. Cancer 25:586-600, 1970 (38) MURAD TM: A proposed histochemical and electron microscopic classification of human breast cancer according to cell of origin. Cancer 27:288-299, 1971 (39) DUNN TB: Morphology of mammary tumors in mice. In The Physiopathology of Cancer (Hornberger F, ed), 2d ed. New York: Paul B. Hoeber, 1959, pp 238-292 (40) HARRIS M, PERSAUD V: Carcinosarcoma of the breast. J Pathol 112:99-105, 1974 (41) HAMPERL H: The myothelia (myoepithelial cells) normal state, regressive changes, hyperplasia, tumors. Curr Top Pathol 53: 162-220, 1970 (42) HENDRICK LC, FRANCHIMONT P: Rdaioimmunoassay of casein in the serum of normal subjects and of patients with various malignancies. Eur J Cancer 10:725-730, 1974 (43) TOOZE J: The molecular biology of tumor viruses, chapt 2. Cold Spring Harbor Laboratory Press, New York, 1973, pp 74-172
VOL. 58, NO.6, JUNE 1977
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(29) HACKETT AJ: Monitoring for presence of oncogenic virus in tissue culture. In Tissue Culture Methods and Applications (Kruse PF, Patterson MK, eds). New York: Academic Press, 1973, pp 539-548 (30) RIGGS JL: Application of fluorescent antibody techniques to viral infections. Industr Med Surg 34:269-277, 1965 (31) NELSON-REES WA, HOOSER LE, HACKETT AJ: Chronic poliovirus infection of co-cultivated monkey cells harboring the MasonPfizer monkey virus. J Natl Cancer Inst 49:713-725, 1972 (32) OWENS RB, HACKETT AJ: Tissue culture studies of mouse mammary tumor cells and associated virus. J Natl Cancer Inst 49:1321-1332, 1972 (33) RIGGS JL, McALLISTER RM, LENNETTE EH: Immunofluorescent studies of RD114 virus replication in cell culture. J Gen Virol 25:21-29, 1974 (34) WRIGHT BS, LASFARGUES EY: Multiplication of the Rauscher leukemia virus in a mixed culture of mouse spleen and thymus cells. Proc Am Assoc Cancer Res 5:70, 1964 (35) MCGRATH CM: Replication of mammary tumor virus in tumor cell cultures: Dependence on hormone-induced cellular organization. J Natl Cancer Inst 47:455-467, 1971
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I.-Histopathologic section of tumor of origin (HT860). Infiltrating ductular carcinoma with prominent fibrous stromal reaction. Hyperchromatic sarcoma-like cells could be malignant mesenchymal cells, in which case this could be considered carcinosarcoma.
FIGURE
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FIGURE
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2.-Light micrographs of monolayer cultures: A) Hs578T, passage 22; B) Hs578Bst, passage 7.
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FIGURE 3.-EM of Hs578T cells showing ultrastructural markers associated with cancer (A-C) and mammary epithelial cells (D-F). A) Nucleus
with 4 nucleoli with dense nucleonemal network and spotty pars amorpha. X 8,800. B) Microvillus-lined intracytoplasmic duct-like vacuole containing fibers and cell debris. x 11,000. C) Triad and duplex of confronting cisternae. x 40,000. D) Two cells with well-formed desmosomal junctions and tonofibrils. x 30,000. E) Perinuclear vesicular Golgi (g), lipid droplet (I), aberrant mitochondria (m). X 7,000. F) Compact protein granule (casein) in vacuole near cell surface. x 60,000
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4.-EM of multilayered Hs578Bst cells. One cell has dilated RER (R); another is filled with 60 A microfilaments with regional electrondense condensation (c) and slender elongated mitochondria (M). Threads, similar to elastin (arrow), contribute to extracellular matrix resembling basement membrane. X 24,000
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5.-Colony formation on CIM of epithelial cells (A) and fibroblasts (B). Note colony of Hs578T cells growing on epithelial CIM (top, center). Dishes, top row, mock. Middle row, Hs578T cells seeded at two dilutions, 100 and 1,000 cells/dish. Hs578Bst cells were seeded similarly (bottom row).
FIGURE
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Downloaded from http://jnci.oxfordjournals.org/ at Cornell University Library on November 23, 2012
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6.-Karyotype of Hs578T cell, passage 24, with 68 chromosomes. At least one complement of normal human chromosomes, including X, is present, but most chromosomes are in multiple copies. Rearranged "marker" chromosomes (bottom row), including X-autosome translocation, are labeled according to presumed origin.
FIGURE
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