[CANCER RESEARCH 32, 2407-24-12, November 1972] Growth of Normal Human Mammary Gland in Vitro1

B. Allen Flaxman and Eugene J. Van Scott The and Cancer Hospital of Philadelphia, Department of Dermatology, Temple University Health Sciences Center, Philadelphia, Pennsvlvania 19140

SUMMARY gland and used as a specific source of epithelial cells, is described. The cells proliferated readily in vitro and retained A method is described whereby ducts of the nonlactating features of differentiation present in vivo. This system should human mammary gland can be identified in vivo and isolated make it possible to conduct baseline studies of normal so as to serve as a specific source of epithelial cells for studies function and behavior that may be of value in understanding in vitro. The method overcomes the inherent difficulty in factors leading to development of human cancer. obtaining epithelial cells from random expiants of nonlactating mammary gland where the system is poorly developed. MATERIALS AND METHODS Ducts from the nipple and subjacent mammary were first dissected from the surrounding connective tissue, The clinically normal nipple and subjacent mammary tissue cut into small fragments and explanted on glass coverslips. was obtained from autopsy specimens of 2 postmenopausal Epithelial cells grew out onto the coverslip as a stratified (ages 59 and 79) females. The tissue was removed aseptically, squamous epithelium in which mitoses were numerous. placed in Gey's balanced salt solution, and then bisected. With Ultrastructurally, cells remaining on the expiant and those in the aid of a dissecting microscope, ducts were seen in the outgrowth were characterized by an abundance of longitudinal or transverse section. The connective tissue was cytoplasmic filaments indicating their origin from filament- gently dissected, thus freeing the ducts which were white, firm containing cells normally found in breast epithelium. There tubes that stood out clearly if the dissection was carefully was no convincing morphological evidence of milk synthesis or performed. Some large ducts were 2 mm in diameter and could . Further exploitation of this system may allow be seen without the aid of a microscope. The epithelium had a correlative studies of function and carcinogenesis in the human distinct yellow-orange tint that also aided in identification. mammary gland in vivo and in vitro, studies heretofore not Ducts were excised, transferred to a new dish of balanced salt possible. solution, and excess connective tissue was removed (Fig. 1). Ducts were opened by a longitudinal slit, placed with the INTRODUCTION epithelial surface up, and cut into pieces approximately 3x3 mm. These expiants were then transferred to glass coverslips Experimental analysis of development, function, and on the bottom of plastic Petri dishes and held in place by a carcinogenesis of the mammary gland has been limited mainly clot comprised of 1 drop each of chick plasma and embryo extract (9). Cultures were immersed in Eagle's minimal to nonhuman species such as the mouse, the rabbit, and the cow. Although breast cancer is a major cause of human essential medium containing 10% fetal calf serum and 100 units each of penicillin, streptomycin, and mycostatin per ml. mortality, there is little experimental information on the Cultures were grown in a high-humidity incubator at 37°in an of normal human mammary tissue. While numerous attempts have been made to culture cells from a variety of atmosphere of 5% CO2 in air. Culture fluid was changed every benign and malignant human mammary gland lesions (5, 10, 3 to 4 days. 14, 26), few attempts have been made to grow normal For light microscopy, cultures attached to their coverslips epithelium (18, 19). Both explantation and enzymatic were fixed in 10% buffered formalin. The expiants were digestion methods have been used to obtain tumor cells when removed, embedded in paraffin, sectioned, and stained with large numbers of such cells are present. The lactating hematoxylin and eosin. Epithelial cells remaining on the glass mammary gland would provide an abundant supply of were stained with hematoxylin. epithelial cells, but such material is not readily obtainable for For electron microscopy, cultures were fixed in 2.5% obvious reasons. In the normal, nonlactating mammary gland, glutaraldehyde in 0.1 M cacodylate buffer at room the duct system is not well developed; hence, the number of temperature for 1 hr. They were washed in buffer, postfixed in epithelial cells is minimal and explantation or enzymatic a 1:2 mixture of Os04 in collidine (2), dehydrated through digestion is most likely to yield fibroblasts rather than alcohol and propylene oxide, removed from the glass coverslip, epithelial cells. A method that circumvents this problem, in and embedded in Araldite. which individual ducts were dissected from the mammary

'Supported by Grant l PO l CA 11536 from the National Cancer RESULTS Institute, and Grant l PO l AM 1551-01 from the National Institute of Arthritis and Metabolic Diseases. Duct Epithelium in Dissected Specimens. In sections cut Received June 16, 1972; accepted July 28, 1972. perpendicular to the surface, the epithelium was comprised

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. B. Allen Flaxman and Eugene J. Van Scott predominantly of a double layer of cuboid cells (Fig. 2). In growth of which is oriented toward connective tissue (expiant) sections that seemed to have been cut more tangentially, the and toward a nonliving surface (glass coverslip). This should epithelium appeared thicker. make possible experiments designed to elucidate those aspects Epithelial Outgrowths. Twenty-seven expiants were cultured of normal mammary gland epithelial behavior that are for 10 days. In all cases ductal epithelial cells grew onto the connective-tissue dependent. Epithelial cell outgrowths from surface of the glass coverslip. Sections of cultures showed fragments of normal human mammary gland have been direct continuity of the outgrowing cells with epithelial cells reported before (18, 19), but the exact origin of the cells, on the expiant (Fig. 3). Outgrowing cells formed a ring of whether from terminal ductules or larger ducts, was not epithelium surrounding the expiant (Fig. 4). Within the known. Information regarding the region of the ductal tree epithelium, cells were mainly polygonal and closely adherent from which the epithelium was obtained may be important to one another (Fig. 5). The amount of overlap was variable with respect to an understanding of epithelial carcinogenesis. for, while the epithelium seemed to be monolayer at the Histological and ultrastructural characteristics of the human periphery, centrally it appeared stratified. Mitoses were mammary gland during development and in response to numerous (Fig. 5), and the mitotic index was 32 ±7/1000 in hormonal stimulation have not been adequately studied. As a 10-day-old cultures. The sheet of cells enlarged radially during consequence, there is a lack of baseline information with the 10-day period of growth. In 7 cultures fibroblasts became regard to the "normal" ultrastructural appearance. The ductal numerous but grew mainly outside the ring of epithelium, the system starts at the skin level as large ducts in the nipple, growth of which did not seem to be affected. In the remaining branches downward into smaller ductules and, during cultures, few or no fibroblasts emerged. pregnancy, presumably, develops terminal alveoli. In ultra- Ultrathin sections for electron microscopy cut vertical to structural studies of terminal ductules, different authors have the surface of the outgrowth showed that the cells formed a not been able to agree on the number of cell types comprising stratified squamous epithelium (Fig. 6). The flattened cells the epithelium (22-24). Whether differences are real is not were piled 2 to 5 layers thick. In some regions cell contact was clear, since the hormonal status of the women from whom quite intimate; elsewhere large spaces were present. The material was obtained was not known. In at least one of the plasma membrane often showed short villous projections. Cells published studies (22), however, a cell has been described were connected by fairly numerous desmosomes with attached which contains bundles of cytoplasmic filaments resembling tonofilaments. Similar filaments were also present in the epidermal tonofilaments. These cells were connected by cytoplasm. Mitochondria, smooth and rough endoplasmic desmosomes. The majority of cells observed in the present reticulum, and free ribosomes were present. Round, dense experiments had similar characteristics, and it seems possible bodies, probably representing lipid, were found in some cells. that they were derived from the filament-containing cells of Many vesicles were also present. Evidence of protein secretion the duct. was not found. There was no convincing morphological evidence for Expiants. The behavior of the epithelium was variable. In 2 secretion by cells either on the expiant or in the outgrowth. cases the cells retained a cuboid or columnar shape and a Such evidence of secretion would include the presence of large certain amount of upward proliferation appeared to have amounts of rough and smooth endoplasmic reticulum, vesicles occurred (Fig. 7). Mitoses were present. In the remaining containing milk protein, and lipid (8, 17). While some cells did expiants, the epithelial cells became flattened. The ultra- contain lipid and significant amounts of endoplasmic structure of the cells was not significantly different from those reticulum, no lipid or milk protein droplets were found in the of the outgrowth (Fig. 8). There was no specific evidence for extracellular space (24). Experimental studies of mouse and secretory activity. At the epithelial-connective tissue junction, bovine gland function in vitro have shown that milk protein a basement lamina was seen. The collagen appeared well synthesis must be preceded by mitosis but that mitosis alone is preserved and its periodicity was normal. not sufficient for initiation of synthesis (1, 15, 21). In cultures of mouse mammary tissue, high levels of have been shown to promote mitosis, while prolactin and DISCUSSION hydrocortisone cause the cells to synthesize milk protein (7, 11, 15, 20). Recently, however, a protein fraction from serum The method described in this paper whereby the larger has been shown to stimulate mammary epithelial mitosis at ducts of the human mammary gland were isolated and served physiological levels and it has been proposed that this material, as the source of epithelial cells eliminates the problem of rather than insulin, is the normal physiological promoter of experimentally insufficient epithelial cells in random expiants mitosis in vivo (16). This might explain why mitosis occurs so of gland tissue. readily in cultures of human breast epithelium where the The results show that normal epithelium from the human culture fluid contains 10% calf serum. The failure to observe mammary gland can be readily grown in vitro for up to 10 convincing morphological evidence of function might be days. Cells remaining on the explant showed mitosis and some related to the fact that we did not add exogenous prolactin even retained their cuboid or columnar morphology. and hydrocortisone. In the absence of supplemental , Outwandering cells also divided but became flattened and mouse mammary gland cell cultures also do not show formed a stratified squamous epithelium. That these cells were secretory activity (6, 12, 13). derived from duct epithelium was evident from sections Within the outgrowth, cells at the periphery showed good showing direct continuity with epithelial cells on the explant. contact inhibition of overlap and formed a monolayer. Close The system thus simultaneously provides epithelial cells the to the expiant, however, the cells piled up to form a stratified

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. Human Mammary Gland in Vitro epithelium similar to that described in studies of human 5. Coman, D. R. Human Neoplasms in Tissue Culture. Cancer Res., 2: epidermal cells in vitro (9). This growth pattern, as well as the 618-625, 1942. presence of cytoplasmic filaments resembling tonofilaments, 6. Daniel, C. W., and DeOme, K. B. Growth of Mouse Mammary may reflect the developmental origin of breast epithelial cells Glands in Vivo after Monolayer Culture. Science, 149: 634—636, from epidermis. No keratinized cells were seen, however. The 1965. retention of the cuboidal shape of many cells that remained on 7. Elias, J. J. Effect of Insulin and Cortisol on Organ Cultures of Adult Mouse Mammary Gland. Proc. Soc. Exptl. Biol. Med., 707: the connective tissue suggests that this aspect of cell 500-502, 1959. morphology depends on mesenchymal influences (25). Piling 8. Fiddler, T. J., Birkinshaw, M., -and Falconer, I. R. Effects of up within the outgrowth cannot be considered to represent an Intraductal Prolactin on Some Aspects of the Ultrastructure and abnormal expression of contact inhibition since such behavior Biochemistry of Mammary Tissue in the Pseudopregnant Rabbit. J. appears to be a normal attribute of epithelial cells in vitro (9). Endocrinol., 49: 459-469, 1971. Moreover, contact inhibition is now known to depend on 9. Flaxman, B. A., Lutzner, M. A., and Van Scott, E. J. Cell several factors such as the relative strength of adherence of Maturation and Tissue Orgnization in Epithelial Outgrowths from cells for one another versus the substratum to which they are Skin and Buccal Mucosa in Vitro. J. Invest. Dermatol., 49: attached. Altering properties of the substratum leads to 322-332, 1967. changes in the relation of cells to one another (4). 10. Foley, J. F., and Aftonomos, B. T. Growth of Human Breast Neoplasms in Cell Culture. J. Nati. Cancer Inst., 34: 217-229, Many investigators have grown epithelial cells from a variety 1965. of benign and malignant human breast lesions in vitro (5, 10, 11. Juergens, W. G., Stockdale, F. E., Topper, Y. J., and Elias, J. J. 14, 18, 19, 26). In general, the methods have involved -dependent Differentiation of Mammary Gland in Vitro. culturing expiants of randomly chosen pieces of tissue or Proc. Nati. Acad. Sci., 54: 629-634, 1965. making cell suspensions with the aid of proteolytic enzymes. 12. Lasfargues, E. Y. Cultivation and Behavior in Vitro of the Normal In both methods, it was not possible to know whether the cells Mammary Epithelium of the Adult Mouse. Anat. Record, 727: that survived and grew in vitro were normal, abnormal, or a 117-129,1957. mixture of both. The present method, in which large 13. Lasfargues, E. Y. Cultivation and Behavior in Vitro of the Normal mammary gland ducts were dissected, may well be applicable Mammary Epithelium of the Adult Mouse. II. Observations on the to dissection of smaller ducts, which could then be studied in Secretory Activity. Exptl. Cell Res., 13: 553-562, 1957. 14. Lasfargues, E. Y., and Ozzello, L. Cultivation of Human Breast vitro. This would be important since cancer of the breast arises .J. Nati. Cancer Inst., 21: 1131-1147, 1958. in both large and small ducts, although more commonly in the 15. Lockwood, D. H., Stockdale, F. E., and Topper, Y. J. latter (3). Hormone-dependent Differentiation of Mammary Gland: Sequence The experimental study of normal and abnormal mammary of Action of Hormones in Relation to Cell Cycle. Science, 156: gland behavior has heretofore been primarily limited to 945-946, 1967. nonhuman species. The relevance of findings in these species 16. Majumder, G. C., and Turkington, R. W. Stimulation of Mammary to human breast biology can only be determined when human Epithelial Cell Proliferation in Vitro by Protein Factor(s) Present in material is studied. The present investigation has demonstrated Serum. , 88: 1506-1510, 1971. that human mammary gland cells can be readily propagated in 17. Mills, E. S., and Topper, Y. J. Mammary Alveolar Epithelial Cells: short-term culture and that experiments analogous to those Effect of Hydrocortisone on Ultrastructure. Science, 765: 1127-1129, 1969. performed on other species can now be carried out. 18. Orr, M. F., and McSwain, B. Tissue Culture of Human Breast and Normal Breast Epithelium. Texas Rept. Biol. Med., 12.916-920, 1954. ADDENDUM 19. Orr, M. !•'.,andMcSwain, B. Tissue Culture of Human Breast Since this manuscript was submitted, epithelial cell cultures have Carcinoma. Am. J. Pathol., 31: 125-141, 1955. been obtained in 2 instances from human mammary glands removed 20. Rivera, E. M., and Bern, H. A. Influence of Insulin on Maintenance surgically because of a malignant neoplasm. The nipple and underlying and Secretory Stimulation of Mouse Mammary Tissues by tissue were free of disease. Results were identical to those obtained Hormones in Organ-Culture. Endocrinology, 69: 340-353, 1961. from autopsy material. 21. Stockdale, F. E., and Topper, Y. J. The Role of DNA Synthesis and Mitosis in Hormone-Dependent Differentiation. Proc. Nati. Acad. Sci., U.S., 56: 1283-1289, 1966. REFERENCES 22. Tannenbaum, M., Weiss, M., and Marx, A. J. Ultrastructure of the Human Mammary Ductule. Cancer, 23: 958-978, 1969. 1. Anderson, C. R., and Larson, B. L. Comparative Maintenance of 23. Toker, C. Observations on the Ultrastructure of a Mammary Function in Dispersed Cell and Organ Cultures of Bovine Mammary Ductule. J. Ultrastruct. Res., 27: 9-25, 1967. Tissue. Exptl. Cell Res., 61: 24-30, 1970. 24. Waugh, D., and VanderHoeven, E. Fine Structure of the Human 2. Bennett, H. S., and Luft, J. H. s-Collidine as a Basis for Buffering Adult Female Breast. Lab. Invest., 77: 220-228, 1962. Fixatives. J. Biophys. Biochem. Cytol., 6. 113-114, 1959. 25. Wessells, N. K. Tissue Interactions during Skin Histodifferentiation. 3. Boyd, W. The Breast. In: A Textbook of Pathology, p. 1019. Develop. Biol., 4: 87-107, 1962. Philadelphia: Lea& Febiger, 1970. 26. Whitescarver, J., Recher, L., Sykes, J. A., and Briggs, L. Problems 4. Carter, S. B. Principles of Cell Motility: The Direction of Cell Involved in Culturing Human Breast Tissue. Texas Rept. Biol. Movement and Cancer Invasion. Nature, 208: 1183-1187, 1965. Med.,26. 613-628, 1968.

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i I r1 Kig. 1. Light micrograph of portion of duct dissected from the nipple. Arrow indicates lumen. X 50. Fig. 2. Light micrograph of ductal epithelium which consists of 2 layers of cuboid cells. X 1000. Fig. 3. Light micrograph of cultured tissue removed in its entirety from coverslip. Specimen plastic-embedded. Epithelial cells have migrated down side of explant (£")toform outgrowth (arrow). X 450. Fig. 4. Light micrograph showing outgrowth of epithelial cells derived from ductal expiant. Arrow, leading edge. X 125. Fig. 5. Light micrograph showing epithelial cells in outgrowth at higher magnification. Note mitosis. X 700.

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Fig. 6. Electron micrograph of vertical section of outgrowth from 10-day culture. Cells are flat and form a stratified epithelium. Size of intercellular space and extent of cell contact varies. TV,nucleus; NU, nucleolus; T, tonofilaments;/., lipid droplets;/), desmosome. X 6000. Fig. 7. Light micrograph of plastic-embedded explant from 10-day culture showing epithelium of cuboid cells. Arrow, mitosis. X 400.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. Growth of Normal Human Mammary Gland Epithelium in Vitro

B. Allen Flaxman and Eugene J. Van Scott

Cancer Res 1972;32:2407-2412.

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