Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3401-3405, July 1979
Cell Biology
Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels (mouse mammary cells/human mammary cells/proliferation/long-term culture/primary culture)
JASON YANG, JAMES RICHARDS, PHILLIP BOWMAN, RAPHAEL GUZMAN, JUMPEI ENAMI, KATHLEEN MCCORMICK, SUSAN HAMAMOTO, DOROTHY PITELKA, AND S. NANDI Cancer Research Laboratory and Department of Zoology, University of California, Berkeley, California 94720
Communicated by A. Starker Leopold, April 26, 1979
We have develo ed a method for embedding ABSTRACT cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H]thymidine incorporation during 8 weeks in culture. An increase in DNA content of the cultured cells as a function of time was observed. Mouse mammary tumor cells cultured in the conventional monolayer system failed to show any significant increase in cell number during a culture period of 6 weeks. In addition, in such monolayer systems, cells progressively became detached from the dishes in long-term culture. The mammary epithelial cell origin of the collagen gel cell outgrowths was shown by electron microscopic demonstration of polarized cells containing tight junctions and budding mammary tumor virus particles. In addition, in vivo transplantation of collagen gel outgrowths resulted in the development of mammary adenocarcinoma histologically similar to the donor tumor. Cellular outgrowth patterns resembling those from tumor cells were also seen in similar collagen gel cultures of normal mammary cells from mouse and human and of hyperplastic alveolar nodule cells from mouse. The significance and usefulness of this system in comparison to the conventional monolayer system are discussed. The limited in vitro growth capacity of mammary epithelial cells in conventional primary culture in tissue culture dishes is a well-known phenomenon. The cells generally undergo a few rounds of division, but proliferation cannot be sustained nor can these cells be passaged. Mouse mammary epithelial cells cultured at low density become flattened and multinucleated, and rarely do they attain confluence (1, 2). Cells plated at high density are maintained well but little growth is ever
the growth of mouse mammary tumor cells in such cultures. Preliminary work with normal mouse and human mammary tissues indicates that this technique may also be suitable for growing other types of mammary cells.
MATERIALS AND METHODS Cell Dissociation. Primary mammary tumors from BALB/cfC3HCrgl mice, hyperplastic alveolar nodules (D2) maintained as transplantable lines in vivo in BALB/cCrgl mice (10), and normal mammary gland from C3H/Crgl and BALB/cNIV/Crgl mice were dissociated by a modification of a previously described method (11, 12). Briefly, finely minced mammary tissues were placed in 125- or 250-ml erlenmeyer flasks containing 0.1% collagenase (CLS III; 120-150 units/mg; Worthington) in Hanks' balanced salt solution (10 ml/g of tissue) and swirled on a gyratory water bath shaker (model G76, New Brunswick) at 120-150 rpm at 370C for approximately 90 min or until the suspensions were uniform without macroscopic lumps. The suspension was passed through Nitex cloth (mesh size, 150 ,um); the cells were collected by centrifugation at 80 X g for 5 min, and washed twice with Hanks' solution. The resulting preparation consisted mainly of small clumps of cells. Cell number was estimated by mixing 1 vol of cell suspension with 9 vol of 0.02% crystal violet in 0.1 M citric acid and counting stained nuclei in a hemocytometer. Dissociated human mammary epithelial cells were obtained from excised tissue (reduction mammoplasties and mastectomies) by modification of the above procedure and subsequent gradient centrifugation (details will be reported elsewhere). Culture Procedure. Collagen solution and gel were prepared as originally described (13) and are the same as those used for achieved. floating collagen gel experiments (7-9, 12). Briefly, 1 g of ratMost studies to date dealing with proliferation of mammary tail collagen fibers was sterilized in alcohol overnight and disepithelial cells in vitro have used established cell lines adapted solved in 300 ml of 1:1000 acetic acid in sterile distilled water; to grow in conventional monolayer culture (3-6). Considerable the supernatant after centrifugation at 10,000 X g for 30 min effort is being devoted in several laboratories to analysis of was the stock collagen solution. Eight volumes of stock solution hormone and drug sensitivity in these selected cell lines. An was mixed with 2 vol of 2:1 mixture of 10-fold concentrated inherent limitation of this approach is that such lines may not growth medium and 0.34 M NaOH and kept on ice to prevent be representative of the original cell population. Therefore, it immediate gelation. Dissociated cells were embedded in gel is of considerable importance to improve the conditions for in either a dispersed or a localized pattern. For dispersion, the culture of primary cells. Our demonstration of differentiated appropriate number of cells was added to cold gelation mixture; function in mouse mammary cells cultured on floating collagen 1 ml, containing 5-10 X 104 cells, was pipetted into each well gels, as indicated by levels of casein (7), mammary tumor virus of Falcon plastic Multiwell plates (well diameter, 16 mm) and (8), and prolactin receptor (9), has prompted us to examine allowed to gel at room temperature. For localized embedding, collagen as an appropriate substrate for growth. The most enapproximately 5-10 X 104 cells in 1 ,l were placed on the couraging results were obtained when mammary cells were surface of 0.5 ml of gelled collagen in each well and overlaid embedded within the collagen gel. The present study describes with 3 ,ul of cold gelation mixture so that the diameter of the cell cluster was roughly 5-10% of the well diameter. After this The publication costs of this article were defrayed in part by page layer had gelled, 0.5 ml of collagen was added and allowed to charge payment. This article must therefore be hereby marked "adgel. Cultures were fed every 2 days with F-12 medium vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. (GIBCO) containing 12.5% horse serum, 2.5% fetal calf serum, 3401
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Proc. Natl. Acad. Sci. USA 76 (1979)
each well of Multiwell plates. Cell Number. Cells inside the gel were released by digesting the gel in 1 ml of 0.1% collagenase in Hanks' balanced salt solution in a 10 X 75 mm test tube on a gyratory water bath shaker for approximately 30 min at 370C. The cells were collected by centrifugation at 100 X g for 5 min. Samples were stored frozen until the time of DNA assay. The DNA content was determined by a fluorometric assay (14) utilizing BALB/c spleen cells or BALB/cfC3H mammary tumor cells, counted in a hemocytometer, as a standard. Autoradiography. Cultures were labeled for 24 hr with [3H]thymidine [2.5 ,gCi/ml (1 Ci = 3.7 X 1010 becquerels); 50 Ci/mmol; ICN], fixed in Bouin's fixative, and embedded in paraffin. Sections were cut parallel to the surface of the gel and coated with NTB nuclear track emulsion (Kodak). After 2
RESULTS Freshly dissociated cells were embedded in the collagen gel in either dispersed or localized form. The general growth pattern was monitored by viewing the culture in an inverted microscope. Culturing mammary cells under the described condition led to a characteristic and reproducible pattern of growth. Locally embedded cells aggregated into a single compact mass and produced relatively thick duct-like structures that radiated into the matrix in three dimensions. Cell clumps dispersed through the collagen gel also produced three-dimensional outgrowths but the duct-like structures were not as thick as those produced from locally embedded cells. Such outgrowths of mammary cells from several sources are shown in Figs. 1 and 2. In control experiments, cells obtained from gland-free mammary fat pads and processed in exactly the same manner as above did not give rise to the same three-dimensional ductal structures. The predominant outgrowth pattern seen was one in which the cells grew out as single units producing dense radial patterns (Fig. 3). In addition to these narrow rays, single cells could often be seen wandering through the gel. Although these mesenchymal "fibroblast-like" cells were sometimes seen contaminating cultures derived from mammary tumors, they were never a problem because of their relatively small numbers within the tumor cell populations dissociated by our procedure. Growth was apparent by light microscopy and was confirmed by measuring the total DNA content of outgrowth-containing gels as a function of time. Detailed studies of this growth as well as characteristics of the resulting outgrowth were carried out with the spontaneous tumor from BALB/cfC3H mice. Mammary tumor cells were locally embedded in collagen gel, with a starting cell number of 1-2 X 105 per gel. At weekly intervals, cells were recovered from the gels by collagenase digestion and stored frozen until the time of DNA assay. The results from three separate experiments are shown in Fig. 4. The growth curve in collagen gel revealed an increase in DNA content as a function of culture period. On the other hand, in the monolayer culture, cells seeded at low density progressively became detached from the culture dish, and there was a decrease in cell number, consistent with earlier observation (1, 2). It is unlikely that the increase in DNA content in collagen gel culture was attributable to multinucleation, for several reasons. When similar outgrowths were recovered by collagenase digestion, dissociated, and plated in conventional monolayer culture, the resulting continguous pavement-like sheet of polygonal cells showed no obvious increase in multinucleated cells as compared to mammary tumor cells plated directly in monolayer culture. Histological observation performed in the autoradiographic analysis described below also confirmed that there was no obvious increase in multinucleated cells as a function of culture
weeks, the slides were developed in D-19 developer. Electron Microscopy. Cultures were fixed in 1% formaldehyde/3% glutaraldehyde/0.1 M sodium cacodylate, postfixed in 1% OS04, dehydrated in ethanol, and embedded in Epon. Thick (1-2 tim) sections were stained with methylene blue/ Azure II and photographed with a Zeiss photomicroscope II. Thin sections stained with uranyl acetate and lead citrate were examined in a Siemens Elmiskop 102 electron microscope. In Vivo Transplantation. Approximately 1-mm3 pieces of gel containing visible tissue from the periphery of the duct-like outgrowth were implanted into the gland-free inguinal mammary fat pads of 3-week-old female syngeneic mice (15). Four to 8 weeks after transplantation, the mice were sacrificed and the mammary fat pads were examined for outgrowths. Histological sections of selected outgrowths were prepared and the morphological type was determined.
period. Patterns of DNA synthesis were assessed at weekly intervals by [3H]thymidine incorporation followed by autoradiography. By the end of the first week, most of the locally embedded mammary cells had aggregated into a single relatively compact mass (Fig. 5A). Incorporation of [3H]thymidine was generally not high at this time, usually not exceeding a labeling index of 10%, but was found throughout the mass of cells. By the end of the second week, and throughout the next several weeks, the labeling index increased to about 20-35%, and labeling was localized chiefly at the periphery of the embedded cells. By this time there were numerous duct-like structures that protruded into the gel, and these processes exhibited high labeling indices, exceeding 50% (Fig. SB). Within the central mass, the labeling index tended to decrease with time. Histologically and ultrastructurally, the outgrowths resem-
FIG. 1. Outgrowth from BALB/cfC3H mammary tumor cells embedded locally in collagen gels for 26 days. (Inset) Aggregated clumps at 1 day after embedding 105 cells. (X25.)
and 100 units of penicillin, 100 ,gg of streptomycin, and 2.5 Atg
of amphotericin B per ml. For conventional monolayer culture, 5-10 X 104 cells in 0.5 ml of above medium were plated into
Cell Biology: Yang et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
3403
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FIG. 2. Outgrowth from different sources. D and E were cultured in dispersed form; the rest were in localized form. (A and B) Human tissue from reduction mammoplasty, after 7 days in culture. (A, X18; B, X45.) (C) Human tissue from mastectomy, after 16 days in culture. (X18.) (D) Normal mammary gland from multiparous regressed BALB/cNIV mice, after 14 days in culture. (X18.) (E) Normal mammary gland from C3H virgin mice, after 15 days in culture. (X18.) (F) D2 nodule line cells, after 25 days in culture. (X18.)
bled mammary tumors in vio. They consisted of compact, multilayered cords of cells with frequent, well-defined intercellular cavities (Fig. 6). Electron microscopy showed that such cavities were typical epithelial lumina (Figs. 7 and 8): cells bordering them were morphologically polarized; they were joined by apical tight junctions and desmosomes and bore variable numbers of apical microvilli. Immature particles of mammary tumor virus were abundant, budding from luminal
cell surfaces or into intracytoplasmic vacuoles, and mature particles often lay free within the lumen. Occasional patches of basal lamina were present at the interface between outgrowth and collagen. Duct-like projections were cut from the periphery of the outgrowth and transplanted in vivo. A total of six such outgrowths were transplanted and all produced palpable tumors in 4 weeks. Histological sections of these tumors were morphologically characteristic of mouse mammary adenocarcinoma and were similar to sections of the original donor tumors. Finally, outgrowths from gels were recovered by collagenase digestion, dissociated, and reembedded in other collagen gels for subeultivation. Pieces of peripheral projection from the outgrowth were also cut out and reembedded, in a manner 7
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FIG. 4. Growth curves for BALB/cfC3H mammary tumor cells embedded locally in collagen gels. Three experiments are shown (a, 3, 0), each done with cells pooled from three or four tumors. Each point represents the mean of four to six gels. The negative standard deviation depicted in the curve with open circles was typical of all experiments. (U), Typical curve for the same cells cultured as conventional monolayer.
3404
Cell Biology: Yang et al.
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Proc. Natl. Acad. Sci. USA 76 (1979)
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FIG. 5. Autoradiographs of BALB/cfC3H mammary tumor cells. (A) Embedded locally in collagen gel for 1 week. (X320.) (B) End of a growing projection of BALB/cfC3H tumor cells after 3 weeks in gel. Note the very high labeling index. (X130.)
similar to the in vivo transplantation procedure. In both cases, reembedded mammary tumor cells gave rise to further outgrowth that continued to expand as a duct-like projection.
FIG. 7. Electron micrograph of a thin section from the same outgrowth as Fig. 6. A lumen (L) and two intracellular vacuoles (V) are shown, with numerous dense, spherical, immature virions of mammary tumor virus along the membranes and at the tips of microvilli. (X6000.)
DISCUSSION
Demonstration of proliferation of rodent and human mammary cells in primary monolayer culture heretofore has been limited primarily to [3H]thymidine incorporation (16-20). The major difficulty has been an inability to promote sustained growth in .4
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FIG. 8. Higher magnification of the edge of a lumen in the same area as Fig. 7. Two cells are joined at their apical borders by a typical epithelial junctional complex: tight junction (T), intermediate junction (I), and desmosome (D). In addition to the several immature virus particles at the apical cell surface (the connecting microvilli may be out of the plane of this section), a mature particle, with an eccentric, angular nucleoid, is in the upper right corner. (X40,000.)
Cell Biology: Yang et al. long-term primary culture (1, 2). In contrast, the collagen gel system permits sustained growth of primary mammary tumor epithelial cells, as judged by the [3H]thymidine labeling index in autoradiography during long-term culture as well as by a 10to 15-fold difference in DNA content compared to monolayer systems after 6 weeks in culture. The growth curve for mammary tumor cells derived from locally embedded cultures may have provided us with a rather low estimate of actual cell doubling that takes place in collagen culture. This was because of the fact that radioautography showed most of the DNA synthesis taking place in the periphery. Additionally, histologic analysis showed that most of the cells in the central portion of the locally embedded culture became necrotic, probably owing at least in part to problems with diffusion. These observations, along with the preliminary finding that duct-like outgrowths can be passaged, indicate that considerable growth of cells may be taking place from the peripheral cells after local embedding and it is likely that the growth will be more rapid if the cells are dispersed in small clumps. Sustained growth of primary cells may be attributed to the fact that the hydrated collagen gel matrix allows three-dimensional growth in a manner somewhat similar to the growth of epithelial cells in vivo. Because control cells plated in conventional monolayer culture did not grow, the collagen gel may make cells more receptive to the growth-promoting effect of serum or other agents in culture. Mammary cell shapes are different in collagen gels compared to those in conventional monolayers and it is becoming increasingly evident that shape may play an important role in the control of epithelial cell growth (21, 22) as well as in eliciting hormone responsiveness in epithelial cells (7-9, 12, 23). The following observations strongly indicate that the collagen gel outgrowths are mammary epithelial in origin: (i) when embedded cells were recovered and transferred to monolayer culture, they formed a continuous sheet of polygonal cells that were indistinguishable from primary cultures of mouse mammary epithelial cells; (ii) electron microscopy revealed lumina bordered by cells with epithelial junctional complexes and budding mammary tumor virus particles; (iii) transplantation of embedded mouse mammary tumor cells to parenchyma-free fat pads in vivo resulted in palpable tumors with the characteristic morphology of mouse mammary adenocarcinoma. Although we have limited our detailed analysis to the outgrowth derived from mammary tumor cells, epithelial cells obtained from normal mammary gland and hyperplastic alveolar nodules from mice as well as human mammary cells seem to behave in a similar fashion. Our preliminary observations suggest that the collagen gel matrix supports growth of mammary epithelial cells from both rodents and humans. The development of a primary culture system that allows continuous and prolonged growth of mammary epithelial cells opens up a new approach to studies which heretofore have been limited by use of the conventional primary culture. We have recently succeeded in transforming rat mammary epithelial cells with dimethylbenzanthracene in vitro, but the incidence of transformation was low (24). A higher growth rate of cells in collagen matrix may improve the transformation rate, thereby allowing more intensive studies of transformation in culture. In addition, the hormone-dependent nature of some human breast cancers in primary culture could be investigated by using this system. Evidence of hormone effects in primary culture of the original tumor may be of value from the therapeutic point of view.
Proc. Natl. Acad. Sci. USA 76 (1979)
3405
Finally, the system shows promise for investigating whether hormones and growth factors have direct mitogenic effect on mammary cells. Recent studies (25) indicate, paradoxically, that although estrogens are obligatory for tumor formation in vivo
no direct mitogenic effect of estrogens can be shown in culture on the basis of assay for increase in cell number. In vitro growth studies may help to define the role of hormones in vivo. Note Added in Proof. Growth rate of mammary epithelial cells can be enhanced greatly (from 5 X 104 cells to 30-50 X 104 cells in about 2 weeks) by dispersing the cells throughout the collagen gel and increasing the serum concentration in the medium. These results will be presented in the near future. We thank Drs. J. Elias, N. Petrakis and R. Wellings for their help in obtaining human tissues. We also thank J. Underhill for photographic
assistance, S. Castillo for clerical assistance, and Drs. K. B. DeOme, M. Harris, and R. Shiurba for critically reading the manuscript. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute and by the Cancer Research Coordinating Committee funds from the University of California.
1. Das, N. K., Hosick, H. L. & Nandi, S. (1974) J. Natl. Cancer Inst.
52,849-861.
2. Hosick, H. L. (1974) Cancer Res. 34,259-261. 3. Engel, L. W. & Young, N. A. (1978) Cancer Res. 38, 43274339. 4. Smith, J. A. & King, R. J. B. (1972) Exp. Cell Res. 73, 351359. 5. Butel, J. S., Dudley, J. P. & Medina, D. (1977) Cancer Res. 37,
1892-1900. 6. Dexter, D. L., Kowalski, H. M., Blazar, B. A., Fligiel, Z., Vogel, R. & Heppner, G. H. (1978) Cancer Res. 38,3174-3181. 7. Emerman, J. T., Enami, J., Pitelka, D. R. & Nandi, S. (1977) Proc. Natl. Acad. Sci. USA 74, 4466-4470. 8. Yang, J., Enami, J. & Nandi, S. (1977) Cancer Res. 37, 36443647. 9. Sakai, S., Bowman, P. D., Yang, J., McCormick, K. & Nandi, S. (1979) Endocrinology, 104, 1447-1449. 10. Medina, D. (1973) Methods Cancer Res. 7, 1-54. 11. Enami, J., Nandi, S. & Haslam, S. (1973) In Vitro 8, 405
(abstr.). 12. Enami, J., Yang, J. & Nandi, S. (1979) Cancer Lett. 6, 99-105. 13. Michalopoulos, G. & Pitot, H. C. (1975) Exp. Cell Res. 94, 7078. 14. Hinegardner, R. T. (1971) Anal. Biochem. 39, 197-201. 15. DeOme, K. B., Faulkin, L. J., Jr., Bern, H. A. & Blair, P. B. (1959) Cancer Res. 19, 515-520. 16. Ceriani, R. L. & Blank, E. W. (1977) Mol. Cell. Endocrinol. 8, 95-103. 17. Hallowes, R. C., Rudland, P. S., Hawkins, R. A., Lewis, D. J., Bennett, D. & Durbin, H. (1977) Cancer Res. 37, 2492. 18. Richards, J. & Nandi, S. (1978) J. Natl. Cancer Res. 61, 765771. 19. Gaffney, E. V., Polanowski, F. P., Blackburn, S. E., Lambiase, J. T. & Burke, R. E. (1976) Cell Differ. 5, 69-81. 20. Stoker, M. G. P., Pigott, D. & Taylor-Papadimitriou, J. (1977) Nature (London) 264, 764-767. 21. Folkman, J. & Moscona, A. (1978) Nature (London) 273,345349. 22. Gospodarowicz, D., Greenburg, G. & Birdwell, C. R. (1978) Cancer Res. 38, 4155-4171. 23. Michalopoulos, G., Sattler, G. L. & Pitot, H. C. (1978) Cancer Res. 38, 1550-1555. 24. Richards, J. & Nandi, S. (1978) Proc. Natl. Acad. Sci. USA 75, 3836-3840. 25. Sirbasku, D. A. (1978) Proc. Natl. Acad. Sci. USA 75, 37863790.
Cell Biology
Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels (mouse mammary cells/human mammary cells/proliferation/long-term culture/primary culture)
JASON YANG, JAMES RICHARDS, PHILLIP BOWMAN, RAPHAEL GUZMAN, JUMPEI ENAMI, KATHLEEN MCCORMICK, SUSAN HAMAMOTO, DOROTHY PITELKA, AND S. NANDI Cancer Research Laboratory and Department of Zoology, University of California, Berkeley, California 94720
Communicated by A. Starker Leopold, April 26, 1979
We have develo ed a method for embedding ABSTRACT cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H]thymidine incorporation during 8 weeks in culture. An increase in DNA content of the cultured cells as a function of time was observed. Mouse mammary tumor cells cultured in the conventional monolayer system failed to show any significant increase in cell number during a culture period of 6 weeks. In addition, in such monolayer systems, cells progressively became detached from the dishes in long-term culture. The mammary epithelial cell origin of the collagen gel cell outgrowths was shown by electron microscopic demonstration of polarized cells containing tight junctions and budding mammary tumor virus particles. In addition, in vivo transplantation of collagen gel outgrowths resulted in the development of mammary adenocarcinoma histologically similar to the donor tumor. Cellular outgrowth patterns resembling those from tumor cells were also seen in similar collagen gel cultures of normal mammary cells from mouse and human and of hyperplastic alveolar nodule cells from mouse. The significance and usefulness of this system in comparison to the conventional monolayer system are discussed. The limited in vitro growth capacity of mammary epithelial cells in conventional primary culture in tissue culture dishes is a well-known phenomenon. The cells generally undergo a few rounds of division, but proliferation cannot be sustained nor can these cells be passaged. Mouse mammary epithelial cells cultured at low density become flattened and multinucleated, and rarely do they attain confluence (1, 2). Cells plated at high density are maintained well but little growth is ever
the growth of mouse mammary tumor cells in such cultures. Preliminary work with normal mouse and human mammary tissues indicates that this technique may also be suitable for growing other types of mammary cells.
MATERIALS AND METHODS Cell Dissociation. Primary mammary tumors from BALB/cfC3HCrgl mice, hyperplastic alveolar nodules (D2) maintained as transplantable lines in vivo in BALB/cCrgl mice (10), and normal mammary gland from C3H/Crgl and BALB/cNIV/Crgl mice were dissociated by a modification of a previously described method (11, 12). Briefly, finely minced mammary tissues were placed in 125- or 250-ml erlenmeyer flasks containing 0.1% collagenase (CLS III; 120-150 units/mg; Worthington) in Hanks' balanced salt solution (10 ml/g of tissue) and swirled on a gyratory water bath shaker (model G76, New Brunswick) at 120-150 rpm at 370C for approximately 90 min or until the suspensions were uniform without macroscopic lumps. The suspension was passed through Nitex cloth (mesh size, 150 ,um); the cells were collected by centrifugation at 80 X g for 5 min, and washed twice with Hanks' solution. The resulting preparation consisted mainly of small clumps of cells. Cell number was estimated by mixing 1 vol of cell suspension with 9 vol of 0.02% crystal violet in 0.1 M citric acid and counting stained nuclei in a hemocytometer. Dissociated human mammary epithelial cells were obtained from excised tissue (reduction mammoplasties and mastectomies) by modification of the above procedure and subsequent gradient centrifugation (details will be reported elsewhere). Culture Procedure. Collagen solution and gel were prepared as originally described (13) and are the same as those used for achieved. floating collagen gel experiments (7-9, 12). Briefly, 1 g of ratMost studies to date dealing with proliferation of mammary tail collagen fibers was sterilized in alcohol overnight and disepithelial cells in vitro have used established cell lines adapted solved in 300 ml of 1:1000 acetic acid in sterile distilled water; to grow in conventional monolayer culture (3-6). Considerable the supernatant after centrifugation at 10,000 X g for 30 min effort is being devoted in several laboratories to analysis of was the stock collagen solution. Eight volumes of stock solution hormone and drug sensitivity in these selected cell lines. An was mixed with 2 vol of 2:1 mixture of 10-fold concentrated inherent limitation of this approach is that such lines may not growth medium and 0.34 M NaOH and kept on ice to prevent be representative of the original cell population. Therefore, it immediate gelation. Dissociated cells were embedded in gel is of considerable importance to improve the conditions for in either a dispersed or a localized pattern. For dispersion, the culture of primary cells. Our demonstration of differentiated appropriate number of cells was added to cold gelation mixture; function in mouse mammary cells cultured on floating collagen 1 ml, containing 5-10 X 104 cells, was pipetted into each well gels, as indicated by levels of casein (7), mammary tumor virus of Falcon plastic Multiwell plates (well diameter, 16 mm) and (8), and prolactin receptor (9), has prompted us to examine allowed to gel at room temperature. For localized embedding, collagen as an appropriate substrate for growth. The most enapproximately 5-10 X 104 cells in 1 ,l were placed on the couraging results were obtained when mammary cells were surface of 0.5 ml of gelled collagen in each well and overlaid embedded within the collagen gel. The present study describes with 3 ,ul of cold gelation mixture so that the diameter of the cell cluster was roughly 5-10% of the well diameter. After this The publication costs of this article were defrayed in part by page layer had gelled, 0.5 ml of collagen was added and allowed to charge payment. This article must therefore be hereby marked "adgel. Cultures were fed every 2 days with F-12 medium vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. (GIBCO) containing 12.5% horse serum, 2.5% fetal calf serum, 3401
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Proc. Natl. Acad. Sci. USA 76 (1979)
each well of Multiwell plates. Cell Number. Cells inside the gel were released by digesting the gel in 1 ml of 0.1% collagenase in Hanks' balanced salt solution in a 10 X 75 mm test tube on a gyratory water bath shaker for approximately 30 min at 370C. The cells were collected by centrifugation at 100 X g for 5 min. Samples were stored frozen until the time of DNA assay. The DNA content was determined by a fluorometric assay (14) utilizing BALB/c spleen cells or BALB/cfC3H mammary tumor cells, counted in a hemocytometer, as a standard. Autoradiography. Cultures were labeled for 24 hr with [3H]thymidine [2.5 ,gCi/ml (1 Ci = 3.7 X 1010 becquerels); 50 Ci/mmol; ICN], fixed in Bouin's fixative, and embedded in paraffin. Sections were cut parallel to the surface of the gel and coated with NTB nuclear track emulsion (Kodak). After 2
RESULTS Freshly dissociated cells were embedded in the collagen gel in either dispersed or localized form. The general growth pattern was monitored by viewing the culture in an inverted microscope. Culturing mammary cells under the described condition led to a characteristic and reproducible pattern of growth. Locally embedded cells aggregated into a single compact mass and produced relatively thick duct-like structures that radiated into the matrix in three dimensions. Cell clumps dispersed through the collagen gel also produced three-dimensional outgrowths but the duct-like structures were not as thick as those produced from locally embedded cells. Such outgrowths of mammary cells from several sources are shown in Figs. 1 and 2. In control experiments, cells obtained from gland-free mammary fat pads and processed in exactly the same manner as above did not give rise to the same three-dimensional ductal structures. The predominant outgrowth pattern seen was one in which the cells grew out as single units producing dense radial patterns (Fig. 3). In addition to these narrow rays, single cells could often be seen wandering through the gel. Although these mesenchymal "fibroblast-like" cells were sometimes seen contaminating cultures derived from mammary tumors, they were never a problem because of their relatively small numbers within the tumor cell populations dissociated by our procedure. Growth was apparent by light microscopy and was confirmed by measuring the total DNA content of outgrowth-containing gels as a function of time. Detailed studies of this growth as well as characteristics of the resulting outgrowth were carried out with the spontaneous tumor from BALB/cfC3H mice. Mammary tumor cells were locally embedded in collagen gel, with a starting cell number of 1-2 X 105 per gel. At weekly intervals, cells were recovered from the gels by collagenase digestion and stored frozen until the time of DNA assay. The results from three separate experiments are shown in Fig. 4. The growth curve in collagen gel revealed an increase in DNA content as a function of culture period. On the other hand, in the monolayer culture, cells seeded at low density progressively became detached from the culture dish, and there was a decrease in cell number, consistent with earlier observation (1, 2). It is unlikely that the increase in DNA content in collagen gel culture was attributable to multinucleation, for several reasons. When similar outgrowths were recovered by collagenase digestion, dissociated, and plated in conventional monolayer culture, the resulting continguous pavement-like sheet of polygonal cells showed no obvious increase in multinucleated cells as compared to mammary tumor cells plated directly in monolayer culture. Histological observation performed in the autoradiographic analysis described below also confirmed that there was no obvious increase in multinucleated cells as a function of culture
weeks, the slides were developed in D-19 developer. Electron Microscopy. Cultures were fixed in 1% formaldehyde/3% glutaraldehyde/0.1 M sodium cacodylate, postfixed in 1% OS04, dehydrated in ethanol, and embedded in Epon. Thick (1-2 tim) sections were stained with methylene blue/ Azure II and photographed with a Zeiss photomicroscope II. Thin sections stained with uranyl acetate and lead citrate were examined in a Siemens Elmiskop 102 electron microscope. In Vivo Transplantation. Approximately 1-mm3 pieces of gel containing visible tissue from the periphery of the duct-like outgrowth were implanted into the gland-free inguinal mammary fat pads of 3-week-old female syngeneic mice (15). Four to 8 weeks after transplantation, the mice were sacrificed and the mammary fat pads were examined for outgrowths. Histological sections of selected outgrowths were prepared and the morphological type was determined.
period. Patterns of DNA synthesis were assessed at weekly intervals by [3H]thymidine incorporation followed by autoradiography. By the end of the first week, most of the locally embedded mammary cells had aggregated into a single relatively compact mass (Fig. 5A). Incorporation of [3H]thymidine was generally not high at this time, usually not exceeding a labeling index of 10%, but was found throughout the mass of cells. By the end of the second week, and throughout the next several weeks, the labeling index increased to about 20-35%, and labeling was localized chiefly at the periphery of the embedded cells. By this time there were numerous duct-like structures that protruded into the gel, and these processes exhibited high labeling indices, exceeding 50% (Fig. SB). Within the central mass, the labeling index tended to decrease with time. Histologically and ultrastructurally, the outgrowths resem-
FIG. 1. Outgrowth from BALB/cfC3H mammary tumor cells embedded locally in collagen gels for 26 days. (Inset) Aggregated clumps at 1 day after embedding 105 cells. (X25.)
and 100 units of penicillin, 100 ,gg of streptomycin, and 2.5 Atg
of amphotericin B per ml. For conventional monolayer culture, 5-10 X 104 cells in 0.5 ml of above medium were plated into
Cell Biology: Yang et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
3403
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FIG. 2. Outgrowth from different sources. D and E were cultured in dispersed form; the rest were in localized form. (A and B) Human tissue from reduction mammoplasty, after 7 days in culture. (A, X18; B, X45.) (C) Human tissue from mastectomy, after 16 days in culture. (X18.) (D) Normal mammary gland from multiparous regressed BALB/cNIV mice, after 14 days in culture. (X18.) (E) Normal mammary gland from C3H virgin mice, after 15 days in culture. (X18.) (F) D2 nodule line cells, after 25 days in culture. (X18.)
bled mammary tumors in vio. They consisted of compact, multilayered cords of cells with frequent, well-defined intercellular cavities (Fig. 6). Electron microscopy showed that such cavities were typical epithelial lumina (Figs. 7 and 8): cells bordering them were morphologically polarized; they were joined by apical tight junctions and desmosomes and bore variable numbers of apical microvilli. Immature particles of mammary tumor virus were abundant, budding from luminal
cell surfaces or into intracytoplasmic vacuoles, and mature particles often lay free within the lumen. Occasional patches of basal lamina were present at the interface between outgrowth and collagen. Duct-like projections were cut from the periphery of the outgrowth and transplanted in vivo. A total of six such outgrowths were transplanted and all produced palpable tumors in 4 weeks. Histological sections of these tumors were morphologically characteristic of mouse mammary adenocarcinoma and were similar to sections of the original donor tumors. Finally, outgrowths from gels were recovered by collagenase digestion, dissociated, and reembedded in other collagen gels for subeultivation. Pieces of peripheral projection from the outgrowth were also cut out and reembedded, in a manner 7
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FIG. 4. Growth curves for BALB/cfC3H mammary tumor cells embedded locally in collagen gels. Three experiments are shown (a, 3, 0), each done with cells pooled from three or four tumors. Each point represents the mean of four to six gels. The negative standard deviation depicted in the curve with open circles was typical of all experiments. (U), Typical curve for the same cells cultured as conventional monolayer.
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Proc. Natl. Acad. Sci. USA 76 (1979)
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FIG. 5. Autoradiographs of BALB/cfC3H mammary tumor cells. (A) Embedded locally in collagen gel for 1 week. (X320.) (B) End of a growing projection of BALB/cfC3H tumor cells after 3 weeks in gel. Note the very high labeling index. (X130.)
similar to the in vivo transplantation procedure. In both cases, reembedded mammary tumor cells gave rise to further outgrowth that continued to expand as a duct-like projection.
FIG. 7. Electron micrograph of a thin section from the same outgrowth as Fig. 6. A lumen (L) and two intracellular vacuoles (V) are shown, with numerous dense, spherical, immature virions of mammary tumor virus along the membranes and at the tips of microvilli. (X6000.)
DISCUSSION
Demonstration of proliferation of rodent and human mammary cells in primary monolayer culture heretofore has been limited primarily to [3H]thymidine incorporation (16-20). The major difficulty has been an inability to promote sustained growth in .4
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FIG. 8. Higher magnification of the edge of a lumen in the same area as Fig. 7. Two cells are joined at their apical borders by a typical epithelial junctional complex: tight junction (T), intermediate junction (I), and desmosome (D). In addition to the several immature virus particles at the apical cell surface (the connecting microvilli may be out of the plane of this section), a mature particle, with an eccentric, angular nucleoid, is in the upper right corner. (X40,000.)
Cell Biology: Yang et al. long-term primary culture (1, 2). In contrast, the collagen gel system permits sustained growth of primary mammary tumor epithelial cells, as judged by the [3H]thymidine labeling index in autoradiography during long-term culture as well as by a 10to 15-fold difference in DNA content compared to monolayer systems after 6 weeks in culture. The growth curve for mammary tumor cells derived from locally embedded cultures may have provided us with a rather low estimate of actual cell doubling that takes place in collagen culture. This was because of the fact that radioautography showed most of the DNA synthesis taking place in the periphery. Additionally, histologic analysis showed that most of the cells in the central portion of the locally embedded culture became necrotic, probably owing at least in part to problems with diffusion. These observations, along with the preliminary finding that duct-like outgrowths can be passaged, indicate that considerable growth of cells may be taking place from the peripheral cells after local embedding and it is likely that the growth will be more rapid if the cells are dispersed in small clumps. Sustained growth of primary cells may be attributed to the fact that the hydrated collagen gel matrix allows three-dimensional growth in a manner somewhat similar to the growth of epithelial cells in vivo. Because control cells plated in conventional monolayer culture did not grow, the collagen gel may make cells more receptive to the growth-promoting effect of serum or other agents in culture. Mammary cell shapes are different in collagen gels compared to those in conventional monolayers and it is becoming increasingly evident that shape may play an important role in the control of epithelial cell growth (21, 22) as well as in eliciting hormone responsiveness in epithelial cells (7-9, 12, 23). The following observations strongly indicate that the collagen gel outgrowths are mammary epithelial in origin: (i) when embedded cells were recovered and transferred to monolayer culture, they formed a continuous sheet of polygonal cells that were indistinguishable from primary cultures of mouse mammary epithelial cells; (ii) electron microscopy revealed lumina bordered by cells with epithelial junctional complexes and budding mammary tumor virus particles; (iii) transplantation of embedded mouse mammary tumor cells to parenchyma-free fat pads in vivo resulted in palpable tumors with the characteristic morphology of mouse mammary adenocarcinoma. Although we have limited our detailed analysis to the outgrowth derived from mammary tumor cells, epithelial cells obtained from normal mammary gland and hyperplastic alveolar nodules from mice as well as human mammary cells seem to behave in a similar fashion. Our preliminary observations suggest that the collagen gel matrix supports growth of mammary epithelial cells from both rodents and humans. The development of a primary culture system that allows continuous and prolonged growth of mammary epithelial cells opens up a new approach to studies which heretofore have been limited by use of the conventional primary culture. We have recently succeeded in transforming rat mammary epithelial cells with dimethylbenzanthracene in vitro, but the incidence of transformation was low (24). A higher growth rate of cells in collagen matrix may improve the transformation rate, thereby allowing more intensive studies of transformation in culture. In addition, the hormone-dependent nature of some human breast cancers in primary culture could be investigated by using this system. Evidence of hormone effects in primary culture of the original tumor may be of value from the therapeutic point of view.
Proc. Natl. Acad. Sci. USA 76 (1979)
3405
Finally, the system shows promise for investigating whether hormones and growth factors have direct mitogenic effect on mammary cells. Recent studies (25) indicate, paradoxically, that although estrogens are obligatory for tumor formation in vivo
no direct mitogenic effect of estrogens can be shown in culture on the basis of assay for increase in cell number. In vitro growth studies may help to define the role of hormones in vivo. Note Added in Proof. Growth rate of mammary epithelial cells can be enhanced greatly (from 5 X 104 cells to 30-50 X 104 cells in about 2 weeks) by dispersing the cells throughout the collagen gel and increasing the serum concentration in the medium. These results will be presented in the near future. We thank Drs. J. Elias, N. Petrakis and R. Wellings for their help in obtaining human tissues. We also thank J. Underhill for photographic
assistance, S. Castillo for clerical assistance, and Drs. K. B. DeOme, M. Harris, and R. Shiurba for critically reading the manuscript. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute and by the Cancer Research Coordinating Committee funds from the University of California.
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