A possible mammary stem cell line

Cell,

Vol. 15. 283-298,

September

A Possible

1978,

Copyright

Mammary

0 1978 by MIT

Stem Cell Line

Dorothy C. Bennett, Linda A. Peachey, Helga Durbin and Philip S. Rudland Imperial Cancer Research Fund P. 0. Box 123 Lincoln’s Inn Fields London WC2A 3PX, England Summary The cell line Rama 25 is derived from a mammary tumor induced in a rat by dimethylbentanthracene. During rapid proliferation, Rama 25 cells appear as a single undifferentiated epithelial type; at high cell densities, however, small numbers of two other cell types are formed, which respectively resemble secretory and myoepithelial cells of the mammary gland, as judged by light and electron microscopy and immunofluorescent staining for casein (milk proteins). These additional cell types cannot be explained as contaminating cell populations since the cell line has been cloned several times; furthermore, the proportion of either can be increased by dimethylsulphoxide under different conditions. Specific epithelial features are seen by histological and ultrastructural examination of tumors formed by Rama 25 cells in immunodeficient mice. A line of the myoepithelial-like cells, Rama 29, isolated from a Rama 25 culture by cloning, is also described. We propose that the undifferentiated cell type is a form of mammary stem cell which can differentiate in culture. Introduction The epithelial components of the mammary gland, which are embedded in fatty stroma or connective tissue, consist of a branching system of ducts terminating, if fully developed, in clusters of alveoli, which secrete milk during lactation. Three main types of mammary epithelial cell are usually distinguished: those lining the alveoli, those lining the ducts, arid the myoepithelial cells which form a continuous or discontinuous layer around both ducts and alveoli, and which contract during suckling. Differences between these three cell types are seen in sections of the gland with certain histological or histochemical stains (Dempsey, Bunting and Wislocki, 1947; Schlotke, 1976) or by transmission electron microscopy (Hollman, 1974; Pitelka et al., 1973). It has also been suggested that different specific types of tumors originate in each of the three cell types (Murad, 1971; Slemmer, 1974). It is unclear, however, whether these cell categories are absolutely distinct, since structural intermediates or gradations have been reported between myoepithelial and “lining” (ductal or alveolar)

epithelial cells (Tannenbaum, Weiss and Marx, 1969; Ozzello, 1971) and between ductal and alveolar cells (Young and Hallowes, 1973; Pitelka et al., 1973). The techniques of cell and tissue culture provide one approach to cell classification: if two or more allegedly different cell populations can be physically separated and then cultured, one can observe whether one type can change into another, at least under culture conditions. The range of mammary cell culture systems in use has been reviewed by Kerkof and Abraham (1974). In relation to the problem of isolating individual cell types, numerous mammary epithelial cell lines (see also Discussion) have been initiated from normal or neoplastic mammary tissue. Some of these appear to consist of a single type of cell, resembling either lining epithelial cells as in MCF7 (Soule et al., 1973), NMuMg (Owens, Smith and Hackett, 1974) and some lines described by Butel, Dudley and Medina (1977), or myoepithelial cells in one case (Hackett et al., 1977). Without long-term cell culture, Kraehenbuhl (1977) and Schroeder, Chakraborty and Soloff (1977) have achieved some direct separation of myoepithelial from lining epithelial cells. We have developed a method for the partial separation of two cell populations, believed to be myoepithelial and lining epithelial cells, in cultures made from normal rat mammary epithelium or from DMBA-induced rat mammary tumors, which usually contain both types of cell (Young and Hallowes, 1973). We completed this separation by cloning cells from both populations. Among the first clones, which were from a tumor, we found a type of epithelial cell, here termed cuboidal, which could give rise to two other morphological types, elongated and droplet cells. This ability was retained after repeated cloning. The morphological resemblance of elongated and droplet cells to those believed to be myoepithelial and lining epithelial cells in our short-term cultures, and the findings that their formation could be predicted, prevented or chemically promoted (see below), all suggested that we were observing differentiation rather than genetic instability. We now describe the isolation and characterization of Rama 25, a clonal line of these cuboidal cells, and Rama 29, a subclone derived from Rama 25 and consisting of elongated cells. We discuss the connections between cuboidal, elongated and droplet cells and the cell types in the mammary gland. Results Culture Media Two supplemented throughout. “Routine

culture medium”

media were used was 90% (v/v) DEM

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(Dulbecco’s modified Eagle’s medium), 10% FCS (fetal calf serum), 50 ng ml-’ of cortisol and 50 ng ml-’ of bovine insulin. “Conditioned medium” was 80% DEM and 20% FCS, conditioned for 1 day by mouse 3T3 cells as described by Rheinwald and Green (1975), and supplemented just before use with cortisol and insulin as above, 500 ng ml-’ of ovine prolactin, 100 ng ml-’ of progesterone and 100 ng ml-’ of bovine growth hormone. Derivation of Cell Lines Rama 25 and Rama 29 A DMBA-induced rat mammary tumor was dissociated by the method described in Experimental Procedures (Hallowes et al., 1977; Rudland et al., 1977) to yield primary cultures consisting almost entirely of clumps of epithelial cells. 5 days after plating in routine medium, many of these clumps remained in suspension, having formed hollow spheres and branching tubes, which suggested that this tumor was unusual since such tumor cell clumps normally attached to the culture dishes within 1 day. The cell clumps were now replated in conditioned medium. After a further 16 days, all clumps had attached and spread to form cell colonies. Many of the colonies consisted of an upper layer of small isometric cells on a wider basal layer of larger elongated cells. Ultrastructural studies of cultures from similar tumors (R. C. Hallowes, D. C. Bennett and L. A. Peachey, unpublished data) suggested that the upper layer was of lining epithelial cells and the basal cells were myoepithelial. Upper layer cells were now separated by a brief incubation in EDTA solution (Experimental Procedures), then suspended in conditioned medium, passed through a 27pm Polymon mesh to remove cell clumps and replated (approximately lo5 cells). After 8 days, some 20 colonies of proliferating cells were seen, resembling sheets of low cuboidal epithelium (Figure la) (for terms, see Bloom and Fawcett, 1975). This type of cell is called “cuboidal” here. Cells from a number of these colonies were transferred separately to 16 mm culture wells using steel cloning rings. The results of this and subsequent rounds of cloning are summarized in Figure 2. Cells in four of the wells continued to proliferate, and each such culture was passaged/subcuItured (Experimental Procedures) twice further to yield 106-10’ cells. All four of these cell strains, however, were by now composed predominantly of “elongated” cells, as shown in Figure 1 b, and resembled the basal cells described above. In one of the strains, L2, colonies of cuboidal cells were seen growing on top of the confluent elongated cells. One such colony grew onto a fragment of glass coverslip placed near it and thence onto a dish to which the glass was transferred. Clones were made from this culture by a

rigorous method, always used subsequently, in which individual cells were transferred from a suspension to 6 mm culture wells (Experimental Procedures). Of 32 cells picked, 21 formed colonies, 13 being of elongated cells and eight of cuboidal cells after 5 days. Cell lines were initiated from several clones of each type (Figure 2). [All cell lines described here are primary rather than established lines in the nomenclature of Paul (1975), in that none has been subcultured for 160 population doublings, the equivalent of his 70 subcultures at 1:5, and none is used beyond a given number of subcultures.] While no detectable phenotypic change was seen in any line of elongated cells within about 50 population doublings, all eight clones derived from cuboidal cells gave rise to a mixture of cuboidal and elongated cells either within the cloning well or after one passage. The proportion of elongated cells in these mixtures was found to be lowest when the cells were subcultured frequently. One of the mixed cell lines, Rama (Rat mammary) 22, was therefore recloned, and by subculturing every 2 days, two cuboidal cell lines, Rama 25 and Rama 26, were grown to yield sufficient cells for frozen storage (lo’-108) without appreciable contamination by elongated cells, as estimated in the following test. Cells were plated at IO4 cells cm-*, allowed to grow to saturation density (see below), fixed and stained with Giemsa. The number of foci of elongated cells (Figure lc) then visible to the naked eye was taken as an estimate of the number of elongated cells present at plating. Stocks of Rama 25 cells frozen at the sixth to ninth passages after cloning (or 19th to 22nd since primary culture) contained under 1 in lo5 elongated cells by this criterion. This number was much higher when a dense culture was replated and tested. Clones of elongated cells were obtained from Rama 25 as follows. Rama 25 cells were plated sparsely, grown to saturation density, plated sparsely again and cloned upon reaching a moderate density. 50 cells were picked and yielded 21 colonies of cuboidal and 17 of elongated cells. Three of the latter were selected and grown to give the cell lines Rama 29, 30 and 31, of which Rama 29 was chosen for detailed study. A few of the cuboidal cell clones were also subcultured, and again all yielded mixtures of both cell types. Behavior of Cuboidal and Elongated Cells Living cells in all clones from this tumor were observed frequently by phase-contrast microscopy, and particular cell lines are mentioned here only as examples. Cell type-specific behavior was most easily seen in colonies of like cells as in Figure 1 (a, b and d), where cell colonies from Rama 25 and 29 are compared with one from a clonal line of

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Figure

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Cell Line

of Different

Cell Types

Living cells shown by phase-contrast optics. (a, b and d) 5 day old colonies of (a) cuboidal cells (Rama 25), (b) elongated cells (Rama 29), and (d) mammary stromal cells (Rama 27). (c) Focus of elongated cells in confluent Rama 25 culture after passage from a dense culture. (e) Cuboidal cells growing over area of elongated cells in mixed culture (Rama 22). (f) Rama 25, patch of droplet cells, showing defined edge and several small domes (arrowheads). Bars = 100 fim.

cells from the stroma of normal rat mammary gland, Rama 27. These are strictly Iipoblasts, since some of them differentiated shortly after cloning into fat cells, like those described and illustrated by Russell and Ho (1976). It can be seen that both cuboidal and elongated cells formed continuous

colonies with well defined edges, unlike the stromal cells. The cuboidal cells invariably grew as a single layer (monolayer), whereas the cytop!asm of adjacent elongated or stromal cells tended to overlap. Cuboidal cells would also proliferate as a monolayer on top of confluent elongated cells,



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Tumour Culture EDTA-released fraction

I

0

O

1

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ORama 22



Rama

I

1

1 ORama 25

O O

Rama 29

Figure 2 . Cloning of Rama 25 and Related Cell Lines Each symbol represents a clonal cell strain or line of cuboidal cells (Q) or elongated cells (p) . Further cell strains and/or lines of the same types have been omitted (---) . The origin of each generation of clones and repeated production of elongated from cuboidal cells is shown . Names of cell lines are given where relevant to the text .

whose orientation they then tended to adopt (Figure le) . We found this invariably in a number of combinations of these cell lines, whereas we have never seen epithelial cells grow on the upper surface of stromal cells in primary cultures or combinations of cell lines . Cuboidal and stromal cells instead formed clearly delineated areas, as observed in the "type 2" outgrowths from bovine mammary epithelium described by Ebner et al . (1961) . No cell type would grow on confluent cuboidal cells . The behavior of cuboidal cells at high local density, including the centers of large cell colonies, was complex . In some regions, elongated cells were formed ; in a growing colony, these could form a patterned, branching network beneath the cuboidal cell layer, but in very dense cultures, they tended to be preferentially shed into the culture medium . In other regions, occasional groups of "droplet cells" (Figure 1f) were observed . These cells were small in area, polygonal and dark, with many pale droplets or vacuoles at their edges . They were found in roughly circular patches with well defined edges (Figure 1f), and these patches frequently contained domes, hemispherical blisters formed by local detachment of the monolayer and suggesting active transport of fluid (McGrath, 1975) . Cuboidal cells did not form domes, even at high cell density . In Rama 25 cultures the pale droplets were not stained by Oil Red 0, a nonspecific stain for lipid, although very fine particles which did

stain were seen in all cells in sparse or dense cultures . The cells of some, though not all, domes were stained more brightly than those in the monolayer, however, due to the presence of more numerous fine particles . At maximum proliferation rate in routine medium, Rama 25 had a population doubling time of about 10 hr and Rama 29 of about 14 hr from determinations of cell numbers . The saturation cell density in this medium was 3 x 10 5 cells cm -2 in Rama 25 and 1 .8 x 10 5 cells cm -2 in Rama 29 . Confluency, or the covering of all available substrate, occurred in both lines at a cell density well below the maximum . From rough comparisonsfor example, time required between subculturesproliferation rates varied quite widely among different lines of either cell type . Karyology Chromosome spreads (Rothfels and Siminovitch, 1958) were made from Rama 25 cells at passage 11 after cloning and Rama 29 at passage 8 after cloning . Chromosome numbers in both lines showed a sharp mode at 46, with slightly more spread in Rama 25 . The diploid number for Rattus norvegicus is 42, but the chromosome morphology was readily recognizable as that of the rat . Effects of Dimethylsulphoxide The behavior of the cuboidal cells at high density suggested a form of differentiation . We therefore investigated the effects of agents, primarily dimethylsulphoxide (DMSO), known to promote differentiation in some other cell lines, including Friend erythroleukemic cells (Friend et al ., 1971) and neuroblastoma cells (Palfrey et al ., 1977) . When 250 mM DMSO (1 .8%, v/v) in fresh routine medium was added to Rama 25 and Rama 29 cultures at saturation cell density, no effect was seen in Rama 29 cells ; in 2 days, however, at least 95% of the Rama 25 cells had become droplet cells, with a progressive increase in the number of domes present . This stimulation of dome formation was largely dependent upon the cortisol and/or insulin present in routine medium, as the following results show . Various media were added to Rama 25 cells grown to saturation density in DEM and 10% FCS . 36 hr later, the number of domes per microscope field (28 mm 2 ) was found to be 0 ± 0 in routine medium and 67 ± 6 in routine medium with DMSO, but only 4 ± 4 in routine medium with DMSO and lacking cortisol and insulin (means ± standard errors of ten fields, five from each of two dishes) . For such experiments, FCS was dialyzed extensively against EGTA ; the omission of hormones had much less effect in the presence of undialyzed FCS . Both hormones were reported to promote dome formation in mouse mammary tumor cells (McGrath,

A Possible 287

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Cell Line

1975). Lower concentrations of DMSO (50 mM, 100 mM) produced patches of droplet cells after 1 day which spread progressively. Domes were seen only within the patches. In preliminary experiments, similar effects were observed with two other artificial inducers of differentiation in erythroleukemic and neuroblastoma cells, butyric acid and hexamethylene bisacetamide (Reuben et al., 1976; Palfrey et al., 1977). The effects of DMSO were not stable; its removal caused a further change, seen most clearly in sparse cultures. When very sparse Rama 25 cultures were exposed to 250 mM DMSO from 4 until 7 days after plating, the cell colonies ceased to grow, and most appeared to be composed of droplet cells, although these were much flatter than usual. [Domes were formed, sometimes at local cell densities-about 1 x lo5 cells cm-2-unusually low for doming (Hosick, 1974).] Such cultures could be maintained in DMSO with a low death rate, but when DMSO was removed and fresh routine medium added, more than half the colonies were dead or partly dead within 48 hr. The remaining cells resumed proliferation, but a high proportion were now elongated cells, the rest being cuboidal. In this way, DMSO could be used to produce cultures consisting largely of elongated cells. This was probably by selective survival rather than through an effect on the formation of elongated cells, since when either a lower DMSO concentration or a lower serum concentration was used, many more of the cells survived, and a correspondingly higher proportion of cuboidal, rather than elongated, cells was observed. These experiments also raised the possibility that droplet cells were terminally differentiated, as did the finding that when a dense culture consisting of at least 97% droplet cells (prolonged exposure to 50 mM DMSO) was subcultured, the plating efficiency (number of colonies of one or more cells after 5 days) was about one tenth that of Rama 25 stocks. Colonies formed after a lag, and most were of cuboidal cells. As a rigorous test of division in droplet cells, dense cultures which had been maintained with or without 100 mM DMSO were therefore “wounded” with a blade, and time-lapse films were made of cells spreading toward the wounds. Whether DMSO had been present or not, more than 40 of the 50 observed droplet cells divided viably within 70 hr, sometimes after loss of their droplets, which later reappeared. Only 5% of the observed cells died. Hence, while droplet cells might not be immediately capable of a high proliferation rate, it appeared that they could readapt, apparently reverting to the cuboidal cell type if the stimulus was sufficiently gradual (spreading) or small (low serum; see above)-that is, if they were not terminally differentiated.

Tumors Formation in Nude Mice: Histology of the Tumors To test whether Rama 25 and Rama 29 cells were tumorigenic in nude (immunodeficient) mice, 2 x lo6 cells of either line were injected subcutaneously and ventrally into young females. In all ten mice injected with Rama 25 cells, one or two tumors were palpated at the injection site by the sixth week after injection. After 8 weeks, all mice were killed and the tumors were examined by histology and electron microscopy (see below). No tumors were formed by Rama 29 cells in 19 mice killed and autopsied between the sixth and tenth weeks after injection. Nevertheless, at high cell densities, Rama 29 was among the most slowly proliferating of the elongated cell clones; hence the lack of tumorigenicity in these cells was not necessarily a result of the change from the cuboidal to the elongated form. Most of the Rama 25 tumors had invaded the skin and/or body wall, although the livers and lungs of all mice were free of macroscopic metastases. Sections from all tumors were stained with hematoxylin and eosin, and some for reticulin (Bloom and Fawcett, 1975). The histological pattern varied little from tumor to tumor, the center being necrotic and hard, and the periphery cellular. The organization (Figure 3) was largely sarcomalike (spindle cells) but with regions of adenocarcinoma usually comprising cords and clumps of cells surrounded by extracellular material. Occasional duct-like structures were seen, some with eosinophilic contents (Figure 3). Both carcinomatous and sarcomatous areas had portions showing an epithelial pattern when stained for reticulin-that is, reticular fibers surrounding cell groups rather than individual cells (Evans, 1958); furthermore, both

Figure Cells

3. Histological

Section

of

Tumor

Formed

by Rama

25

Hematoxylin and eosin stain. Showing both sarcoma-like (S) and carcinoma-like (C) patterns and invasion between muscle fibers (M) of body wall. Duct-like profiles (D) are visible. The split in section (between M-M) is an artifact of the histological preparation. Bar = 100 pm.

Cell 288

were composed predominantly of rat cells-that is, Rama 25 cells-as shown by the fluorescent stain Hoechst 33258, which demonstrates a characteristic spotty pattern of heterochromatin in mouse cell nuclei (Moser, Dorman and Ruddle, 1975) . This can be used on conventionally fixed and sectioned tissue (B . Hogan, unpublished results), although the pattern can be ambiguous in small, dense nuclei . Small, fibroblastic cells of probable host origin were seen, singly or in groups, throughout the tumors and around them, and occasional host blood vessels were present . Both epithelioid and spindle cells were invading host tissue, and both showed numerous mitoses at the tumor periphery . Recently, more such tumors were fixed in methacarn, sectioned and stained with TP-Levanol Fast Cyanine (TPL), a stain for myosin-like proteins (Schlotke, 1976) . In normal rat mammary gland fixed and sectioned in parallel, stain was concentrated only in the myofilaments of the myoepithelial cells and vascular smooth muscle, and in erythrocytes . In the tumors, the cells of many of the sarcomatous regions showed intracytoplasmic staining, sometimes in discrete streaks, while those in other such regions showed long branching processes which stained . Hence at least some of these cells were both muscle-like and epitheliallike (from the reticulin pattern ; see above), suggesting that some or most of the spindle cells were myoepithelial, with varying degrees of differentiation . Electron Microscopy Cells from Rama 25, Rama 29 and related cell lines were prepared for transmission electron microscopy as described in Experimental Procedures, and were grown on glass coverslips for this purpose unless otherwise stated . Sections were also prepared from tumors formed by Rama 25 cells in nude mice . See Fawcett (1966) for ultrastructural terms in the following . The ultrastructure of Rama 25 cells varied greatly with external conditions . Proliferating cuboidal cells (Figure 4a) were relatively featureless with little cytoplasm and few organelles . These cells were very flat, with sparse stubby microvilli on the surface facing the culture medium ; adjacent cells were generally linked by a poorly developed functional complex of occluding and intermediate junctions . Cuboidal cells grown on plastic (not shown) appeared similar but less flattened . (In Rama 25, both the saturation cell density and the cell density in growing colonies were higher on plastic than on borosilicate glass .) Figure 4b shows droplet cells from a confluent Rama 25 culture grown on plastic and exposed to DMSO . These had more cytoplasmic organelles and microvilli than the growing cuboidal cells, as did confluent cuboidal cells (not

shown) . The droplet cells also contained large intra- and intercellular vacuoles, usually without osmiophilic or electron-dense contents, and presumably the droplets visible by light microscopy . Examination of Figure 4b suggests that these droplets could sometimes be released beneath cells and could have mediated dome formation . There was little ultrastructural evidence of secretion of protein or lipid . Similar features were observed in sections of droplet cells formed in a Rama 22 culture (see Figure 2) grown on glass and in the absence of DMSO . The ultrastructure'of the elongated cells differed from that of both cuboidal and droplet cells in many respects . Confluent Rama 29 cells are shown in Figures 4c and 4d . Many layers of cells were present ; microvilli and junctional complexes were not seen, and the cytoplasm was often rich in rough endoplasmic reticulum (RER) with electronlucent contents of fibrillar appearance . Extracellular material resembling basal lamina was common, and was connected to some cells by hemisome-like junctions . The material showed microfibrillar substructure at high magnifications . Cells often contained peripheral myofilaments, or bundles of cytoplasmic filaments containing scattered electrondense foci or condensations (Murad and Von Haam, 1968) . Groups of "pinocytotic" vesicles (Figure 4c) were common, forming broad arrays in sections parallel and close to the membrane (not shown) . Rama 4 cells (see Figure 2) were also examined (not shown) ; these cells were larger but displayed the same specific features . The significance of these findings is considered in the Discussion . A marked increase in the expression of secretory characteristics has been described in primary cultures of normal mouse mammary cells when maintained, in the presence of cortisol, insulin and prolactin, on floating gels prepared from rat-tail collagen rather than on plastic (Emerman et al ., 1977) . We therefore examined the ultrastructure of Rama 25 cells maintained in the same way, in the presence of the same three hormones and DMSO (Figure 4e) . Like the mouse cells, Rama 25 now formed a cuboidal to columnar epithelium, usually of a single layer of cells, which showed numerous apical microvilli and well developed junctional complexes . Slight secretory activity was suggested by an apical Golgi apparatus in most cells, with lateral RER (sometimes stacked), but this development was much less than that observed by Emerman et al . (1977), which mimicked lactation in the mammary gland . Groups of spindly cells were also seen in the Rama 25 cultures, migrating into the gel in chains or spikes . The ultrastructure of these was similar to that of Rama 29 and Rama 4 cells (elongated cells) grown on glass .

A Possible Mammary Stem Cell Line 289

Epithelial cells could be firmly identified by electron microscopy of the tumors formed by Rama 25 cells in nude mice . Carcinoma-like and sarcomalike regions were both selected for study . Figure 5 shows cells from a carcinoma-like area with moderate ductal organization . In this area, arrangement of cells around a lumen could often be seen (Figure 5a), as could myoepithelial-like cells (Figures 5b and 5c) and well developed desmosomes (Figure 5d) . Banded collagen and finer extracellular fibers surrounded groups of cells (Figure 5a), but basal lamina was usually absent . The spindly cells comprising the sarcoma-like regions (not shown) displayed no lumina nor microvilli, some intermediate and (rarely) desmosome-like junctions, frequently prominent RER and abundant extracellular material usually resembling multiple, disorganized basal lamina . Some banded collagen was also seen, suggesting the presence of host fibroblasts . Nevertheless, the ultrastructure of the spindly cells, together with the patterns of reticulin and chromatin staining (see above), suggested that they were related to the elongated cells seen in culture . Clear myofilaments were not seen in these preparations, although myofilaments were apparently stained in the spindle cells of a different set of Rama 25induced tumors by the TPL stain (above) . It is possible that the specimens examined by electron microscopy were less differentiated and/or more degenerated . Cells of unhealthy or necrotic appearance were common in both sarcomatous and carcinomatous regions . Searches in all cell lines and tumors studied failed to detect either virus-like particles or the Weibel-Palade bodies (Weibel and Palade, 1964) typical of endothelial cells of species including the rat . Immunofluorescent Staining for Casein Caseins are acidic phosphoproteins found in milk (Feldman and Ceriani, 1970) ; they have been detected by immunofluorescent staining in mammary secretory cells but rarely in immature mammary gland (Young and Nelstrop, 1970), and also in primary mammary cell cultures (Feldman and DeOme, 1975) . We therefore tested our cultures for casein as a possible marker of mammary differentiation using indirect immunofluorescence (Roitt, 1977 ; Experimental Procedures) . All results in Table 1 were obtained with an antiserum (serum 2 ; Experimental Procedures) characterized as follows . Reactivity against casein was detected by immunodiffusion tests and by immunoprecipitation of components resembling two rat caseins in electrophoretic mobility (on SDSpolyacrylamide gels), both from mixed solutions of purified rat caseins (after Feldman and Ceriani, 1970) and from rat milk (autoradiographic detec-

tion ; rats injected with radioactive amino acids) . These components were not detectably precipitated in either experiment by serum from unimmunized rabbits ["normal rabbit serum" (NRS)] at the same concentration, while several other purified proteins were precipitated neither by the antiserum nor by NRS . The possibility of the presence of antibodies other than against casein, but specific to the antiserum, was tested by immunofluorescent staining of sections of a number of different rat organs . "Specific fluorescence" was defined as fluorescence of greater intensity in specimens stained for casein than that seen in parallel preparations in which NRS had been substituted for anti-casein serum . As shown in Table 1 a, specific fluorescence was observed only in sections from mammary gland among the organs tested, and this fluorescence increased with secretory activity in the gland . The specific fluorescence appeared to be localized in the apical (luminal) parts of epithelial cells (Figure 6) and inside some lumina, as seen by Young and Nelstrop (1970) . This specific fluorescence could be abolished by preadsorption of the antiserum with 1 mg/mI of purified rat caseins (Table 1a) . Hence any specific antibodies against components other than casein would have to be against antigens present only in milk-secreting cells and their products, and also in the casein preparation . We now tested the antiserum on cultured cells of various lines : Rama 25, Rama 29, Rama 27 (rat mammary stromal cells ; see above), CSG 122/17 (mouse salivary epithelial cells ; Knowles and Franks, 1977) and CMT 64 (mouse lung epithelial cells ; Franks et al ., 1976) . The effect of a supplement of three hormones-cortisol, insulin and prolactin -was also tested ; this combination produced the greatest observed stimulation of casein synthesis in cultured mammary gland from mice (Turkington, Lockwood and Topper, 1967) or rats (except corticosterone replaced cortisol ; Hallowes, Wang and Lewis, 1973) . The results are summarized in Table 1b and illustrated in Figure 7 . No specific fluorescence was seen in Rama 29, Rama 27 or the salivary or lung cell lines under any conditions . Rama 25 cells, however, treated with cortisol, insulin, prolactin and DMSO gave bright specific fluorescence . This was particularly apparent in cells in domes, which were numerous in such cultures (see above), but this could be explained by folding of the cell sheet where a dome had collapsed (Figure 7b) . Definitely positive fluorescence was also seen in Rama 25 cultures when exposed to DMSO without hormones and (probably) vice versa (Table 1b), but when cells were grown without either, definite specific fluorescence was seen in only one of three experiments . There was some association between positive flu-

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Figure

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29 Cells

in Culture

(a-d) Vertical sections through cultures grown on glass or plastic, in routine medium. Cell surfaces facing medium are uppermost. (a) Rama 25, from a colony of cuboidal cells on glass. (b) Rama 25. culture at saturation cell density on plastic: 250 mM DMSO present for 2 days. (c and d) Rama 29, confluent cultures on glass; further cell layers not shown. Note intracellular bundles of filaments in longitudinal (F) and transverse (F’) section, both with electron-dense condensations; groups of plasma membrane-associated vesicles (V) and basal lamina-like material (B) with hemisome-like junctions to cells (H). (e) Rama 25, culture grown on collagen gel, fixed 6 days after release of gel from plastic. Routine medium with 500 ng ml-’ of ovine prolactin present for 4 (2 + 2) days and 250 mM DMSO for 2 days. Part of section through complete duct, or similar structure with lumen (L). Collagen fibers (C) visible in gel. Bars = 2 pm.

orescence and the presence of domes in a culture, but this has not yet been studied systematically. Variability in the intensity of fluorescence seen in Rama 25 cells (Table 1 b) may have been due both to the subjectivity of the assay and to hormonal differences in batches of FCS. Positive fluorescence has again been obtained both in mammary gland sections and in Rama 25 cultures with another anti-casein serum, serum 4 (Experimental Procedures). Additional evidence for casein synthesis by Rama 25 cells after exposure to DMSO and hormones has been obtained from preliminary experiments (P. S. Rudland and H. Durbin), in which a protein with the electrophoretic mobility of the rat casein component p (Experimental Procedures) has been precipitated from culture medium removed from these cells, and from cell extracts, both before and after preabsorption with NRS. A small number of other

components were also seen after preabsorption which did not migrate with rat caseins. It is possible that breakdown products and/or casein precursors were present. Preliminary experiments have also been carried out with a specific radioimmune assay for casein -that is, for material capable of competing with ‘*Y rat casein for binding to the antiserum, after Emerman et al. (1977). Significant amounts of casein were detected in medium removed from Rama 25 cells after treatment with DMSO and hormones according to the legend of Table 1, whereas no detectable amount (cl ng ml-‘) was present before the addition of DMSO (M. J. Warburton, unpublished results). Discussion Our central conclusion, that the cuboidal cell

summarized in Figure 8. is of Rama 25 is a type of



A Possible Mammary Stem Cell Line 293

Table 1 . Immunofluorescence Intensities in Tissue Sections and Cell Cultures Specimen

Normal Serum

Anti-Casein Serum

+ 0 + 0

+ 0 + 0 0,(+) + +++, +++, ++ ++, ++, +++ 0,0

(a) Tissue Sections Liver Spleen Salivary gland Lung Prepubertal rat (32 days) Mature virgin (58 days) Mammary gland

Mid-pregnant rat Lactating rat Lactating rat/serum preadsorbed with casein

0,0 0 0, 0, 0 0, 0, 0 ND

(b) Cell Cultures

Rama 29 Rama 27 CSG 122/17

No supplement DMSO Hormones DMSO + hormones DMSO + hormones DMSO + hormones DMSO + hormones

CMT 64

DMSO + hormones

Rama 25

(+), 0, +

+, (+)

(+),0, (+) (+), (+), 0, (+), (+), + 0,(+) 0,(+) 0,0 0

++, (+) ++, ++ +(+), +, ++ (+),

++, ++, ++(+), ++, ++, +++ 0,(+) 0,(+) 0,0 0

Tissues, all from female rats, were fixed and sectioned ; cultures were fixed on plastic coverslips . Specimens were incubated with either normal or anti-casein rabbit serum, and then with fluorescein-conjugated anti-rabbit globulin serum (Experimental Procedures) . Intensity of fluorescence was scored as follows : [0] definitely negative ; [+] indifferent ; [++] definitely positive ; [+++] strongly positive ; [(+)] intermediate between [0] and [+] . (ND) not done . Repeated scores represent different experiments . ("Hormones") cultures exposed to cortisol, insulin and prolactin for 3 + 2 days ; ("DMSO") cultures exposed to DMSO for 2 days (Experimental Procedures) . Rama 27, CSG 122/17 and CMT 64 are cell lines of mammary stromal cells, salivary epithelial cells and lung epithelial cells, respectively (see text) . Rama 29 and the last three cell lines were uniformly scored as [0], with normal or anti-casein serum, after exposure to DMSO alone or hormones alone (not shown) . Nonspecific fluorescence in liver and salivary gland sections was yellow, suggesting autofluorescence .

mammary cell capable of forming two other cell types, the droplet cell and the elongated cell . We now discuss possible relationships between these three types and normal cell types in vivo (see Introduction), with the reservation that either tumor cells or cultured cells may on occasion be so altered as to resemble no identifiable cell type . We first propose that the cuboidal cells are a type of mammary epithelial cell for the following reasons . These cells are derived from a mammary tumor which appeared almost entirely epithelial in culture . By light microscopy, cuboidal cells too have the form and behavior of epithelial cells . They can give rise-to epithelial cells (in the tumors) and to cells with specific characteristics of mammary secretory cells (see below) . It could be argued that since no specific mammary properties have been demonstrated in cuboidal cells themselves, they could be a cell type normally occurring very early

in development and competent to form mammary epithelium as well as-for example, fibroblasts . This is highly improbable : mammary epithelium develops directly from the embryonic epidermis, so that any less committed cell might be expected to form at least epidermal (keratinizing) cells . In particular, cells capable of forming both epithelium and mesenchyme should be so developmentally primitive as to form a large number of different cell types . Since the cuboidal cells seem capable of the formation of only two further cell types, and given their origin and epithelial behavior, it then seems highly probable that they are committed as mammary epithelial cells . A number of findings suggest that droplet cells are related to mammary secretory cells . First, droplet cells can form domes, a property believed to be specific in mouse mammary cell cultures to alveolar-like cells (McGrath, 1975) . Second, lipid, a com-

Figure 5 . Electron Micrographs of Parts of Tumor Formed by Rama 25 Cells Ultrathin sections from same tumor shown in histological section (Figure 3) . (a) Cells surrounding a lumen (L), showing microvilli and junctional complexes ; an apparently intracellular lumen is present in one cell . (b) Myoepithelial-like cell, with electron-dense nucleus and cytoplasm and triangular profile ; abutting small lumen (L) . (c) Enlarged portion of cell in (b), as outlined . It is improbable that the cell's dark appearance is due only to moribundity (but note swollen mitochondria), since the basal portion is filled with fibrils (here sectioned near transversely) and contains membrane-associated vesicles (arrowheads) as in myoepithelial cells . (d) Desmosome, from a region near that shown in (a) ; central lamina clearly shown ; part of nearby lumen seen on right . (a and b) bars = 2 µm ; (c and d) bars = 0 .5 µm .

Cell 294

Figure

6. lmmunofluorescent

Staining:

Sectio Ins of Lactating

Rat Mammary

Gland

See the text for methods. (a and b) Anti-case in serum; same field viewed by (a) phase-co1 ntrast and (b) fluorescence exposure rabbit serum; adjacent section of gland; flue ,rescence optics; identical illumination and photographic fluorescence in (b) located chiefly in epithelial cell apices, next to lumen. Bars = 50 pm.

ponent of milk, accumulates in the cells of some domes, although the presence of milk-specific lipids has not been tested. Finally, Rama 25 cultures consisting almost entirely of droplet cells (after 2 days of exposure to DMSO and hormones) appear to synthesize casein-like material. The apparent lack of well developed secretory cell ultrastructure in DMSO-treated Rama 25 cultures, even on collagen gels, might be due either to a genetic defect in the cells, affecting casein secretion more than its synthesis, or to deficient culture conditions (for example, absence of a hormone or another cell type). Droplet cells do appear to be sensitive to cortisol and/or insulin, in that while casein-like material was synthesized with or without addition of these hormones, dome formation in the presence of DMSO was greatly stimulated by them. The shape of the elongated cells resembles that of cultured “fibroblasts”, while each of their ultrastructural features is seen in one or more types of mesenchymal cell (Franks and Cooper, 1972). Of these types, however, elongated cells differ from endothelial cells in their elongated form, manylayered growth and lack of Weibel-Palade bodies, Figure

7. lmmunofluorescent

Staining:

optics. (c) Normal time to (b). Specific

and from fibroblasts or pericytes in their formation of tightly cohesive colonies, in the ability of epithelial cells to grow over them, and in the apparent production of basal lamina and hemisomes in culture. They cannot be distinguished by ultrastructure or behavior from smooth muscle cells, since we do not know how the latter interact with epithelial cells. The elongated cells, however, arise from repeatedly cloned cuboidal cells, which are very probably mammary and epithelial (see above). All these findings are explained by the hypothesis that elongated cells are mammary myoepithelial cells. The latter form a second layer beneath lining epithelial cells in vivo and are probably the cells which do so in primary cultures (Results). There is then no difficulty in explaining the formation of elongated cells from mammary epithelial cells or their ultrastructural resemblance to smooth muscle. Myoepithelial cells possess myofilaments, pinocytotic vesicles and hemisomes, and are believed to synthesize basal lamina (Murad and Von Haam, 1968; Ozzello, 1971; Hollman, 1974). In two features only, their relatively sparse myofilaments and prominent endoplasmic reticulum, elongated cells

Cell Cultures

See the text for methods. All specimens from same experiment; typical fields selected. (a-c) Rama 25 cells exposed to cortisol, insulin and prolactin (3 + 2 days) and DMSO (2 days). (d-f) Rama 25 cells maintained without hormones or DMSO; medium changed on same days as above. (g-i) Rama 29 cells exposed to hormones and DMSO as above. (a-i) First two photographs in each row show the same field stained with anti-casein serum, viewed by phase-contrast and fluorescence optics. The last photograph in each row shows a duplicate culture stained with normal rabbit serum, viewed by fluorescence optics. Identical illumination and exposure times were used for all six fluorescence photographs. Note in (b) the granular pattern of stain in some cells and the amplified brightness produced by folding of cell sheet where a dome has collapsed during drying. Bars = 50 pm.

A Possible 295

Mammary

Stem

Cell Line

Cell 296

classification becomes meaningless ; again, epithelial cultures which obviously contain elongated fibroblast-like cells are likely to be discarded . Finally, the rigorous cell cloning necessary to prove pluripotency has, until recently, been difficult to apply to epithelial cells . Clonal analysis of this kind could evidently be useful in determining clearly how many types of cells participate in forming the developing and the mature mammary gland and its tumors . As the first reported line of mammary stem cells, Rama 25 should prove to be an interesting and useful tool in the study of mammary development and differentiation . Figure 8 . Summary : Model for Behavior of Rama 25 Cells Symbols represent the cuboidal, droplet and elongated cells ; possible pathways are represented by arrows . Note the differences between droplet cells and elongated cells : droplet cells cannot be cloned, but appear to be capable of turning back into cuboidal cells .

differ ultrastructurally from mature myoepithelial cells in vivo . These two features, however, can be found with all the above both in the cells of the human breast "myoepithelial" cell line Hs578Bst (Hackett et al ., 1977) and in the "immature" or "premyoepithelial" cells observed in the salivary gland by Tandler (1965) . It is often stated that cells tend to differentiate less in culture than in vivo, and indeed, more obviously myoepithelial-like cells were seen, both by electron microscopy and with the TPL stain, in the tumors arising from Rama 25 cells . In summary, it seems that the cuboidal cells are a type of pluripotent mammary epithelial cell, or stem cell, and that the two further cell types which they can form are probably milk-secretory and myoepithelial (or premyoepithelial) cells . If such stem cells exist in normal mammary epithelium, then they probably appear early in development, since myoepithelial cells can be found in the mammary rudiment before birth (Salazar and Tobon, 1974 ; Schlotke, 1976) . Again if such pluripotent cells exist, and unless they are rare in adult mammary gland and in mammary tumors, it is perhaps surprising that they have not previously been seen in culture and reported . It may merely be that they have not been recognized . For example, in several reports of lines of mammary epithelial cells, either cloned or otherwise purified, fibroblast-like cells are described as reappearing persistently, either in culture or in tumors formed after reimplantation (Sanford et al ., 1961 ; Lasfargues and Moore, 1971 ; Owens and Hackett, 1972 ; Yagi, 1973 ; Cohen, Tsuang and Chan, 1974 ; Butel et al ., 1977) . In other cases and from our own experience, epithelial cells may have responded poorly to culture conditions, producing giant cells and other variants so that morphological

Experimental Procedures Culture Conditions All cultures were grown at 37°C in an incubator containing 10% CO 2 and 90% air . Rat mammary cell lines were grown in routine medium (see Results) unless otherwise stated ; mouse lung and salivary cell lines were grown in the same medium, but with DEM replaced by Waymouth's medium (MB752/1) . For passage or subculture, cultures were washed twice in warm PBSA (Dulbecco's phosphate-buffered saline lacking MgCl 2 and CaCl 2), and once in PBSA containing 200 µg ml - ' EDTA and 500 ng ml - ' trypsin, and then incubated until the cells had detached . Cells were suspended by pipetting in warm medium and transferred to new dishes . Rama 25 cultures were passaged every 2 days from one plate to eight or ten, and Rama 29 once per week, from one plate to 50 ; plating densities were approximately 5 x 10 3 and 3 x 10 3 cells cm -2 , respectively . These lines were passaged only 6 and 5 times, respectively, after thawing from frozen stocks . Cell strains, and cell lines during and just after cloning, were grown in conditioned medium (see Results), and passaged as above but using DEM-EDTA (calcium- and magnesium-free DEM containing 200 µg ml - ' EDTA) in place of the EDTA and trypsin solution . Incubations for up to 40 min were required for cell detachment in the earliest passages after primary culture . Conditioned medium appeared to improve cell survival and proliferation at very low cell densities, such as cloning density ; this was particularly important during early passages when epithelial cell plating efficiency could be very low . Five hormones reported to increase proliferation in cultured rat mammary cells (Hallowes, et al ., 1977 ; Rudland et al ., 1977) were included in the conditioned medium . Three others were omitted : estradiol because of a suspected inhibitory effect on this culture, and EGF and OGF because of the quantities required for routine culture . All hormones were purchased from Sigma, except prolactin and growth hormone, which were donated by the Pituitary Hormone Distribution Program of the National Institute of Arthritis and Metabolic Diseases (Bethesda, Maryland) . Hexamethylene bisacetamide was a gift from Dr . W . Ostertag . Primary Cultures from Rat Mammary Tumors Mammary tumors appeared within 2 months in female SpragueDawley rats fed 7,12 dimethylbenz-a-anthracene by stomach tube when 50 days old (Huggins, Grand and Brillantes, 1961) . For primary culture (Hallowes et al ., 1977 ; Rudland et al ., 1977), a small growing tumor was selected, chopped and digested with collagenase and hyaluronidase to yield a mixture of cell clumps (predominantly epithelial) and single cells (mostly stromal) . The mixture was suspended in routine medium and incubated in culture dishes . The single cells adhered most rapidly to the dishes ; to select for epithelium, the unattached cells were transferred to new dishes after 90 min and again after 180 min . Selection for Upper Layer Epithelial Cells Epithelial cell colonies obtained by the above method were rinsed

A Possible Mammary Stem Cell Line 297

twice in warm PBSA and once in warm DEM-EDTA (see above) . After incubation for 3 min, an upper layer of small epithelioid cells became partly detached from a basal cell layer . The detached cells were suspended by very gentle pipetting in warm conditioned medium and replated separately . The same method could be applied to cultures of normal mammary epithelium from adult virgin female rats, prepared according to Hallowes et al . (1977) . Cloning by Cell Selection A cell suspension was obtained as for subculture (see above) . This was diluted greatly in conditioned medium and viewed with a microscope ; single cells were picked with a drawn-out Pasteur pipette and plugged mouth tube, and each was transferred to a 6 mm culture well containing 100 µl of conditioned medium . After 4-8 days of incubation, selected cell colonies were passaged to 16 mm wells, and thereafter as necessary . Histology and Electron Microscopy Tissue for histological study was fixed in neutrally buffered formaldehyde (4%), embedded in paraffin wax and cut into 5 µm sections . The fluorochrome Hoechst 33258 was purchased from Riedel de Haen AG, Seelze, West Germany . For electron microscopy, cells grown on glass coverslips were fixed and processed as described previously (Rudland et al ., 1977), with Durcupan substituted for Araldite . When cells were fixed on plastic dishes, acetone was omitted during dehydration and the Durcupan was initially polymerized for about 24 hr at 37°C ; the plastic was then broken away and, after further polymerization for about 24 hr at 70°C, replaced by Durcupan . The whole was finally polymerized for about 24 hr at 70°C . Collagen gels and tumor tissues were processed essentially by the same method as cells on glass, with more time allowed for permeation of solutions at each stage . Collagen gels were made using the method of Emerman et al . (1977) . Preparation of Anti-Casein Antisera Milk was collected from Sprague-Dawley rats by the procedure of Young and Nelstrop (1970) . Milk protein micelles were separated by centrifugation (27,000 x g for 30 min) and resuspended in water . Whole casein was isolated by repeated isoelectric precipitation ; individual caseins were then purified by chromatography on DEAE-cellulose columns (Rosen, Woo and Comstock, 1975) . The three main components obtained, designated a, (3 and y in order of elution, were rechromatographed individually . Recoveries, with apparent molecular weights (estimated by discontinuous SDS-polyacrylamide gel electrophoresis ; Laemmli, 1970) were 62 mg (31,000), 86 mg (43,000) and 13 mg (26,000), respectively, from an initial 200 ml of milk . These molecular weights resemble those of bands 2, 1 and 3 of Rosen et al . (1975) . Antisera were each raised by injection of a rabbit at multiple sites with a total of 1 mg each of components a, f3 and y (three rabbits) or 5 mg each of a and (3 (three rabbits) . This procedure was repeated every 2 weeks until anti-casein titers appeared to increase no further (after 2 months) in Ouchterlony immunodiffusion tests (Roitt, 1977) . The two sera of highest titer (serum 2 raised with a, (3 and y, and serum 4 with a and )3) were used in these experiments ; both gave detectable precipitation bands only against components a and (3 . FITC-conjugated goat anti-rabbit IgG was purchased from Miles-Yeda . Immunofluorescence Assay Tissues were fixed and sectioned as for histology (see above), while cultured cells were grown on polystyrene plastic coverslips, since Rama 25 grew relatively sparsely on glass coverslips . All cells were grown to saturation density in routine medium (see Results), with Waymouth's medium substituted for DEM throughout for the salivary and lung cells . The medium was then changed to DEM and 10% FCS, with or without cortisol (50 ng ml -1 ), bovine insulin (50 ng ml -1 ) and ovine prolactin (500 ng ml -1 ) . After 3 days, these media were renewed, together with 250 mM DMSO where required . After 2 further days, cultures were fixed in

formaldehyde (5%) in Dulbecco's phosphate-buffered saline, followed by methanol at -20°C . After air-drying, coverslips were cut in half, and the duplicate halves (or two serial tissue sections) were incubated, respectively, with anti-casein serum and normal rabbit serum (20 1d), both diluted one tenth in PBSA, followed by 20 µl of FITC-conjugated anti-rabbit IgG serum (one fifth) . Both incubations were for 30 min at 37°C . Specimens were washed and mounted wet in glycerol buffered at pH 8 .2, and then examined on a Zeiss model RA fluorescence microscope using excitation filter I and barrier filter 47 . Photographs were taken on Kodak TriX Panchromatic film exposed for 10 min (fluorescence) or 1 sec (phase-contrast) . Scoring for fluorescence was usually performed blind (where possible) . Parallel scoring by two independent observers showed slight, but not systematic, differences . Acknowledgments We are particularly indebted to Mel Greaves, Dick Hallowes, Chris Marshall and Brigid Hogan for their advice and help ; to Renato Dulbecco for stimulating discussions ; and to Ken Miller and Pam Kirby for skilled technical assistance . The costs of publication of this article were defrayed in part by the payment of page charges . This article must therefore be hereby marked "advertisement" in accordance with 18 U .S .C . Section 1734 solely to indicate this fact . Received March 15, 1978 ; revised June 12, 1978 References Bloom, W . and Fawcett, D . W . (1975) . A Textbook of Histology, tenth edition (Philadelphia : W . B . Saunders) . Butel, J . S ., Dudley, J . P . and Medina, D . (1977) . Cancer Res . 37, 1892-1900 . Cohen, L . A ., Tsuang, J . and Chan, P . C . (1974) . In Vitro 10, 5162 . Dempsey, E . W ., Bunting, H . and Wislocki, G . B . (1947) . Am . J . Anat .81, 309-341 . Ebner, K . E ., Hoover, C . R ., Hageman, E . C . and Larson, B . L . (1961) . Exp . Cell Res . 23, 373-385 . Evans, R . W . (1958) . Histological Appearances of Tumours (Edinburgh : Churchill Livingstone) . Emerman, J . T ., Enami, J ., Pitelka, D . R . and Nandi, S . (1977) . Proc . Nat . Acad . Sci . USA 74, 4466-4470 . Fawcett, D . W . (1966) . An Atlas of Fine Structure . The Cell, Its Organelles and Inclusions (Philadelphia : W . B . Saunders) . Feldman, M . K . and Ceriani, R . L . (1970) . Comp . Biochem . Physiol . 37, 421-427 . Feldman, M . K . and DeOme, K . B . (1975) . Histochem . J . 7, 411418 . Franks, L . M . and Cooper, T . W . (1972) . Int . J . Cancer 9, 19-29 . Franks, L . M ., Carbonell, A . W ., Hemmings, V . J . and Riddle, P . N . (1976) . Cancer Res . 36, 1049-1055 . Friend, C ., Scher, W ., Holland, J . G . and Sato, T . (1971) . Proc . Nat . Acad . Sci . USA 68, 378-382 . Hackett, A . J ., Smith, H . S ., Springer, E . L ., Owens, R . B ., NelsonRees, W . A ., Riggs, J . L . and Gardner, M . B . (1977) . J . Nat . Cancer Inst . 58, 1795-1806 . Hallowes, R . C ., Wang, D . Y . and Lewis, D . J . (1973) . J . Endocrinol . 57, 253-264 . Hallowes, R . C ., Rudland, P . S ., Hawkins, R . A ., Lewis, D . J ., Bennett, D . C . and Durbin, H . (1977). Cancer Res . 37, 2492-2504 . Hollman, K . H . (1974) . In Lactation : a Comprehensive Treatise, 1, B . L . Larson and V . R . Smith, eds . (New York : Academic Press), pp . 3-95 . Hosick, H . L . (1974) . Cancer Res . 34, 259-261 . Huggins, C ., Grand, L . C . and Brillantes, F . P . (1961). Nature 189, 204-207 .

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