Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo and influence of medium

J. TAYLOR-PAPADIMITRIOU1*, M. STAMPFER2, J. BARTER1, A. LEWIS1, M. BOSHELL1, E. B. LANE3 and I. M. LEIGH4

'imperial Cancer Research Fund Laboratories, PO Box 123, Lincoln's Inn Fields, London WC2A3PX, UK zLawrence Berkeley Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA 3Imperial Cancer Research Fund, Clare Hall laboratories, Blanche Lane, South Minims, Potters Bar, Hertfordshire Ei\!6 3LD, UK 4The London Hospital, 56 Ashfield Street, London El 2BL, UK

* Author for correspondence

Summary

The luminal and basal epithelial cells in the human moplasty organoids, cells from the basal layer mammary gland can be distinguished in tissue proliferate, while in MM medium the basal pheno- sections on the basis of the pattern of they type dominates, but a few cells with the luminal express. Moreover, the invasive cells in primary are found. Around passage 3, in medium show a profile that corresponds MCDB 170, most cells senesce and a subpopulation to that of the dominant luminal cell (7, 8, 18, 19). of cells proliferates on further passage. These cells When homogeneous populations of luminal epi- retain expression of the basal epithelial keratins but thelial cells from milk or from meta- also express some features characteristic of luminal stases are cultured the profile of keratin expression epithelial cells, suggesting that the basal layer may seen in vivo is maintained. We have therefore used contain a that can develop along the monospecific reactive with individual luminal lineage. In culture, however, they do not keratins to examine the phenotype of cells cultured express , which in vivo is a feature of the in three different media from reduction mammo- fully differentiated luminal cell. The cells cultured plasty tissue that contains both luminal and basal from primary breast cancer in medium MCDB 170 cells. The phenotype of cells cultured from primary have a similar keratin profile to that of the normal breast cancers in one of these media (MCDB170) cells cultured in this medium. They do not express has also been examined. In characterizing cell keratin 19, even though the invasive cells in pri- phenotypes, antibodies to a polymorphic epithelial mary cancers homogeneously express this keratin mucin (PEM) expressed in vivo by luminal cells, in vivo. The invasive phenotype, which in its kera- and to smooth muscle (a) , expressed in vivo tin profile corresponds to the differentiated luminal by basal cells, have also been used. Our results cell and that of the metastatic cancer lines, cannot show that proliferation of different cell phenotypes be cultured from primary breast cancers using MX, is selected for in different media. In milk mix (MX) which supports proliferation of the corresponding developed for growth of luminal cells from milk, normal cell. only the luminal cell phenotype proliferates (for only 1 or 2 passages). In medium MCDB 170, which was developed for long-term growth of human mammary epithelial cells from reduction mam- Key words: keratin, mammary , malignancy.

Introduction Breast cancer represents one of the most common carci- nomas in women in the Western world, and clearly The mammary gland is a complex tissue containing a develops from an epithelial cell found in the breast. In variety of cell types including the two major epithelial order to be able to study this cell and its transformation in phenotypes (basal and luminal) that line the ductal tree. culture, it is important to try to define the phenotypes in Journal of Cell Science 94, 403-413 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 403 both the normal tissue and the malignant tumours, and (large and small ducts, and TDLU) and contain both also the cells that can be cultured from them. Character- basal and luminal epithelial cells. These complex orga- ization of cell types /'// vivo can be done readily, using noids have been grown in a variety of media, and immunohistochemical techniques and monoclonal anti- although the cells cultured have been clearly identified as bodies on tissue and tumour sections. In this context, the epithelial (Bartek et al. 1985a; Curschellas et al. 1987; epithelial keratins have been found to be extremely useful Dairkee et al. 1986), their detailed phenotype particularly markers. In the human, the soft tissue keratins have been in terms of keratin expression has not been defined. In characterized and classified on the basis of molecular the serum-free medium MCDB 170 (Hammond et al. weight and charge (Moll et al. 1982) and the profile of 1984), the cells can be cultured for 10-20 passages. In the expression of these components is characteristic of a complex medium MM, containing some serum and particular cell type (Sun et al. 1984). Recently, mono- conditioned medium (Stampfer, 1982), fewer passages specific antibodies reactive with a single keratin have (5-7) are obtained, while in medium MX developed for been developed, and these are being successfully applied growth of luminal epithelial cells from milk (Taylor- in to the identification of keratins Papadimitriou et al. 1980), only 1-2 passages are ob- expressed in tissues and tumours at the single cell level tained. Cells cultured in these three media have been (Ramaekerse/fl/. 1983«,6; Osbornetal. 1985; Morgans/ examined for expression of the epithelial keratins 7, 8, 14, al. 1987). Using such tools it is possible to distinguish the 18 and 19 (and in some cases 5), and also for expression of two major epithelial cell types in the adult mammary the mucin expressed in vivo by luminal cells and cv-actin gland, since the basal cells express keratins typically expressed //; vivo by basal cells. In addition, the keratin expressed by the basal cells in stratified epithelia, while profile of cells cultured in medium MCDB 170 from the luminal cells express smaller keratins typical of simple primary breast cancers has been examined. The results epithelia (Altmannsberger et al. 1986; Taylor-Papadimi- indicate that growth of different cell types is selected for triou and Lane, 1987; Dairkee et al. 1988; Nagle et al. by different media, and that long-term growth is shown 1986). Some subclassification has also been possible on in medium MCDB 170 by a minority population that the basis of keratin expression and position in the appears to be derived from the basal layer. mammary tree (Bartek e? a/. 1985o, 19866). Immunohis- tochemical staining of breast cancers shows that in their profile of keratin expression, and in their expression of a Materials and methods polymorphic epithelial mucin (PEM; Gendler et al. 1988), they resemble the luminal epithelial cells (Taylor- Culture of human mammary epithelial cells Papadimitriou and Lane, 1987). Breast tissues were obtained from reduction mammoplasties and mastectomies and digested to epithelial organoids as When breast tissue or primary breast cancers are used previously described (Stampfer et al. 1980; Stampfer, 1985). as starting material for culture it is difficult to ascertain The organoids were placed into culture in either the serum-free whether the in vivo markers are retained in culture, medium MCDB 170 (Hammond et al. 1984), medium MM because of the heterogeneous cell population in the (Stampfer, 1982) or MX (milk mix; Taylor-Papadimitriou et al. sample. A metastatic breast cancer on the other hand 1980), and subcultured as described (Stampfer, 1985). In represents a relatively homogeneous cell population, and medium MCDB 170 bovine pituitary extract is used as an many cell lines have been developed from metastases, additive in place of serum while medium MM contains medium mainly from cells in pleural effusions. Similarly, milk conditioned by other cell lines and a low concentration of foetal represents a source of normal luminal epithelial cells, calf serum. These two media were developed for growth of cells uncontaminated by fibroblasts, or epithelial cells from from reduction mammoplasty tissue. Medium MX was devel- oped for growing cells from human milk and contains 10% the basal epithelial layer (Buehring, 1972). Data reported human serum as well as 10% foetal calf serum. The MM in the literature (Bartek et al. 1985«; Chang and Taylor- medium and the milk mix contained cholera toxin and, where Papadimitriou, 1983; Taylor-Papadimitriou et al. 1987) indicated, the MCDB 170 medium contained 10~ M-isoproter- and in this paper indicate that the profile of keratin enol. For immunohistochemical analysis, cells were seeded onto expression in the cultured milk cells and in breast cancer cleaned glass coverslips and fixed when subconfluent with 1: 1 cell lines is found to be remarkably consistent, corre- (v/v) cold methanol/acetone for ISmin at room temperature. sponding to that of the differentiated luminal cell. Since The coverslips were rinsed with phosphate-buffered saline some of the breast cancer cell lines have been in culture (PBS), air-dried and stored at -20°C until use. for many years in different laboratories, it would appear In this study, cells from a reduction mammoplasty (specimen that the expression of keratins 8, 18 and 19 and sometimes no. 161) were examined at different passages. The phenotypes 7 is a stable feature of these cells that is not altered by of the cells emerging after long-term culture were examined at culture. We have therefore used antibodies to these passage 6-9 for three other specimens (172R, 184 and 48R) from individuals with no apparent breast pathology, and for keratins together with other immunological markers to three specimens taken from breast cancers (203T, 192T, try to characterize cells cultured from breast tissue and 186TL). For comparison of phenotypes cultured in different tumours. medium, reduction mammoplasty specimens are indicated in An important source of non-malignant mammary epi- the relevant experiment. thelial cells is the tissue removed at reduction mammo- plasty surgery. The epithelial organoids that are isolated Preparation of tissue sections after enzymic digestion of the tissue (Stampfer et al. Frozen sections from tissues and tumours obtained from Dr 1980) come from different parts of the mammary tree Rosemary Millis at the ICRF Breast Cancer Unit, Guy's

404 jf. Taylor-Papadimitriou et al. Table 1. Monoclonal antibodies used to detect chemical staining with the antibodies listed in Table 1 is expression of specific keratins shown diagrammatically in Fig. 1. With the exception of Target Reference the very large ducts, where some expression is seen in luminal cells (Purkis et al. 1989), only basal cells LL001 Keratin 14 Leigh el at. (1988) express keratin 14, a keratin expressed by basal cells in C43 Bartek Keratin 7 Bartekef o/. (1987) cells express keratins 7, 8 and 18, which are normally C35 Keratin 7 Bartekef o/. (1989) associated with simple epithelia. In the TDLU where the CK7 Keratin 7 Ramaekers el a!. (1987) overall level of keratin expression is lower, especially in RCK105 Keratin 7 Ramaekers el at. (1987) the basal cells, the distinction between luminal and basal LE61 Lane (1982) C04 Keratin 18 Bartek e/o/ (1989) cells is most clear cut; keratin 14 is not expressed in the BA16 Keratin 19 Bartek el at. (1985) luminal cells (see Fig. 1; also Dairkee et al. 19856) and BA17 Keratin 19 Bartek protein by basal cells of the mammary primary cultures and third and sixth passage of reduction gland, has recently been reported (Guelstein et al. 1988). mammoplasty specimen 161 grown in medium MCDB 170 and This report is in contrast to earlier observations on in secondary cultures of specimen 184 in medium 170 and in human mammary gland, which did not detect vimentin MM with and without cholera toxin. These were: 312C8 (Dairkee et al. 1986) and LL002 (Leigh et al. 1988) to keratin expression (Dairkee et al. 1986). We have found vimentin 14; RCK105 (Ramaekers et al. 1987) and CK7 (Tolle et al. to be undetectable in the basal cells of the mammary 1985) to keratin 7; RCK106 (Ramaekers et al. 1987) and CK5 gland, using the commercially available antibody VIM (Tolle et al. 1983) to keratin 18, KM4.62 (Gigi-Leitner and 13.2 (see Materials and methods). Geiger, 1986) and A53-B/A2 (Karsten et al. 1985) to keratin 19. Breast cancers. The profile of keratin expression by A polyclonal rabbit antiserum to (Rba5: E. B. Lane, invasive breast cancer cells, as seen by immunohisto- unpublished) was also used to stain specimens cultured in MM chemical staining of tissue sections, resembles that of the (secondary culture of specimen 184) and medium MCDB 170 differentiated luminal epithelial cell (Table 2). With a (first, third and sixth passage of specimen 161). few exceptions, keratin 14 is not detected in the invasive The expression of vimentin was examined with antibody cells by antibody staining (see Fig. 2E; and Dairkee et al. VIM 13.2, which was obtained from ICN Immunobiologicals 1987; Wetzels et al. 1989). Where remnants of structure and in some cultures with antibody V9 from DAKO. Fibronec- tin expression was detected using antibody FN-3, which reacts are visible some expression of keratin 14 can occasionally with cell-associated but not plasma fibronectin (Keen et al. be detected, but in contrast to the situation in the normal 1984). The expression of a polymorphic epithelial (PEM) gland the expressing cells are lining lumens (see Fig. 2E). mucin (Gendler et al. 1988) was detected with antibodies The keratin profile of cells in these areas resembles that HMFG-1 and HMFG-2 (Taylor-Papadimitriou et al. 19816; seen in benign tumours, where keratin 14 is detected and Burchell et al. 1983), and smooth muscle

Keratin expression in mammary epithelial cells 405 KERATIN 7 Predominantly lumenal Some expression in basal cells

KERATINS 8 & 18 Lumenal

KERATIN 19 Predominantly lumenal Some unstained cells in end structures Some staining of basal cells in large ducts

KERATIN 14 Predominantly basal Some staining of lumenal cells in large ducts

Key: ••H Strong staining t. .i *..• j Less intense staining r •*•' •' Heterogeneous staining

Fig. 1. Diagrammatic representation of distribution of keratins in the human mammary gland. and malignant epithelial cells showing the luminal pheno- and 18, and the majority (85 %) also express keratin 19 type vivo were examined. The differentiated 19+ luminal homogeneously (Bartek et al. 1985a). The major differ- epithelial cell is the dominant cell in the lactating breast ence seen in the cultured milk cells compared to the and is shed into milk. Cultured in medium MX, these luminal phenotype seen in vivo is expression of keratin cells continue to express keratins 7, 8, 18 and 19. All the 14, which is seen in around 30 % of colonies (see colonies show homogeneous expression of keratins 7, 8 Table 2).

406 J. Taylor-Papadimitriou et al. l^^^i p^y

B

v».V

Fig. 2. Immunoperoxidase staining of sections of normal tissue (A,B), fibroadenoma (C,D) and infiltrating ductal (E,F) with antibody 001 directed to keratin 14 (A,C,E) or antibody BA17 to keratin 19 (B,D,F). A,C,E, x200; B,E,F, X300.

Table 2. Expression of keratins by normal luminal epithelial cells and invasive breast cancer cells Keratin

Tissue or culture l 8 18 19 14 PEM< Normal luminal epithelial cells +t (+)* in sections of TDLU Shed luminal epithelial cells ( + ) () () (+)t cultured from milk in (>95 %) (85 %) (30%) medium MX Invasive breast cancer cells in sections of primary carcinomas Metastatic breast cancer cells in N.D.§ (+)* sections of L/N metastases Cultured breast cancer cell lines from metastases MCF-7 T47D (+)* ZR-75.1 (+)*

•Polymorphic epithelial mucin. •f +, Indicates homogeneously positive staining; (+) some cells stained, some not. The values in parenthesis indicate the proportion of positive cells. ^Proportion of cells staining varies from 50 to 100%, depending on antibody used and culture conditions. § Not done.

Cell lines derived from metastatic cancers have been After this 'self selection' a cell population with a cobble- developed in several laboratories. Table 2 shows that the stone epithelial morphology can be passaged for an profile of keratins expressed in three such lines corre- additional 7-20 passages depending on the individual sponds to the profile seen in situ. Since these lines have specimen (Stampfer, 1985). The morphological appear- been in culture for many years, again this suggests that ance of the cells at different passages is illustrated in the expression of the simple epithelial keratins 8, 18 and Fig. 3. We first examined the primary cultures and 19 is maintained in culture, and in this case, keratin 14 is subsequent passages from a single specimen (161) and a not induced. summary of our findings is presented in Table 3. In primary cultures, two dramatically different cell types can be seen, which in their keratin expression Keratin expression in mammary epithelial cells cultured appear to correspond to the basal and luminal cells seen in from reduction mammoplasty tissue vivo, and it is the cells with the basal phenotype that Phenotypes cultured in medium MCDB 170. Medium appear to proliferate. The basal-like cells, which appear MCDB 170 was developed by Hammond and colleagues to form the bottom layer, show many mitoses (see (1984) for the clonal and long-term culture of cells in Fig. 4B), express high levels of keratins 5 and 14 epithelial organoids derived by enzyme digestion from (Fig. 4A), no keratin 18 or 19 (Fig. 4B), moderate levels reduction mammoplasty (RM) tissue (Stampfer et al. of keratin 7 and variable but detectable levels of keratin 8. 1980). These organoids contain both basal and luminal The superficial luminal-like cells on the other hand cells from pieces of ducts and from TDLU. In medium express high levels of keratins 7, 8, 18 and 19 (Fig. 4B). MCDB 170, most cells cultured from reduction mam- These cells also stain with the antibody (HMFG-1) to the moplasty organoids develop a flattened irregular-edged polymorphic epithelial mucin (Gendler et al. 1988), morphology and cease proliferation around passage 3. which is a marker for luminal epithelial cells (Taylor- Table 3. Profile of keratin expression in cells from reduction mammoplasty organoids (specimen 161) proliferating in medium MCDB 170* Keratin Smooth muscle Passage 18 19 14 o-actin 1 3 ( + + )* 6 (+)t4 ( + + + )* H—> + + 13 N.D.11 N.D. + H—> + + + N.D.

* For composition see Hammond et al. (1984). In these experiments isoproterenol was added. •(•With antibody C43, heterogeneous staining was seen, while with antibody M20 most cells showed positive staining. t( + ), some cells stained, some not. § ±—* + + , ranged from weak to moderately strong staining. 1[ N.D., not done.

Keratin expression in mammary epithelial cells 407 \

4 B >vr-.\-;

Fig. 3. Morphological appearance of cells cultured from reduction mammoplasty (RM) 161 in medium MCDB 170. A. Primary culture. B. Cells at third passage showing large senescing cells and smaller, post-selection cells. C. Cells at passage 9 (post- selection cells). D. Cells senescing at passage 16. A,B, X40; C,D, X130.

Papadimitriou et al. 19816; data not shown). Although levels of vimentin, and reduced but detectable levels of small patches of these cells may migrate out some smooth muscle

408 jf. Taylor-Papadiniitriou et al. Fig. 4. Phenotype of cells in primary and secondary cultures of reduction mammoplasty organoids grown in different media. Cultures were prepared as described in Materials and methods and stained in an indirect immunoperoxidase assay, using anti- keratin antibodies (A-C,E,F) or an antibody to vimentin (D). A,B- Primary cultures of RM 161 in medium MCDB 170 stained with antibodies monospecific for keratin 14 (A), and keratin 18 (B). C. Primary culture of RM 17N grown in milk mix and stained with antibody to keratin 19. D. Secondary culture of RM 184 grown in medium MCDB 170 and stained with an antibody to vimentin. E,F. Secondary cultures of RM 184 grown in MM and stained with antibodies to keratin 14 (E) or keratin 18 (F). A-C, X200; D-F, X300. Bffi Fig. 5. Phenotvpe of cells selected from reduction niammoplasty (A,B) or primary breast cancers (C,D) for long-term proliferation in medium MCDB 170. Cultures were prepared and passaged in medium MCDB 170, fixed at the passages indicated and stained with the indirect immunoperoxidase method using antibodies monospecific for keratins. A. RM 172, ninth passage, stained with antibody LL001 to keratin 14. B. RM 48, eighth passage, stained with antibody C04 to keratin 18. C. Primary cancer 203, seventh passage stained with antibody LL-001 to keratin 14. D. Primary cancer 203, seventh passage, stained with antibody C35 to keratin 7. X200. Table 4. Keratin expression in human mammary epithelial cells cultured in different media Keratin expression Pattern of Medium Culture growth 18 19 14 PEM* MCDB 170 RM184 (+)t4 Passage 2 MM§ RM184 Open Passage 2 MM§ RM184 Closed Passage 2 Milk MixU RM17N ( + )t (MX) Primary Culture

* Polymorphic epithelial mucin, detected by staining with antibody HMFG-1. t( + )i some cells stained, some not. % Staining weaker and more heterogeneous with antibody C43, than with M20. § For composition, see Stampfer (1982). H For composition, see Taylor-Papadimitriou et at. (1980).

Three other reduction mammoplasties from indi- medium contains human serum as well as foetal calf viduals with no apparent epithelial cell pathology were serum, and allows the milk cells to go through approxi- examined at passages 6-8 and the pattern of staining seen mately 15-20 divisions (1-2 passages). All of the media with 161 was confirmed, i.e. all cells stained with contain cyclic AMP-elevating agents (see Materials and antibodies to keratins 7 and 14 and positively but variably methods). and heterogeneously with antibodies to 8 and to 18 (see Table 4 lists the phenotypes seen in primary or second- Fig. 5A,B). No evidence for expression of keratin 19 was ary cultures made from reduction mammoplasty tissue seen, using three different antibodies. Vimentin con- using medium MM, and the MX medium and compares tinued to be expressed in the post-selection cells; how- them with those seen in cultures grown in medium ever, the intensity of staining was generally lower, but MCDB 170. Fig. 4 illustrates some of the staining appeared to vary with both the antibody and the tissue patterns. In early passages of cells grown in medium specimen, the strongest staining being observed with MM, there appear to be two rather well-defined morpho- specimen 161 (passage 6) and the antibody V9. logical phenotypes. The minority phenotype forms com- Interestingly, the fibronectin in these cells shows the pact colonies (closed) and is surrounded by the dominant punctate pattern previously reported for epithelial cells phenotype, which has looser packing and gives an 'open' grown from milk (Taylor-Papadimitriou et al. 1981a), appearance (see Fig. 4E,F). The closed colonies stain and smooth muscle cr-actm was not detectable. In later strongly with antibodies to keratins 7, 8 and 18 (see passages (passage 13, specimen 161), when the cells were Fig. 4F), and heterogeneously with keratin 19 anti- beginning to senesce, cr-actm could again be detected and bodies. Keratin 14 may or may not be expressed low levels of the mucin could be detected with the (Fig. 4E); some mucin is detected with antibody antibody HMFG-1. HMFG-1 and vimentin is not detected. Thus, these The above results are consistent with the interpretation small groups of cells resemble the /'// vivo luminal that medium MCDB 170 supports the proliferation of phenotype. The dominant phenotype on the other hand basal cells for two to three passages, at which point these resembles the basal cells in its expression of keratin 5 and cells senesce and a cell type expressing some properties of 14 (Fig. 4E) and although keratin 7 is homogeneously both basal and luminal cells is selected and continues to expressed and keratin 8 somewhat more heterogeneously, proliferate. 18 and 19 are not detected (Fig. 4F). Vimentin, the protein normally found in mes- Keratin expression in mammary epithelial cells cultured enchymal cells, is however expressed at a high level, as from reduction mammoplasty tissue was noted by Dairkee and colleagues (1985«). Subculture in the same medium does not result in a selection for Phenotypes cultured in other media. Medium MCDB another cell type as is observed with MCDB 170, but 170 was developed to obtain the maximum number of cell rather both phenotypes seem to be maintained until the divisions in cells cultured from reduction mammoplasty cultures senesce, although the small colonies of cells of tissue. Before the development of MCDB 170 epithelial the luminal phenotype formed only 1-5% of the popu- cells from reduction mammoplasty tissue were cultured lation after the second passage of reduction mammoplasty in a medium (MM) containing serum and medium 184. conditioned by other cell types (Stampfer et al. 1980). In MM medium the cells can be subcultured for fewer In the medium MX, developed for growth of luminal passages. Medium MX (milk mix), which was developed cells shed into milk, fewer cells attach from the orga- to culture human mammary epithelial cells from milk noids, and although considerable cell division occurs in (Taylor-Papadimitriou et al. 1980), can also be used to primary culture, giving rise to large colonies, further culture cells from reduction mammoplasty tissue. This passage is difficult. In primary culture, the dominant

Keratin expression in mammary epithelial cells 409 phenotype resembles that of the differentiated luminal so that the effect of this agent on keratin expression in cell. These cells form closed colonies that stain strongly cells expressing the luminal phenotype in culture could with antibodies to the simple epithelial keratins 7, 8 and not be examined. 18 and to the mucin; most of these colonies also express keratin 19 (see Fig. 4C). MX medium therefore appears to select for the differentiated luminal phenotype that has Discussion a limited life span. As with the luminal cells cultured from milk, however, some expression of keratin 14 is In this paper, we have examined the expression of seen. A similar phenotype can be grown in MX medium keratins by mammary epithelial cells cultured in different from fibroadenomas, but we have been unsuccessful in media, using antibodies that are monospecific for a single culturing any epithelial cells from primary carcinomas in keratin and compared the phenotypes with those seen in this medium. basal and luminal cells in vivo. Antibodies directed to other markers of basal and luminal epithelial cells were Keratin expression in cells cultured in medium MCDB also used to define the cultured cells. It is very clear from 170 from primary breast carcinomas this study that the phenotype of the cultured cells varies To see whether there was any difference in the phenotype dramatically with the medium used, and can, where of cells cultured in MCDB 170 from malignant and non- subculture is possible, also change with passage. malignant breast tissue, cultures at passages 7-8 from We chose to use the epithelial keratins as markers, since three tumour specimens were also examined. Again a the profile of keratin expression is tissue specific, and can homogeneous positive staining was seen with antibodies be used to define subpopulations of cells in complex to 7 and 14 (Fig. 5C,D), the cells stained variably and tissues such as the mammary gland. Moreover, where heterogeneously with antibodies to keratins 8 and 18, and relatively homogeneous populations of cells have been no staining could be detected with antibodies to keratin cultured, as with metastatic breast cancer cell lines, and 19. These studies suggest that in medium MCDB 170, luminal epithelial cells from milk, the observed keratin the keratin profile of the cells cultured from breast expression is similar to that observed in vivo. In the case cancers is similar to that of the cells cultured from the of the cell lines, the phenotype is identical to that of the normal breast. The phenotype of this cell differs from invasive breast cancer cell in vivo (8, 18, 19 and that of the invasive cancer cell identifiable in tissue sometimes 7). There is one difference in the cultured sections (Fig. 2) and in cell lines derived from metastatic milk cells in that, in addition to the simple epithelial breast cancers (Table 2), which express keratins 8, 18 and keratins 7, 8, 18 and 19, some cells express keratin 14. In 19 (and sometimes 7), but do not usually express keratin vivo, although keratin 14 is associated only with basal 14 (Vojtesek and Bartek, unpublished data; Curschellas cells in the smaller ducts, some expression is seen also in + etal. 1987). luminal cells in the large ducts. Thus the 14 cells may come from the larger ducts. It is possible however that Effect of cyclic AMP-elevating agents on keratin the cholera toxin present in the MX medium used for expression culturing milk cells induces keratin 14 expression. An In medium MCDB 170 some reduction mammoplasty important conclusion that can be drawn, however, is that specimens require isoproterenol to move through the expression of keratin 7, 8, 18 and 19 is not lost in culture 'self-selection' period, which occurs around third pass- using the widely different media used for growing breast age. Other specimens (e.g. specimen 161), however, cancer cell lines and in medium MX used for growing grow equally well in medium MCDB 170 in the absence normal cells from milk. Therefore the absence of any of of an added cyclic AMP-elevating agent. We therefore these keratins in cultured mammary epithelial cells examined the effect of isoproterenol on keratin expression suggests that the cells came from a cell that was not in specimen 161. We observed that expression of all the expressing the keratin in vivo. keratins was lower in cells grown in the absence of cyclic Unlike the cells in milk, the organoids derived by AMP-elevating agents and keratin 14 expression was enzymic digestion of reduction mammoplasty tissue con- dramatically increased by addition of isoproterenol. We tain both basal and luminal cells, and the total digest also examined secondary cultures of reduction mammo- contains structures coming from all parts of the gland. plasty 184 grown in MM with and without cholera toxin. Using the medium MX developed for luminal cell The intensity of staining of the majority of cells with culture, the luminal cell phenotype selectively prolifer- antibody LL001 to keratin 14, and with antibodies CK7 ates, and the basal cells appear to die. As was seen with and RCK105 to keratin 7, was greatly increased in cells the luminal cells cultured from milk, some expression of cultured in medium containing cholera toxin. Moreover, keratin 14 is seen in addition to keratins 7, 8, 18 and 19. most of the compact colonies showed stronger staining With the two other media used in this study (MCDB 170 with antibodies to keratins 18 and 19. There was also an and medium MM), the basal cells proliferate and initially apparent increase in the numbers of cells stained with express keratins 5, 7 and 14 homogeneously (in a few cells these antibodies, including several more-dispersed cells keratin 8 can be detected) as well as high levels of seen as 'strings' of cells maintaining contact with each vimentin and smooth muscle actin. In this context it is of other or as single cells around the islands. In the MX interest that medium MCDB 170 does not support the medium, cell proliferation is much reduced in the ab- proliferation of the epithelial cells found in milk (Ham- sence of the cyclic AMP-elevating agent (cholera toxin), mond, S. and Taylor-Papadimitriou, J., personal com-

410 jf. Taylor-Papadimitriou et al. munication). Small colonies of cells with the luminal cultured from reduction mammoplasty in this medium. phenotype can be seen in early passages of MM cultured The tumour-derived cells could be normal or dysplastic cells but their numbers are reduced with passage. Both of basal cells associated with but unrelated to the malignant these media therefore favour the proliferation of the basal component. In support of this idea is the observation that cell phenotype. It seems likely that basal cells are cells cultured in a similar medium or in medium MM are expressing low levels of vimentin in vivo as reported by diploid, even when cultured from aneuploid tumours Guelstein and colleagues (1988) and that the levels are (Wolman et al. 1985; Zhang et al. 1989). Alternatively, increased by culture (Franke et al. 1979). they may represent more primitive neoplastic cells, which Of great interest is the observation that the cells that can differentiate in vivo to give the luminal phenotype of emerge from self-selection in medium MCDB 170 after the invasive breast cancer cell. This would suggest that passage 3 show a different phenotype to early passage the original carcinogenic event occurred in a cell with a cultures, in that they show increased expression of high proliferative potential, but that the invasive function keratins 8 and 18 (more heterogeneously expressed than is expressed by the more differentiated phenotype, which keratins 7, 5 and 14). They also show a reduced ex- in malignancy acquires proliferative potential. Why it is pression of fibronectin, O"-actin and vimentin. They do so difficult to culture the invasive cancer cells from not however express keratin 19, and generally produce primary breast carcinomas is not clear. Only a small only small or undetectable levels of the mucin recognised fraction of these tumours grow as xenografts in the nude by antibody HMFG-1 (although mucin and cv-actin levels mouse. Certainly, immortality appears to be a late event can increase when the cells eventually senesce). The only seen in late-stage patients, generally in pleural observations suggest that a minority cell type with high effusion metastases (Smith et al. 1987). It seems quite proliferative potential is selected for, with passage in likely that the phenotype that will become the invasive medium MCDB 170. This cell appears to acquire some cell can initially go through only a limited number of cell features of the luminal cell (expression of keratin 8 and divisions, many of which will have been exhausted in 18) but does not express keratin 19 and does not lose the vivo. basal cell-related keratins that were detected in early It will be of great importance to examine the behav- passage. Transferring MCDB 170 grown cells (passage 8) ioural properties of the cells that can be cultured from to medium MM or to the same medium supplemented primary breast cancers in medium MCDB 170 to see if with serum, resulted in some increase in the level of they show any malignant features. Some studies have expression of keratins 7, 8 and 18, and in a low expression been done with cells grown from primary cancers in of the PEM mucin, but keratin 19 expression was not medium MM and although these are diploid even when induced in any of the cells (data not shown). grown from tumours exhibiting aneuploidy they do show It is difficult to relate the cells proliferating at later invasive features (Smith et al. 1985) and are more passages in medium MCDB 170 to the basal and luminal sensitive to tumour necrosis factor (TNF) than the epithelial cells seen in vivo. It may be related to the corresponding normal cells (Dollbaum et al. 1988). It keratin 19~ luminal cell that is associated with ductal will also be important to study the effect of oncogenic branching at puberty, and which shows a less differen- transformation on the different phenotypes that can be tiated phenotype than the 19+ luminal cell (does not cultured from the normal gland. Some immortalized lines produce secretory component) in the adult breast (Bartek have been developed using chemical carcinogens and et al. 1989). The keratin 19~ luminal cell does not retroviruses to transform MCDB 170-grown cells however, express the basal epithelial keratins in vivo. (Stampfer and Bartley, 1985; Clark et al. 1988) and Conceivably, a stem cell found in the basal layer, is simian virus 40 to transform luminal epithelial cells precursor to the 19~ luminal cell in vivo, and it is this grown from milk (Chang et al. 1982). We are now precursor that proliferates and shows partial differen- developing immortalized lines that accurately maintain tiation in long-term culture in medium MCDB 170. This the phenotype and keratin profile of the luminal epithelial would imply that not all the cells in the basal layer that cell, in order to examine the effect of a variety of express keratins 5 and 14 are committed myoepithelial oncogenes on this cell type. cells. If this is the case, as has also been suggested for the rat mammary gland (Allen et al. 1984), it should be The authors are grateful to Ms P. Purkis and Ms J. Steel for possible to identify markers that would distinguish the their contribution to the development of the antibody LL001 directed to keratin 14. They also thank Linda Hayashi for small populations of putative stem cells from the majority technical assistance and Dr Rosemary Millis for supplies of of basal cells. tissues and tumours. Part of this work vas supported by NIH The observations of keratin expression in cells cultured grant CA-24844 and by the Office of Health and Environmental from primary tumours are rather surprising. As indi- Research, US Department of Energy, under contract DE- cated, the invasive cancer cell seen in primary and AC03SF00098. metastatic lesions has a keratin profile identical to that of the differentiated luminal epithelial cell. Using medium MX, which can be used to grow this cell type from the References normal gland, no cells can be cultured from primary ALLEN, R., DULBECCO, R., SYKA, P., BOWMAN, M. AND ARMSTRONG, carcinomas. The phenotype of the cells that are cultured B. (1984). Developmental regulation of in cells of the from cancers in medium MCDB 170, however, is the rat mammary gland studied with monoclonal antibodies. Ptvc. same in its keratin profile as the phenotype of the cells natn.Acad. Sci. U.S.A. 81, 1203-1207.

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