ARTICLE |
Correspondence to: Matthew J. Smalley, Section of Cell Biology and Experimental Pathology, The Breakthrough Toby Robins Breast Cancer Research Centre, Inst. of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK. E-mail: matthew@icr.ac.uk
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Summary |
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We have previously demonstrated that purified virgin mouse mammary luminal epithelial and myoepithelial cells promiscuously express cell type-specific cytokeratins when they are cloned in vitro. Changes in cytokeratin expression may be indicators of the loss or change of the differentiated identity of a cell. To investigate the factors that may be responsible for the maintenance of differentiated cellular identity, specifically cellcell and cellmatrix interactions, we cloned flow-sorted mouse mammary epithelial cells on the extracellular matrix (ECM) derived from the EngelbrethHolmSwarm murine sarcoma (EHS matrix). Changes in cell differentiation on EHS, compared with culture on glass, were analyzed by comparing patterns of cytokeratin expression. The results indicate that ECM is responsible for maintenance of the differentiated identity of basal/myoepithelial cells and prevents the inappropriate expression of luminal antigens seen on glass or plastic. Luminal cell identity in the form of retention of luminal markers and absence of basal/myoepithelial antigens, on the contrary, appears to depend on homotypic cellcell contacts and interactions. The results also show that luminal cells (or a subpopulation of them) can generate a cell layer that expresses only basal cytokeratin markers (and no luminal cytokeratin markers) and may form a pluripotent compartment. (J Histochem Cytochem 47:15131524, 1999)
Key Words: mouse mammary epithelium, luminal, myoepithelium, extracellular matrix, cytokeratin, flow cytometry
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Introduction |
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To fully understand how the development and differentiation of the mammary gland parenchyma and of the different cell types within it are regulated, it is necessary to be able to study separately the behavior of the individual cell types within the gland. The mature rodent mammary epithelium consists of two cell populations, the luminal epithelial cells and the basal/myoepithelial cells. The luminal epithelial cells are cuboidal or low columnar cells that line the ducts and alveoli and are responsible for milk secretion. Primary and secondary ducts may be composed of several layers of luminal epithelial cells, which become fewer with higher orders of branching. Most terminal ducts have only one layer of luminal cells (
We have previously described (
Cytokeratins are important in tissue architecture and cell organization in vivo (
There are at least two possibilities for the extracellular signals that could reinforce cellular identity in vivo. First, each cell type could require contact with, or paracrine signals from, other cells, either of the same type or of different types. Thus, myoepithelial cells might require signals from luminal cells, and vice versa. When put in separated cultures and lacking signals from the other cell type, the cells could then begin to dedifferentiate. The second possibility is that mouse cells are especially sensitive to the presence or absence of extracellular matrix (ECM). ECM not only provides a support for cells in vivo but its components also act as ligands that interact with cells via cell surface molecules which include the integrins (
To test the hypothesis that maintenance of the differentiated identity of mouse mammary epithelial cells requires ECM, we cultured separated virgin mouse mammary luminal epithelial and myoepithelial cells on EHS matrix at clonal density. The morphology of the resulting clones was examined and the expression of cytokeratins was used as an index of whether the in vivo differentiated identity of the isolated cells was maintained by culture on the EHS matrix. The results show that, in the presence of ECM, mouse mammary myoepithelial cells formed flat clones that maintained a more differentiated phenotype, with a marked reduction in the extent to which inappropriate antigens were expressed. Mouse mammary luminal epithelial cells under similar conditions formed either flat clones, some of which generated a layer of cells with basal/myoepithelial characteristics, or spheroidal clones ("mammospheres") in which the central mass of cells, which had no contact with ECM but were surrounded in all directions by other cells of luminal origin, retained a more differentiated phenotype and did not express basal markers. The outer layer of mammospheres still expressed both luminal and basal markers and interestingly it was only this outer cell layer which produced ß-casein in response to lactogenic hormones.
These results demonstrate that the maintenance of luminal phenotype in mouse mammary cells is likely to be a function of homotypic cellcell interactions, whereas ECM signaling is likely to be primarily responsible for the maintenance (and generation) of the basal/myoepithelial phenotype, although it also appears to be a requirement for the functional differentiation of luminal cells. The results also provide evidence for the existence of a pluripotent compartment within the adult mouse mammary luminal epithelium.
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Materials and Methods |
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Isolation of Mouse Mammary Epithelial Cells
This has previously been described in detail (
Immunofluorescence Staining of Cell Surface Antigens for Flow Cytometry
Mouse mammary luminal epithelial or myoepithelial cells were isolated from the prepared cell suspension by flow cytometry, as previously described (
Cell type-specific antibodies used were rat monoclonal antibodies (MAbs) obtained from Dr. A. Sonnenberg. MAb 33A10, which is specific for a milk fat globule membrane antigen, was used to specifically isolate luminal epithelial cells. MAb JB6, which in our hands uniformly stains the basal/myoepithelial layer of the mammary epithelium in Parkes mice (
Flow Cytometry
Flow cytometry was used for separation of cell samples stained with antibodies against cell surface antigens. This enabled the isolation of purified individual epithelial cells that were either positive or negative for a particular surface antigen, because they were first analyzed and gated as single cells on the basis of forward and orthogonal light scatter before identification and separation of fluorescent cells.
An Ortho 50H Cytofluorograf cell sorter equipped with a 2150 data analysis system and an argon ion laser tuned to give 50 mW at a wavelength of 488 nm was used. Recorded parameters were light-scattered orthogonally and at a narrow forward angle at 488 nm and green fluorescence at 520 nm [from fluorescein isothiocyanate (FITC) excited at 488 nm]. Cells were sorted from a flow stream of sterile PBS, and using anti-coincidence circuitry one drop (containing one cell) was deflected for each sort command.
Mammary Epithelial Cell Cloning
Primary mouse mammary epithelial clone cultures were established on ethanol-sterilized 13-mm glass coverslips in Nunc (Roskilde, Denmark) 24-well plates. Coverslips were either left uncoated or were coated with 250 µl of EHS murine sarcoma matrix (Collaborative Research; Cambridge, UK). This was allowed to set into a thick gel by incubation at 37C for 1 h before medium was added to the wells.
Flow-sorted mouse mammary luminal epithelial and myoepithelial cells were cultured using a feeder layer of irradiated (20 Gy from a 60Co source) 3T3-L1 preadipocyte cells at a density of 20 cells mm-2 and a growth medium consisting of a 1:1 mixture of DMEM and Ham's F12 with 10% FCS, 5 µg ml-1 insulin, and 10 ng ml-1 cholera toxin. Freshly disaggregated mouse epithelial cells were plated at a density of 2.5 cells mm-2 (equal to approximately 500 cells per well) and cultures were maintained at 37C in an atmosphere consisting of 5% (v/v) oxygen, 5% (v/v) CO2, and 90% (v/v) nitrogen. These growth conditions had previously been determined as optimal for clonal culture of virgin mouse mammary epithelium (
To examine functional differentiation, clones grown on glass or on EHS matrix were cultured in the basic growth medium or in medium containing ovine prolactin at 5 µg/ml and hydrocortisone at 1 µg/ml ("lactation medium";
Immunofluorescence Staining of Epithelial Clones In Vitro
After 8 days in culture, epithelial colonies were observed, fixed and stained. Table 1 gives a list of mouse MAbs used for staining of mouse mammary cells in vitro. LE61, LE65, LLOO2, and LP2K were obtained from Professor E.B. Lane (-isoform smooth muscle actin was obtained from Sigma (Poole, Dorset, UK). RßC-1, a mouse anti-rat ß-casein MAb that crossreacts with mouse casein, was obtained from Dr. C. Kaetzel (
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Clones on glass coverslips were methanol-fixed and stained in the wells of the 24-well plates in which they had been grown, as previously described (
Phase-contrast and Fluorescence Microscopy
A Zeiss Axiovert 100 infinity-corrected microscope fitted with a Zeiss MC80 photomicrograph apparatus was used for phase-contrast and fluorescence microscopy. A mercury vapor lamp was used for epifluorescent illumination and the microscope was also fitted with a triple filter block apparatus containing wedge-free emission filters for FITC (Zeiss filter set 10) and TX (Zeiss filter set 00, also used for TRITC) as well as a filter for AMCA (Zeiss filter set 02). So that AMCA fluorescence could be observed and photographed in multiply stained samples without breakthrough from the FITC or TX/TRITC, the microscope was also fitted with an extra emission filter (transmission cut-off at 500 nm) for use with AMCA only.
The majority of phase-contrast images were photographically recorded using a x10 Achrostigmat (NA 0.25) objective. Fluorescence images were recorded using a x25 water immersion Neofluar (NA 0.80) objective. Phase-contrast photomicrographs were taken on Kodak Technical Pan 35-mm film (100 ASA). Black-and-white fluorescence photomicrographs were taken using Kodak T-Max Professional P3200 35-mm film.
Confocal Microscopy
For observation of certain samples stained by immunofluorescence, a Bio-Rad MRC 600 confocal imaging system equipped with a kryptonargon laser was used in conjunction with a Nikon Optiphot fluorescence microscope. Samples were observed using a x60 plan Apo objective lens.
Scanning Electron Microscopy
Coverslips for scanning electron microscopy (SEM) were prepared by standard protocols (
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Results |
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Myoepithelium-derived Clones: Morphology
Ten-week-old virgin Parkes mouse mammary myoepithelial cells separated by flow cytometry using MAb JB6 were plated at clonal density onto either uncoated glass coverslips or coverslips coated with EHS gel and were observed after 8 days in culture. Morphological comparisons between myoepithelium-derived clones on uncoated and EHS-coated coverslips were made on clones stained by multiple immunofluorescence for cytoskeletal markers, because clones cultured on EHS were difficult to observe directly by phase-contrast microscopy. This was a result of the flattened nature of the cells comprising these clones, the granular background of the underlying matrix, and optical limits of phase-contrast microscopy in the wells of 24-well plates, especially at the edges of such wells.
The majority of clones that formed (>90%) on EHS matrix had a very similar morphology, being monolayers composed of large, flat cells (Figure 1C and Figure 1D), similar in appearance to myoepithelium-derived clones on tissue culture plastic or glass (Figure 1A and Figure 1B) (
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Myoepithelium-derived Clones: Staining for Cytoskeletal Antigens
All cells of all myoepithelium-derived clones on EHS gel were strongly stained by an antibody (LLOO2) to cytokeratin 14 but only approximately 10% or fewer of the cells in such clones reacted with LE61, an anti-cytokeratin 18 antibody, as demonstrated by double indirect immunofluorescence (Figure 1C and Figure 1D). This was in contrast to the myoepithelial clones on glass, which showed similar levels of cytokeratin 14 reactivity but a much higher proportion of cytokeratin 18-positive cells (about 50%), as we have previously described (Figure 1A and Figure 1B) (
The strictly basal nature of the myoepithelium-derived clones on EHS gel was confirmed by triple indirect immunofluorescence staining using the antibody combination LLOO2 (anti-cytokeratin 14), LE65 (a second anti-cytokeratin 18 antibody), and RCK107 (a second anti-cytokeratin 14 antibody). All cells in all such clones were strongly stained with both cytokeratin 14 antibodies (LLOO2 and RCK107), whereas none stained with LE65 (not shown). Over 80% of cells also stained for smooth muscle actin (antibody 1A4; not shown), whereas no cells stained for cytokeratin 19 (antibody LP2K; not shown). This staining pattern is characteristic of mouse myoepithelial cells in vivo (
Comparable results were obtained from two separate JB6 sorts and 13 separate unsorted cell populations prepared from different batches of mice, each of which was simultaneously plated with and without EHS matrix. Clonal identities were established solely on the basis of sorted cells, and unsorted clones were used merely to increase the numbers of analyzable colonies. Unsorted populations were not used to define clonal identities.
Luminal Epithelium-derived Clones: Morphology
Ten-week-old virgin Parkes mouse mammary luminal epithelial cells separated by flow cytometry were plated at clonal density onto uncoated glass coverslips or coverslips coated with EHS gel at clonal density and observed after 8 days in culture.
After this time, the three types of clone previously described (
The morphology of the flat clones varied such that at one extreme they consisted of cells forming distinct colonies but with variable morphologies and varying degrees of contact between them, similar to the clones observed on glass (Figure 2A), whereas at the other extreme they were composed of very tightly packed cuboidal/polygonal cells (Figure 2B). The mammospheres were often simple spheroids but were also observed with either long and thin (Figure 2C) or short and club-shaped (Figure 2D) outgrowths, or both. After 8 days of culture, it appeared that the spheroids were partially enveloped by EHS gel and that the club-shaped outgrowths were growing into the surrounding ECM where it was densest. The observation that the spheroids were growing partly surrounded by a layer of the gel rather than on top of the matrix was confirmed by SEM studies. Figure 3 shows a scanning electron micrograph of an 8-day-old mammosphere seen to be growing in cup of fibrous matrix, some of which forms a thin layer extending up and over the sides and top of the sphere.
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No further morphological changes were visible under phase-contrast after 8 days in culture, apart from an increase in clone size. The more refractile nature of the cells comprising these clones meant that there was little difficulty in observing them by phase-contrast, especially in the flattened clones composed of cuboidal/polygonal cells and in the three-dimensional clones.
Luminal Epithelium-derived Clones: Staining for Cytoskeletal Antigens
Luminal epithelial colonies grown on uncoated glass coverslips and on coverslips coated with EHS matrix gel were principally characterized by double indirect immunofluorescence staining for cytokeratin 14 (antibody LLOO2) and cytokeratin 18 (antibody LE61).
Clones on uncoated glass coverslips had the same promiscuous staining pattern as previously described (
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Type II clones formed a small minority of flat clones (approximately 10%) that were seen only on a well-defined matrix layer. However, they were the most interesting because they were multilayered, although this was apparent only after staining (Figure 4C and Figure 4D). Furthermore, the basal layer, in direct contact with the matrix, consisted of cells that were cytokeratin 14-positive but cytokeratin 18-negative. The upper layer consisted of compact cuboidal cells that were a mixture of cytokeratin 14-negative/18-positive as well as cells positive for both antigens.
The emergence of cells within lumen-derived clones that retained a specifically luminal phenotype (i.e., cytokeratin 14-negative/18-positive) was seen even more clearly in the mammospheres, which could be divided into two types on the basis of their cytokeratin staining patterns (Figure 5). The first type consisted of double staining (cytokeratin 14-positive/18-positive) of all the cells of the mammosphere (Figure 5A and Figure 5B). The second type of staining pattern, which was the most common seen in mammospheres (>70%), was typified by a central mass of cells in the colony staining strongly for cytokeratin 18 but not cytokeratin 14, whereas only the outermost cell layer double stained for both cytokeratins 14 and 18 (Figure 5C and Figure 5D). This segregation of phenotypes within mammospheres was clearly seen by confocal microscopy (Figure 5E and Figure 5F). A complete loss of cytokeratin 18 staining in the outer layer of the mammospheres, to give a cytokeratin 14 positive/18 negative layer comparable to the basal layer seen in the Type II (multilayer) lumen-derived flat clones was, however, not observed in cultures maintained for up to 8 days.
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Lumen-derived clones on EHS matrix were also triple stained for cytokeratin 14, cytokeratin 18, and cytokeratin 19, and for cytokeratin 14, cytokeratin 18, and -isoform smooth muscle actin. The anti-cytokeratin 19 antibody LP2K stains all mouse luminal epithelial cells in vivo but only stains lumen-derived clones heterogeneously in vitro (
The results showed that approximately 5090% of the cytokeratin 18-positive cells in Type I and Type II colonies on EHS were weakly or moderately stained for cytokeratin 19 (not shown). In mammospheres, all cytokeratin 18-positive cells were strongly cytokeratin 19-positive, which again confirms their specific luminal identity (not shown). Lumen-derived clones showed little or no staining for -smooth muscle actin (not shown).
Comparable results were obtained from nine separate 33A10 sorts from different batches of mice, each of which was simultaneously plated with and without EHS matrix, and from 13 separate preparations of unsorted populations of virgin mouse mammary epithelial cells. As stated above, clonal identities were established solely on the basis of sorted cells, and unsorted clones were used merely to increase the numbers of analyzable colonies. Unsorted populations were not used in the definition of clonal identities. Furthermore, all clone types that were observed in the sorted lumen-derived cultures were seen in the unsorted populations, and there were no extra colony types seen in unsorted populations that were not observed in either the sorted lumen-derived or myoepithelial populations.
The cytokeratin staining patterns of the main clone types observed are summarized in Table 2 and shown schematically in Figure 6.
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Functional Differentiation of Lumen-derived Colonies
The above results show that the cloned luminal cells on EHS matrix developed a structure and differentiated pattern of cytoskeletal antigen expression that resembled that of intact mouse mammary parenchyma, i.e., a cell layer in Type II (multilayer) flat clones with strictly basal characteristics and inner cells in mammospheres with a strictly luminal phenotype. The capacity of these structures to respond to lactational stimuli was therefore examined by ß-casein staining of clones cultured for 8 days on glass or on EHS matrix in the basic growth medium or in lactation medium (see Materials and Methods). Only clones in the cultures grown on EHS in lactation medium were stained by the antibody to this protein. Furthermore, staining was seen only in mammospheres, not in flat clones, and only in the outer layer of the spheres nearest the gel, not in the center of the mammosphere (Figure 7).
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Discussion |
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We have used flow sorting, cell cloning, and multiple indirect immunofluorescence analysis as a novel method of investigating the role of ECM in the definition and maintenance of the differentiated cellular identity of virgin mouse mammary luminal epithelial and myoepithelial cells. It has previously been observed (
Culture of separated myoepithelial cells as clones on EHS matrix resulted in retention of the myoepithelial identity of the cells (as defined by cytokeratin 14 expression) and to a large degree prevented the expression of an in vivo marker of luminal epithelial cells (cytokeratin 18), which is seen in corresponding clones cultured on glass. There was no evidence in this study that the adult myoepithelial cells could generate a luminal cell type in vitro; structures resembling Type II clones were never seen in sorted myoepithelium cultures. In contrast to the uniformity in phenotypes seen with the myoepithelium-derived clones, those derived from purified luminal epithelial cells showed diverse morphologies and staining patterns, indicating a greater complexity in the factors that determine the specific phenotypes of cells in these clones. In particular, the Type II (multilayer) clones generated de novo a cell layer with a cytokeratin staining pattern (cytokeratin 14-positive/18-negative) that is typical of basal cells.
The basal cell layer of the Type II clones is unlikely to have been derived from myoepithelial cells contaminating the 33A10-positive luminal cell population. Any myoepithelium-derived cells in this situation can have arisen only as contaminants in the luminal sorts. Such contamination could occur only in two ways. First, the single-cell gating, which is the preliminary stage of the cell sorting, would have to be inefficient such that it allowed through doublets of cells (which must further have been composed of one luminal epithelial and one myoepithelial cell). However, preliminary experiments from when the system was being established showed that the flow cytometer sorted single-cells with a greater than 99% purity. The second possibility is that the pure single-cell populations contained contaminating single myoepithelial cells among the single luminal epithelial cells. Our previous data (-isoform smooth muscle actin, whereas sorted, myoepithelium-derived clones were positive for this marker.
A different pattern of segregation and differentiation of cells was seen in the mammosphere type of luminal clone. In the majority of these, an exclusively luminal phenotype (cytokeratin 18-positive/19-positive/14-negative) was seen in the central mass of cells. The outer layer of the mammospheres, however, retained the promiscuous cytokeratin expression and still stained with both luminal and basal markers (i.e., they were cytokeratin 18-positive/19-positive/14-positive), in contrast to the basal layer of the Type II (multilayer) flat clones. The cytokeratin 14/18 staining patterns of the major clone types are summarized schematically in Figure 6. It is clear that sorted single luminal epithelial cells from the virgin mouse mammary parenchyme can generate both luminal and basal epithelial cells, depending on the manner in which they interact with each other and the surrounding matrix. Luminal epithelium-derived clones could not generate fully differentiated myoepithelial cells with both basal cytokeratins and -isoform smooth muscle actin, however, at least under the culture conditions used. For this study we limited the culture conditions to a single set of parameters that we had previously shown to allow maximal clonal growth of mouse mammary epithelium (
We propose that the difference between the cytokeratin 18-positive/14-negative phenotype of the innermost cells in the mammospheres and the principally double-positive cells of the flat clones shows that homotypic cellcell interactions reinforce and maintain the luminal epithelial phenotype. It may be that the topography or extent of these interactions is important in the maintenance of the luminal epithelial phenotype in this system. It is unlikely that the EHS matrix takes an active role (e.g., via ligandreceptor interactions) in the maintenance of the luminal cell identity as the central mass of cells in the mammospheres, which exclusively expressed the luminal cell markers, were not in contact with it. However, it does appear that the extracellular matrix has an active role in promoting the generation of de novo basal epithelial cells (in the Type II flat clones).
At first sight it appears paradoxical that the outermost cells of the mammospheres are apparently less well differentiated as basal cells than their counterparts in the Type II (multilayer) flat clones. However, as shown by SEM, most of the basal cell layer of the mammospheres was covered only by a very thin matrix layer and in some areas was not covered by matrix at all. The Type II (multilayer) flat clones, in contrast, were always observed on a well-defined matrix layer, under which conditions a cytokeratin 14-positive/18-negative basal layer formed, and it is therefore likely that active ECM signaling played a role in the generation of this layer.
This study provides evidence, therefore, that mouse mammary luminal epithelial cells are, or contain, a compartment that is pluripotent, as shown by their ability to generate clones that possess a basal cell layer lacking luminal cytoskeletal markers, although it does not demonstrate conversion from luminal epithelial cells to fully differentiated myoepithelial cells. These findings support the work of
The above model, in which cellcell interactions reinforce luminal cell identity and contact with an optimal amount of ECM is required to generate basal epithelial cells de novo from luminal epithelial cells, must also accommodate the observation that culture of mammospheres in a lactation medium resulted in ß-casein production only in the outermost cell layer, in which we have demonstrated stains for both the luminal epithelial and basal markers, and not in the more fully luminally differentiated central cells of the mammospheres. It is known that mouse mammary luminal epithelial cells isolated in vitro require contact with ECM for functional differentiation (
We have demonstrated in this study the utility of single cell sorting and clone-by-clone analysis of cell type-specific markers in the dissection of the differentiative capacity of the component cells of a complex tissue, and we have provided in vitro evidence from primary mouse mammary epithelial cells for the existence of pluripotential cells within the mammary luminal epithelial but not the myoepithelial cell compartment. Our study supports the work of
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Footnotes |
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1 Current address: LICR/UCL Breast Cancer Laboratory, Department of Surgery, University College London, London, UK.
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Acknowledgments |
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Supported by the Cancer Research Campaign.
We would like to thank Professor Barry Gusterson for his support.
Received for publication April 23, 1999; accepted July 13, 1999.
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