Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland

Differentiation (1993) 5 5 : 13-18

Differentiation O n t g n y , Neoplda and Diliemtiation Therapy

0 Springer-Verlag1993

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland Anna Sapino ', Luigia Macri ', Patrizia Gugliotta Donatella Pacchioni', Yong-Jian Liu Daniel Medina 2,

I,

2,

Gianni Bussolati I

' Department of Biomedical Sciences and Oncology, University of Torino, Italy 'Department of Cell Biology, Baylor College of Medicine, Houston, Texas, USA Accepted in revised form June 24, 1993

Abstract. The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells. We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically - detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.

Introduction The mouse mammary gland achieves the complete development of the parenchymal component in the virgin mouse through budding of solid cellular bulb-like structures at the apex of elongating ducts. The bulb-like strucCorrespondence to: G. Bussolati, Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Anatomia Patologica, Via Santena 7, 1-10126 Torino, Italy

tures, termed end buds, later cavitate giving origin to secondary ductular branches localized within the fat pad PI. From the earliest stages of development, the structure of ducts is arranged as a dual layer of luminal epithelial and basally located myoepithelial cells : these two cell types have different functions and morphologies, as recognized by specific markers. It remains to be established if epithelial and myoepithelial cells originate from a common precursor and later differentiate into different cell types, or if they follow separate lines of proliferation and differentiation from the very early stages of development. In line with the latter hypothesis, in a recent study on the proliferating cell types in the mouse mammary gland, we obtained evidence of proliferative activity in myoepithelial cells localized in ducts and ductules [22]. In the end buds, representing the most primitive developmental site and characterized by the highest proliferation rate within the mammary gland, no myoepithelial cells can be recognized: the solid bulk of epithelial cells, body cells, is surrounded by a layer of highly proliferating cells of undetermined nature and apparently undifferentiated. It has been suggested that these cells, termed cap cells, might represent the common precursor of both epithelial and myoepithelial cells [18, 311. We report here an extensive immunohistochemical characterization of the cellular components of the developing mouse mammary gland. Divergent expression of the various classes of keratins in different cell types has in fact been observed in the developing mouse mammary gland and demonstrated to be regulated by environmental factors [2].

Methods Mammary glands were collected from 3 to 5-week-old BALB/c female mice (Charles River, Calco, Italy). In some experiments, in order to obtain fully differentiated glands, 3-week-old animals were injected for 9 days with 0.1 ml of a water solution containing 1 pg of 17B-estradiol (Sigma, St. Louis, Mo., USA) and 1 mg of progesterone (Sigma). Organ cultures of these glands were set up

14 Table 1. Specific cytoplasmic markers

Marker

Reagent

Ref.

Source

Dilution

Cell spec.

Cytokeratin (19, 16, 15, 14, 10) Cytokeratin (18, 8, 7,6. 5.4, 3. 2. 1) Cytokeratin (8) Cytokeratin (14, 10, 5 , 1) Cytokeratin (19) Keratin (predominantly 11, 9) K6 K14 z-Smooth muscle actin Vimentin

AE1 a (mAb)

[l, 29, 321

Cambridge Research (Cambridge, Mass., USA) Cambridge Research

1 :200

Epithelial

1 :40

Epithelial Myoepi thelial

Enzo Biochem (New York, N.Y.) Enzo Biochem

1 :500

Epithelial

1 :400

1:5

Vimentin

3B4c (mAb)

Amersham International (Amersham, UK) Dakopatts (Glostrup, Denmark) D. Roop (Houston, Tex., USA) D. Roop Sigma (St. Louis, Mo., USA) Euro-Diagnostic (Apeldoorn, Holland) Dakopatts

Epithelial Myoepithelial Epithelial

Collagen type IV

Antiserum"

Hey1 (Berlin, Germany)

1 :250

AE3" (mAb)

358H11 a (mAb) 348 E 12" 1 165a (mAb)

Antiserum"

~ 3 1 code no. A575

Antiserum Antiserum 1A4" (mAb)

[261 [261 ~ 4 1

Antiserum"

Best reactivity on: a metacharn fixed tissues;

[31

1 :500

1 : 1000 1 : 1000 1 : 1000 1 : 300

1:lOO

Epithelial Myoepithelial Epithelial M yoepithelial M yoepithelial Fibroblast Adipocyte Fibroblast Adipocyte Cap cell Myoepithelial

alcohol fixed tissues; c formalin fixed tissues. after trypsin digestion

Cell type epithelial

€El myoepithelial

KERATIN

ALPHA ACTIN

COLLAGEN IV

+ +

+

+ +

EB [4]. The glands were maintained in a humidified incubator (Forma Scientific, Marietta, Ohio, USA) at 37" C in 50% O2 and 5% C 0 2 . The Waymouth MB 751 (Gibco. Grand Island. N.Y.) culture medium was supplemented with 5 pg/ml insulin. 1 pg/ml progesterone, 0.001 pg/ml 17/?-estradiol,5 pg/ml prolactin and 3 ng/ml epidermal growth factor. Three different fixation methods were performed: buffered formalin for 3 h at room temperature, methanol-carnoy (methacarn) for 1.5 h a t room temperature [19] or cold 100% ethanol overnight. Glands were then embedded in paraffin. The avidin biotin peroxidase (ABC) staining method was used to detect specific cytoplasmic marker proteins [12]. The expression of keratin subtypes was evaluated by five different monoclonal antibodies and three different antisera, the specificities of which are listed in Table 1. Different markers for nonepithelial cells were used (Table 1). The experiments were performed independently in our two laboratories. To obtain information on the estrogen dependency we employed an immunocytochemical procedure on paraffin sections us-

Fig. 1. Schematic representation of differentiation processes and different immunophenotypes in the mouse mammary gland at different developmental stages. The three main cell types are illustrated as myoepithelial, epithelial and cap cells in their respective locations. EB, end bud; D, duct; AB. alveolar bud

ing anti-estrogen receptor monoclonal antibodies (Abbott Lab, Diagnostic Division, Abbott Park, Ill., USA; kit dilution) according to Hiort et al. (lo), modified by substituting the trypsin treatment with a 5 min incubation in Proteinase K (Merck, Darmstadt, Germany) 0.25 mg/ml in PBS at room temperature.

Results In animals from 3 to 5 weeks old the gland morphology was characterized by ducts and end buds. In vitro prolactin stimulation induced gland development, with duct elongation and appearance of alveolar structures. The expression of different antigens in normal mammary epithelial cells at different stages of development is illustrated in Fig. 1.

Fig. 2. Mammary gland from 5-week-old mouse. Immunocytochemical staining for keratin 6 is strongly positive in the majority of body cells of the end bud while peripheral cap cells are negative. Nuclear counterstained with haemalum. Ear, 100 pm

Fig. 3. End-buds from 5-week-old mouse mammary glands. a Staining for keratins with monoclonal AE3 is positive in both epithelial and myoepithelial cells present along the duct. Cap cells are negative. b Basally-located myoepithelial, but not epithelial cells, are positive for a-smooth muscle actin. The cap cells are weakly positive. Ear, 50 pm

Fig. 4. Reactivity for monoclonal antibody 34BE12 in different histological structures. Almost all the body cells of an end bud (a) and the myoepithelial cells of a duct from the mammary gland

of a virgin mouse (b) are positive. In ducts of cultured glands (c) both the myoepithelial cells and endoluminal epithelial papillary structures are stained. Ear, 100 pm

16

Keratin 14 was detected only in myoepithelial cells and was not observed in the cap or body cells of the growing end buds. In complete contrast, keratin 6 was detected only in body cells of the end bud, but not i n the cap cells o r any other cell type in the gland from 5-week-old mice (Fig. 2). At least 30 individual end buds

from 10 different unprimed mice were evaluated and keratin expression was not detected in the cap cells by any of these antibodies. In all of the mammary structures, the acidic keratins recognized by AEI, 35PH11 and 1165 were present only in the epithelial cells. Monoclonal antibodies AE3, 34flE12 and Dako polyclonal antikeratin antiserum, stained both epithelial and myoepithelial cells of buds and ducts (Fig. 3a). An exception was monoclonal antibody 34PE12, detecting both acidic and basic cytokeratins (see Table 1), which stained the body cells of the end buds and the myoepithelial cells in virgin mice, while the luminal epithelial cells of ducts were negative (Fig. 4a, b). On the other hand, this antibody stained the epithelial cells of alveolar buds and the epithelial papillary structures present in ducts of cultured glands (Fig. 4c). Several non-keratin antibodies were used to evaluate the different cell types in the mammary gland. Monoclonal antibody against a-smooth-muscle actin was markedly positive in all myoepithelial cells, while a weak positivity was detected in cap cells of end buds only in 5-week-old animals but not in younger ones. The epithelial cells were negative (Fig. 3b). Collagen type IV was detected in the myoepithelial cells of ducts and in the cap cells of the end buds (Fig. 5). Vimentin antibodies were positive in fibroblasts and adipocytes while negative in epithelial cells. Estrogen receptors were identified in the nuclei of the epithelial cells of ducts and in the nuclei of the body cells of end buds in glands of 3-week-old virgin mice (Fig. 6a, b). In hormonally stimulated mice the estrogen

Fig. 6. Gland of 3-week-old virgin mouse. Immunocytochemical staining for estrogen receptor shows specific reactivity on the nuclei of ductal epithelial cells (a) and of some body cells of an end bud (b). Bar, 100 pm

receptors were present also in the epithelial cells of the alveolar buds. Myoepithelial and cap cells were uniformly negative.

Discussion

Fig. 5. Immunocytochemical staining for collagen type IV strongly decorates the basal lamina and the cytoplasm of myoepithelial and cap cells around an end bud. Bar, 100 pm

The immunophenotypic characterization of cell types in the mammary gland provides information on : (a) differentiation processes, (b) cell dynamics and interactions and (c) the effects of hormones in the developing mammary gland. The results described herein relate to these topics. Previous efforts to characterize the cell components of the developing mammary tree have mainly been focused on the identification of the specific keratin subtypes [2, 6, 26,27,28]. In general, keratin subtypes were detected by immunohistochemistry and/or two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis in both myoepithelial and luminal epithelial cells during all stages of development of the mouse mammary gland including prepubertal. Our immunocytochemical results were consistent with the absence of keratin in the cap cells and the presence of this cytoskeletal protein in the body cells of end

17

buds. These data differ from those of Sonnenberg et al. [27] who studied the expression of keratin proteins specifically in the end bud using a polyclonal antiserum. Their results suggested the presence of keratin proteins in both cap cells and body cells of the end bud; however, the keratin specificity of the antiserum was not defined. The precursors of the luminal cells, i.e. the body cells of the end buds, express a high number of tonofilament protein types, as recognized by their reactivity to all the five anti-keratin mAbs here employed, as well as to the Dako polyclonal and the anti-keratin 6 (K6) antiserum. Such reactivity never appeared homogeneous, being restricted to part of the cell population. Body cells are rapidly proliferating [22] and the observed heterogeneous staining might be related to asynchronous differentiation steps. Keratin 6, corresponding to a 57 kD protein, was not expressed in any other cell type in the ducts. Smith et al. [26] reported K6 staining in cells of the ducts of the 3 to 4-week-old C3H female mice. However, cellular localization of immunofluorescence staining on frozen sections can be difficult to interpret and structures interpreted as ductal could have been tangential sections of end buds. As in the study by Smith et al. [26], we did detect K6/K 14 coexpression in preneoplastic hyperplasias and neoplasias of the mammary gland [16]. A similar, though not fully overlapping phenomenon, was the reactivity of 348E12 mAb with the body cells and with the myoepithelial cells in the ducts. With hormonal stimulation of the glands the 348E12 mAb stained selectively the epithelial papillary structures in the ducts. A similar reactivity of this mAb with hyperplastic epithelium was described in the human breast by Raju [20]. We observed that the localization of estrogen receptor and, consequently, one immediate target of estrogen stimulation was restricted to the body cells of the end buds and to the luminal epithelial cells of the alveolar buds and ducts. These results, in agreement with previous observations on the enhancing effect of estrogen administration on the proliferation of epithelial body cells [22], concur in suggesting that such cells are estrogen-dependent and give rise to the progeny of estrogen - dependent luminal cells along the mammary tree. The absence of estrogen receptors in the cap cells shows that these cells are a unique cell population and are clearly distinct from luminal epithelial and body epithelial cells. Regardless, these data do not argue against the fact that estrogen may affect the growth of the cap cells by indirect means or that local growth factors (i.e. epidermal growth factor, transforming growth factor a) are important for luminal epithelial cells growth. The hypothesis that the cap cell is the totipotent stem cell for the mammary gland was proposed by Williams and Daniel [31] on the basis of cell kinetic, time-lapse photography and electron microscopic studies. In agreement with their ultrastructural data, we failed to detect either keratins or vimentin. The observed lack in intermediate filaments is a characteristic which cap cells seem to share with few other cell types as reported in the literature [9, 14, 15, 301. The hypothesis that cap cells might be true totipotent mammary cells was also sup-

ported by the immunohistochemical results of Sonnenberg et al. [27, 281 and Daams et al. [6] which showed that the basal cell layer of the end buds and ducts were universally positive for monoclonal antibodies JB6 and JsE3, whereas luminal epithelial cells were negative for these markers. Other factors concur against the hypothesis that the cap cell is the only totipotent stem cell. In particular, it is firmly documented that all portions of the mammary ducts and alveoli contain totipotent cells which can repopulate the entire mammary parenchyma [7, 11, 251. Additionally, proliferation in the developing mammary gland is not limited to undifferentiated cap cells, but observed in luminal epithelial and myoepithelial cells [22]. In fact, high levels are observed in the keratin positive, estrogen-receptor positive body cells of the end buds [22]. In our opinion, the data presented here and especially the presence of collagen IV allow another interpretation of the nature and significance of the cap cells. Although cap cells may be viewed as precursors to myoepithelial cells, a second and equally important function is their specialized role in paving the way to ductal elongation. Definitive identification of mammary stem cells will depend on experiments demonstrating the ability of a purified population of cells to produce a normal mammary epithelial tree in the mammary fat pad with full developmental and differentiative potential. Acknowledgements. We thank Mr. Nino Ferraro for technical assistance with photography. Work supported by a grant of the ACRO project of the Consiglio Nazionale delle Ricerche (Roma).

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