Transplanted mammary epithelium grows in association with host stroma: aging of serially transplanted mammary gland is intrinsic to epithelial cells

Mouse mammary gland displays an irreversible decline in growth rate when propagated by serial transplantation in gland-free mammary fat pads of isogeneic mice. Because transplanted fragments of gland contain both mammary epithelial and stromal elements, the present study was undertaken to distinguish between two possibilities: (1) stromal cells in the implants proliferate in coordination with epithelium as the mammary ductal tree regenerates at each passage, or (2) transplanted epithelial tissue interacts exclusively with host stroma. Mammary xenografts from 18-week-old virgin Sprague-Dawley rats were implanted into gland-free mammary fat pads of athymic Balb/cNu/Nu mice. These rat xenografts regenerated chimeric mammary ductal outgrowths. When sectioned and stained with Hoechst dye 33258, a procedure that provides for unambiguous identification of mouse cell nuclei, rat mammary epithelium was found to be associated with mouse stromal cells; only at the site of transplantation were occasional rat stromal nuclei observed. This indicates that as mouse epithelial tissue becomes progressively aged during serial transfer in young mice, the stromal components are refreshed during each passage. The primary lesion underlying the mammary aging phenomenon must therefore be intrinsic to the epithelial cells.     

Diet‐induced obesity disrupts ductal development in the mammary glands of nonpregnant mice

Mammary glands develop postnatally in response to the hypothalamic‐pituitary‐gonadal axis. Obesity‐induced changes in the local environment, however, retard mammary gland development during late pregnancy and lactation. To clarify the effects of obesity on fundamental duct development, we compared the mammary glands of nulliparous nonpregnant obese mice fed a high‐fat diet with those of lean mice fed a normal diet. Obese mice had enlarged mammary glands, reflecting fat pad size, whereas the ducts in obese mice showed a less dense distribution with less frequent branching. Additionally, the ducts were surrounded by thick collagen layers, and were incompletely lined with myoepithelium. Because leptin receptors were localized in the epithelium region and leptin that was highly expressed in the obese glands suppressed mammary epithelial cell proliferation in vitro, the present results suggest that obesity disrupts mammary ductal development, possibly by remodeling the mammary microenvironment and promoting the expression of such paracrine factors as leptin. Developmental Dynamics 238:1092–1099, 2009. © 2009 Wiley‐Liss, Inc.     

Immunolocalization of the human basal epithelial marker monoclonal antibody 312C8-1 in normal tissue and mammary tumours of rodents

Summary Using immunoperoxidase staining of monoclonal antibody 312C8-1 against 51 000 dalton human keratin polypeptide, immunolocalization was observed in frozen sections of normal tissue and mammary tumours of adult female mice and rats. In normal tissue, the epitope was recognized in myoepithelial cells of the mammary, sweat and salivary glands, and in basal and suprabasal cells of the epidermis. However, the antibody did not react with luminal epithelial cells of the above glands or with mesenchymal cells. In spontaneous mammary tumours of mice, marker-positive tumour cells were distributed only in the outer layer of adenocarcinoma Type A, while they were scattered in some foci of adenocarcinoma Type B, and encircled the epithelial foci of pregnancy dependent tumours (plaque). All layers of epidermoid structures in adenoacanthoma revealed positivity. In rat mammary tumours induced by local dusting with 7, 12-dimethylbenz()anthracene (DMBA) powder, the staining pattern of benign tumours was comparable to that of the normal mammary gland. But, in addition to basally situated cells, marker-positive tumour cells were found scattered in the foci of adenocarcinoma, and were not restricted to basal cells in squamous cell carcinoma. The marker was not found in sarcomatous tissue. This antibody can therefore also be applied to rodents, and the staining pattern can be used to identify the epithelial subclass specific marker in normal tissue and in mammary tumours.     

Immunohistochemical localisation of keratin and luminal epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours

Rabbit antisera to human 40-63 000 MW epidermal keratin, one batch with restricted distribution of reactivity from an initial (aK1) and one with "broad spectrum" distribution of reactivity from a late bleeding (aK), and to "luminal epithelial antigen" (aLEA) were applied to formalin fixed paraffin embedded sections of human normal and neoplastic mammary and salivary glands using an indirect immunoperoxidase method. aK1 reacted with myoepithelial cells, aLEA with luminal epithelial cells and aK with both cell types in normal mammary and salivary gland. In breast carcinomas the majority of intraluminal and infiltrating carcinoma cells reacted with aLEA but not with aK1 which reacted only with surrounding myoepithelial cells. aK reacted with both myoepithelial cells and with intraluminal and infiltrating tumour cells. In the salivary gland adenomas the majority of cells reacted with aK, and those cells arranged in a tubular fashion reacted with aLEA.     

Differential mammary morphogenesis along the anteroposterior axis in Hoxc6 gene targeted mice.

Mammary epithelial cell proliferation, branching, and differentiation span from the appearance of the mammary bud in midgestation through to the cycling mammary gland in adulthood. Here, we show that females homozygous for a targeted disruption of the Hoxc6 homeobox gene produce thoracic mammary glands that are slightly under-developed at birth and completely cleared of epithelium by adulthood, and inguinal mammary ducts that are dilated and fail to regress in response to ovariectomy. Mammary buds are detected in E12.5 Hoxc6 homozygous embryos. However, in newborn Hoxc6 homozygous females, branching ductal structures and fat pad development are reduced. Whole-mount and histologic analyses of mammary glands from adult Hoxc6 homozygous females show the absence of mammary epithelium in thoracic glands and dilated ducts in inguinal glands at 100% penetrance. Histologic analysis of inguinal mammary glands from ovariectomized Hoxc6 homozygous females demonstrates no signs of the expected regression of epithelium, suggesting that these glands are not responsive to the loss of ovarian hormone signals. We further observe repression of Hoxc6 expression specifically within mammary stroma by estrogen and progesterone. Hoxc6 homozygous mice also exhibit a homeotic transformation of the second thoracic vertebra into the first (T2 to T1 conversion with 60% penetrance), corresponding to both the gene's anterior boundary of expression and the most extreme appearance of mammary defects. The position-specific phenotypes observed and the potential role for Hoxc6 in mediating hormone-regulated ductal expansion and regression in the adult female are discussed.     

Immunohistochemical expression of monoclonal antibodies (epi-1 and myo-1) derived from human breast cancer against rat mammary tumors.

Immunohistochemical expression of monoclonal antibodies epi-1 and myo-1 derived from human breast cancer cell line (HBC-4W) was examined for DMBA-induced rat mammary tumors. Antibody epi-1 reacted with luminal epithelial cells while antibody myo-1 reacted with myoepithelial cells of the mammary glands in rats, respectively. The reactions with both antibodies were markedly visible, in particular, in the normal mammary gland, tumor-like lesions and benign epithelial mammary tumors in rats, which showed clear two-cell-type structures. Among malignant mammary tumors, adenocarcinoma was strongly positive with antibodies epi-1 and myo-1. However, squamous cell carcinoma and adenoacanthoma mainly reacted with antibody epi-1. On the other hand, the intercellular matrices of pleomorphic cell sarcoma and stromal areas of the normal mammary gland or epithelial tumors were positive with antibody myo-1.     

Mammary gland proliferation in female rats: Effects of the estrous cycle,pseudo-pregnancy and age

Assessment of mammary gland proliferation in rats is an important endpoint in preclinical safety studies of pharmaceutical compounds. However, existing data on mammary gland proliferation in rats during the estrous cycle is conflicting, and it is unknown whether mammary gland proliferation differs between young and mature female virgin rats. Additionally, it is unclear which of the commonly applied markers of proliferating cells that is optimal for assessment of rat mammary gland proliferation. In this study the caudal thoracic, the abdominal and the cranial inguinal (i.e., the 3rd the 4th and the 5th) mammary gland were collected from 29 young and 26 mature non-treated, virgin female Sprague Dawley rats. Estrous cycle stage was determined from repeated vaginal smears and histological examination of the reproductive organs. Proliferation of mammary epithelium was assessed by immunohistochemistry using three markers: PCNA, Ki67, and BrdU. Proliferation of the mammary epithelium occurred mainly in the terminal end buds in the young animals. Epithelial proliferation was significantly increased during metestrus compared to the other phases. Mammary gland proliferation in pseudo-pregnant females was increased compared to proestrus, estrus and diestrus, but not metestrus. Except during estrus no difference in mammary gland proliferation was observed between young and mature female rats, and no significant differences was observed between different mammary glands. The percentages of PCNA-, Ki67- and BrdU-positive epithelial cells were significantly correlated. In conclusion, the variation in normal proliferation between estrous cycle stages and animals with an irregular estrous cycle should be considered in toxico-pathological studies of mammary gland proliferation.     

In situ phosphorylation of Akt and ERK1/2 in rat mammary gland, colon, and liver following treatment with human insulin and IGF-1

High doses of insulin and the insulin analog AspB10 have been reported to increase mammary tumor incidence in female rats likely via receptor-mediated mechanisms, possibly involving enhanced IGF-1 receptor activation. However, insulin and IGF-1 receptor functionality and intracellular signaling in the rat mammary gland in vivo is essentially unexplored. The authors investigated the effect of a single subcutaneous dose of 600 nmol/kg human insulin or IGF-1 on Akt and ERK1/2 phosphorylation in rat liver, colon, and mammary gland. Rat tissues were examined by Western blotting and immunohistochemistry by phosphorylation-specific antibodies. Insulin as well as IGF-1 caused Akt phosphorylation in mammary epithelial cells, with myoepithelial and basal epithelial cells being most sensitive. IGF-1 caused stronger Akt phosphorylation than insulin in mammary gland epithelial cells. Phosphorylation of ERK1/2 was not influenced by insulin or IGF-1. Rather, in liver and mammary gland P-ERK1/2 appeared to correlate with estrous cycling, supporting that ERK1/2 has important physiological roles in these two organs. In short, these findings supported that the rat mammary gland epithelium expresses functional insulin and IGF-1 receptors and that phosphorylation of Akt as well as ERK1/2 may be of value in understanding the effects of exogenous insulin in the rat mammary gland and colon.     

Current theories on the nature of myoepithelial cells and their role in the morphogenesis of dyshormonal hyperplasia and breast neoplasms

An analysis of current concepts on the origin and potentials of neoplastic transformation of myoepithelial cells (MC) of the mammary glands is presented. The epithelial and smooth-muscle nature of these cells is discussed. The hypothesis of mixed (epithelial and myoepithelial) nature of mammary gland carcinoma and possibilities of neoplastic transformation of poorly-differentiated forms of MC are considered. Methods for identification of cells of the myoepithelial origin are described.     

Site of lodination in rat mammary gland

The ability of the mammary gland to take up and organically bind radioiodide was studied in non-pregnant, pregnant, and lactating rats. Autoradiography was used to determine whether duct cells or alveolar cells are responsible for iodination in the rat mammary gland. Iodination was not detected in mammary glands from non-pregnant rats, but occurred late in the twelfth day of gestation and continued throughout pregnancy and lactation. Protein-containing vacuoles in alveolar cells and casein-like proteins in milk were the major sites where iodination occurred within the gland. Milk proteins in the lumens of ductules adjacent to alveoli were also iodinated. In contrast, ducts, myoepithelial cells, fat cells, blood vessels and other histological components of the gland did not show iodinating capability. Cytochemistry was also used to identify endogenous mammary peroxidase activity in the same glands, and it was found that the presence and location of this enzyme was correlated with the ability to iodinate.     

Chemically induced mammary gland adenomyoepitheliomas and myoepithelial carcinomas of mice. Immunohistochemical and ultrastructural features.

Myoepithelial cell tumors of the mammary gland have been observed in several mammalian species and are composed of a single cell type (myoepithelium) or, more often, present as a biphasic process including neoplastic ductal epithelial cells. In dogs, these are common tumors, but in humans they are rare neoplasms of the breast, and little is yet known of their pathogenesis, particularly with respect to myoepithelial origin. The present report describes bicellular mammary gland tumors arising from the duct epithelium that were induced in (C57BL/6NCr X DBA/2NCr)F1 (B6D2F1) mice by four weekly oral applications of 1 mg 7,12-dimethylbenz[a]anthracene (DMBA) starting at 8 weeks of age. Mammary tumors developed 7 to 8 months later in 14 of 57 mice, and most showed great morphologic resemblance to human adenomyoepitheliomas and myoepithelial carcinomas. Ultrastructurally, the induced tumors were composed of cuboidal epithelium with a microvillous border originating from the lining duct epithelium and plump oval or highly elongated cells that were identified as myoepithelial in origin. These spindle cells contained abundant microfilaments in parallel orientation, some with focal densities and intermediate filaments that frequently formed loose bundles or compact tonofibrils. The myoepithelial cells possessed well-developed desmosomes and plasma membrane caveolae and were regularly bordered by single or reduplicated basement membranes. By immunohistochemistry, strong immunoreactivity was observed for actin in the myoepithelial tumor component only, whereas cytokeratin was variably present in both duct epithelium and myoepithelium. Neoplastic myoepithelial cells stained purple with phosphotungstic acid hematoxylin (PTAH) and brilliant red with Masson's trichrome. It is suggested that DMBA-induced mouse mammary gland adenomyoepitheliomas and myoepithelial carcinomas may serve as very useful animal models to study myoepithelial tumorigenesis.     

Cell size and shape changes in the myoepithelium of the mammary gland during differentiation

We have studied changes in myoepithelial cell size and shape during different stages of mouse mammary gland differentiation by using the fluorescent probe for actin NBD-phallacidin. Pieces of mammary tissue were fixed, mounted on slides, permeabilized with cold acetone (-20 degrees C), and then treated with nitrobenzoxadiazole-phallacidin. Myoepithelial cells lining ducts of glands at all stages of development are spindle-shaped structures oriented parallel to the long axis of the duct at the base of the luminal epithelium. In virgin animals, myoepithelial cells also occur as linear tracts oriented parallel to the long axis of small projections along the sides of ducts and terminal end buds. In early pregnancy, small stellate-shaped cells begin to appear around presumptive secretory units. By late pregnancy, larger star-shaped units of intense fluorescence appear at the base of alveoli. During lactation, both cell bodies and cell processes further enlarge as these interlacing stellate-shaped cells encompass the expanded alveoli. In regressing glands, cell size decreases and the processes appear to retract. Although alveoli are virtually absent in the multipartate resting gland, myoepithelial cells remain around lateral buds of ducts. These myoepithelial cells have two distinct shapes: small star-shaped cells capping the buds and spindle-shaped cells oriented parallel to the long axis of the buds. A comparison of myoepithelial cell shape in virgin mice and nulliparous women indicates a more developed cell in the human gland at this stage of development. Intact segments of mammary gland combined with NBD-phallacidin as a probe for actin provide an ideal system for future studies of the control of myoepithelial cell size and shape and their influence on cell functions.     

The role of myoepithelial cells in the morphogenesis of induced mammary tumours

Summary The localization and cytomorphology of myoepithelial (ME) cells and their role in the morphogenesis of the mammary gland tumours of Wistar rats induced by 7,12-Dimethylbenz-a-anthracene-DMBA- were studied. Cells which do not participate in secretion and contain cytoplasmic myofibrillar bundles in a typical arrangement are considered to be of ME origin. In the histogenesis of induced mammary gland tumours no definite role can be attributed to mature ME cells or their precursors. Decreased differentiation is associated with reduced numbers of ME cells. No ME cells can be detected in the anaplastic, stromafree portions of the solid tumour. The sarcomatous component of the induced carcinosarcomas originates from connective tissue. ME cells may give rise to leiomyoma-like tumours comparable with the human benign mammary myoepithelioma. The atrophic areas of mammary gland tumours consisted mostly of preserved ME cells. The ME cells of induced mammary gland tumours were, in every respect, identical with the normal ME cells of control mammary glands.     

Immunohistochemical study of human breast tumors using monoclonal antibodies to intermediate filament proteins (nonproliferating epithelial structures in breast dysplasia)

An immunohistochemical analysis of nonproliferating epithelial structures was carried out in 10 samples of human breast dysplasia and in 4 samples of tissue surrounding mammary gland carcinoma. Monoclonal mouse antibodies against individual prekeratins of rat monolayer epithelial antibodies of clone C12 against rat prekeratin with the molecular mass 49 kilodalton and antibodies of clone E3 against rat prekeratin with the molecular mass 40 kilodalton-monoclonal antibodies against vimentin (clone 30), as well as polyclonal antibodies against smooth muscle myosin and against the basement membrane glycoprotein laminin were used. The lining epithelium of all glandular structures reacted only with C12 antibodies. Two variants of myoepithelial cells containing myosin were detected. Variant I contains myosin and vimentin and is localized in intralobular ducts. Variant 2 contains myosin and prekeratin, recognized by E3 antibodies and is found in extralobular ducts.     

A quantitative histotopologic analysis of the variation in lobulo-alveolar mitotic activity in the Lewis/Mai rat mammary gland during the estrous cycle

Quantitative microscopic techniques were employed to determine relative mitotic activity of lobulo-alveolar tissue in thick sections obtained from the inguinal mammary glands of colchicine-treated post-pubertal Lewis/Mai rats sacrificed at different phases of the estrous cycle. It was found that the magnitude of lobulo-alveolar cell mitotic activity and the proportion of the lobulo-alveolar tissue with mitotic activity undergo significant fluctuations during the estrous cycle. The frequency distribution of mitotic activity in lobulo-alveolar tissue was different and heterogenous at each phase of the estrous cycle. The frequency distribution of nuclei in lobulo-alveolar tissue during the estrous cycle showed only minor variations and could not explain the variation in mitotic activity. Similarly, no morphological differences were observed in the mammary gland which could account for the variation observed in mitotic activity. It was concluded that the variation in mitotic activity of lobulo-alveolar tissue is probably related to alterations in the generation time of lobulo-alveolar cells imposed by cyclical variation in ovarian hormones during the estrous cycle.     

Glucose-6-phosphate dehydrogenase isoenzymes in acinar epithelial cells separated from the mammary gland of the rat at intervals during the course of lactation.

Acinar epithelial cells were purifed from suspensions of cells from the lactating mammary glands of rats. As described previously, this purififaction was accomplished by sedimentation in an isokinetic gradient of Ficoll (polysucrose) in tissue culture medium, and the purity of the separated cells was established by electron microscopy and by histochemical markers. Isoenzymes of glucose-6-phosphate dehydrogenase were investigated at various intervals during lactation in separated populations of stromal and acinar cells. Acinar cells contained three- to eightfold more glucose-6-phosphate dehydrogenase activity than did stromal cells. The proportions of the respective isoenzymes varied during the course of lactation, and the observed changes were parallel in purified acinar cells and in the lactating mammary glands from which the cell suspensions were obtained. The availability of purified acinar cells in the investigation of interactions between hormones and cells from the mammary gland permits a greater degree of specificity than has been possible in the study of mammary cell suspensions which contain myoepithelial cells, duct cells, acinar cells, endothelial cells, fibroblasts, fat cells, mast cells, plasma cells, and blood cells.     

Nonrandom frequency distribution of mitoses in rat lobuloaveolar mammary gland epithelium.

A quantitative microscopic technique was employed to examine the distribution of mitotic activity in the rat mammary gland. The frequency distribution of mitoses per unit volume of lobuloalveolar mammary gland epithelium in virgin Lewis/Mai rats at each phase of the estrous cycle were determined and compared to the expected Poisson frequency distributions, assuming random mitotic activity. Both pooled data and data from individual rats were compared to expected Poisson distributions. At each phase of the estrous cycle, the pooled observed distributions deviated significantly from Poisson distributions. Sixty-seven percent (72/108) of the observed frequency distributions obtained from individual rats also deviated significantly from expected Poisson distributions. These data indicate a nonrandom distribution of mitoses in rat lobuloalveolar mammary gland epithelium. This observation suggests that local cell products and/or a variation in the extent of replicative synchrony of lobuloalveolar cell populations may determine in part the pattern of mitotic activity in this tissue. A nonrandom distribution of mitoses in mammary epithelium may have significance in relation to the genesis of hyperplastic and neoplastic lesions of the mammary gland.     

Keratinocyte Growth Factor Causes Cystic Dilation of the Mammary Glands of Mice: Interactions of Keratinocyte Growth Factor, Estrogen, and Progesterone In Vivo

Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell proliferation that has been reported to induce marked proliferation of mammary epithelium in rats. In this study, systemic administration of KGF into naive and oophorectomized mice causes mammary gland proliferation, as evidenced histologically by the appearance of cysts lined by a single layer of epithelium and by hyperplastic epithelium. Whole mount preparations of the mammary glands reveal that the histologically noted cysts are actually ducts that are dilated along much of their length. The histology of the mammary glands of KGF-treated mice is similar to the histology of fibrocystic disease in the buman female breast. The response in mice differs significantly from the appearance of the mammary glands in KGF-treated rats in which ductal epithelial proliferation is most prominent. Estrogen and progesterone when administered in combination but not alone cause the development of numerous endbuds in the mouse mammary gland. KGF in estrogen- and progesterone-pretreated mice causes the growth of dilated ducts, hyperplastic epithelium within ducts and endbuds, and a fibrous metamorphosis of periductal adipose tissue. The mammary epithelial hyperplasia caused by KGF is rapidly reversible in both mice and rats after cessation of KGF treatment. The spectrum of KGF-, estrogen-, and progesterone-induced mammary histopathology in mice provides a model for the study of fibrocystic and hyperplastic breast disease.     

Involution of the sheep mammary gland

Changes in the ovine mammary gland epithelium during initiated involution were studied by light and electron microscopy. Apoptosis of the duct and alveolar epithelial cells was first identified at 2 d after weaning, reached a peak at 4 d and then progressed gradually thereafter. Apoptotic cells were phagocytosed by intraepithelial macrophages and alveolar epithelial cells. Occasional apoptotic epithelial cells were observed in the alveolar and duct lumina. The highly vacuolated cells in the alveolar and duct lumina were confirmed to be macrophages as they were CD45⁺, MHC class II⁺. Changes in myoepithelial cells involved shrinkage and extension of cytoplasmic processes into the underlying stroma and no apoptosis was observed. Regression of the blood capillaries was also by apoptosis. The resulting apoptotic bodies were either taken up by adjacent endothelial cells or were shed into the capillary lumen to be phagocytosed later by mural endothelial cells or blood monocytes. The mammary glands were completely involuted by 30 d after weaning. It was concluded that the mammary gland involutes by apoptosis, a process which allows deletion of cells without the loss of the basic architecture and the integrity of the epithelial lining of the gland.