Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland

Using antisera to specific proteins, the localization of the rat mammary parenchymal cells (both epithelial and myoepithelial), the basement membrane, and connective tissue components has been studied during the four physiological stages of the adult rat mammary gland, viz. resting, pregnant, lactating, and involuting glands. Antisera to myosin and prekeratin were used to localize myoepithelial cells, antisera to rat milk fat globule membrane for epithelial cells, antisera to laminin and type IV collagen to delineate the basement membrane and antisera to type I collagen and fibronectin as markers for connective tissue. In the resting, virgin mammary gland, myoepithelial cells appear to form a continuous layer around the epithelial cells and are in turn surrounded by a continuous basement membrane. Antiserum to fibronectin does not delineate the basement membrane in the resting gland. The ductal system is surrounded by connective tissue. Only the basal or myoepithelial cells in the terminal end buds of neonatal animals demonstrate cytoplasmic staining for basement membrane proteins, indicating active synthesis of these proteins during this period. In the secretory alveoli of the lactating rat, the myoepithelial cells no longer appear to form a continuous layer beneath the epithelial cells and in many areas the epithelial cells appear to be in contact with the basement membrane. The basement membrane in the lactating gland is still continuous around the ducts and alveoli. In the lactating gland, fibronectin appears to be located in the basement membrane region in addition to being a component of the stroma. During involution, the alveoli collapse, and appear to be in a state of dissolution. The basement membrane is thicker and is occasionally incomplete, as also are the basket-like myoepithelial structures. Basement membrane components can also be demonstrated throughout the collapsed alveoli.     

The human mammary gland basement membrane is integral to the polarity of luminal epithelial cells

We show that myoepithelial cell basement membrane derived E3 and E8 domains of laminin-1 are capable of polarizing luminal epithelial cells with regard to epithelial membrane antigen localization. This event is dependent on the alpha6 integrin and results in aggregation and phosphorylation of the tyrosine residues of the focal adhesion kinase complex. We also demonstrate that uncultured normal luminal epithelial cells synthesize normal levels of beta and gamma laminin chains and reduced levels of alpha chains mRNA in common with malignant epithelial cells. In contrast normal myoepithelial cells synthesize all three constituent chains of laminin-1. Therefore in breast cancer the absence of myoepithelial cells could result in a lack of laminin alpha chains which may contribute to loss of polarity of malignant epithelial cells.     

The Role of the Microenvironment in Mammary Gland Development and Cancer

The mammary gland is composed of a diverse array of cell types that form intricate interaction networks essential for its normal development and physiologic function. Abnormalities in these interactions play an important role throughout different stages of tumorigenesis. Branching ducts and alveoli are lined by an inner layer of secretory luminal epithelial cells that produce milk during lactation and are surrounded by contractile myoepithelial cells and basement membrane. The surrounding stroma comprised of extracellular matrix and various cell types including fibroblasts, endothelial cells, and infiltrating leukocytes not only provides a scaffold for the organ, but also regulates mammary epithelial cell function via paracrine, physical, and hormonal interactions. With rare exceptions breast tumors initiate in the epithelial compartment and in their initial phases are confined to the ducts but this barrier brakes down with invasive progression because of a combination of signals emitted by tumor epithelial and various stromal cells. In this article, we overview the importance of cellular interactions and microenvironmental signals in mammary gland development and cancer.The mammary gland is composed of a combination of multiple cell types that together form complex interaction networks required for the proper development and functioning of the organ. The branching milk ducts are formed by an outer myoepithelial cell layer producing the basement membrane (BM) and an inner luminal epithelial cell layer producing milk during lactation. The ducts are surrounded by the microenvironment composed of extracellular matrix (ECM) and various stromal cell types (e.g., endothelial cells, fibroblasts, myofibroblasts, and leukocytes). Large amount of data suggest that cell-cell and cell-microenvironment interactions modify the proliferation, survival, polarity, differentiation, and invasive capacity of mammary epithelial cells. However, the molecular mechanisms underlying these effects are poorly understood. The purification and comprehensive characterization of each cell type comprising normal and neoplastic human breast tissue combined with hypothesis testing in cell culture and animal models are likely to improve our understanding of the role these cells play in the normal functioning of the mammary gland and in breast tumorigenesis. In this article, we overview cellular and microenvironmental interactions that play important roles in the normal functioning of the mammary gland and their abnormalities in breast cancer.     

Mammary gland development: Role of basal myoepithelial cells

Mammary epithelium is organized as a bilayer with a layer of luminal secretory cells and a layer of basal myoepithelial cells. To dissect the specific functions of these two major compartments of the mammary epithelium in mammary morphogenesis we have used genetically modified mice carrying transgenes or conditional alleles whose expression or ablation were cell-type specific. Basal cells are located in close proximity to mammary stroma and directly interact with the extracellular matrix (basement membrane) during all their lifespan. On the contrary, luminal secretory cells during early stages of the postnatal mammary development have only limited contacts with basement membrane and become exposed to the extracellular matrix only during late developmental stages at the end of pregnancy and in lactation. Consistently perturbation of beta1-integrin function specifically in the luminal layer of the mammary epithelium, did not interfere with mammary morphogenesis until the second part of pregnancy but led to impaired secretory differentiation and lactation. On the contrary, ablation of beta1-integrin gene in the basal mammary epithelial cells resulted in a more precocious phenotype: disorganized branching in young virgin animals and a complete arrest of lobuloalveolar development. Further, a constitutive activation of beta-catenin signaling due to expression of N-terminally truncated (stabilized) beta-catenin specifically in basal myoepithelial cells resulted in accelerated differentiation of luminal secretory cells in pregnancy, precocious postlactational involution, increased angiogenesis and development of mammary tumors. Altogether these data suggest that basal mammary epithelial cells can affect growth and differentiation of luminal secretory cells, have an impact on the epithelium-stroma relationships and, thereby, play an important role in the process of mammary morphogenesis and differentiation.     

Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.     

Three-dimensional culture models of mammary gland

The mammary gland is a complex tissue comprised of a branching network of ducts embedded within an adipocyterich stroma. The ductal epithelium is a bi-layer of luminal and myoepithelial cells, the latter being in contact with a basement membrane. During pregnancy, tertiary branching occurs and lobuloalveolar structures, which produce milk during lactation, form in response to hormonal and cytokine signals. Postlactational regression is characterized by extensive cell death and tissue remodeling. These complex developmental events have been difficult to mimic in cell culture although many useful culture models exist. Recently, considerable advances in three-dimensional modelling of the mammary gland have been made with the use of collagen and other biomaterials for the study of branching morphogenesis and tumorigenesis, techniques which may enable rapid advances in our understanding of both basic biology and the study of cancer therapeutics.     

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.     

Establishment and characterization of a novel human myoepithelial cell line and matrix-producing xenograft from a parotid basal cell adenocarcinoma

Summary Myoepithelial cells exert important paracrine effects on epithelial morphogenesis and mitogenesis through direct cell-cell interactions and through synthesis of a basement membrane extracellular matrix. To study these effects further, this study established the first immortalized human myoepithelial cell line, HMS-1, and transplantable xenograft, HMS-X, from the rare parotid basal cell adenocarcinoma. The cell line exhibited a fully differentiated myoepithelial phenotype and the xenograft exhibited the rare property of accumulating an abundant extracellular matrix composed of both basement membrane and nonbasement membrane components with the latter predominating. With HMS-1 as a feeder layer, dramatic and specific induction of epithelial morphogenesis (sheroid formation) occurred with selected normal epithelial and primary carcinoma target cells. HMS-1 and HMS-X provide distinct advantages over the conventional murine matrices in existence. They will be invaluable in future studies of human tumor-myoepithelial and matrix interactions important for tumor cell growth, invasion, and metastasis.     

Distribution and synthesis of type V collagen in the rat mammary gland

In the 100-day-old virgin and lactating rat mammary glands, type V collagen is mainly present in the interstitial connective tissue and in association with blood vessels. It is not present in the basement membrane region surrounding the ducts in mature virgin glands but is present in this region in neonatal and lactating glands. Ultrastructural localization of type V collagen reveals that it is mainly located on the basal surface (i.e., the surface in contact with the basement membrane) of epithelial but not myoepithelial cells. In addition, type V collagen is located on some interstitial collagen fibers and on a large number of granules that are in close proximity to the basal surface of both epithelial and myoepithelial cells. Immunofluorescence and biochemical studies indicate that several clonal mammary fibroblastic cell lines synthesize type V collagen in vitro. In some cell lines, type V collagen is secreted as an extensive fibrillar meshwork on the surface of the cells, whereas in other cell lines, it is secreted beneath the cells around their periphery. A number of mammary epithelial and myoepithelial-like cells, however, do not synthesize type V collagen in vitro.     

Down-regulation of laminin-5 in breast carcinoma cells.

BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three ln-5 mRNAs was up-regulated. CONCLUSION: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that ln-5 functions to control tumor progression in the breast.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

Three-dimensional culture models of mammary gland

The mammary gland is a complex tissue comprised of a branching network of ducts embedded within an adipocyte-rich stroma. The ductal epithelium is a bi-layer of luminal and myoepithelial cells, the latter being in contact with a basement membrane. During pregnancy, tertiary branching occurs and lobuloalveolar structures, which produce milk during lactation, form in response to hormonal and cytokine signals. Postlactational regression is characterized by extensive cell death and tissue remodeling. These complex developmental events have been difficult to mimic in cell culture although many useful culture models exist. Recently, considerable advances in three-dimensional modelling of the mammary gland have been made with the use of collagen and other biomaterials for the study of branching morphogenesis and tumorigenesis, techniques which may enable rapid advances in our understanding of both basic biology and the study of cancer therapeutics.Key words: mammary gland, models, extracellular matrix, cell culture, cell-lines, scaffolds, tissue engineering, epithelium, adipocytes     

On the source of uterine 'luminal fluid' proteins in the mouse

Examination by light-, transmission electron- and scanning electron-microscopy showed that flushing the lumen of the mouse uterus with small volumes of fluid damaged the endometrium by rupturing and removing luminal epithelial cells, splitting the epithelial basement membrane and connective tissue stroma, and rupturing and leaching stromal cells and blood vessels. The damage increased with increasing progestation of the uterus and between Days 4 and 5 of pregnancy. I conclude that many so-called 'luminal fluid' proteins originate from luminal and stromal cells, intercellular fluid and blood and that apparent changes in luminal fluid protein content during early pregnancy may largely reflect alterations in the extent and type of damage produced by flushing, as a consequence of changes in the physical state of the uterus induced by hormones and the presence of blastocysts.     

Immunocytochemical localization of the ectoenzyme aminopeptidase N in the human breast.

The ectoenzyme aminopeptidase N (APN) was localized in the normal human breast at both the light microscopic and the ultrastructural level. APN was expressed on intralobular and interlobular fibroblasts and on the apical surface of some luminal epithelial cells. This enzyme was not detected on either myoepithelial cells and their associated basement membrane or capillary endothelium. Furthermore, the staining pattern was maintained in benign and malignant breast disease. APN belongs to a family of enzymes that hydrolyze peptides in the extracellular space. As with other ectoenzymes present in the breast, APN expression is restricted to specific cell types. This pattern of expression may indicate a role for these enzymes in the biology of the normal breast.     

Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development

The salivary epithelium initiates as a solid mass of epithelial cells that are organized into a primary bud that undergoes morphogenesis and differentiation to yield bilayered acini consisting of interior secretory acinar cells that are surrounded by contractile myoepithelial cells in mature salivary glands. How the primary bud transitions into acini has not been previously documented. We document here that the outer epithelial cells subsequently undergo a vertical compression as they express smooth muscle α-actin and differentiate into myoepithelial cells. The outermost layer of polarized epithelial cells assemble and organize the basal deposition of basement membrane, which requires basal positioning of the polarity protein, Par-1b. Whether Par-1b is required for the vertical compression and differentiation of the myoepithelial cells is unknown. Following manipulation of Par-1b in salivary gland organ explants, Par-1b-inhibited explants showed both a reduced vertical compression of differentiating myoepithelial cells and reduced levels of smooth muscle α-actin. Rac1 knockdown and inhibition of Rac GTPase function also inhibited branching morphogenesis. Since Rac regulates cellular morphology, we investigated a contribution for Rac in myoepithelial cell differentiation. Inhibition of Rac GTPase activity showed a similar reduction in vertical compression and smooth muscle α-actin levels while decreasing the levels of Par-1b protein and altering its basal localization in the outer cells. Inhibition of ROCK, which is required for basal positioning of Par-1b, resulted in mislocalization of Par-1b and loss of vertical cellular compression, but did not significantly alter levels of smooth muscle α-actin in these cells. Overexpression of Par-1b in the presence of Rac inhibition restored basement membrane protein levels and localization. Our results indicate that the basal localization of Par-1b in the outer epithelial cells is required for myoepithelial cell compression, and Par-1b is required for myoepithelial differentiation, regardless of its localization.     

Antigenic subsets of human breast epithelial cells distinguished by monoclonal antibodies

Three monoclonal antibodies raised to the human milk fat globule membrane bind, within the normal breast, to the surface of the luminal epithelial cells but not to the surrounding myoepithelial, connective tissue, or blood vessel cells. These antibodies distinguish three subsets of the epithelial cells that are not distinguishable by conventional histology. To show the arrangement of the cells in two dimensions over the sheet of epithelium, ducts were dissected out of normal breast tissue, opened up and laid flat as sheets of epithelium. The apical faces of the cells were strained, unfixed, using two-color immunofluorescence to contrast the subsets of cells stained by the different antibodies. The epithelium was then seen to be a mosaic of cells that express different surface antigens. The grouping and appearance of the cells stained by the different antibodies was characteristic. This may be just a random heterogeneity of antigen expression but alternatively the different cells may be in different physiological states. Regardless of its biological significance, the observation has practical consequences for the use of such antibodies in identifying cells and the study of antigenic heterogeneity in tumors.