The Cellular Organization of the Mammary Gland: Insights From Microscopy

Despite rapid advances in our knowledge of the cellular heterogeneity and molecular regulation of the mammary gland, how these relate to 3D cellular organization remains unclear. In addition to hormonal regulation, mammary gland development and function is directed by para- and juxtacrine signaling among diverse cell-types, particularly the immune and mesenchymal populations. Precise mapping of the cellular landscape of the breast will help to decipher this complex coordination. Imaging of thin tissue sections has provided foundational information about cell positioning in the mammary gland and now technological advances in tissue clearing and subcellular-resolution 3D imaging are painting a more complete picture. In particular, confocal, light-sheet and multiphoton microscopy applied to intact tissue can fully capture cell morphology, position and interactions, and have the power to identify spatially rare events. This review will summarize our current understanding of mammary gland cellular organization as revealed by microscopy. We focus on the mouse mammary gland and cover a broad range of immune and stromal cell types at major developmental stages and give insights into important tissue niches and cellular interactions.

     

Stem/Progenitor Cells in Mouse Mammary Gland Development and Breast Cancer

Breast cancer is a genetically and clinically heterogeneous disease. It is unclear whether different target cells contribute to this heterogeneity and which cell types are most susceptible to oncogenesis. Stem cells are speculated to be the cellular origin of at least a subset of human breast cancers. To begin to address these issues, we have isolated and characterized cell populations enriched in normal mammary stem/progenitors and have studied the expression of putative stem/progenitor markers in tumors derived from genetically engineered mice. Specifically, transgenic activation of Wnt signaling in the mammary gland induces tumors comprised of epithelial and myoepithelial cells harboring the same genetic defect implying that the tumor arose from transformation of a bipotent progenitor cell. On the other hand, transgenic activation of Neu signaling induces tumors comprising cells of more limited lineage capacity. Thus, the heterogeneity of different breast cancers may reflect the activation of different oncogenic pathways, different cellular targets in which these genetic changes occur, or both.     

microRNAs and EMT in Mammary Cells and Breast Cancer

MicroRNAs are master regulators of gene expression in many biological and pathological processes, including mammary gland development and breast cancer. The differentiation program termed the epithelial to mesenchymal transition (EMT) involves changes in a number of microRNAs. Some of these microRNAs have been shown to control cellular plasticity through the suppression of EMT-inducers or to influence cellular phenotype through the suppression of genes involved in defining the epithelial and mesenchymal cell states. This has led to the suggestion that microRNAs maybe a novel therapeutic target for the treatment of breast cancer. In this review, we will discuss microRNAs that are involved in EMT in mammary cells and breast cancer.     

Stem Cells and the Developing Mammary Gland

The mammary gland undergoes dynamic changes throughout life. In the mouse, these begin with initial morphogenesis of the gland in the mid-gestation embryo followed by hormonally regulated changes during puberty and later in adulthood. The adult mammary gland contains a hierarchy of cell types with varying potentials for self-maintenance and differentiation. These include cells able to produce complete, functional mammary glands in vivo and that contain daughter cells with the same remarkable regenerative potential, as well as cells with more limited clonogenic activity in vitro. Here we review how applying in vitro and in vivo methods for quantifying these cells in adult mammary tissue to fetal mammary cells has enabled the first cells fulfilling the functional criteria of transplantable, isolated mammary stem cells to be identified a few days before birth. Thereafter, the number of these cells increases rapidly. Populations containing these fetal stem cells display growth and gene expression programs that differ from their adult counterparts but share signatures characteristic of certain types of breast cancer. Such observations reinforce growing evidence of important differences between tissue-specific fetal and adult cells with stem cell properties and emphasize the merits of investigating their molecular basis.     

KLK5基因在基底细胞样乳腺癌组织中的差异表达

目的：探索KLK5基因作为诊断基底细胞样乳腺癌（BLBC）的分子标志和治疗靶点的可能。方法：通过包含48804个探针的人类mRNA基因表达谱芯片比较48例各亚型乳腺癌和6例正常乳腺组织基因表达谱,并通过荧光实时定量PCR法进行结果验证,分析BLBC与其他各亚型乳腺癌基因之间的差异表达。结果：分析乳腺癌各亚型和正常乳腺组织的基因表达谱,在BLBC中有99个基因上调4倍以上（2倍以上有意义）,其中KLK5基因显著上调（5倍以上）,但KLK5在LuminalA、LuminalB、HER-2过表达的基因表达谱中下调超过2倍,它们之间的差异表达具有统计学意义（P〈005）;并通过RT—PCR检测得到进一步验证。结论：KLK5可以作为诊断基底细胞样乳腺癌的标志基因和治疗靶点。     

EGF-Related Peptides in the Pathophysiology of the Mammary Gland

Normal mammary gland development is the result of complex interactions between a number of hormones and growth factors. Normal and malignant human mammary epithelial cells are able to synthesize and to respond to various different, locally acting growth factors and growth inhibitors. Among these, the EGF-related peptides play an important role in regulating the proliferation and differentiation of human mammary epithelial cells. EGF⁴ and TGF are able to stimulate the lobulo-alveolar development of the mammary gland in vivo as well they are involved in the pathogenesis of human breast cancer. Experimental evidence suggests that estrogen-induced proliferation of breast carcinoma cells is mediated in part by EGF-related growth factors. It has also been demonstrated that activation of certain cellular protooncogenes such as c-Ha-ras in human mammary epithelial cells results in cellular transformation and in an increased production of several EGF-related growth factors such as TGF and amphiregulin. Coexpression of both EGF-related peptides and their own receptors frequently occurs in human breast carcinomas and in human breast cancer cell lines, suggesting that an autocrine pathway of uncontrolled cell growth sustains neoplastic transformation.     

Cell Polarity in Motion: Redefining Mammary Tissue Organization Through EMT and Cell Polarity Transitions

Epithelial to mesenchymal transition (EMT) and its reversion via mesenchymal to epithelial transition (MET), represent a stepwise cycle of epithelial plasticity that allows for normal tissue remodelling and diversification during development. In particular, epithelial-mesenchymal plasticity is central to many aspects of mammary development and has been proposed to be a key process in breast cancer progression. Such epithelial-mesenchymal plasticity requires complex cellular reprogramming to orchestrate a change in cell shape to an alternate morphology more conducive to migration. During this process, epithelial characteristics, including apical-basal polarity and specialised cell-cell junctions are lost and mesenchymal properties, such as a front-rear polarity associated with weak cell-cell contacts, increased motility, resistance to apoptosis and invasiveness are gained. The ability of epithelial cells to undergo transitions through cell polarity states is a central feature of epithelial-mesenchymal plasticity. These cell polarity states comprise a set of distinct asymmetric distributions of cellular constituents that are fashioned to allow specialized cellular functions, such as the regulated homeostasis of molecules across epithelial barriers, cell migration or cell diversification via asymmetric cell divisions. Each polarity state is engineered using a molecular toolbox that is highly conserved between organisms and cell types which can direct the initiation, establishment and continued maintenance of each asymmetry. Here we discuss how EMT pathways target cell polarity mediators, and how this EMT-dependent change in polarity states impact on the various stages of breast cancer. Emerging evidence places cell polarity at the interface of proliferation and morphology control and as such the changing dynamics within polarity networks play a critical role in normal mammary gland development and breast cancer progression.     

Extracellular Proteolysis in Transgenic Mouse Models of Breast Cancer

Growth and invasion of breast cancer require extracellular proteolysis in order to physically restructure the tissue microenvironment of the mammary gland. This pathological tissue remodeling process depends on a collaboration of epithelial and stromal cells. In fact, the majority of extracellular proteases are provided by stromal cells rather than cancer cells. This distinct expression pattern is seen in human breast cancers and also in transgenic mouse models of breast cancer. The similar expression patterns suggest that transgenic mouse models are ideally suited to study the role of extracellular proteases in cancer progression. Here we give a status report on protease intervention studies in transgenic models. These studies demonstrate that proteases are involved in all stages of breast cancer progression from carcinogenesis to metastasis. Transgenic models are now beginning to provide vital mechanistic insight that will allow us to combat breast cancer invasion and metastasis with new protease-targeted drugs.     

ErbB/EGF Signaling and EMT in Mammary Development and Breast Cancer

Activation of the ErbB family of receptor tyrosine kinases via cognate Epidermal Growth Factor (EGF)-like peptide ligands constitutes a major group of related signaling pathways that control proliferation, survival, angiogenesis and metastasis of breast cancer. In this respect, clinical trials with various ErbB receptor blocking antibodies and specific tyrosine kinase inhibitors have proven to be partially efficacious in the treatment of this heterogeneous disease. Induction of an embryonic program of epithelial-to-mesenchymal transition (EMT) in breast cancer, whereupon epithelial tumor cells convert to a more mesenchymal-like phenotype, facilitates the migration, intravasation, and extravasation of tumor cells during metastasis. Breast cancers which exhibit properties of EMT are highly aggressive and resistant to therapy. Activation of ErbB signaling can regulate EMT-associated invasion and migration in normal and malignant mammary epithelial cells, as well as modulating discrete stages of mammary gland development. The purpose of this review is to summarize current information regarding the role of ErbB signaling in aspects of EMT that influence epithelial cell plasticity during mammary gland development and tumorigenesis. How this information may contribute to the improvement of therapeutic approaches in breast cancer will also be addressed.     

Epithelial-Mesenchymal Transition in Cancer: Parallels Between Normal Development and Tumor Progression

From the earliest stages of embryonic development, cells of epithelial and mesenchymal origin contribute to the structure and function of developing organs. However, these phenotypes are not always permanent, and instead, under the appropriate conditions, epithelial and mesenchymal cells convert between these two phenotypes. These processes, termed Epithelial-Mesenchymal Transition (EMT), or the reverse Mesenchymal-Epithelial Transition (MET), are required for complex body patterning and morphogenesis. In addition, epithelial plasticity and the acquisition of invasive properties without the full commitment to a mesenchymal phenotype are critical in development, particularly during branching morphogenesis in the mammary gland. Recent work in cancer has identified an analogous plasticity of cellular phenotypes whereby epithelial cancer cells acquire mesenchymal features that permit escape from the primary tumor. Because local invasion is thought to be a necessary first step in metastatic dissemination, EMT and epithelial plasticity are hypothesized to contribute to tumor progression. Similarities between developmental and oncogenic EMT have led to the identification of common contributing pathways, suggesting that the reactivation of developmental pathways in breast and other cancers contributes to tumor progression. For example, developmental EMT regulators including Snail/Slug, Twist, Six1, and Cripto, along with developmental signaling pathways including TGF-β and Wnt/β-catenin, are misexpressed in breast cancer and correlate with poor clinical outcomes. This review focuses on the parallels between epithelial plasticity/EMT in the mammary gland and other organs during development, and on a selection of developmental EMT regulators that are misexpressed specifically during breast cancer.     

The Cell of Origin of <Emphasis Type="Italic">BRCA1</Emphasis> Mutation-associated Breast Cancer: A Cautionary Tale of Gene Expression Profiling

Breast tumours are highly heterogeneous with several distinct sub-types recognised according to their histological and molecular features. The biological basis for this heterogeneity is largely unknown, although there are some distinct phenotype–genotype correlations. These include BRCA1 mutation-associated breast cancers, which are typically high grade invasive ductal carcinomas of no special type (IDC-NSTs) with pushing margins that do not express estrogen receptor (ER), progesterone receptor (PR) or the HER2 receptor tyrosine kinase (‘triple negative’). Gene expression analysis of these tumours has grouped them with so called ‘basal-like’ breast cancers and this, together with evidence that knock-down of BRCA1 in vitro blocked luminal differentiation, led to speculation that these tumours arose from the normal basal stem cells within the mammary gland. Recently, however, human breast tissue from BRCA1 mutation carriers was shown to contain an expanded population of luminal progenitor cells which have increased in vitro clonogenic ability. In the mouse, targeted deletion of Brca1 in luminal ER negative progenitors resulted in the formation of mammary tumours which phenocopied human BRCA1 breast tumour pathology, while the deletion of Brca1 in basal stem cells resulted in the formation of tumours which neither resembled human BRCA1 tumours or sporadic basal-like breast tumours. Importantly, however, both sets of mouse tumours were classified as ‘basal-like’ by methods used for human tumour classification based on gene expression profiles. This demonstrates that, as it stands, expression profiling is poor at distinguishing tumour histological subtypes and is also a poor guide to the cell of tumour origin. These human and rodent studies support an origin of BRCA1-mutation associated breast cancer (and indeed of the majority of sporadic basal-like breast cancers) in a luminal ER negative mammary epithelial progenitor. This is a key finding, as identification of the cells of origin in breast cancer subtypes makes possible the identification of key processes associated with initiation, progression and maintenance of each tumour subtype, the development of novel targeted therapies and, potentially, of new preventative approaches in high risk groups.     

Immune Cell Location and Function During Post-Natal Mammary Gland Development

Post-natal mammary gland development requires complex interactions between the epithelial cells and various cell types within the stroma. Recent studies have illustrated the importance of immune cells and their mediators during the various stages of mammary gland development. However, the mechanisms by which these immune cells functionally contribute to mammary gland development are only beginning to be understood. This review provides an overview of the localization of immune cells within the mammary gland during the various stages of post-natal mammary gland development. Furthermore, recent studies are summarized that illustrate the mechanisms by which these cells are recruited to the mammary gland and their functional roles in mammary gland development.     

Myoepithelial Cells in the Control of Mammary Development and Tumorigenesis: Data From Genetically Modified Mice

Until recently, myoepithelial cells—the second major cell population in the mammary epithelium—were not considered to play an important role in the morphogenetic events during gland development. Mouse mutants with changes in the gene expression pattern characteristic of the basal myoepithelial cell layer have been generated and used to show that these cells influence the proliferation, survival and differentiation of luminal cells, modulate stromal–epithelial interactions and actively participate in mammary morphogenesis. Various cellular and molecular mechanisms may underlie the observed phenotypes. These include an unbalanced expression of matrix degrading metalloproteinases (MMPs) and their inhibitors, leading to changes in the composition and organization of the (extracellular matrix) ECM, the production of soluble growth factors affecting stromal and epithelial cell growth and differentiation and direct signaling through cell–cell contacts between the myoepithelial and luminal cell layers.     

Mammary Gland Growth and Development from the Postnatal Period to Postmenopause: Ovarian Steroid Receptor Ontogeny and Regulation in the Mouse

Ovarian steroid hormones play a critical role inregulating mammary gland growth and development. Themammary gland sequentially acquires and cyclicallyexhibits proliferative responses to estrogen and/or progesterone from birth to postmenopause. Thefocus of this review is to presentour currentunderstanding of estrogen and progesterone receptordistribution in epithelial and stromal cells and theirfunctions in relation to mammary gland development.Insights gained from the study of the normal mammarygland are relevant to our understanding of theconditions which may predispose women to the developmentof breast cancer as well as to alterations inhormonal regulation that occur in breastcancer.     

Role of Ets transcription factors in mammary gland development and oncogenesis

PEA3 is the founding member of a subfamily of closely related ets genes that includes ER81 and ERM. PEA3 is expressed in the epithelial cells of mammary buds at the time that these first appear during mouse embryogenesis, and it is differentially expressed during postnatal mammary gland development. PEA3 expression is highest at the onset of puberty and during early pregnancy, times of extensive epithelial outgrowth and branching. PEA3 is expressed in undifferentiated epithelial cap cells of terminal end buds, and in differentiated myoepithelial cells of ducts and alveoli. Loss-of-function mutations in the PEA3 gene compromise mammary ductal branching at the onset of puberty and early during pregnancy. PEA3 is overexpressed in the vast majority of human breast tumors and in nearly all of the HER2-positive subclass of such tumors. PEA3 is similarly overexpressed in transgenic mouse models of this malignancy. Expression of dominant-negative PEA3 in the mouse mammary gland of MMTV-HER2 transgenic mice dramatically delays the onset and reduces the incidence of mammary tumors. Hence PEA3 and/or its close relatives play key regulatory roles in both mammary gland development and oncogenesis.     

The Tension Mounts: Mechanics Meets Morphogenesis and Malignancy

The tissue microenvironment regulates mammary gland development and tissue homeostasis through soluble, insoluble and cellular cues that operate within the three dimensional architecture of the gland. Disruption of these critical cues and loss of tissue architecture characterize breast tumors. The developing and lactating mammary gland are also subject to a plethora of tensional forces that shape the morphology of the gland and orchestrate its functionally differentiated state. Moreover, malignant transformation of the breast is associated with dramatic changes in gland tension that include elevated compression forces, high tensional resistance stresses and increased extracellular matrix stiffness. Chronically increased mammary gland tension may influence tumor growth, perturb tissue morphogenesis, facilitate tumor invasion, and alter tumor survival and treatment responsiveness. Because mammary tissue differentiation is compromised by high mechanical force and transformed cells exhibit altered mechanoresponsiveness, malignant transformation of the breast may be functionally linked to perturbed tensional-homeostasis. Accordingly, it will be important to define the role of tensional force in mammary gland development and tumorigenesis. Additionally, it will be critical to identify the key molecular elements regulating tensional-homeostasis of the mammary gland and thereafter to characterize their associated mechanotransduction pathways.     

Cell Cycle Genes in a Mouse Mammary Hyperplasia Model

Human mammary epithelial cells emerge spontaneously from senescence, exhibiting eroding telomeric sequences, and ultimately enter crisis to generate the type of chromosomal abnormalities seen in early stages of breast cancer. In a mouse mammary tumor model, the spontaneous escape of senescence can be observed as an increase in DNA synthesis that is reflected by alterations in the cell cycle profile and increases in the expression levels and activities of cell cycle molecular components. This review provides an overview of gene alterations in the cell cycle components in mouse mammary hyperplasia.     

Adipose-Depleted Mammary Epithelial Cells and Organoids

Analysis of genes and proteins involved in lipid biosynthesis in mammary epithelial cells (MECs) is complicated by the presence of adipose tissue in the mammary gland, which may be predominant in whole tissue lysates depending upon developmental stage. We have developed a method based on protocols used to establish primary mammary epithelial cell cultures that allows for analysis of MECs depleted of adipose. Adipose depletion yields enriched MECs that are suitable for gene expression profiling and protein analysis from a single mouse. Additionally, the phosphorylation of proteins is maintained, allowing investigation of signal transduction events. Application of this method to the analysis of MECs from genetically modified mice will aid in the identification of factors controlling tissue-specific events in the mammary gland. In contrast to other methods such as laser capture microdissection, the MEC enrichment method described here is performed using standard lab supplies, equipment, and techniques.