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Introduction

Extensive mammographic density in women is associated with increased risk for breast cancer. Mouse models provide a powerful approach to the study of human diseases, but there is currently no model that is suited to the study of mammographic density.

Methods

We performed individual manipulations of the stromal, epithelial and matrix components of the mouse mammary gland and examined the alterations using in vivo and ex vivo radiology, whole mount staining and histology.

Results

Areas of density were generated that resembled densities in mammographic images of the human breast, and the nature of the imposed changes was confirmed at the cellular level. Furthermore, two genetic models, one deficient in epithelial structure (Pten conditional tissue specific knockout) and one with hyperplastic epithelium and mammary tumors (MMTV-PyMT), were used to examine radiographic density.

Conclusion

Our data show the feasibility of altering and imaging mouse mammary gland radiographic density by experimental and genetic means, providing the first step toward modelling the biological processes that are responsible for mammographic density in the mouse.

Background

Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue.

Methods

We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging.

Results

In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice.

Conclusion

In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary.

We investigated mammary gland differentiation and cell proliferation in rats after acute exposure to xenoestrogens. Pubertal female Sprague-Dawley rats (six/group) were treated for 1 week with diethylstilbestrol (DES), genistein, o,p'-DDT, Aroclor 1221, Aroclor 1254, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or the vehicle, sesame oil. Animals were killed 18 hr after the last treatment. Analysis of mammary whole-mounts revealed that exposure to DES, genistein, and o,p'-DDT resulted in enhanced gland differentiation and increased epithelial cell proliferation as measured by proliferating cell nuclear antigen immunohistochemistry, TCDD treatment inhibited cell proliferation and gland development. Aroclor 1221 and Aroclor 1254 treatments had slight but not statistically significant effects on cell proliferation and mammary gland development. We conclude that DES, genistein, and o,p'-DDT given to pubertal rats act as morphogens; i.e., they increase cell proliferation, which promotes maturation of the undifferentiated terminal end buds to more differentiated lobular terminal ductal structures.

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Introduction

Keratin 6 (K6) has previously been identified as a marker of early mammary gland development and has also been proposed to be a marker of mammary gland progenitor cells. However, the function of K6 in the mammary gland was not known, so we examined the expression pattern of the protein during both embryonic and postnatal mammary development, as well as the mammary gland phenotype of mice that were null for both K6a and K6b isoforms.

Method

Immunostaining was performed to determine the expression pattern of K6a throughout mammary gland development, from the embryonic mammary bud to lactation. Double immunofluorescence was used to co-localize K6 with known markers of mammary gland development. Wild-type and K6ab-null mammary tissues were transplanted into the cleared fat pads of nude mice and the outgrowths were analyzed for morphology by whole-mount staining and for markers of mammary epithelium by immunostaining. Finally, progesterone receptor (PR) and bromodeoxyuridine co-localization was quantified by double immunofluorescence in wild-type and K6ab-null mammary outgrowths.

Results

Here we report that K6 is expressed earlier than described previously, by embryonic day 16.5. K6a is the predominant isoform expressed in the mammary gland, localized in the body cells and luminal epithelial cells but not in the cap cells or myoepithelial cells. Co-localization studies showed that most K6a-positive cells express steroid receptors but do not proliferate. When both the K6a and K6b genes are deleted, mammary gland development appears normal, with similar expression of most molecular markers examined in both the pubertal gland and the mature gland. Loss of K6a and K6b, however, leads to an increase in the number of steroid-receptor-positive cells, and increased co-localization of steroid receptor expression and proliferation was observed.

Conclusion

Although K6a was not essential for mammary gland development, loss of both K6a and K6b resulted in an increase in PR-positive mammary epithelial cells and decreased proliferation after exposure to steroid hormones. There was also increased co-localization of PR and bromodeoxyuridine, suggesting alterations in patterning events important for normal lobuloalveolar development.

Preclinical models that accurately reproduce specific aspects of human disease etiology are invaluable for the initial development and evaluation of chemopreventive agents. We developed a novel, short-term prevention model, which is particularly useful for assessing a compound’s efficacy to prevent hormonally responsive and non-responsive in situ carcinomas. In this model, carcinogenesis is induced by a high titer of neu-containing, replication-defective retrovirus. The multiplicity and size of the resulting in situ carcinomas are scored in whole-mounted, aluminum carmine-stained mammary glands at 15 days post-infusion.

These in situ carcinomas represent a distinct biological time-point in the development of neu-induced mammary cancer in the rat. They are characterized by high rates of proliferation (40.0%, p<0.0001) and apoptosis (2.8%, p<0.005), compared to mammary carcinomas. The majority of in situ carcinomas regress spontaneously after 20 days post-infusion.

The in situ carcinomas at 15 days post-infusion exhibit hormonal responsiveness. The effects of the chemoprevention agents tamoxifen, celecoxib and targretin on hormonally responsive and non-responsive in situ carcinomas recapitulate those observed on mammary carcinomas at 12 and 18 weeks post-infusion for intact and ovariectomized rats respectively.

Neu-induced in situ carcinomas in the rat represent etiologically relevant intermediate time points of mammary carcinogenesis. Our prevention model represents a cost-efficient in vivo system to determine whether a compound’s preventive effects extend to hormonally non-responsive mammary lesions, for which new chemoprevention approaches are needed.

Nonimmortalized mouse mammary epithelial cells expressing Escherichia coli beta-galactosidase from a murine amphotropic packaged retroviral vector were injected into the epithelium-divested mammary fat pads of syngeneic mice. Mammary glands formed from the injected mammary epithelial cells contained ductal and lobular cells, both of which expressed beta-galactosidase when examined in situ more than 12 months later. These results indicate that stable recombinant gene expression can be achieved in vivo in the mammary gland without altering the growth properties of normal mammary epithelium.

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Peptidylarginine Deiminases (PADs) convert arginine residues on substrate proteins to citrulline. Previous reports have documented that PAD2 expression and activity varies across the estrous cycle in the rodent uterus and pituitary gland, however, the expression and function of PAD2 in mammary tissue has not been previously reported. To gain more insight into potential reproductive roles for PAD2, in this study we evaluated PAD2 expression and localization throughout the estrous cycle in canine mammary tissue and then identified possible PAD2 enzymatic targets. Immunohistochemical and immunofluorescence analysis found PAD2 expression is low in anestrus, limited to a distinct, yet sparse, subset of epithelial cells within ductal alveoli during estrus/early diestrus, and encompasses the entire epithelium of the mammary duct in late diestrus. At the subcellular level, PAD2 is expressed in the cytoplasm, and to a lesser extent, the nucleus of these epithelial cells. Surprisingly, stimulation of canine mammary tumor cells (CMT25) shows that EGF, but not estrogen or progesterone, upregulates PAD2 transcription and translation suggesting EGF regulation of PAD2 and possibly citrullination in vivo. To identify potential PAD2 targets, anti-pan citrulline western blots were performed and results showed that citrullination activity is limited to diestrus with histones appearing to represent major enzymatic targets. Use of site-specific anti-citrullinated histone antibodies found that the N-terminus of histone H3, but not H4, appears to be the primary target of PAD activity in mammary epithelium. This observation supports the hypothesis that PAD2 may play a regulatory role in the expression of lactation related genes via histone citrullination during diestrus.

Introduction

Retinoic acid signaling pathways are disabled in human breast cancer suggesting a controlling role in normal mammary growth that might be lost in tumorigenesis. We tested a single receptor isotype, RARα1 (retinoic acid receptor isotype alpha, isoform 1), for its role in mouse mammary gland morphogenesis and mouse mammary tumor virus (MMTV)-wingless-related MMTV integration site 1 (wnt1)-induced oncogenesis.

Methods

The role of RARα1 in mammary morphogenesis was tested in RARα1-knockout (KO) mice and in mammary tumorigenesis in bi-genic (RARα1/KO crossed with MMTV-wnt1) mice. We used whole mounts analysis, stem cells/progenitor quantification, mammary gland repopulation, quantitative polymerase chain reaction (Q-PCR), test of tumor-free survival, tumor fragments and cell transplantation.

Results

In two genetic backgrounds (129/Bl-6 and FVB) the neo-natal RARα1/KO-mammary epithelial tree was two-fold larger and the pubertal tree had two-fold more branch points and five-fold more mature end buds, a phenotype that was predominantly epithelial cell autonomous. The stem/progenitor compartment of the RARα1/KO mammary, defined as CD24low/ALDHhigh activity was increased by a median 1.7-fold, but the mammary stem cell (MaSC)-containing compartment, (CD24low/CD29high), was larger (approximately 1.5-fold) in the wild type (wt)-glands, and the mammary repopulating ability of the wt-gland epithelium was approximately two-fold greater. In MMTV-wnt1 transgenic glands the progenitor (CD24low/ALDHhigh activity) content was 2.6-fold greater than in the wt and was further increased in the RARα1/KO-wnt1 glands. The tumor-free survival of RARα1/KO-wnt1 mice was significantly (P = 0.0002, Kaplan Meier) longer, the in vivo growth of RARα1/KO-wnt1 transplanted tumor fragments was significantly (P = 0.01) slower and RARα1/KO-wnt1 tumors cell suspension produced tumors after much longer latency.

Conclusions

In vitamin A-replete mice, RARα1 is required to maintain normal mammary morphogenesis, but paradoxically, also efficient tumorigenesis. While its loss increases the density of the mammary epithelial tree and the content of luminal mammary progenitors, it appears to reduce the size of the MaSC-containing compartment, the mammary repopulating activity, and to delay significantly the MMTV-wnt1-mammary tumorigenesis. Whether the delay in tumorigenesis is solely due to a reduction in wnt1 target cells or due to additional mechanisms remains to be determined. These results reveal the intricate nature of the retinoid signaling pathways in mammary development and carcinogenesis and suggest that a better understanding will be needed before retinoids can join the armament of effective anti-breast cancer therapies.

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

Introduction

The experiments reported here address the question of whether a short-term hormone treatment can prevent mammary tumorigenesis in two different genetically engineered mouse models.

Methods

Two mouse models, the p53-null mammary epithelial transplant and the c-neu mouse, were exposed to estrogen and progesterone for 2 and 3 weeks, respectively, and followed for development of mammary tumors.

Results

In the p53-null mammary transplant model, a 2-week exposure to estrogen and progesterone during the immediate post-pubertal stage (2 to 4 weeks after transplantation) of mammary development decreased mammary tumorigenesis by 70 to 88%. At 45 weeks after transplantation, analysis of whole mounts of the mammary outgrowths demonstrated the presence of premalignant hyperplasias in both control and hormone-treated glands, indicating that the hormone treatment strongly affects the rate of premalignant progression. One possible mechanism for the decrease in mammary tumorigenesis may be an altered proliferation activity as the bromodeoxyuridine labeling index was decreased by 85% in the mammary glands of hormone-treated mice. The same short-term exposure administered to mature mice at a time of premalignant development also decreased mammary tumorigenesis by 60%. A role for stroma and/or systemic mediated changes induced by the short-term hormone (estrogen/progesterone) treatment was demonstrated by an experiment in which the p53-null mammary epithelial cells were transplanted into the cleared mammary fat pads of previously treated mice. In such mice, the tumor-producing capabilities of the mammary cells were also decreased by 60% compared with the same cells transplanted into unexposed mice. In the second set of experiments using the activated Her-2/neu transgenic mouse model, short-term estradiol or estradiol plus progesterone treatment decreased mammary tumor incidence by 67% and 63%, and tumor multiplicity by 91% and 88%, respectively. The growth rate of tumors arising in the hormone-treated activated Her-2/neu mice was significantly lower than tumors arising in non-hormone treated mice.

Conclusion

Because these experiments were performed in model systems that mimic many essential elements of human breast cancer, the results strengthen the rationale for translating this prevention strategy to humans at high risk for developing breast cancer.

Whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionally-localized phenotypes that would be difficult to detect or analyze using other techniques. We present three newly-developed methods for preparing whole mounts of mammary glands from genetically-engineered mice expressing fluorescent proteins, as well as using either neutral red or a variety of fluorescent dyes. Unlike traditional hematoxylin- or carmine-stained preparations, neutral red-stained, and some fluorescent preparations, can be used for several common downstream analyses.

Introduction

Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland in a macrophage-depleted fat pad.

Methods

Two different in vivo models were used to deplete macrophages from the mouse mammary fat pad, allowing us to examine the effect of macrophage deficiency on the mammary repopulating activity of MaSCs. Both the Csf1op/op mice and clodronate liposome-mediated ablation models entailed transplantation studies using the MaSC-enriched population.

Results

We show that mammary repopulating ability is severely compromised when the wild-type MaSC-enriched subpopulation is transplanted into Csf1op/op fat pads. In reciprocal experiments, the MaSC-enriched subpopulation from Csf1op/op glands had reduced regenerative capacity in a wild-type environment. Utilizing an alternative strategy for selective depletion of macrophages from the mammary gland, we demonstrate that co-implantation of the MaSC-enriched subpopulation with clodronate-liposomes leads to a marked decrease in repopulating frequency and outgrowth potential.

Conclusions

Our data reveal a key role for mammary gland macrophages in supporting stem/progenitor cell function and suggest that MaSCs require macrophage-derived factors to be fully functional. Macrophages may therefore constitute part of the mammary stem cell niche.

We generated a transgenic (Tg)-mouse model expressing a dominant negative-(DN)-RARα, (RARαG303E) under adipocytes-specific promoter to explore the paracrine role of adipocyte retinoic acid receptors (RARs) in mammary morphogenesis. Transgenic adipocytes had reduced level of RARα, β and γ, which coincided with a severely underdeveloped pubertal and mature ductal tree with profoundly decreased epithelial cell proliferation. Transplantation experiments of mammary epithelium and of whole mammary glands implicated a fat-pad dependent paracrine mechanism in the stunted phenotype of the epithelial-ductal tree. Co-cultures of primary adipocytes, or in vitro differentiated adipocyte cell line, with mammary epithelium showed that when activated, adipocyte RARs contribute to generation of secreted proliferative and pro-migratory factors. Gene expression microarrays revealed a large number of genes regulated by adipocyte-RARs. Among them, pleiotrophin (PTN) was identified as the paracrine effectors of epithelial cell migration. Its expression was found to be strongly inhibited by DN-RARα, an inhibition relieved by pharmacological doses of all-trans retinoic acid (atRA) in culture and in vivo. Moreover, adipocyte-PTHR, another atRA responsive gene, was found to be an up-stream regulator of PTN. Overall, these results support the existence of a novel paracrine loop controlled by adipocyte-RAR that regulates the mammary ductal tree morphogenesis.

Over nearly half a century, transplantation methods have been employed to regenerate the mammary gland in vivo. Recent highly cited reports claim to have demonstrated the regeneration of an entire functional mammary gland from a single mammary epithelial cell. Nevertheless, re-examination of the literature on the transplantation biology of mammary gland regeneration reveals that a complex, combinatorial interaction between variously differentiated mammary epithelial cells and the mammary fat pad stroma is indispensable to this process. In the present article, these issues are reviewed and discussed to provide a greater understanding of the complexity of these multiplex interactions.

During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the capacities to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG−/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.

The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When mouse mammary tumor virus (MMTV)-neu-induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium- free mammary fat pads, they were redirected to noncarcinogenic cell fates by interaction with untransformed MECs during regenerative growth. In the presence of nontransformed MECs (50:1), tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu- transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells show the capacity to differentiate into normal mammary cell types, including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals, combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth.

The mammary gland is unique in its requirement to develop in close association with a depot of adipose tissue that is commonly referred to as the mammary fat pad. As discussed throughout this issue, the mammary fat pad represents a complex stromal microenvironment that includes a variety of cell types. In this article we focus on adipocytes as local regulators of epithelial cell growth and their function during lactation. Several important considerations arise from such a discussion. There is a clear and close interrelationship between different stromal tissue types within the mammary fat pad and its adipocytes. Furthermore, these relationships are both stage- and species-dependent, although many questions remain unanswered regarding their roles in these different states. Several lines of evidence also suggest that adipocytes within the mammary fat pad may function differently from those in other fat depots. Finally, past and future technologies present a variety of opportunities to model these complexities in order to more precisely delineate the many potential functions of adipocytes within the mammary glands. A thorough understanding of the role for this cell type in the mammary glands could present numerous opportunities to modify both breast cancer risk and lactation performance.

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent, i.e., may be generated from a single mammary epithelial stem cell. Early studies showed that any portion of an intact murine mammary gland containing epithelium could recapitulate an entire mammary epithelial tree on transplantation into an epithelium-free mammary fat pad. More recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium and that their behavior and maintenance is dependent on signals generated both locally and systemically. In this review, we have attempted to develop the scientific saga surrounding the discovery and characterization of the murine mammary stem/progenitor cell hierarchy and to suggest further approaches that will enhance our knowledge and understanding of these cells and their role in both normal development and neoplasia.

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent, i.e., may be generated from a single mammary epithelial stem cell. Early studies showed that any portion of an intact murine mammary gland containing epithelium could recapitulate an entire mammary epithelial tree on transplantation into an epithelium-free mammary fat pad. More recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium and that their behavior and maintenance is dependent on signals generated both locally and systemically. In this review, we have attempted to develop the scientific saga surrounding the discovery and characterization of the murine mammary stem/progenitor cell hierarchy and to suggest further approaches that will enhance our knowledge and understanding of these cells and their role in both normal development and neoplasia.

A single mammary epithelial stem cell is capable of generating an entire mammary gland. In vivo, systemic estrogen and locally generated paracrine factors such as amphiregulin regulate stem cell cycling and differentiation.

Treatment with aryl hydrocarbon receptor (AhR) agonists can slow or reverse the growth of primary mammary tumors in rodents, which has fostered interest in developing selective AhR modulators for treatment of breast cancer. However, the major goal of breast cancer therapy is to inhibit metastasis, the primary cause of mortality in women with this disease. Studies conducted using breast cancer cell lines have demonstrated that AhR agonists suppress proliferation, invasiveness, and colony formation in vitro; however, further exploration using in vivo models of metastasis is warranted. To test the effect of AhR activation on metastasis, 4T1.2 mammary tumor cells were injected into the mammary gland fat pad of syngeneic Balb/c mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Primary tumor growth was monitored for 4 weeks, at which time metastasis was determined. TCDD treatment suppressed metastasis by approximately 50%, as measured both in the lung and in mammary glands at sites distant from the primary tumor. Primary tumor growth was not suppressed by TCDD exposure nor was proliferation of 4T1.2 cells affected by TCDD treatment in vitro. Taken together, these results suggest that the protective effect of AhR activation was selective for the metastatic process and not simply the result of a direct decrease in tumor cell proliferation or survival at the primary site. These observations in immunologically intact animals warrant further investigation into the mechanism of the protective effects of AhR activation and support the promise for use of AhR modulators to treat breast cancer.

Conjugated linoleic acid (CLA) is a class of commercially available fatty acids that have been associated with anticancer properties in rodent models of chemical carcinogenesis. We conducted a pilot study to examine the antitumor effect of dietary CLA in a polyoma virus-middle T antigen (PyMT) mouse model of invasive breast cancer. Virgin 4-week-old PyMT mice were administered a mixed-isomer CLA diet (1% wt/wt) or control AIN-93G diet for 4 weeks (N = 6 and 5, respectively) and tumor burden was assessed at 8 weeks of age. Thoracic mammary glands were prepared as whole mounts with other glands being formalin fixed and paraffin embedded for histology and immunohistochemistry (IHC). Total RNA was prepared for microarray and real-time reverse transcription–polymerase chain reaction analysis. Western blots were performed for protein expression analysis. Tumor incidence was significantly increased in CLA-treated animals compared with controls (P = 0.009) and occurred with extensive lobular–alveolar expansion and loss of mammary adipose tissue. More than 100 genes were downregulated ≥2-fold in the CLA-treated group compared with controls, including adipose-specific markers, as wells as cytoskeletal and adhesion-related genes. This was supported by dramatic decreases in the epithelial adherens E-cadherin and β-catenin as demonstrated by IHC. Taken together, these results suggest that dietary CLA affects the mammary stromal environment, leading to tumor progression and cellular expansion in the PyMT mouse model. Further studies of the potential for cancer promotion are needed, especially because mixed-isomer CLA formulations are sold commercially as a nutritional supplement.

Introduction

The receptor ErbB3/HER3 is often over-expressed in human breast cancers, frequently in conjunction with over-expression of the proto-oncogene ERBB2/HER2/NEU. Although the prognostic/predictive value of ErbB3 expression in breast cancer is unclear, ErbB3 is known to contribute to therapeutic resistance. Understanding ErbB3 functions in the normal mammary gland will help to explain its role in cancer etiology and as a modulator of signaling responses to the mammary oncogene ERBB2.

Methods

To investigate the roles of ErbB3 in mouse mammary gland development, we transplanted mammary buds from ErbB3-/- embryos into the cleared mammary fat pads of wild-type immunocompromised mice. Effects on ductal outgrowth were analyzed at 4 weeks, 7 weeks and 20 weeks after transplantation for total ductal outgrowth, branch density, and number and area of terminal end buds. Sections of glands containing terminal end buds were analyzed for number and epithelial area of terminal end buds. Terminal end buds were also analyzed for presence of mitotic figures, apoptotic figures, BrdU incorporation, and expression of E-cadherin, P-cadherin, α-smooth muscle actin, and cleaved caspase-3.

Results

The mammary ductal trees developed from ErbB3-/- buds only partly filled the mammary fat pad. In contrast to similar experiments with ErbB2-/- mammary buds, this phenotype was maintained through adulthood, pregnancy, and parturition. In addition, and in contrast to similar work with ErbB4-/- mammary buds, lobuloalveolar development of ErbB3-/- transplanted glands was normal. The ErbB3-/- mammary outgrowth defect was associated with a decrease in the size of the terminal end buds, and with increases in branch density, in the number of terminal end buds, and in the number of luminal spaces. Proliferation rates were not affected by the lack of ErbB3, but there was an increase in apoptosis in ErbB3-/- terminal end buds.

Conclusions

Endogenous ErbB3 regulates morphogenesis of mammary epithelium.

Eph receptor tyrosine kinases, including EphA2, are expressed in the mammary gland. However, their role in mammary gland development remains poorly understood. Using EphA2-deficient animals, we demonstrate for the first time that EphA2 receptor function is required for mammary epithelial growth and branching morphogenesis. Loss of EphA2 decreased penetration of mammary epithelium into fat pad, reduced epithelial proliferation, and inhibited epithelial branching. These defects appear to be intrinsic to loss of EphA2 in epithelium, as transplantation of EphA2-deficient mammary tissue into wild-type recipient stroma recapitulated these defects. In addition, HGF-induced mammary epithelial branching morphogenesis was significantly reduced in EphA2-deficient cells relative to wild-type cells, which correlated with elevated basal RhoA activity. Moreover, inhibition of ROCK kinase activity in EphA2-deficient mammary epithelium rescued branching defects in primary three-dimensional cultures. These results suggest that EphA2 receptor acts as a positive regulator in mammary gland development, functioning downstream of HGF to regulate branching through inhibition of RhoA. Together, these data demonstrate a positive role for EphA2 during normal mammary epithelial proliferation and branching morphogenesis.

The tissue microenvironment directs stem/progenitor cell behavior. Cancer cells are also influenced by the microenvironment. It has been shown that, when placed into blastocysts, cancer cells respond to embryonic cues and differentiate according to the tissue type encountered during ontological development. Previously, we showed that the mouse mammary gland was capable of redirecting adult mouse testicular and neural stem/progenitor cells toward a mammary epithelial cell fate during gland regeneration. Here, we report that human embryonal carcinoma cells proliferate and produce differentiated mammary epithelial cell progeny when mixed with mouse mammary epithelial cells and inoculated into the epithelium-free mammary fat pads of athymic nude mice. Fluorescence in situ hybridization confirmed the presence of human cell progeny in the mammary outgrowths for human centromeric DNA, as well as immunochemistry for human-specific breast epithelial cytokeratins and human-specific milk proteins in impregnated transplant hosts. It was found that the number of human cells increased by 66- to 660-fold during mammary epithelial growth and expansion as determined by human cytokeratin expression. All features found in primary outgrowths were recapitulated in the secondary outgrowths from chimeric implants. These results show that human embryonal carcinoma–derived progeny interact with mouse mammary cells during mammary gland regeneration and are directed to differentiate into cells that exhibit diverse mammary epithelial cell phenotypes. This is the first demonstration that human cells are capable of recognizing the signals generated by the mouse mammary gland microenvironment present during gland regeneration in vivo.