Previously, we characterized a parity-induced mammary epithelial cell population that possessed the properties of pluripotency and self-renewal upon transplantation. These cells were lineally marked by the expression of ␤-galactosidase (LacZ) as a result of mammary-specific activation of a reporter gene through Cre-lox recombination during pregnancy. We used this experimental model to determine whether testicular cells would alter their cell fate upon interaction with the mammary gland microenvironment during pregnancy, lactation, and involution. Adult testicular cells, isolated from seminiferous tubules, were mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads. The host mice were bred 6 -8 weeks later and examined 20 -30 days postinvolution. This approach allowed for the growth of mammary tissue from the injected cells and transient activation of the whey acidic protein promoter-Cre gene during pregnancy and lactation, leading to Cre-lox recombination and constitutive expression of LacZ from its promoter. Here we show that cells from adult seminiferous tubules interact with mammary epithelial cells during regeneration of the gland. They adopt mammary epithelial progenitor cell properties, including self-renewal and the production of cell progeny, which differentiate into functional mammary epithelial cells. Our results provide evidence for the ascendancy of the tissue microenvironment over the intrinsic nature of cells from an alternative adult tissue.transdifferentiation ͉ transplantation ͉ niche ͉ stem cell S omatic stem cells are maintained and regulated by their surrounding microenvironment (niche). A tissue-specific niche is a restricted locale that supports self-renewing division of stem cells and prevents them from differentiating. A simple stem cell niche has three components: localized signaling cells and extracellular matrix-controlling stem cell behavior, a specified range of signaling, and stem cell(s) (1). Any portion of the mouse mammary gland can regenerate an entire functional gland upon transplantation into a cleared mammary fat pad (2-4). This capacity remains undiminished regardless of the donor's age or reproductive history (5). Therefore, mammary stem cells are stably maintained within specific microenvironments throughout the gland for life. Mammary regeneration also occurs when dispersed epithelial cells from the glands are transplanted, suggesting that complete mammary epithelial stem cell niches may be reconstituted de novo (6-8). Stepwise (limiting) dilution of dispersed mammary cells results in a reduction of the percentage of inoculated fat pads positive for mammary epithelial growth, implying reduction in the number of mammary epithelial stem cells (7, 9). We hypothesized that the remaining cells comprised the epithelial signaling components and might support glandular regeneration if supplied with an extraneous source of stem/progenitor cells when injected into the mammary stroma. To test this hypothesis, we iso...

transplantation ͉ niche ͉ plasticity ͉ trans-differentiation ͉ regeneration S omatic stem cells are defined by how they function physiologically in the presence of heterologous cells, that is, the microenvironment or stem cell niche that balances protecting stem cells from exhaustion and protecting the host from unregulated stem cell growth. The dominance of the niche over the stem cell's autonomous phenotype has been demonstrated in several reports involving cells crossing lineage ''boundaries'' to regenerate ''foreign'' tissues. We previously demonstrated that cells isolated from the seminiferous tubules of the mature testis, when mixed together with normal mammary epithelial cells (MECs), would cooperate with these cells and contribute robust numbers of epithelial progeny to normally growing mammary glands in transplanted mammary fat pads (1). In these experiments, the testicular cells included Ϸ10% germinal stem cells (spermatogonia ␣ and ␤), Ϸ20% Sertoli cells, and the remainder primary and secondary spermatocytes and other cells at various stages of spermatogenic cell differentiation. Interstitial and vascular tissue was separated from the seminiferous tubules before dissociation. In these experiments, we were unable to distinguish whether all or just a subpopulation of the isolated seminiferous cells contributed progeny to the mammary epithelial outgrowths.In the present study, to more directly demonstrate the activity of multipotent cells (i.e., cells with the capacity to differentiate into multiple cell types) in this experimental model, we opted to begin with carefully characterized and stable multipotent stem cell populations that could be maintained indefinitely in vitro and could differentiate into multiple cell types in culture after the addition of serum factors and the removal of culture components responsible for stem cell maintenance. Toward this end, neural stem cells (NSCs) from fetal and adult WAP-Cre/Rosa26R mice were isolated and propagated in serum-free medium. Subsequently, these NSCs were mixed with equal numbers of MECs and inoculated immediately into epithelium-divested mammary fat pads of prepubertal female mice. Both fetal and adult NSCs were shown to interact with MECs on transplantation and to robustly contribute mammary epithelial-specific progeny to normal mammary outgrowths.

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.

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.

IntroductionStem cells of somatic tissues are hypothesized to protect themselves from mutation and cancer risk through a process of selective segregation of their template DNA strands during asymmetric division. Mouse mammary epithelium contains label-retaining epithelial cells that divide asymmetrically and retain their template DNA.MethodImmunohistochemistry was used in murine mammary glands that had been labeled with [3H]thymidine during allometric growth to investigate the co-expression of DNA label retention and estrogen receptor (ER)-α or progesterone receptor (PR). Using the same methods, we investigated the co-localization of [3H]thymidine and ER-α or PR in mammary tissue from mice that had received treatment with estrogen, progesterone, and prolactin subsequent to a long chase period to identify label-retaining cells.ResultsLabel-retaining epithelial cells (LRECs) comprised approximately 2.0% of the entire mammary epithelium. ER-α-positive and PR-positive cells represented about 30–40% of the LREC subpopulation. Administration of estrogen, progesterone, and prolactin altered the percentage of LRECs expressing ER-α.ConclusionThe results presented here support the premise that there is a subpopulation of LRECs in the murine mammary gland that is positive for ER-α and/or PR. This suggests that certain mammary LRECs (potentially stem cells) remain stably positive for these receptors, raising the possibility that LRECs comprise a hierarchy of asymmetrically cycling mammary stem/progenitor cells that are distinguished by the presence or absence of nuclear steroid receptor expression.

Remodeling of the airways, as occurs in asthmatic patients, is associated with the continual presence of inflammatory mediators and Th2 cytokines, especially interleukin (IL)-13, during cycles of epithelial injury and repair. In this study, we examined the effect of IL-13 on well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture. IL-13 induced proliferation of NHBE cells after 24 h exposure, as reflected by [(3)H]thymidine uptake and cell counts. The effects of IL-13 were mediated through the epidermal growth factor receptor (EGFR), as proliferation was attenuated by AG1478, an EGFR tyrosine kinase inhibitor. Proliferation appeared to be mediated by transforming growth factor (TGF)-alpha, a potent ligand for EGFR, which was released rapidly from NHBE cells in response to IL-13. Neutralizing antibody to TGF-alpha, but not antibodies against other potentially important growth factors (EGF, heparin binding epidermal growth factor-like growth factor [HB-EGF], platelet-derived growth factor [PDGF]), inhibited the mitogenic response to IL-13. This study provides the first experimental evidence that IL-13 can initiate a proliferative response of human airway epithelium in the absence of inflammatory cells or other cell types. The results are consistent with a mechanism whereby IL-13 induces release of TGF-alpha from the epithelial cells, which in turn binds via an autocrine/paracrine-type action to the EGFR, initiating proliferation. IL-13-induced airway remodeling in vivo may involve this epithelium-driven response.

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.

Research efforts investigating the potential of natural compounds in the fight against cancer are growing. Tannic acid (TA) belongs to the class of hydrolysable tannins and is found in numerous plants and foods. TA is a potent collagen cross-linking agent; the purpose of this study was to generate TA-cross-linked beads and assess the effects on breast cancer cell growth. Collagen beads were stable at body temperature following crosslinking. Exposure to collagen beads with higher levels of TA inhibited proliferation and induced apoptosis in normal and cancer cells. TA-induced apoptosis involved activation of caspase 3/7 and caspase 9 but not caspase 8. Breast cancer cells expressing the estrogen receptor were more susceptible to the effects of TA. Taken together the results suggest that TA has the potential to become an anti-ER+ breast cancer treatment or preventative agent.