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Cellular pathways that contribute to adult human mammary gland architecture and lineages have not been previously described. In this study, we identify a candidate stem cell niche in ducts and zones containing progenitor cells in lobules. Putative stem cells residing in ducts were essentially quiescent, whereas the progenitor cells in the lobules were more likely to be actively dividing. Cells from ducts and lobules collected under the microscope were functionally characterized by colony formation on tissue culture plastic, mammosphere formation in suspension culture, and morphogenesis in laminin-rich extracellular matrix gels. Staining for the lineage markers keratins K14 and K19 further revealed multipotent cells in the stem cell zone and three lineage-restricted cell types outside this zone. Multiparameter cell sorting and functional characterization with reference to anatomical sites in situ confirmed this pattern. The proposal that the four cell types are indeed constituents of an as of yet undescribed stem cell hierarchy was assessed in long-term cultures in which senescence was bypassed. These findings identify an adult human breast ductal stem cell activity and its earliest descendants.

The human breast epithelium is a branching ductal system composed of an inner layer of polarized luminal epithelial cells and an outer layer of myoepithelial cells that terminate in distally located terminal duct lobular units (TDLUs). While the luminal epithelial cell has received the most attention as the functionally active milk-producing cell and as the most likely target cell for carcinogenesis, attention on myoepithelial cells has begun to evolve with the recognition that these cells play an active part in branching morphogenesis and tumor suppression. A major question that has been the subject of investigation pertains to how the luminal epithelial and myoepithelial lineages are related and precisely how they arise from a common putative stem cell population within the breast. Equally important is the question of how heterotypic signaling occurs between luminal epithelial and surrounding myoepithelial cells in normal breast morphogenesis and neoplasia. In this review we discuss data from our laboratories and from others regarding the cellular origin of human myoepithelial cells, their function in maintaining tissue polarity in the normal breast, and their role during neoplasia.

A breast carcinoma biopsy showed cytochemical evidence of epithelial mesenchymal transition and an ␣-smooth muscle actin-positive stromal reaction. To study the lineage, and the nature of the cells in the stromal reaction, we derived a novel cell line, HBFL-1, from the explanted biopsy. HBFL-1 cells are immortal and exhibit a shared non-random X-chromosome inactivation pattern with the epithelial tumor of origin. Yet they closely resemble normal, finite-life-span fibroblasts by morphology, lack of tumor formation in nude mice, marker expression profile, protein pattern using two-dimensional gel electrophoresis and the ability to undergo myofibroblast conversion. HBFL-1 interacts reciprocally with tumor cells in collagen gel to induce activation of MMP2, leading to tumor-like behavior of epithelial colonies. In vivo, HBFL-1 cells resembled normal-derived myofibroblasts and conferred a significant 3.5-to 7-fold increase in MCF-7 tumor size in nude mice. However, that they were indeed not normal fibroblasts was revealed by residual keratin expression and formation of epithelial microfoci in a reconstituted basement membrane and in nude mice. We conclude that breast cancer can generate its own nonmalignant stroma and that one function for this is that of a reciprocal interac- Epithelial mesenchymal transition (EMT) was originally described as a normal developmental process. 1,2 Later, it was adopted as an explanation for mesenchymal conversion in a number of cultured epithelial cells. 3,4 In cancer, EMT generally depicts a more aggressive behavior of the tumor cells. 5,6 In breast cancer, EMT has been estimated to occur in as much as 18% of tumors in vivo. [7][8][9] Under these conditions EMT is defined as the occurrence of a variable proportion of tumor cells that express mesenchymal markers such as vimentin, tenascin and stromelysin-3. 7,10 In its most elaborate form, EMT-derived cells of mixed epithelial-mesenchymal breast tumors may be difficult to distinguish from resident normal stromal cells. 11 These tumors which are also referred to as carcinosarcomas or metaplastic carcinomas in particular offer an excellent opportunity to study the nature and the consequence of this subset of EMT. 5,6 Such studies, however, have been hampered by lack of representative cell lines most likely due to low frequency of overtly metaplastic carcinomas but also to difficulties in culturing breast cancer cells in general. In the present study we succeeded in isolating a mesenchymal-like cell line from a metaplastic human breast carcinoma. Clonality analysis revealed that the cell line and the epithelial tumor cells of origin had a common ancestor. Even though the cells were immortal and severely aneuploid, and exhibited a rudimentary epithelial phenotype in terms of keratin expression and formation of microfoci in Matrigel and in vivo, they nevertheless behaved remarkably like normal resident fibroblasts. In particular they responded to transforming growth factor-␤ (TGF-␤) by having ␣-smooth muscle actin (␣-sm actin) induced and t...

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44high/CD24low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

BackgroundLungs develop from the fetal digestive tract where epithelium invades the vascular rich stroma in a process called branching morphogenesis. In organogenesis, endothelial cells have been shown to be important for morphogenesis and the maintenance of organ structure. The aim of this study was to recapitulate human lung morphogenesis in vitro by establishing a three dimensional (3D) co-culture model where lung epithelial cells were cultured in endothelial-rich stroma.MethodsWe used a human bronchial epithelial cell line (VA10) recently developed in our laboratory. This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human umbilical vein endothelial cells (HUVECs), to mimic the close interaction between these cell types during lung development. Morphogenesis and differentiation was monitored by phase contrast microscopy, immunostainings and confocal imaging.ResultsWe found that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like structures, suggesting that lung epithelial branching is facilitated by the presence of endothelial cells. The VA10 derived epithelial structures display various complex patterns of branching and show partial alveolar type-II differentiation with pro-Surfactant-C expression. The epithelial origin of the branching VA10 colonies was confirmed by immunostaining. These bronchioalveolar-like structures were polarized with respect to integrin expression at the cell-matrix interface. The endothelial-induced branching was mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2) and sprouty-2 were expressed at the growing tips of the branching structures and the branching was inhibited by the FGFR-small molecule inhibitor SU5402.DiscussionIn this study we show that a human lung epithelial cell line can be induced by endothelial cells to form branching bronchioalveolar-like structures in 3-D culture. This novel model of human airway morphogenesis can be used to study critical events in human lung development and suggests a supportive role for the endothelium in promoting branching of airway epithelium.

The prevalence of coronary artery disease (CAD) in patients with peripheral arterial disease (PAD) has been well defined. However, the prevalence of PAD in hospitalized patients with CAD has not been defined. The ankle-brachial index (ABI) is a useful non-invasive tool to screen for PAD. The aim of our study was to assess the prevalence of PAD in hospitalized patients with CAD by measuring the ABI. The study was conducted at the University of Wisconsin Hospital and Clinics inpatient Cardiovascular Medicine Service. Medically stable patients with CAD were invited to participate prior to hospital discharge. Data regarding cardiovascular risk factors, history of previous PAD, physical examination, and ABI were collected. An ABI less than or equal to 0.9 or a history of previous lower extremity vascular invention was considered to be indicative of significant PAD. A total of 100 patients (66 men and 34 women) were recruited. Forty patients were found to have PAD (mean ABI in non-revascularized patients with PAD = 0.67). By measuring the ABI, 37 (25 men) were positive for PAD and three had an ABI corrected with previous revascularization. Of these patients, 21 (52.5%) had previously documented PAD. Patients with PAD were older (p = 0.003), had a greater smoking history (p = 0.002), were more likely to have diabetes (p = 0.012), hypertension (p = 0.013) and a trend towards more dyslipidemia (p = 0.055). In conclusion, hospitalized patients with CAD are likely to have concomitant PAD. Risk factors for PAD in this patient population include advanced age, history of smoking, diabetes, hypertension, dyslipidemia and abnormal pulse examination. Identification of patients with PAD by measuring the ankle-brachial index is easily done.

Tight junctions (TJs) play a key role in maintaining bronchial epithelial integrity, including apical-basolateral polarity and paracellular trafficking. Patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) often suffer from chronic infections by the opportunistic Gram-negative bacterium Pseudomonas aeruginosa, which produces multiple virulence factors, including rhamnolipids. The macrolide antibiotic azithromycin (azm) has been shown to improve lung function in patients with CF without reducing the bacterial count within the lung. However, the mechanism of this effect is still debated. It has previously been shown that azm increased transepithelial electrical resistance (TER) in a bronchial epithelial cell line. In this study we used an air-liquid interface model of human airway epithelia and measured TER, changes in TJ expression and architecture after exposure to live P. aeruginosa PAO1, and PAO1-Deltarhl which is a PAO1 mutant lacking rhlA and rhlB, which encode key enzymes for rhamnolipid production. In addition, the cells were challenged with bacterial culture medium conditioned by these strains, purified rhamnolipids, or synthetic 3O-C(12)-HSL. Virulence factors secreted by P. aeruginosa reduced TER and caused TJ rearrangement in the bronchial epithelium, exposing the epithelium to further bacterial infiltration. Pretreatment of the bronchial epithelium with azm attenuated this effect and facilitated epithelial recovery. These data suggest that azm protects the bronchial epithelium during P. aeruginosa infection independent of antimicrobial activity, and could explain in part the beneficial results seen in clinical trials of patients with CF.