Thorarinn Gudjonsson

Evidence for a stem cell hierarchy in the adult human breast

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.

Myoepithelial Cells: Their Origin and Function in Breast Morphogenesis and Neoplasia

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.

The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neoplastic breast epithelial cells may be substantially more plastic in their differentiation repertoire than previously anticipated. Thus, along with an increasing availability of markers for the myoepithelial lineage, at least a partial differentiation towards this lineage is being revealed frequently. It has also become clear that conversions towards the mesenchymal lineage actually occur, referred to as epithelial to mesenchymal transitions. Indeed, some of the so-called myofibroblasts surrounding the tumor may have an epithelial origin rather than a mesenchymal origin. Because myoepithelial cells, epithelial to mesenchymal transition-derived cells, genuine stromal cells and myofibroblasts share common markers, we now need to define a more ambitious set of markers to distinguish these cell types in the microenvironment of the tumors. This is necessary because the different microenvironments may confer different clinical outcomes. The aim of this commentary is to describe some of the inherent complexities in defining cellular phenotypes in the microenvironment of breast cancer and to expand wherever possible on the implications for tumor suppression and progression.

To create the correct microenvironment: three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia.

The normal human breast comprises an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells separated from the connective tissue stroma by an intact basement membrane. In breast cancer, tumor cells are in direct contact with the surrounding highly activated collagenous stroma, with little or no discernible myoepithelial fence from the original double-layered structure. To understand the evolution of these two scenarios, we took advantage of a three-dimensional hydrated collagen gel approach. The contribution of myoepithelial cells to normal morphogenesis was studied by ablation and rescue experiments, and genes regulated on tumor cell-fibroblast interaction were identified in a tumor environment assay. In normal breast morphogenesis, the ability to correctly polarize sialomucin to the luminal membrane of emerging acini was used as a criterion for apical polarity and functional differentiation. In the assay of breast neoplasia, the consequence of reciprocal tumor cell-fibroblast interaction was addressed morphologically as well as by a differential display approach. Normal breast epithelial cells were purified immunomagnetically and an established cell line, MCF-7, was used as a surrogate tumor cell. With regard to the importance of myoepithelial cells in normal breast epithelial morphogenesis, the collagen gel assay elucidated the following subtleties: In contrast to culturing in basement membrane gels, luminal epithelial cells when cultured alone made structures that were all inversely polarized. This aberrant polarity could be rescued by co-culture with myoepithelial cells. The molecular activity of myoepithelial cells responsible for correct morphogenesis was narrowed down to the laminin-1 component of the basement membrane. As for the consequence of interaction of tumor cells with connective tissue fibroblasts, the assay allowed us to identify a hitherto undescribed gene referred to as EPSTI1. The relevance of the assay-based identification of regulated genes was confirmed in a series of breast carcinomas in which EPSTI1 was highly upregulated compared with normal breast. Few if any of these observations would have been possible on two-dimensional tissue culture plastic.

Novel Effects of Azithromycin on Tight Junction Proteins in Human Airway Epithelia

ABSTRACT The macrolide antibiotic azithromycin improves lung function and prognosis among patients with cystic fibrosis or diffuse panbronchiolitis, independently of bacterial eradication. Anti-inflammatory effects have been implicated, but data from in vivo studies are scarce, and the link between abnormal electrolyte content in airway surface liquid and bronchial infections remains uncertain. In the present study, we treated human airway epithelia on filter supports with azithromycin and monitored transepithelial electrical resistance. We found that azithromycin increased transepithelial electrical resistance of airway epithelia in a dose-dependent manner. Immunocytochemistry and Western blotting showed that addition of azithromycin changed the locations of proteins in cell cultures and induced processing of the tight junction proteins claudin-1 and claudin-4, occludin, and junctional adhesion molecule-A. These effects were reversible, and no effect was seen when cells were treated with penicillin or erythromycin. The data indicate that azithromycin increases the transepithelial electrical resistance of human airway epithelia by changing the processing of tight junction proteins. The results are novel and may help explain the beneficial effects of azithromycin in patients with cystic fibrosis, diffuse panbronchiolitis, and community-acquired pneumonia.

Airway branching morphogenesis in three dimensional culture

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.

Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia

Abstract.  The majority of human breast carcinomas exhibit luminal characteristics and as such, are most probably derived from progenitor cells within the luminal epithelial compartment. This has been subdivided recently into at least three luminal subtypes based on gene expression patterns. The value of knowing the cellular origin of individual tumours is clear and should aid in designing effective therapies. To do this, however, we need strategies aimed at defining the nature of stem and progenitor cell populations in the normal breast. In this review, we will discuss our technical approach for delineating the origin of the epithelial cell types. A major step forward was the purification of each cell type by the application of immunomagnetic cell sorting based on expression of lineage‐specific surface antigens. We then developed chemically defined media that could support either the luminal epithelial or the myoepithelial cell phenotype in primary cultures. Having succeeded in continuous propagation presumably without loss of markers, we could show that a subset of the luminal epithelial cells could convert to myoepithelial cells, signifying the possible existence of a progenitor cell population. By combining the information on marker expression and in situ localization with immunomagnetic sorting and subsequent immortalization, we have identifed and isolated a cytokeratin 19‐positive suprabasal putative precursor cell in the luminal epithelial compartment and established representative cell lines. This suprabasal‐derived epithelial cell line is able to generate both itself and differentiated luminal epithelial and myoepithelial cells, and in addition, is able to form elaborate terminal duct lobular unit (TDLU)‐like structures within a reconstituted basement membrane. As more than 90% of breast cancers arise in TDLUs and more than 90% are also cytokeratin 19‐positive, we suggest that this cell population contains a breast‐cancer progenitor.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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.

Azithromycin Maintains Airway Epithelial Integrity during Pseudomonas aeruginosa Infection

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.

Characterization of a Novel Breast Carcinoma Xenograft and Cell Line Derived from a BRCA1 Germ-Line Mutation Carrier

A human tumor xenograft (L56Br-X1) was established from a breast cancer axillary lymph node metastasis of a 53-year-old woman with a BRCA1 germ-line nonsense mutation (1806C>T; Q563X), and a cell line (L56Br-C1) was subsequently derived from the xenograft. The xenograft carries only the mutant BRCA1 allele and expresses mutant BRCA1 mRNA but no BRCA1 protein as determined by immunoprecipitation or Western blotting. The primary tumor, lymph node metastasis, and xenograft were hypodiploid by DNA flow cytometry, whereas the cell line displayed an aneuploidy apparently developed via polyploidization. Cytogenetic analysis, spectral karyotyping, and comparative genomic hybridization of the cell line revealed a highly complex karyotype with numerous unbalanced translocations. The xenograft and cell line had retained a somatic TP53 missense mutation (S215I) originating from the primary tumors, as well as a lack of immunohistochemically detectable expression of steroid hormone receptors, epidermal growth factor receptor, human epidermal growth factor receptor 2 (HER-2), and keratin 8. Global gene expression analysis by cDNA microarrays supported a correlation between the expression profiles of the primary tumor, lymph node metastasis, xenograft, and cell line. We conclude that L56Br-X1 and L56Br-C1 are useful model systems for studies of the pathogenesis and new therapeutic modalities of BRCA1-induced human breast cancer.