Cellular Plasticity and Heterotypic Interactions during Breast Morphogenesis and Cancer Initiation.

The human breast gland is a unique organ as most of its development occurs postnatally between menarche and menopause, a period ranging from 30 to 40 years. During this period, the monthly menstruation cycle drives the mammary gland through phases of cell proliferation, differentiation, and apoptosis, facilitated via a closely choreographed interaction between the epithelial cells and the surrounding stroma preparing the gland for pregnancy. If pregnancy occurs, maximal differentiation is reached to prepare for lactation. After lactation, the mammary gland involutes to a pre-pregnant state. These cycles of proliferation, differentiation, and involution necessitate the presence of epithelial stem cells that give rise to progenitor cells which differentiate further into the luminal and myoepithelial lineages that constitute the epithelial compartment and are responsible for the branching structure of the gland. Maintaining homeostasis and the stem cell niche depends strongly on signaling between the stem and progenitor cells and the surrounding stroma. Breast cancer is a slowly progressing disease whose initiation can take decades to progress into an invasive form. Accumulating evidence indicates that stem cells and/or progenitor cells at different stages, rather than terminally differentiated cells are the main cells of origin for most breast cancer subgroups. Stem cells and cancer cells share several similarities such as increased survival and cellular plasticity which is reflected in their ability to switch fate by receiving intrinsic and extrinsic signals. In this review, we discuss the concept of cellular plasticity in normal breast morphogenesis and cancer, and how the stromal environment plays a vital role in cancer initiation and progression.

Ventilator-induced lung injury results in oxidative stress response and mitochondrial swelling in a mouse model.

BACKGROUND: Mechanical ventilation is a life-saving therapy for critically ill patients, providing rest to the respiratory muscles and facilitating gas exchange in the lungs. Ventilator-induced lung injury (VILI) is an unfortunate side effect of mechanical ventilation that may lead to serious consequences for the patient and increase mortality. The four main injury mechanisms associated with VILI are: baro/volutrauma caused by overstretching the lung tissues; atelectrauma, caused by repeated opening and closing of the alveoli resulting in shear stress; oxygen toxicity due to use of high ratio of oxygen in inspired air, causing formation of free radicals; and biotrauma, the resulting biological response to tissue injury, that leads to a cascade of events due to excessive inflammatory reactions and may cause multi-organ failure. An often-overlooked part of the inflammatory reaction is oxidative stress. In this research, a mouse model of VILI was set up with three tidal volume settings (10, 20 and 30 mL/kg) at atmospheric oxygen level. Airway pressures and heart rate were monitored and bronchoalveolar lavage fluid (BALF) and lung tissue samples were taken. RESULTS: We show a correlation between increased inflammation and barrier failure, and higher tidal volumes, evidenced by increased IL-6 expression, high concentration of proteins in BALF along with changes in expression of adhesion molecules. Furthermore, swelling of mitochondria in alveolar type II cells was seen indicating their dysfunction and senescence-like state. RNA sequencing data present clear increases in inflammation, mitochondrial biogenesis and oxidative stress as tidal volume is increased, supported by degradation of Keap1, a redox-regulated substrate adaptor protein. CONCLUSIONS: Oxidative stress seems to be a more prominent mechanism of VILI than previously considered, indicating that possible treatment methods against VILI might be identified by impeding oxidative pathways.

Ventilator-induced lung-injury in mouse models: Is there a trap?

Ventilator-induced lung injury (VILI) is a serious acute injury to the lung tissue that can develop during mechanical ventilation of patients. Due to the mechanical strain of ventilation, damage can occur in the bronchiolar and alveolar epithelium resulting in a cascade of events that may be fatal to the patients. Patients requiring mechanical ventilation are often critically ill, which limits the possibility of obtaining patient samples, making VILI research challenging. In vitro models are very important for VILI research, but the complexity of the cellular interactions in multi-organ animals, necessitates in vivo studies where the mouse model is a common choice. However, the settings and duration of ventilation used to create VILI in mice vary greatly, causing uncertainty in interpretation and comparison of results. This review examines approaches to induce VILI in mouse models for the last 10 years, to our best knowledge, summarizing methods and key parameters presented across the studies. The results imply that a more standardized approach is warranted.

Azithromycin ameliorates sulfur dioxide-induced airway epithelial damage and inflammatory responses.

BACKGROUND: The airway epithelium (AE) forms the first line of defence against harmful particles and pathogens. Barrier failure of the airway epithelium contributes to exacerbations of a range of lung diseases that are commonly treated with Azithromycin (AZM). In addition to its anti-bacterial function, AZM has immunomodulatory effects which are proposed to contribute to its clinical effectiveness. In vitro studies have shown the AE barrier-enhancing effects of AZM. The aim of this study was to analyze whether AE damage caused by inhalation of sulfur dioxide (SO2) in a murine model could be reduced by pre-treatment with AZM. METHODS: The leakiness of the AE barrier was evaluated after SO2 exposure by measuring levels of human serum albumin (HSA) in bronchoalveolar lavage fluid (BALF). Protein composition in BALF was also assessed and lung tissues were evaluated across treatments using histology and gene expression analysis. RESULTS: AZM pre-treatment (2 mg/kg p.o. 5 times/week for 2 weeks) resulted in reduced glutathione-S-transferases in BALF of SO2 injured mice compared to control (without AZM treatment). AZM treated mice had increased intracellular vacuolization including lamellar bodies and a reduction in epithelial shedding after injury in addition to a dampened SO2-induced inflammatory response. CONCLUSIONS: Using a mouse model of AE barrier dysfunction we provide evidence for the protective effects of AZM in vivo, possibly through stabilizing the intracellular microenvironment and reducing inflammatory responses. Our data provide insight into the mechanisms contributing to the efficacy of AZM in the treatment of airway diseases.

Melflufen, a peptide-conjugated alkylator, is an efficient anti-neo-plastic drug in breast cancer cell lines.

Melphalan flufenamide (hereinafter referred to as "melflufen") is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins Æ´H2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.

ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion.

The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial-endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.

YKL-40/CHI3L1 facilitates migration and invasion in HER2 overexpressing breast epithelial progenitor cells and generates a niche for capillary-like network formation.

Epithelial to mesenchymal transition (EMT) is a developmental event that is hijacked in some diseases such as fibrosis and cancer. In cancer, EMT has been linked to increased invasion and metastasis and is generally associated with a poor prognosis. In this study, we have compared phenotypic and functional differences between two isogenic cell lines with an EMT profile: D492M and D492HER2 that are both derived from D492, a breast epithelial cell line with stem cell properties. D492M is non-tumorigenic while D492HER2 is tumorigenic. Thus, the aim of this study was to analyze the expression profile of these cell lines, identify potential oncogenes, and evaluate their effects on cellular phenotype. We performed transcriptome and secretome analyses of D492M and D492HER2 and verified expression of selected genes at the RNA and protein level. One candidate, YKL-40 (also known as CHI3L1), was selected for further studies due to its differential expression between D492M and D492HER2, being considerably higher in D492HER2. YKL-40 has been linked to chronic inflammation diseases and cancer, yet its function is not fully understood. Knock-down experiments of YKL-40 in D492HER2 resulted in reduced migration and invasion as well as reduced ability to induce angiogenesis in an in vitro assay, plus changes in the EMT-phenotype. In summary, our data suggest that YKL-40 may provide D492HER2 with increased aggressiveness, supporting cancer progression and facilitating angiogenesis.

Azithromycin induces epidermal differentiation and multivesicular bodies in airway epithelia.

BACKGROUND: Azithromycin (Azm) is a macrolide recognized for its disease-modifying effects and reduction in exacerbation of chronic airway diseases. It is not clear whether the beneficial effects of Azm are due to its anti-microbial activity or other pharmacological actions. We have shown that Azm affects the integrity of the bronchial epithelial barrier measured by increased transepithelial electrical resistance. To better understand these effects of Azm on bronchial epithelia we have investigated global changes in gene expression. METHODS: VA10 bronchial epithelial cells were treated with Azm and cultivated in air-liquid interface conditions for up to 22 days. RNA was isolated at days 4, 10 and 22 and analyzed using high-throughput RNA sequencing. qPCR and immunostaining were used to confirm key findings from bioinformatic analyses. Detailed assessment of cellular changes was done using microscopy, followed by characterization of the lipidomic profiles of the multivesicular bodies present. RESULTS: Bioinformatic analysis revealed that after 10 days of treatment genes encoding effectors of sterol and cholesterol metabolism were prominent. Interestingly, expression of genes associated with epidermal barrier differentiation, KRT1, CRNN, SPINK5 and DSG1, increased significantly at day 22. Together with immunostaining, these results suggest an epidermal differentiation pattern. We also found that Azm induced the formation of multivesicular and lamellar bodies in two different airway epithelial cell lines. Lipidomic analysis revealed that Azm was entrapped in multivesicular bodies linked to different types of lipids, most notably palmitate and stearate. Furthermore, targeted analysis of lipid species showed accumulation of phosphatidylcholines, as well as ceramide derivatives. CONCLUSIONS: Taken together, we demonstrate how Azm might confer its barrier enhancing effects, via activation of epidermal characteristics and changes to intracellular lipid dynamics. These effects of Azm could explain the unexpected clinical benefit observed during Azm-treatment of patients with various lung diseases affecting barrier function.

Innovative in vitro method to study ventilator induced lung injury.

Mechanical ventilation (MV) is a life-saving therapy for critically ill patients, alleviating the work of breathing and supporting adequate gas exchange. However, MV can cause ventilator induced lung injury (VILI) by baro/volu- and atelectrauma, even lead to acute respiratory distress syndrome (ARDS), and substantially augment mortality. There is a need for specific biomarkers and novel research platforms for VILI/ARDS research to study these detrimental disorders and seek ways to avoid or prevent them. Previous in vitro studies on bronchial epithelium, cultured in air-liquid interface (ALI) conditions, have generally utilized static or constant pressure.  We have developed a Cyclical Pressure ALI Device (CPAD) that enables cyclical stress on ALI cultured human bronchial cells, with the aim of mimicking the effects of MV. Using CPAD we were able to analyze differentially expressed VILI/ARDS and innate immunity associated genes along with increased expression of associated proteins. CPAD provides an easy and accessible way to analyze functional and phenotypic changes that occur during VILI and may provide a platform for future drug testing.

Application of the D492 Cell Lines to Explore Breast Morphogenesis, EMT and Cancer Progression in 3D Culture.

The human female breast gland is composed of branching epithelial ducts that extend from the nipple towards the terminal duct lobular units (TDLUs), which are the functional, milk-producing units of the gland and the site of origin of most breast cancers. The epithelium of ducts and TDLUs is composed of an inner layer of polarized luminal epithelial cells and an outer layer of contractile myoepithelial cells, separated from the vascular-rich stroma by a basement membrane. The luminal- and myoepithelial cells share an origin and in recent years, there has been increasing understanding of how these cell types interact and how they contribute to breast cancer. Accumulating evidence links stem/or progenitor cells in the mammary/breast gland to breast cancer. In that regard, much knowledge has been gained from studies in mice due to specific strains that have allowed for gene knock out/in studies and lineage tracing of cellular fates. However, there is a large histologic difference between the human female breast gland and the mouse mammary gland that necessitates that research needs to be done on human material where primary cultures are important due to their close relation to the tissue of origin. However, due to difficulties of long-term cultures and lack of access to material, human cell lines are of great importance to bridge the gap between studies on mouse mammary gland and human primary breast cells. In this review, we describe D492, a breast epithelial progenitor cell line that can generate both luminal- and myoepithelial cells in culture, and in 3D culture it forms branching ducts similar to TDLUs. We have applied D492 and its daughter cell lines to explore cellular and molecular mechanisms of branching morphogenesis and cellular plasticity including EMT and MET. In addition to discussing the application of D492 in studying normal morphogenesis, we will also discuss how this cell line has been used to study breast cancer progression.

Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum.

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

Inhibition of PTP1B disrupts cell-cell adhesion and induces anoikis in breast epithelial cells.

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.

Pathological changes in basement membranes and dermal connective tissue of skin from patients with hereditary cystatin C amyloid angiopathy.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a genetic disease caused by a mutation in the cystatin C gene. Cystatin C is abundant in cerebrospinal fluid and the most prominent pathology in HCCAA is cerebral amyloid angiopathy due to mutant cystatin C amyloid deposition with associated cerebral hemorrhages, typically in young adult carriers. Analyses of post-mortem brain samples shows that pathological changes are limited to arteries and regions adjacent to arteries. The severity of pathological changes at post-mortem has precluded the elucidation of the evolution of histological changes. Mutant cystatin C deposition in carriers is systemic and has, for example, been described in the skin, suggesting similar pathological mechanisms both in the brain and outside of the central nervous system. The aim of this study was to use skin biopsies from asymptomatic and symptomatic carriers to study intermediate events in HCCAA pathogenesis. We found that cystatin C deposition in minimally affected samples was limited to the basement membrane (BM) between the dermis and epidermis. When the deposits were more advanced, they extended to other BM regions in the skin. Our results showed that the immunoreactivity of the BM protein COLIV was increased to a similar extent in all carrier biopsies and cystatin C deposits were in close association with COLIV. The density of fibroblasts in the upper dermis of carrier skin was increased, whereas the distribution of other cell types examined did not differ compared with control biopsies. COLIV and cystatin C immunoreactivity in carrier biopsies was closely associated with the fibroblasts. The results of this study, in conjunction with our previous results regarding pathological BM changes in leptomeningeal arteries of patients, suggest that BM changes are early and important events in HCCAA pathogenesis that could facilitate cystatin C deposition and aggregation.

Epithelial Plasticity During Human Breast Morphogenesis and Cancer Progression.

Understanding the complex events leading to formation of an epithelial-based organ such as the breast requires a detailed insight into the crosstalk between epithelial and stromal compartments. These interactions occur both through heterotypic cellular interactions and between cells and matrix components. While in vivo models may partially capture these complex interactions, there is a need for in- vitro models to study these events. In this review we discuss cell-cell interactions in breast development focusing on the stem cell niche and branching morphogenesis. Given the recent understanding that the basic developmental events underlying branching morphogenesis are closely related to pathways important to cancer progression, i.e. epithelial plasticity and epithelial to mesenchymal transition (EMT), we will also discuss aspects relevant to cancer progression. In cancer, the adoption of mesenchymal phenotype by the malignant cells allows stromal invasion and subsequent intravasation to blood- or lymphatic vessels, a route that is a prerequisite for metastasis. A number of publications have demonstrated that tumor initiating cells, sometimes referred to as cancer stem cells adapt an EMT phenotype that renders them more resistant to apoptosis and drug therapy. The mechanism behind this phenomenon is currently unknown but this may partially explain relapse in breast cancer patients. Increased understanding of branching morphogenesis in the breast gland and the regulation of EMT and its reverse process mesenchymal to epithelial transition (MET) may hold the keys for future development of methods/drugs that neutralize the invading properties of cancer cells.

Context-Dependent Function of Myoepithelial Cells in Breast Morphogenesis and Neoplasia.

Myoepithelial cells (MEPs) are specialized cells derived from epithelial progenitor cells, yet they also express the contractile machinery of smooth muscle cells. MEPs are prominent in glandular tissues where their function is to help expel secretions generated by the glandular epithelial cells. In the breast, MEPs are part of the bi-layered breast epithelium that line ducts and alveoli positioned perpendicular to the luminal epithelial cells (LEPs), separated from the surrounding stroma by the basement membrane. Researchers have recognized MEPs as important regulators of structural and functional behavior of LEPs, namely having role in polarization of LEPs, and regulating milk production. Furthermore, they have also been proposed to act as tumor suppressors as their presence inhibits invasion of cancer cells into the surrounding stroma. There is, however, accumulating evidence that MEPs in normal breast, carcinoma in situ and in invasive breast cancer differ significantly in terms of marker expression and this may truly interfere with their ability to behave as tumor suppressors. The term myoepithelial cell is often used synonymously with basal cell. While all MEPs, due to their position, can be referred to as basal cells, some basal cells do not fulfill the criteria of being MEPs. Synonymous use of these terms may hold true under normal conditions but careful interpretation of these terms should be used in breast cancer. In recent years, partial myoepithelial differentiation and epithelial to mesenchymal transition (EMT) have been shown to be associated with, and in some cases, necessary for cancer invasion and metastasis. In this review, we will discuss the context-dependent role of MEPs in breast morphogenesis, tumor suppression, and also the appearance of basal or partial myoepithelial differentiation in aggressive forms of breast cancer.

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

Expression and functional role of sprouty-2 in breast morphogenesis.

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.

Selection for EGFR gene amplification in a breast epithelial cell line with basal-like phenotype and hereditary background.

An epithelial cell line, referred to as A163, was established from breast carcinoma derived from a patient with a strong family history of breast cancer but no known breast cancer susceptibility mutation. A163 was propagated in a serum-free culture medium including the epidermal growth factor. Immunophenotypic characterization demonstrated a mixed luminal and basal-like phenotype. When epidermal growth factor was excluded from the culture medium, A163 entered a quiescent period followed by a period of increased cell proliferation in a subpopulation of the cells. The epidermal growth factor-independent subpopulation retained the basal-like phenotype of the parental cell line. Karyotype and fluorescent in situ hybridization analysis showed an amplification of epidermal growth factor receptor on 7q in A163-S1 only, resulting in high expression of total and phosphorylated epidermal growth factor receptor. The A163-S1 sub-line piles up in culture, indicating a loss of contact inhibition. When grown on transwell filters, A163 shows basal expression of P63 and cytokeratin 14, whereas A163-S1 expresses P63 ubiquitously, and has lost the basal specific expression of cytokeratin 14, indicating a loss of polarity. Furthermore, when cultured in reconstituted basement membrane matrix, A163 form polarized normal like acini. In contrast, A163-S1 form large disorganized structures with lack of polarity. These cell lines may prove useful to understand molecular changes in breast cancer progression, in particular basal-like breast cancer subtype with bad prognosis and no current treatment options.

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture.

BACKGROUND: Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D) co-culture assay. METHODS: Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. RESULTS: In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. CONCLUSION: Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

Evanescent-wave fluorescence microscopy using symmetric planar waveguides.

We describe a new evanescent-wave fluorescence excitation method, ideally suited for imaging of biological samples. The excitation light propagates in a planar optical waveguide, consisting of a thin waveguide core sandwiched between a sample in an aqueous solution and a polymer with a matching refractive index, forming a symmetric cladding environment. This configuration offers clear advantages over other waveguide-excitation methods, such as superior image quality, wide tunability of the evanescent field penetration depth and compatibility with optical fibers. The method is well suited for cell membrane imaging on cells in culture, including cell-cell and cell-matrix interaction, monitoring of surface binding events and similar applications involving aqueous solutions.