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Edward E Morrisey,
Wellington V Cardoso,
Robert H Lane,
Marlene Rabinovitch,
Steven H Abman,
Xingbin Ai,
Kurt H Albertine,
Richard D Bland,
Harold A Chapman,
William Checkley, [......],
Lawrence S Prince,
Jeff Reese,
Janet Rossant,
Wei Shi,
Xin Sun, Zena Werb,
Jeffrey A Whitsett,
Dorothy Gail,
Carol J Blaisdell,
Qing S Lin
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ABSTRACT: Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.
Annals of the American Thoracic Society. 04/2013; 10(2):S12-6.
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ABSTRACT: Despite advances in our understanding of breast cancer, patients with metastatic disease have poor prognoses. GATA3 is a transcription factor that specifies and maintains mammary luminal epithelial cell fate, and its expression is lost in breast cancer, correlating with a worse prognosis in human patients. Here, we show that GATA3 promotes differentiation, suppresses metastasis and alters the tumour microenvironment in breast cancer by inducing microRNA-29b (miR-29b) expression. Accordingly, miR-29b is enriched in luminal breast cancers and loss of miR-29b, even in GATA3-expressing cells, increases metastasis and promotes a mesenchymal phenotype. Mechanistically, miR-29b inhibits metastasis by targeting a network of pro-metastatic regulators involved in angiogenesis, collagen remodelling and proteolysis, including VEGFA, ANGPTL4, PDGF, LOX and MMP9, and targeting ITGA6, ITGB1 and TGFB, thereby indirectly affecting differentiation and epithelial plasticity. The discovery that a GATA3-miR-29b axis regulates the tumour microenvironment and inhibits metastasis opens up possibilities for therapeutic intervention in breast cancer.
Nature Cell Biology 01/2013; · 19.49 Impact Factor
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ABSTRACT: Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, and hair follicle, as well as in kidney nephrons. Here, we demonstrate the outstanding regenerative potential of a rare population of Lgr5-expressing (Lgr5(+)) mammary epithelial cells (MECs). We found that Lgr5(+) cells reside within the basal population, are superior to other basal cells in regenerating functional mammary glands (MGs), are exceptionally efficient in reconstituting MGs from single cells, and exhibit regenerative capacity in serial transplantations. Loss-of-function and depletion experiments of Lgr5(+) cells from transplanted MECs or from pubertal MGs revealed that these cells are not only sufficient but also necessary for postnatal mammary organogenesis.
Cell reports. 01/2013;
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ABSTRACT: Mitra and colleagues analyzed microRNA expression profiles of fibroblasts isolated from ovarian cancer patients, searching for dysregulated microRNAs in the stromal compartment of human cancer. They found that decreased miR-31 and miR-214 and increased miR-155 expression can reprogram normal fibroblasts into tumor-promoting cancer-associated fibroblasts. They identified CCL5, a protumorigenic chemokine that is highly expressed in tumors, as a key target of miR-214, thus showing that microRNA perturbation in the stromal microenvironment can affect tumor growth. Cancer Discov; 2(12); 1078-80. ©2012 AACR.
Cancer discovery. 12/2012; 2(12):1078-80.
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Xiaodong Wang,
Yan Yiu Yu,
Shirley Lieu,
Frank Yang,
Jeffrey Lang,
Chuanyong Lu, Zena Werb,
Diane Hu,
Theodore Miclau,
Ralph Marcucio,
Céline Colnot
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ABSTRACT: Like other tissue injuries, bone fracture triggers an inflammatory response, which plays an important role in skeletal repair. Inflammation is believed to have both positive and negative effects on bone repair, but the underlying cellular mechanisms are not well understood. To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. These results suggested that the inflammatory response could influence skeletal cell differentiation after fracture. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, and which we previously showed is a potential regulator of cell fate decisions during fracture repair. Mmp9(-/-) mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9(-/-) compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9(-/-) mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9(-/-) stroma and periosteum. Following transplantation, Mmp9(-/-) mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Moreover, Mmp9(-/-) periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9(-/-) hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9(-/-) mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. Taken together, these results show that MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation.
Bone 09/2012; 52(1):111-119. · 4.02 Impact Factor
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ABSTRACT: Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transcriptome analysis of our 3D cultures revealed few ECM-dependent changes in RNA expression. However, we observed many differences between normal and malignant epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene could induce sustained dissemination of nontransformed cells into collagen I. We found that deletion of P-cadherin was sufficient for sustained dissemination, but exclusively into collagen I. Our data reveal that metastatic tumors preferentially disseminate in specific ECM microenvironments. Furthermore, these data suggest that breaks in the basement membrane could induce invasion and dissemination via the resulting direct contact between cancer cells and collagen I.
Proceedings of the National Academy of Sciences 08/2012; 109(39):E2595-604. · 9.68 Impact Factor
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Laurie E Littlepage,
Adam S Adler,
Hosein Kouros-Mehr,
Guiqing Huang,
Jonathan Chou,
Sheryl R Krig,
Obi L Griffith,
James E Korkola,
Kun Qu,
Devon A Lawson,
Qing Xue,
Mark D Sternlicht,
Gerrit J P Dijkgraaf,
Paul Yaswen,
Hope S Rugo,
Colleen A Sweeney,
Colin C Collins,
Joe W Gray,
Howard Y Chang, Zena Werb
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ABSTRACT: The transcription factor ZNF217 is a candidate oncogene in the amplicon on chromosome 20q13 that occurs in 20% to 30% of primary human breast cancers and that correlates with poor prognosis. We show that Znf217 overexpression drives aberrant differentiation and signaling events, promotes increased self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and represses an adult tissue stem cell gene signature downregulated in cancers. By in silico screening, we identified candidate therapeutics that at low concentrations inhibit growth of cancer cells expressing high ZNF217. We show that the nucleoside analogue triciribine inhibits ZNF217-induced tumor growth and chemotherapy resistance and inhibits signaling events [e.g., phospho-AKT, phospho-mitogen-activated protein kinase (MAPK)] in vivo. Our data suggest that ZNF217 is a biomarker of poor prognosis and a therapeutic target in patients with breast cancer and that triciribine may be part of a personalized treatment strategy in patients overexpressing ZNF217. Because ZNF217 is amplified in numerous cancers, these results have implications for other cancers. SIGNIFICANCE: This study finds that ZNF217 is a poor prognostic indicator and therapeutic target in patients with breast cancer and may be a strong biomarker of triciribine treatment efficacy in patients. Because previous clinical trials for triciribine did not include biomarkers of treatment efficacy, this study provides a rationale for revisiting triciribine in the clinical setting as a therapy for patients with breast cancer who overexpress ZNF217.
Cancer discovery. 06/2012; 2(7):638-51.
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Elizabeth S Nakasone,
Hanne A Askautrud,
Tim Kees,
Jae-Hyun Park,
Vicki Plaks,
Andrew J Ewald,
Miriam Fein,
Morten G Rasch,
Ying-Xim Tan,
Jing Qiu,
Juwon Park,
Pranay Sinha,
Mina J Bissell,
Eirik Frengen, Zena Werb,
Mikala Egeblad
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ABSTRACT: Little is known about the dynamics of cancer cell death in response to therapy in the tumor microenvironment. Intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death, and tumor-stroma interactions. We observed associations between vascular leakage and response to doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. Thus, live imaging can be used to gain insights into drug responses in situ.
Cancer cell 04/2012; 21(4):488-503. · 25.29 Impact Factor
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ABSTRACT: The nature and site of tumor-antigen presentation to immune T cells by bone-marrow-derived cells within the tumor microenvironment remains unresolved. We generated a fluorescent mouse model of spontaneous immunoevasive breast cancer and identified a subset of myeloid cells with significant similarity to dendritic cells and macrophages that constitutively ingest tumor-derived proteins and present processed tumor antigens to reactive T cells. Using intravital live imaging, we determined that infiltrating tumor-specific T cells engage in long-lived interactions with these cells, proximal to the tumor. In vitro, these cells capture cytotoxic T cells in signaling-competent conjugates but do not support full activation or sustain cytolysis. The spatiotemporal dynamics revealed here implicate nonproductive interactions between T cells and antigen-presenting cells on the tumor margin.
Cancer cell 03/2012; 21(3):402-17. · 25.29 Impact Factor
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ABSTRACT: Glioblastoma (GBM), a uniformly lethal brain cancer, is characterized by diffuse invasion and abnormal activation of multiple receptor tyrosine kinase (RTK) signaling pathways, presenting a major challenge to effective therapy. The activation of many RTK pathways is regulated by extracellular heparan sulfate proteoglycans (HSPG), suggesting these molecules may be effective targets in the tumor microenvironment. In this study, we demonstrated that the extracellular sulfatase, SULF2, an enzyme that regulates multiple HSPG-dependent RTK signaling pathways, was expressed in primary human GBM tumors and cell lines. Knockdown of SULF2 in human GBM cell lines and generation of gliomas from Sulf2(-/-) tumorigenic neurospheres resulted in decreased growth in vivo in mice. We found a striking SULF2 dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. Interestingly, in a survey of SULF2 levels in different subtypes of GBM, the proneural subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. Therefore, in addition to its potential as an upstream target for therapy of GBM, SULF2 may help identify a subset of GBMs that are more dependent on exogenous growth factor-mediated signaling. Our results suggest the bioavailability of growth factors from the microenvironment is a significant contributor to tumor growth in a major subset of human GBM.
The Journal of clinical investigation 03/2012; 122(3):911-22. · 15.39 Impact Factor
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ABSTRACT: The local microenvironment, or niche, of a cancer cell plays important roles in cancer development. A major component of the niche is the extracellular matrix (ECM), a complex network of macromolecules with distinctive physical, biochemical, and biomechanical properties. Although tightly controlled during embryonic development and organ homeostasis, the ECM is commonly deregulated and becomes disorganized in diseases such as cancer. Abnormal ECM affects cancer progression by directly promoting cellular transformation and metastasis. Importantly, however, ECM anomalies also deregulate behavior of stromal cells, facilitate tumor-associated angiogenesis and inflammation, and thus lead to generation of a tumorigenic microenvironment. Understanding how ECM composition and topography are maintained and how their deregulation influences cancer progression may help develop new therapeutic interventions by targeting the tumor niche.
The Journal of Cell Biology 02/2012; 196(4):395-406. · 10.26 Impact Factor
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ABSTRACT: The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28-30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.
Journal of Neuroscience 11/2011; 31(44):15894-903. · 7.11 Impact Factor
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ABSTRACT: The extracellular matrix (ECM) serves diverse functions and is a major component of the cellular microenvironment. The ECM is a highly dynamic structure, constantly undergoing a remodeling process where ECM components are deposited, degraded, or otherwise modified. ECM dynamics are indispensible during restructuring of tissue architecture. ECM remodeling is an important mechanism whereby cell differentiation can be regulated, including processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. In contrast, abnormal ECM dynamics lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer. Understanding the mechanisms of ECM remodeling and its regulation, therefore, is essential for developing new therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine.
Cold Spring Harbor perspectives in biology 09/2011; 3(12). · 9.40 Impact Factor
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Jeong-Soo Hong,
Kendra J Greenlee,
Ramanan Pitchumani,
Seung-Hyo Lee,
Li-zhen Song,
Ming Shan,
Seon Hee Chang,
Pyong Woo Park,
Chen Dong, Zena Werb,
Akhil Bidani,
David B Corry,
Farrah Kheradmand
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ABSTRACT: A localized and effective innate immune response to pathogenic bacterial invasion is central to host survival. Identification of the critical local innate mediators of lung defense against such pathogens is essential for a complete understanding of the mechanism(s) underlying effective host defense. In an acute model of Streptococcus pneumoniae lung infection, deficiency in matrix metalloproteinase (MMP)2 and MMP9 (Mmp2/9(-/-)) conferred a survival disadvantage relative to wild-type mice treated under the same conditions. S. pneumoniae-infected Mmp2/9(-/-) mice recruited more polymorphonuclear leukocytes to the lung but had higher bacterial burdens. Mmp2/9(-/-) mice showed significantly higher levels of IL-17A, IP-10, and RANTES in the lung. Although MMP2-dependent cleavage partially inactivated IL-17A, MMP9 was critical for effective bacterial phagocytosis and reactive oxygen species generation in polymorphonuclear neutrophils. These data demonstrate critical nonredundant and protective roles for MMP2 and MMP9 in the early host immune response against S. pneumoniae infection.
The Journal of Immunology 06/2011; 186(11):6427-36. · 5.79 Impact Factor
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Zena Werb
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ABSTRACT: It has been known for some time that cell-cell adhesion mediated by E-cadherin and catenins is important for development and cancer in epithelial tissues. Although β-catenin is upregulated in many cancers, p120-catenin is downregulated in most human cancers. Before this study, the molecular mechanism underlying β-catenin function in tumorigenesis was well on its way to being worked out, but little was known about p120. This paper piqued my interest because it showed that p120 was a major regulator of E-cadherin stability in the salivary gland. Its loss caused a major decrease in E-cadherin with severe defects in cell-cell adhesion and tissue morphology resembling intraepithelial hyperplasia. I liked this paper because it made me think about how looking at development gives important insights into cancer and about how two related molecules that bind to E-cadherin could behave in opposite ways to get the initial phases of tumorigenesis started. This PaperPick refers to "Blocked Acinar Development, E-Cadherin Reduction, and Intraepithelial Neoplasia upon Ablation of p120-Catenin in the Mouse Salivary Gland," by M.A. Davis and A.B. Reynolds, published in January 2006. VIDEO ABSTRACT:
Developmental cell 05/2011; 20(5):e3. · 13.36 Impact Factor
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ABSTRACT: Macrophages and polymorphonuclear cells (PMNs) represent an essential part of the innate immune system. These cells mediate a wide spectrum of immunological functions including bacterial defense, immune modulation, and inflammation; they are necessary for tissue homeostasis and also contribute to pathologies such as malignancy, autoimmunity, and chronic inflammation. Both macrophages and PMNs express a set of matrix metalloproteinases (MMPs), zinc-dependent endopeptidases that are involved in a variety of biological functions such as the turnover of extracellular matrix (ECM) components, angiogenesis, and the regulation of inflammation. Given the link between unregulated MMP function and diseases such as chronic inflammation or cancer, it is not surprising that these enzymes have been implicated as key effectors in clinical studies. Thus, it is important to widen our knowledge about the role of these enzymes in macrophage and PMN biology. Here, we briefly discuss the general role of inflammatory cell-derived MMPs and describe methods to analyze their activity in macrophages and PMN.
Current protocols in immunology / edited by John E. Coligan ... [et al.] 04/2011; Chapter 14:Unit14.24.
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ABSTRACT: The regulation of energy homeostasis integrates diverse biological processes ranging from behavior to metabolism and is linked fundamentally to numerous disease states. To identify new molecules that can bypass homeostatic compensatory mechanisms of energy balance in intact animals, we screened for small-molecule modulators of Caenorhabditis elegans fat content. We report on several molecules that modulate fat storage without obvious deleterious effects on feeding, growth and reproduction. A subset of these compounds also altered fat storage in mammalian and insect cell culture. We found that one of the newly identified compounds exerts its effects in C. elegans through a pathway that requires previously undescribed functions of an AMP-activated kinase catalytic subunit and a transcription factor previously unassociated with fat regulation. Thus, our strategy identifies small molecules that are effective within the context of intact animals and reveals relationships between new pathways that operate across phyla to influence energy homeostasis.
Nature Chemical Biology 03/2011; 7(4):206-13. · 14.69 Impact Factor
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ABSTRACT: Amplification of 8p11-12 in human breast cancers is associated with increased proliferation and tumor grade and reduced metastasis-free patient survival. We identified Zeppo1 (zinc finger elbow-related proline domain protein 1) (FLJ14299/ZNF703) within this amplicon as a regulator of cell adhesion, migration, and proliferation in mammary epithelial cells. Overexpression of Zeppo1 reduces cell-cell adhesion and stimulates migration and proliferation. Knockdown of Zeppo1 induces adhesion and lumen formation. Zeppo1 regulates transcription, complexing with Groucho and repressing E-cadherin expression and Wnt and TGFβ reporter expression. Zeppo1 promotes expression of metastasis-associated p120-catenin isoform 1 and alters p120-catenin localization upon cell contact with the extracellular matrix. Significantly, Zeppo1 overexpression in a mouse breast cancer model increases lung metastases, while reducing Zeppo1 expression reduces both tumor size and the number of lung metastases. These results indicate that Zeppo1 is a key regulator of breast cancer progression.
Genes & development 02/2011; 25(5):471-84. · 12.08 Impact Factor
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ABSTRACT: INTRODUCTION Genetic studies and tumor biopsies have shown the importance of stromal components for cancer progression, but much remains to be learned about the dynamic interactions among the distinct tumor components within live animals. One challenge of studying cell behavior in progressively developing tumors has been the difficulty of maintaining live mice on the microscope stage. To prepare mice for long-term intravital imaging, auxiliary equipment is necessary to enable and to control anesthesia (such as the anesthesia gas mixer itself, a gas humidifier, indwelling lines for saline, and heat blanket). The other important component is to gain optical access to the mammary gland. This protocol describes a surgical technique that creates a skin flap with the mammary gland. The method is relatively easily taught, does not compromise the peritoneal cavity or any major blood vessels, and is generally well tolerated by the mice. There is minimal inflammatory response to the surgery itself if the solutions and tools are sterile, the surgical work area is clean, and aseptic techniques are used. This protocol works well for a single long-term image session, but does not enable repeated imaging sessions. For such approaches, methods for implanting imaging windows over the inguinal mammary gland should be used instead.
Cold Spring Harbor Protocols 01/2011; 2011(2):pdb.prot5562. · 4.63 Impact Factor
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ABSTRACT: INTRODUCTION Anesthesia protocols for mice have been optimized, as described here, to achieve long-term imaging (up to 40 h) and facilitate survival through careful monitoring of the mice during anesthesia. Isoflurane anesthesia is the preferred method, because it can be adjusted quickly as needed during the experiment. Critical for the long survival times under anesthesia is the use of the lowest possible dose of anesthesia, which is identified by corneal reflex and monitoring of breath and heart rate, blood-oxygenation levels, and vascular distension using an oximeter probe. It is critical that the carrier gas for isoflurane is humidified. In addition, it is essential to keep mice warm and to compensate for loss of fluid by supplementing with saline. Alternative approaches rely on injectable anesthetics, which do not require dedicated equipment or high-ventilation rates in the imaging room. However, injectable anesthetics are harder to dose for image sessions of >6-10 h.
Cold Spring Harbor Protocols 01/2011; 2011(2):pdb.prot5563. · 4.63 Impact Factor
