Modeling lymphangiogenesis in a three-dimensional culture system

Bruyère, Françoise; Melen-Lamalle, Laurence; Blacher, Silvia; Roland, Guy; Thiry, Marc; Moons, Lieve; Frankenne, Françis; Carmeliet, Peter; Alitalo, Kari; Libert, Claude; Sleeman, Jonathan; Foidart, Jean-Michel; Noël, Agnès

doi:10.1038/nmeth.1205

Cited by 119 publications

(138 citation statements)

References 31 publications

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“…Previously, several studies were performed based on in vitro compound screens with blood vascular endothelial cells, including proliferation assays of human umbilical vein endothelial cells (HUVEC) (16), HUVEC/vascular smooth muscle cell cocultures, human microvascular endothelial cell tube formation assays (17,18), and HUVEC spheroid sprouting assays (19). While the tube formation assay has been considered as a model of vasculogenesis rather than angiogenesis (20), the other assays attempt to measure different aspects of blood vessel formation. However, a number of studies from our group as well as from other investigators have clearly demonstrated that lymphatic endothelial cells differ from blood vascular endothelial cells with regard to their morphology, differentiation, and function, as well as to the molecular mechanisms inducing their growth (lymphangiogenesis and angiogenesis, respectively) (5).…”

Section: Discussionmentioning

confidence: 99%

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Schulz

Reisen

Zgraggen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds ðLOPACÞ 1280 collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.high-content screening | small compound screening | lymphatic endothelium | chemical genetics

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Section: Discussionmentioning

confidence: 99%

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Schulz

Reisen

Zgraggen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Medium containing 20 ng/ml VEGF-A for aortas and 300 ng/ml VEGF-C (R&D Systems) for thoracic ducts was added to each well of gelled Matrigel (26,29). The length of microvessel-like sprouting was measured with MetaMorph imaging software (version 7.5).…”

Section: Methodsmentioning

confidence: 99%

Deletion of Tetraspanin CD9 Diminishes Lymphangiogenesis in Vivo and in Vitro

Iwasaki

Takeda

Maruyama

et al. 2013

Journal of Biological Chemistry

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Background: The molecular mechanisms regulating lymphangiogenesis remain unclear. Results: Tetraspanin CD9 modulates molecular organization of integrins in LEC, thereby supporting several functions required for lymphangiogenesis. Conclusion: Deletion of CD9 diminished lymphangiogenesis in mice and humans. Significance: Given that CD9 mediates inflammation and tumor progression, CD9 might be a key component not only in tumor metastasis, but also in inflammation.

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“…In vitro modeling is appropriate for dissecting various mechanisms involved in cancer cell invasion and stromal cell 1991; Wolf et al, 2003) are frequently used as ECM substrates in invasion assays. The ideal model would allow for easy manipulation, quantification by digital analysis, morphotypic and morphometric studies, further downstream biochemical assays, and close recapitulation of the in vivo situation (Bruyère et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Modeling and quantification of cancer cell invasion through collagen type I matrices

Wever¹,

Hendrix²,

Boeck³

et al. 2010

Int. J. Dev. Biol.

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Tumor invasion is the outcome of a complex interplay between cancer cells and the stromal environment. Considering the contribution of the stromal environment, we developed a membrane-free single-cell and spheroid based complementary model to study cancer invasion through native collagen type-I matrices. Cell morphology is preserved during the assays allowing real time monitoring of invasion-induced changes in cell structure and F-actin organization. Combining these models with computerized quantification permits the calculation of highly reproducible and operator-independent data. These assays are versatile in the use of fluorescent probes and have a flexible kinetic endpoint. Once the optimal experimental conditions are empirically determined, the collagen type-I invasion assays can be used for preclinical validation of small-molecule inhibitors targeting invasion. Initiation and monitoring of the single-cell and spheroid invasion model can be achieved in 8 h (over 3 days) and in 14 h (over 8 days) respectively.

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Modeling lymphangiogenesis in a three-dimensional culture system

Cited by 119 publications

References 31 publications

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Deletion of Tetraspanin CD9 Diminishes Lymphangiogenesis in Vivo and in Vitro

Modeling and quantification of cancer cell invasion through collagen type I matrices

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