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Modeling lymphangiogenesis in a three-dimensional culture system

Abstract

A lack of appropriate in vitro models of three-dimensional lymph vessel growth hampers the study of lymphangiogenesis. We developed a lymphatic ring assay—a potent, reproducible and quantifiable three-dimensional culture system for lymphatic endothelial cells that reproduces spreading of endothelial cells from a pre-existing vessel, cell proliferation, migration and differentiation into capillaries. In the assay, mouse thoracic duct fragments are embedded in a collagen gel, leading to the formation of lumen-containing lymphatic capillaries, which we assessed by electron microscopy and immunostaining. We developed a computerized method to quantify the lymphatic network. By applying this model to gene-deficient mice, we found evidence for involvement of the matrix metalloproteinase, MMP-2, in lymphangiogenesis. The lymphatic ring assay bridges the gap between two-dimensional in vitro models and in vivo models of lymphangiogenesis, can be used to exploit the potential of existing transgenic mouse models, and rapidly identify regulators of lymphangiogenesis.

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Figure 1: Lymphatic ring assay.
Figure 2: Lymphatic endothelial cell outgrowth from mouse thoracic duct.
Figure 3: Characterization of three-dimensional lymphatic ring cultures.
Figure 4: Modulation of lymphatic vessel outgrowth by growth factors and inhibitors.
Figure 5: Modulation of lymphatic vessel outgrowth by proteases and their inhibitors.

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Acknowledgements

We thank S. Itohara (RIKEN brain Science Institute) for providing MMP-2–deficient mice; A. Bredan for scientific advice; and M.L. Alvarez, I. Dasoul, E. Feyereisen, L. Poma, P. Gavitelli and F. Olivier for technical assistance. This work was supported by grants from the EU Framework Program projects (FP6 and FP7), the Fonds de la Recherche Scientifique Médicale, the Fonds National de la Recherche Scientifique (FNRS, Belgium), the Fondation contre le Cancer, the Fonds spéciaux de la Recherche (University of Liège), the Centre Anticancéreux près l'Université de Liège, the Fonds Léon Fredericq (University of Liège), the Direction Generale des Technologies et de la Recherche en Region Wallonne (DGTRE), the Interuniversity Attraction Poles Program—Belgian Science Policy (Brussels, Belgium). F.B. is recipient of a Televie-FNRS grant. This work was also supported by a grant from the Deutsche Forschungsgemeinschaft to J.P.S. under the auspices of Schwerpunktprogramm (SPP 1190 “The tumor-vessel interface”).

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Authors and Affiliations

Authors

Contributions

F.B. wrote the draft of the manuscript and performed most of the experiments with L.M.-L.; S.B. did all quantification work based on image analyses; G.R. contributed in setting up the model; and M.T. did the electron microscopy analysis. L.M., F.F., P.C., K.A., C.L., J.P.S. contributed to the work by either scientific advices or by providing essential materials or reagents. A.N. conceived the study and participated in its design and coordination. J.-M.F. contributed to work supervision. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Agnès Noël.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Methods (PDF 2424 kb)

Supplementary Video 1

Lymphatic thoracic duct dissection: step 1. Thoracic ducts are obtained by microsurgery using very thin microdissecting forceps and Vannas scissors. The periaortic fibroadipose tissue is carefully dissected. (MOV 18796 kb)

Supplementary Video 2

Lymphatic thoracic duct dissection: step 2. Fat tissue surrounding the lymphatic ring was removed and care was taken not to damage the lymphatic wall. (MOV 24793 kb)

Supplementary Video 3

Lymphatic thoracic duct dissection: removal of fat tissue. (MOV 22940 kb)

Supplementary Video 4

Lymphatic thoracic duct dissection: step 3. Both edges of the thoracic duct are cut and the duct is immediately transferred to a culture dish. (MOV 15518 kb)

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Bruyère, F., Melen-Lamalle, L., Blacher, S. et al. Modeling lymphangiogenesis in a three-dimensional culture system. Nat Methods 5, 431–437 (2008). https://doi.org/10.1038/nmeth.1205

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