A three-dimensional lymphatic endothelial cell tube formation assay to identify novel kinases involved in lymphatic vessel remodeling

T. Jessica Gambino, Steven P. Williams, Carol Caesar, Daniel Resnick, Cameron Nowell, Rae Farnsworth, Marc G. Achen, Steven A. Stacker, Tara Karnezis

Research output: Contribution to journalArticleResearchpeer-review

3 Citations (Scopus)


The lymphatic system is a series of vessels that transport cells and excess fluid from tissues to the blood vascular system. Normally quiescent, the lymphatics can grow or remodel in response to developmental, immunological, or cells pathological stimuli. Lymphatic vessels comprise lymphatic endothelial cells (LECs) that can respond to external growth factors by undergoing proliferation, migration, adhesion, and tube and lumen formation into new vessel structures, a process known as lymphangiogenesis. To understand the key gene and signaling pathways necessary for lymphangiogenesis and lymphatic vessel remodeling, we have developed a three-dimensional LEC tube formation assay to explore the role of kinase signaling in these processes. The collagen-overlay-based assay was used with primary human adult dermal LECs to investigate a library of 60 tyrosine kinase (TK) and TK-like genes by siRNA knockdown. Nine candidate genes were identified and characterized for their ability to modify key parameters of lymphatic tube formation, including tube length, area, thickness, branching, and number of blind-ended sacs. Four genes - ZAP70, IRAK4, RIPK1, and RIPK2 - were identified as high-confidence hits after tertiary deconvolution screens and demonstrate the utility of the assay to define LEC genes critical for the formation of tube structures. This assay facilitates the identification of potential molecular targets for novel drugs designed to modulate the remodeling of lymphatics that is important for the metastatic spread of cancer and other pathologies.

Original languageEnglish
Pages (from-to)30-43
Number of pages14
JournalAssay and Drug Development Technologies
Issue number1
Publication statusPublished - 1 Jan 2017


  • lymphatics
  • endothelium
  • kinases
  • tube formation

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