title: A Three-Dimensional Lymphatic Endothelial Cell Tube Formation Assay to Identify Novel Kinases Involved in Lymphatic Vessel Remodeling.
abstract: 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.
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .
authors.forename: T Jessica
authors.initials: TJ
authors.lastname: Gambino
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .
authors.forename: Steven P
authors.initials: SP
authors.lastname: Williams
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .
authors.forename: Carol
authors.initials: C
authors.lastname: Caesar
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .
authors.forename: Daniel
authors.initials: D
authors.lastname: Resnick
authors.affiliation: 2 Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, Victoria, Australia .
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Nowell
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .
authors.forename: Rae H
authors.initials: RH
authors.lastname: Farnsworth
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .|3 Sir Peter MacCallum Department of Oncology, University of Melbourne , Melbourne, Victoria, Australia .|4 Department of Surgery, Royal Melbourne Hospital, University of Melbourne , Parkville, Victoria, Australia .
authors.forename: Marc G
authors.initials: MG
authors.lastname: Achen
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .|3 Sir Peter MacCallum Department of Oncology, University of Melbourne , Melbourne, Victoria, Australia .|4 Department of Surgery, Royal Melbourne Hospital, University of Melbourne , Parkville, Victoria, Australia .
authors.forename: Steven A
authors.initials: SA
authors.lastname: Stacker
authors.affiliation: 1 Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre , Melbourne, Victoria, Australia .|3 Sir Peter MacCallum Department of Oncology, University of Melbourne , Melbourne, Victoria, Australia .|5 O'Brien Institute , a Department of St. Vincent's Institute, Fitzroy, Victoria, Australia .
authors.forename: Tara
authors.initials: T
authors.lastname: Karnezis
mesh_terms: D018929:Cell Culture Techniques; D042783:Endothelial Cells / Q000737:chemistry / Q000502:physiology*; D061307:Human Umbilical Vein Endothelial Cells / Q000737:chemistry / Q000502:physiology; D006801:Humans; D042583:Lymphangiogenesis / Q000502:physiology*; D042601:Lymphatic Vessels / Q000737:chemistry / Q000166:cytology* / Q000502:physiology*; D034741:RNA, Small Interfering / Q000502:physiology; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; D053422:Receptor-Interacting Protein Serine-Threonine Kinases
keywords: endothelium; kinases; lymphatics; tube formation