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Evaluation of Angiogenesis Inhibitors Using the HUVEC Fibrin Bead Sprouting Assay   

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Original research article

A brief version of this protocol appeared in:
Cancer Research
Oct 2015

Abstract

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a critical process that occurs during normal development and tumor formation. Targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated some clinical benefit; nevertheless there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. Here, we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints.

Background

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a physiological process that occurs during wound healing and normal development. Angiogenesis is a complex and highly regulated process involving the tight coordination of endothelial cell proliferation, differentiation, migration, matrix adhesion, and cell-to-cell signaling. Angiogenesis is also critically involved in tumor development and metastasis. Indeed, targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated clinical benefit. Since tumors do eventually develop resistance to VEGF-targeted therapy, there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) (Nakatsu et al., 2007; Nakatsu and Hughes, 2008; Nehls and Drenckhahn, 1995) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. This assay recapitulates key aspects of angiogenesis such as lumen formation, endothelial cell polarization and dependency on stromal cells, and is correlative with the activities of angiogenesis inhibitors as observed in in vivo tumor studies (Figures 1 and 2) (Eichten et al., 2013; Holash et al., 2012; Kuhnert et al., 2015; Noguera-Troise et al., 2006). Here we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints, such as the number of endothelial cells or branch points, as well as sprout length and area (Figure 3). To illustrate the spectrum of treatment outcomes in the FBA, the effects of three different angiogenesis inhibitors [aflibercept, Dll4 blocking monoclonal antibody (Dll4 MAB) and anti-Integrin a6 antibody GOH3] on endothelial sprouting have been included in the protocol.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Winters, L., Thambi, N., Andreev, J. and Kuhnert, F. (2016). Evaluation of Angiogenesis Inhibitors Using the HUVEC Fibrin Bead Sprouting Assay. Bio-protocol 6(19): e1947. DOI: 10.21769/BioProtoc.1947.
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