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Assessment of the Anti-angiogenic Effect of VEGFR2 siRNA in Clonetics™ HUVEC using the Lonza 4D-Nucleofector™ System
Poster Title: Assessment of the Anti-angiogenic Effect of VEGFR2 siRNA in Clonetics™ HUVEC using the Lonza 4D-Nucleofector™ System
Submitted on 26 Apr 2016
Author(s): Srinivasan Kokatam1 , Kanchan Tiwari1 , Jenny Schroeder2 , Andrea Toell2 , Lubna Hussain3, Preeti Kapoor1
Affiliations: 1Lonza India Pvt Ltd, Hyderabad, India; 2Lonza Cologne GmbH, Cologne, Germany; 3Lonza Walkersville, Inc., Walkersville, MD, U.S.A.
This poster was presented at AACR 2016
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Poster Information
Abstract: Angiogenesis is a hallmark of most cancers, and is thus an attractive arget for the treatment of cancer. One of the easiest screening and target validation strategies for antiangiogenic target identification nvolves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation in Corning’s Matrigel™ product.
The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector™ Technology.
In the current study we used the Lonza 4D-Nucleofector™ X-Unit to transfect single-donor Clonetics™ HUVEC cultured in EGM™ 2 Endothelial Cell Growth Media. Cells were transfected with siRNA directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Transfection conditions were fine-tuned using pmaxGFP™ Vector, and it was shown that the Nucleofection process had no deleterious effect on the tube formation potential of HUVEC on Corning’s Matrigel™ product.
Efficient knock-down of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency was observed 24 hours after transfection.
VEGFR2-siRNA transfected cells demonstrated significant inhibition of tube formation on Corning’s Growth Factor Reduced Matrigel™ product in comparison to control samples.
Since the efficient knock-down of the VEGFR2 protein in Clonetics™ HUVEC using the Nucleofector™ Technology can be demonstrated at time points as early as 24 hours after
transfection, this is an efficient approach for target identification, validation and screening of siRNA based anti-angiogenesis therapeutics for cancer treatment.
Summary: In the current study we have used siRNA targeting VEGFR2 as an example to study knockdown of VEGFR2 and subsequent inhibition of tube formation by HUVECs on Growth Factor Reduced Matrigel™ in a 96-well plate format. The same strategy can be used for screening and validating siRNA based inhibitors of the angiogenic process in vitro and thus could be of utility in anti-cancer screening strategies.References: Timar, J. et. al. (2001) Angiogenesis-dependent diseases and angiogenesis therapy. Pathol. Oncol. Res.7 (2): 85-94.
Murga M1, Fernandez-Capetillo O, Tosato G. (2005) Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2. Blood 105 (5): 1992-9.
Yang, S, Xin, X, Zlot, C et. al. (2001) Vascular Endothelial Cell Growth Factor-Driven Endothelial Tube Formation Is Mediated by Vascular Endothelial Cell Growth Factor Receptor-2, a Kinase Insert Domain-Containing Receptor. Arterioscler. Thromb. Vasc. Biol. 21: 1934-1940
Zumbansen M, Altrogge L, Toell A, Leake D, Muller-Hartmann H (2009) First siRNA Library Screening in Difficult-to-Transfect HUVEC and Jurkat Cells. White paper. (“ siRNA_Library_Screening_in_Hard-to-Transfect_HUVEC_and_Jurkat_Cells.pdf”)
Zeitelhofer M, Vessey JP, Xie Y, Tubing F, Thomas S, Kiebler M, Dahm R (2007) High
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