Assessment of the Anti-angiogenic Effect of VEGFR2 siRNA in Clonetics™ HUVEC using the Lonza 4D-Nucleofector™ System
Poster Apr 26, 2016
Srinivasan Kokatam1 , Kanchan Tiwari1 , Jenny Schroeder2 , Andrea Toell2 , Lubna Hussain3, Preeti Kapoor1
Angiogenesis is a hallmark of most cancers, and is thus an attractive target for the treatment of cancer. One of the easiest screening and target validation strategies for antiangiogenic target identification involves 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.