Assessment of the Anti-Angiogenic Effect of VEGFR2 sirna in HUVEC Using the Nucleofector Technology

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1 BioResearch Assessment of the Anti-Angiogenic Effect of VEGFR2 sirna in HUVEC Using the Nucleofector Technology Srinivasan Kokatam 1, Kanchan Tiwari 1, Jenny Schroeder 2, Andrea Toell 2, Lubna Hussain 3, Preeti Kapoor 1. 1 Lonza India Pvt Ltd, Hyderabad, India; 2 Lonza Cologne GmbH, Cologne, Germany; 3 Lonza Walkersville, Inc., U.S.A., Walkersville, MD Abstract Angiogenesis is a hallmark of most cancers, and is thus an attractive phenomenon for the treatment of cancer. One of the easiest screening and target validation strategies for anti-angiogenic target identification involves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation. 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 have used our Nucleofector Technology to transfect single-donor Clonetics HUVEC with sirna directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Efficient knockdown of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency of 90% 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. The efficient knock-down of the VEGFR2 protein in Clonetics HUVEC as early as 24 hours after transfection, shows that the Nucleofector Technology is an efficient approach for screening, target identification and validation. Introduction The key role of angiogenesis in tumor development and cancer metastasis makes inhibition of angiogenesis an attractive strategy to target a number of cancer types (Timar et. al., 2001). VEGF (Vascular Endothelial Growth Factor) is a key cytokine involved in physiological and pathological angiogenesis. While VEGF mediates its effects through multiple cell surface receptors, VEGFR2 appears to be the major mediator of VEGF-induced proliferation and migration of endothelial cells (Murga et. al., 2005), and endothelial morphogenesis into tube-like structures (Yang et. al., 2001). Thus VEGFR2 based pathway inhibition becomes interesting from a standpoint of cancer therapeutic intervention. sirna and shrna based gene silencing is being increasingly used to identify genes and pathways involved in various cellular processes within mammalian cells and to enable target identification and validation. RNAibased approaches are being utilized in the cancer area, for therapeutic approaches, and to identify and characterize cancer genes involved in specific pathways, disease etiology and the progression of cancer (Guo et. al., 2013). Screening with sirna or shrna involves delivery into cells of interest using various approaches. The most commonly used approach is the lipid-mediated transfection methodology; however many of these reagents can be cytotoxic, alter gene expression profiles and induce immunogenic responses. Moreover they demonstrate low transfection efficiency in hard-to-transfect primary cells or suspension cell lines; and do not work for non-dividing cells like neurons, due to their dependence on cell division (Zumbansen et. al., 2009). Thus for applications where high transfection efficiencies and higher cell viabilities are essential, Nucleofection is the method of choice (Gresch O, Altrogge L. 2012; von Levetzow et. al., 2006; Zeitelhofer et. al., 2007). With this optimized electroporation-based technology, a large proportion of the substrate is delivered directly into the nucleus. The Nucleofector Systems from Lonza (Figure 1) enable efficient transfection of primary cells and cell lines using a variety of substrates like DNA and sirna.

2 In the current study we have used sirna specific to VEGFR2 to study its knockdown and subsequent inhibition of tube formation by HUVECs. The same strategy can be used for screening / validating sirna based inhibitors of the angiogenic process in vitro and thus could be of utility in anti-cancer screening strategies. A B Transfection with sirna constructs HUVEC cells were transfected with SMARTpool ON-TARGETplus KDR sirna targeting VEGFR2 (GE Dharmacon, Cat. No. L ) and the ON-TARGETplus Non-targeting Pool (GE Dharmacon, Cat. No. D ) (Neg) using the fine-tuned Nucleofector Program (DG-167) identified from the pmaxgfp Vector transfection studies. A no sirna control set (SC), which was subjected to the transfection process, was also included in the assay. Varying concentrations of sirna (0.1 2 µm) were used to assess the effect of sirna on cell viability and VEGFR2 knockdown in cells. C D E After transfection, cells were plated in parallel for VEGFR2 ELISA and ViaLight Assay in 96 well plates and incubated for varying time points in complete EGM-2 media. Cells were harvested at 24 hours, 48 hours, 72 hours and 96 hours after transfection to determine the time point of most efficient VEGFR2 knockdown. Figure 1. Nucleofector Systems used in this study along with accessories: A. 96-well Shuttle Add-on, B. 96-well Nucleocuvette Plate, C. 4D-Nucleofector System, D. 100 µl Nucleocuvette Vessel and E. 20 µl 16-well Nucleocuvette Strip Methods Cell maintenance Single-donor Clonetics HUVEC cells (Lonza, Cat. No. C2517A; lot# 94182) were maintained in complete EGM -2 Endothelial Growth Medium-2 (Lonza, Cat. No. CC-3162) as per manufacturers instructions. They were maintained at 70 80% confluence and used within passage 5. Cells were passaged at least once using ReagentPack (Lonza, Cat. No. CC-5034) after being removed from liquid nitrogen and before being used for any assay. Transfection of cells using the 4D-Nucleofector System Transfection with pmaxgfp Vector Some fine tuning of transfection conditions was done for this specific lot of HUVEC using the 4D Nucleofector System in combination with the 96-well Shuttle Add-on. Briefly, cells (1 x 10 5 cells) were transfected with 0.4 μg pmaxgfp Vector using the P5 Primary Cell Nucleofector Kits (Lonza, Cat. No. V4SP-5096, V4XP-5032, or V4XP-5012 depending on the format used). Subsequently, 2.5 x 10 4 cells were seeded and transfection efficiency was analyzed 24 hours post-transfection using flow cytometry. Viability was determined in parallel using the ViaLight Plus BioAssay Kit (Lonza, Cat. No. LT ). Transfection efficiencies and viabilities were expressed as percentages of non-transfected, no program control () values. The tube formation potential of the transfected cells was checked following 24 hours after transfection using the previously standardized protocol (Kapoor et. al., 2014). VEGFR2 ELISA Cells were washed and lysed using the M-PER Mammalian Protein Extraction Reagent (Pierce, Cat. No ) as per manufacturer s instructions. Lysates were collected and subjected to the human total VEGFR2/KDR ELISA (R&D Systems, Cat No. DYC1780-5) as per the kit s instructions. Data was normalized to cell numbers obtained from the ViaLight standard curve and then expressed as percentage of no sirna control (SC) values. The detection limit of the kit was 62 pg/ml. ViaLight Assay and cell number assessment Cell viability was determined using the ViaLight Assay Kit (Lonza, Cat. No. LT ) as per the kit protocol. A standard curve was made by plotting RLU (Relative Luminescence Units) values against cell number, and number of cells was determined from the standard curve. Tube formation setup on Growth Factor Reduced Matrigel After transfection with sirna, cells were plated in cell culture flasks and incubated for 4 hours in complete EGM-2 media to recover from the transfection process. Subsequently, medium was changed to starvation media (reduced FBS, no cytokines, no VEGF) and cells were incubated for another 18 hours in starvation media, after which they were harvested, counted and plated onto Phenol-Red Free Growth Factor Reduced Matrigel (Corning, Cat. No ) for tube formation assay in 96 well format using the previously standardized protocol (Kapoor et. al., 2014). Tube formation was observed after 18 hours of incubation under 5X magnification under bright field, and fluorescence images were taken after staining with Calcein AM (Life Technologies, Cat. no. C3100MP) (Kapoor et. al., 2014). 2

3 Results & Discussion HUVECs isolated from a single donor (lot# 94182) were transfected with pmaxgfp Vector and a set of programs using the 4D-Nucleofector System (either 96-well Shuttle Add-on or X Unit) to find the best program for this HUVEC lot regarding transfection efficiency and cell viability in both the 20 µl Nucleocuvette Strip and the single 100 µl Nucleocuvette Vessel. For this specific HUVEC lot, Nucleofector Program DG-167 showed superior results compared to the standard HUVEC program CA-167 (Figure 2). For both programs cell viabilities of >60% compared to a no program control were obtained, but program DG-167 demonstrated higher transfection efficiencies in both strip and cuvette formats. As shown in Figure 3, morphology of the cells was not affected by transfection. Efficient maxgfp Expression was observed as green fluorescent cells at 24 hours after transfection. Higher levels of maxgfp Expression were found in cells transfected with program DG-167 compared to program CA-167. Subsequently it was analyzed whether the transfection process itself had an impact on the tube formation potential of the HUVEC. As shown in Figure 4, tube formation was not adversely impacted by transfection with both the programs and maxgfp Protein was expressed in tubes at higher expression with the DG-167 program in comparison to the CA- 167 program. This unaltered tube formation could be observed for HUVEC transfected in both the strip and cuvette format Figure-5. CA-167 DG-167 Percentage (of Values) Strip-CA-167 % Efficiency-Avg % Viability-Avg Cuvette-CA-167 Strip-DG-167 Cuvette-DG-167 Strip Cuvette Strip Cuvette CA-167 CA-167 DG-167 DG Figure 4. Tube formation capability of pmaxgfp Vector transfected HUVEC at 24 hours assay point. All images were taken under 5X magnification. Strip Cuvette Phase Contrast Image Pre-Calcein Image Calcein Stained Image CA-167 DG-167 Figure 2. Transfection efficiency and cell viability of HUVEC in the 20 µl Nucleocuvette Strip and the 100 µl Nucleocuvette Format with pmaxgfp Vector and optimized transfection conditions in the 4D-Nucleofector X Unit. CA-167 Figure 5. No adverse effect of transfection on tube formation potential of HUVEC observed in both the Nucleocuvette Strip (20 µl per reaction) and the single Nucleocuvette Vessel (100 µl per reaction). All images were taken under 5X magnification after Calcein AM staining. HUVEC were transfected with VEGFR2 sirna, non-targeting pool of sirna (), or no sirna (Substrate control SC) in the 96-well Shuttle Add-on using the optimized program DG-167. The dose response of the sirna was studied from 0.1 µm to 2.0 µm for hours time point to evaluate the optimum concentration and time to observe for the efficient VEGFR2 gene knock down at the protein level while maintaining highest cell viability. DG-167 Bright Field Images Unstained Images Under FITC Filter Figure 3. Morphology and expression of maxgfp Protein 24 hours after transfection of HUVEC in the 20 µl Nucleocuvette Strip. 3

4 %Viability in Clonetics HUVECs Following sirna Transfection - 24 hour Data %Viability in Clonetics HUVECs Following sirna Transfection - 48 hour Data Viability (% of ) Viability (% of ) %Viability in Clonetics HUVECs Following sirna Transfection - 72 hour Data %Viability in Clonetics HUVECs Following sirna Transfection - 96 hour Data Viability (% of ) Viability (% of ) Figure 6. Effect of VEGFR2 sirna and Negative Control sirna on the viability of HUVEC hours after transfection. Viabilities were expressed as percentages of no sirna control values (SC set). Dose and time dependent effect of sirna on cell viability was studied in comparison to no sirna controls (SC). As shown in Figure 6 there was an approximately 40% reduction in cell viability of SC over no program control () values. However, there was no significant adverse effect of VEGFR2 sirna or over SC values at all the concentrations ( µm) and time points used in this study; suggesting that these concentrations of sirna are not cytotoxic to the HUVEC cells. The analysis of the dose and time dependent effect of VEGFR2 sirna on VEGFR2 protein levels in comparison to no sirna controls (SC) demonstrated a considerable reduction of the VEGFR2 protein amount per cell, with best knockdown efficiencies observed at 24 hours, and levels coming back to normal by hours (Figure 7). The 1.0 µm VEGFR2 sirna concentration evidenced maximum knockdown (65 100% at hours time point in comparison to no sirna control), and was thus chosen for all further tube formation studies. The optimized concentration of sirna with maximum VEGFR2 gene knock down at hours time point was chosen to study the effect on tube formation. Optimization of culture conditions with respect to e.g. FBS concentration was needed to optimally see the effects on tube formation as angiogenesis is a complicated process with many pathways acting in concert. HUVEC were transfected with either VEGFR2 sirna, or No sirna (SC) and tube formation assay was set-up hours after transfection in the respective experimental media (Figure 8). Figure 9 depicts tube formation in SC, Negative and VEGFR2 sirna sets in the presence of different concentration of FBS ranging from 0.5% 1.0%. The tube formation potential of VEGFR2 sirna transfected HUVEC cells was significantly reduced at all concentrations of FBS as compared to the SC and. This suggests that the VEGFR2 protein has an important role in tube formation. Interestingly, the entire sample sets demonstrated increased tube formation in response to increasing FBS concentrations, indicating that FBS contains factors promoting tube formation of HUVEC. In a subsequent experiment cytokine responsiveness of cells following sirna transfection was studied. Figure 10 depicts tube formation in no sirna, negative sirna and VEGFR2 sirna sets in the absence or presence of different cytokines and complete media, respectively. All three sample sets showed more pronounced tube formation when cytokines or complete media were used. However, in all cases the tube formation potential of the VEGFR2-siRNA transfected HUVEC was reduced compared to the respective no sirna and negative sirna samples. 4

5 %VEGFR2 Expression in Clonetics HUVECs Following sirna Transfection - 24 hour Data %VEGFR2 Expression in Clonetics HUVECs Following sirna Transfection - 48 hour Data VEGFR2 Expression (% of ) VEGFR2 expression (% of ) %VEGFR2 Expression in Clonetics HUVECs Following sirna Transfection - 72 hour Data %VEGFR2 Expression in Clonetics HUVECs Following sirna Transfection - 96 hour Data VEGFR2 Expression (% of VEGFR2 Expression (% of ) Figure 7. Effect of sirna on VEGFR2 protein levels in HUVEC hours after transfection. VEGFR2 protein levels were normalized to total cell numbers using the ViaLight Assay. VEGFR2 protein levels were expressed as percentages of no sirna control values (SC set). Efficient knock-down of VEGFR2 protein can be observed as early as 24 hours after transfection. Media Additives (to EBM 2 Basal Media) Complete Media Cytokine Media No Cytokine Fetal Bovine Serum (FBS) 2% 0.5% 0.5 1%(as indicated) 0.5% FBS Ascorbic Acid n n n Hydrocortisone n n n Gentamicin/Amphotericin-B (GA) n n n Heparin n n n 0.75% FBS hegf (Human Epidermal Growth Factor) n n hfgf-β (Human Fibroblast Growth Factor-Beta) n n R3-IGF-1 (R3-Insulin-like Growth Factor-1) n n VEGF (Vascular Endothelial Growth Factor) n Added externally (as indicated) Figure 8. Composition of different media used for the tube formation assay. Added externally (as indicated) 1% FBS Substrate Control VEGFR2 sirna Figure 9. Effect of VEGFR2 sirna on HUVEC tube formation in media containing different concentrations of FBS. 5

6 0.5% FBS + No Cytokines + No VEGF 0.5% FBS + Cytokines + No VEGF Complete Media Substrate Control VEGFR2 sirna Figure 10. Effect of VEGFR2 sirna on HUVEC tube formation in the presence or absence of cytokines in the media. Conclusions Transfection of HUVEC cells with small interfering RNA against VEGFR2 caused a significant decrease in VEGFR2 protein levels, and a discernible impairment of tube formation by HUVECs on Growth Factor Reduced Matrigel. The combination of the Nucleofector System, Clonetics HUVECs and FBS reduced cytokine free EGM -2 Media from Lonza turned out to be a valuable tool for establishing an in-vitro anti-angiogenesis model by gene silencing/knock-down process. Efficient knock-down of VEGFR2 protein was observed as early as 24 hours post-transfection while good cell viability and functionality was maintained. In addition, the high-throughput 96-well Shuttle Add-on makes the Nucleofector Technology an attractive methodology for screening large sirna libraries in a single screen in the area of (anti-)angiogenesis research and other research fields. References 1. Tímár, J. et. al. (2001) Angiogenesis-dependent diseases and angiogenesis therapy. Pathol. Oncol. Res.7 (2): Murga M, 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): 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: 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) 5. Zeitelhofer M, Vessey JP, Xie Y, Tubing F, Thomas S, Kiebler M, Dahm R (2007) Highefficiency transfection of mammalian neurons via nucleofection. Nat. Protocols 2: Kapoor R, Reddy V. and Kapoor P. (2014) Tube Formation Assay with Primary Human Umbilical Vein Endothelial Cells. Resource Notes, 2014: ( uploads/tx_mwaxmarketingmaterial/lonza_newsletter_ _lonza_resource_ Notes_Spring.pdf) 7. Gresch O, Altrogge L. (2012) Transfection of difficult-to-transfect primary mammalian cells. Methods Mol Biol. 801: von Levetzow G, Spanholtz J, Beckmann J, Fischer J, Kögler G, Wernet P, Punzel M, Giebel B. (2006) Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells. Stem Cells Dev. Apr; 15(2):

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