Generation and purification of the anti-Tie1 nanobody
The protocol for nanobody generation was adapted from Shlamkovich et al. (37). The selected nanobody (i.e., NB19) was purified using periplasmic extraction, followed by Ni-NTA affinity chromatography and size-exclusion chromatography (SEC), as described in detail in the supplementary information (SI) section.
NB19 binding analysis using surface plasmon resonance (SPR)
The binding interactions between NB19 and Tie1-ECD were analyzed (Proteomics Unit, NIBN, BGU) in real time by surface plasmon resonance (SPR) on a ProteOn XPR36 instrument (Bio-Rad, Hercules, CA, USA). The ProteOn GLC sensor chip was initialized in air, and PBST (PBS, 0.005% Tween) buffer was flushed through the instrument prior to the binding experiments. The purified NB19 protein was immobilized on the surface of the GLC sensor chip by using the amine coupling reagents N-hydroxysuccinimide (0.1 M; sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (0.4 M; EDC; Bio-Rad), as follows. NB19 (10 µg) in 10 mM sodium acetate, pH 4.0, was allowed to flow over an activated GLC sensor chip channel surface, at a flow rate of 30 µL/min, until the target immobilization level was reached. Bovine serum albumin (BSA), 3 µg, in 10 mM sodium acetate, pH 4.5, was then allowed to flow over the activated surface of a control GLC sensor chip channel at a flow rate of 30 µL/min until the NB19 ligand immobilization level was reached. After NB19 immobilization, the chip surface was treated with 1 M ethanolamine HCl at pH 8.5 to deactivate excess reactive esters. All binding experiments were performed at 25°C in a degassed binding buffer (PBS, pH 7.4). Recombinant human Tie1-ECD (R&D Systems, Minneapolis, MN, USA) and recombinant human Tie2 extracellular domain (Tie2-ECD, R&D Systems) at a range of concentrations (10–0.625 nM and 50–6.25 nM, respectively) were allowed to flow over the surface-immobilized NB19 at a flow rate of 30 µL/min for 60 min, and the Tie1-ECD–NB19 and Tie2-ECD–NB19 binding interactions were monitored. After complex dissociation, which was monitored for 60 min, a regeneration step with 50 mM NaOH at a flow rate of 100 µL/min was performed. The analyte sensorgram run was normalized by subtracting the BSA-immobilized channel and the zero-analyte concentration runs. The binding constant (KD) was determined from the sensorgrams of the equilibrium binding phase. Binding kinetics of NB19 were analyzed by fitting to a Langmuir model.
Cell culture
Human embryonic kidney 293 cells (HEK293; ATCC, Manassas, VA, USA) were cultured in complete growth medium composed of DMEM High Glucose (Biological Industries, Kibbutz Beit-Haemek, Israel) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 1% l-glutamine (Biological Industries), and 1% penicillin/streptomycin (Biological Industries) under 5% CO2 at 37°C. HEK293 cells, which lack endogenous Tie receptors, were transiently transfected with full-length HA-tagged Tie1 (pCMV3-TIE1-HA refseq: NM_005424.2) and full-length myc-tagged Tie2 (pCMV3-C-Myc redseq: NM_000459.3). When the cells reached 80–90% confluence in 6-well plates, transfections were performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. Human telomerase-immortalized microvascular endothelium (TIME) cells (ATCC), which endogenously express Tie1 and Tie2, were cultured under 5% CO2 at 37°C, in Vascular Cell Basal Medium (ATCC) supplemented with 10% FBS and Microvascular Endothelial Cell Growth Kit-VEGF (ATCC) according to the manufacturer's instructions. Human glioblastoma (U87-MG) cells were grown under 5% CO2 at 37°C in minimum essential medium supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin.
Cell-binding assay using flow cytometry
Expression of Tie1 and Tie2 and binding of purified NB19 to receptor-expressing cells was analyzed by flow cytometry using TIME or U87-MG cells endogenously expressing Tie1 and Tie2 and HEK293 cells overexpressing Tie1, Tie2 or both receptors. To determine the expression levels of Tie1 and Tie2 in HEK293 cells following transient transfection, cells were centrifuged and resuspended in 100 µL of PBSA with 1:100 allophycocyanin (APC)-labeled anti-human Tie2 antibody (BioLegend) or phycoerythrin (PE)-labeled anti-human Tie1 antibody (R&D Systems), incubated at 4°C for 30 min, and washed three times with 100 µL of PBSA before flow cytometry analysis. Unstained cells were used to determine the background signal. For binding assays, Tie1- and Tie2-expressing TIME (or U87-MG) cells and HEK293 cells transiently transfected with Tie1, Tie2 or both receptors were incubated with NB19 at different concentrations in a total volume of 200 µL of PBSA at 4°C for 1 h with gentle agitation. Thereafter, the cell suspensions were centrifuged at 150 g at 4°C for 5 min and washed three times with 100 µL of PBSA. Cells were then resuspended in 100 µL of PBSA containing a 1:200 dilution of APC-conjugated anti-FLAG antibody (BioLegend) for the detection of NB19 bound to the cells. After 30 min on ice, the cells were washed twice in PBSA and analyzed by flow cytometry with a BD FACSCanto™ II Flow Cytometry System (BD Biosciences, San Jose, CA, USA). Mean fluorescence levels of the background were subtracted, and the data was analyzed using FlowJo (Tree Star Inc., OR, USA) analysis software.
Immunofluorescence microscopy
HEK293 cells were seeded on sterile cover slips and grown to confluence before being transfected using Lipofectamine 2000 (Invitrogen) with plasmids encoding either full-length HA-tagged Tie1 (pCMV3-TIE1-HA; refseq: NM_005424.2) or full-length myc-tagged Tie2 (pCMV3-C-Myc; redseq: NM_000459.3). Untransfected cells were used as the negative control. Twenty-four hours post-transfection, the cells were washed with PBS and then incubated for 30 min at 4°C with 100 nM NB19. The cells were then washed with PBS, fixed for 15 min in 4% paraformaldehyde, and permeabilized for 10 min with 0.5% Triton X-100 )Bio-Lab Ltd, Jerusalem, Israel) in PBS. The cells were blocked for 30 min with gentle shaking at room temperature in 5% BSA in PBS, followed by two washes with PBS. The cells were then incubated in 5% BSA/PBS for 1 h at 4°C with FITC anti-human Tie2 (BioLegend) or PE-labeled anti-human Tie1 (BioLegend), and APC-conjugated anti-FLAG antibody (BioLegend) for the detection of NB19 colocalization with Tie1 and Tie2. Confocal images were captured using a 20× Zeiss Plan-Apochromat dry, 0.8 NA, DIC objective on a laser scanning confocal microscope (Zeiss Axio-Observer Z1 inverted microscope, Ilse Katz Institute for Nanoscale Science & Technology, BGU) and analyzed with Zeiss software.
Inhibition of Tie1 by siRNA
For small interfering RNA (siRNA)-mediated knockdown of Tie1, TIME cells were plated at 80% confluency one day before transfection and then transfected with Tie1 siRNA using the TriFECTa RNAi kit (IDT, San-Jose, CA) with the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions.
Tie1, Tie2, Akt and FAK phosphorylation assays
Confluent TIME cells were cultured on 12-well plates in growth-factor depleted Vascular Cell Basal Medium (ATCC) supplemented with 0.5% FBS for 12 h at 37°C, under 5% CO2. The cells were then washed with PBS, and the medium was exchanged with fresh Vascular Cell Basal Medium depleted of growth factors and serum (ATCC). Following treatment with 1 mM sodium orthovanadate (Na3VO4; Sigma) for 15 min, the cells were co-incubated for 15 min (for Tie1 and Tie2 analysis), or for 30 min (for FAK and Akt analysis), at 37°C with either 500 ng/ml human recombinant Ang1 (R&D systems) as the positive control, or 30 nM (or 300 nM) NB19 combined with 500 ng/ml Ang1. Non-stimulated (in the absence of Ang1) cells served as the negative control. The cells were then washed twice with PBS containing 1 mM Na3VO4 and lysed in ice-cold lysis buffer [20 mM HEPES, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM Na3VO4, and 1× complete protease inhibitor cocktail tablet (Roche, USA)]. The cells were scraped from the culture plate wells, and the lysates were clarified by centrifugation (13,000 rpm for 30 min at 4°C). Protein concentration was measured by the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific), and equivalent amounts of each lysate sample were analyzed by 10% SDS-PAGE transferred to PVDF blotting membranes (BioRad). Blots were blocked (5% BSA, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature and then probed, following an overnight incubation at 4°C, with the following antibodies, all at a 1:1000 dilution: phospho-Tie1 (Tyr1117) specific rabbit polyclonal antibody (Invitrogen), anti-Tie1-specific rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), phospho-Tie2 (Y992) specific rabbit polyclonal antibody (R&D Systems), anti-Tie2-specific rabbit monoclonal antibody (Cell Signaling Technology), anti-phospho-Akt-specific antibody (Cell Signaling Technology), anti-Akt-specific antibody (Cell Signaling Technology), anti-phospho-FAK-specific (Tyr397) antibody (Cell Signaling Technology) or anti-FAK-specific antibody (Cell Signaling Technology). PVDF membranes were washed three times with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and probed with a 1:1000 dilution of HRP-linked anti-rabbit antibodies (Cell Signaling Technology) for 1 h at room temperature. Membranes were washed three times with TBST and then visualized and quantified using chemiluminescence (ECL, Biological Industries) and ImageJ software, respectively. The intensities of the phospho-Tie1, phospho-Tie2, phospho-Akt, and phospho-FAK bands were adjusted for the expression of total Tie1, Tie2, Akt, and FAK, respectively, for each experiment. Blots were stripped and re-probed with a 1:1000 dilution of anti-β-actin antibody (Cell Signaling Technology) followed by an HRP-conjugated anti-mouse antibody (at 1:1000 dilution, Cell Signaling Technology) for further normalization. Each experimental condition was repeated in triplicate. Band intensities of the phosphorylated proteins (Tie1, Tie2, Akt, and FAK) (as measured by ImageJ software) were normalized to the respective total protein levels, and this value was subsequently normalized to the total amount of β-actin for each sample.
Capillary-like tube formation assay
Serum-reduced Matrigel (10 mg/ml; BD Biosciences) was thawed overnight at 4°C, and 150 µL were added to each well of a µ-Slide 8 Well (ibidi, Gräfelfing, Germany) and allowed to solidify for 1 h at 37°C. In each well, the Matrigel was then incubated with 1×105 TIME cells treated with either 500 ng/mL Ang1 or with a combination of 500 ng/mL Ang1 and 30 nM (or 300 nM) NB19; untreated cells were used as the negative control. The cells were incubated for 18 h at 37°C, under 5% CO2, and then washed twice in Hanks’ balanced salt solution (Sigma-Aldrich). Capillary tube formation was observed on confocal images captured using a the Zeiss Plan-Apochromat laser scanning confocal microscope described above and analyzed with Zeiss software. Tube structures were analyzed for the number of junctions generated, and the total tube length was quantified by the analysis of digitized images of the capillary-like structures using ImageJ software and the Angiogenesis Analyzer plugin.
Cell proliferation assay
The effects of NB19 on the growth and survival of TIME or U87-MG cells were assessed by an XTT assay (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay; Biological Industries). TIME or U87-MG cells were seeded (7,500 cells per well) on a human vitronectin-coated 96-well microplate (R&D systems) and incubated in growth medium for 24 h at 37°C, under 5% CO2. The cell growth medium was then replaced with fresh Vascular Cell Basal Medium (ATCC) or minimum essential medium for TIME or U87-MG cells, respectively. In each case the medium was supplemented with 2% FBS and growth factor supplements, and the cells were incubated with either 500 ng/mL Ang1 or with a combination of 500 ng/mL Ang1 and 30 nM (or 300 nM) NB19 for 24 h at 37°C, under 5% CO2. Viable cells from each condition were measured by XTT at UV 450 nm, as described in the manufacturer’s protocol. The UV readings of the cell-only (untreated) control were set at 100% viability, and readings from cells treated with NB19, a mixture of NB19 and Ang1, or Ang1 alone were expressed as % of the control.
Wound healing assay
The in-vitro wound healing assay was performed as previously described with some modifications (38). Briefly, U87-MG cells were seeded in 24-well plates at a density of 1.5×105 cells in each well and allowed to grow for 24 h at 37°C, under 5% CO2, until they reached confluence. A linear scratch was created in each confluent monolayer by gently scraping the surface a with sterile p200 pipette tip (care was taken during scratching process to ensure similar scratches for all samples). The scratched monolayers were then washed twice to remove nonadherent cells. Ang1 (500 ng/mL)-treated cells were used as the positive control, and untreated cells, as the negative control. Test samples included mixtures of 30 nM (or 300 nM) of NB19 and Ang1 (500 ng/mL). Images were taken exactly at the same position prior to and post incubation of the cells with the different treatments or controls so as to document the wound closure process. Wounds were viewed under a light microscope immediately after the scratches were made and again after 6, 12, 18, and 24 h of incubation at 37°C in a 5% CO2 incubator. The experiment was performed in triplicate, and the images were analyzed using ImageJ.
Statistical analysis
Data were analyzed with GraphPad Prism version 9.00 for Windows (La Jolla, CA). SD (standard deviation) was determined from at least three independent experiments. Comparisons between the groups were performed using a t-test (for two independent samples) and with one-way ANOVA (for multiple groups). A probability value of < 0.05 was considered statistically significant.