Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma

Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a “tortuous” vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvβ3 were confirmed by in vivo 99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.


Synthesis of the 99m Tc RGD radioligand
Briefly, 7.5-10 mg of HYNICRGD was incubated with 1 ml of an EDDA/tricine mixture (20 mg/mL, 10 mg/ml EDDA, pH adjusted to 8), 1850 MBq of 99m TcO 4in saline (0.5-1 mL depending on generator calibration) and 20 mg of SnCl 2 (20 mL of a tin(II) solution of 1 mg of SnCl 2 in 1 mL of 0.1 N aqueous HCl) for 10 min at 100 °C. ITLC strips (dark green) were purchased from Biodex (New York, NY, USA), and the solvents used were methyl ethyl ketone and an acetonitrile/water solution (1:1). A sample of 0.1 mCi of the mixture was submitted to thin layer chromatography (TLC) analysis, and the radiochemical purity was estimated with a MiniGita Star Radiochromatograph (Raytest, Straubenhardt, Germany). Methyl ethyl ketone was used as the eluent for the detection of 99m Tc-pertechnetate (R f =1), and an acetonitrile/water solution was the eluent for the determination of reduced hydrolyzed 99m Tc ( 99m Tc colloid, R f =0).

MicroSPECT image analyses
After 2 hour-post [ 99m Tc]HYNIC-RGD injection, SPECT scans were acquired using 64 projections over 360° (ROR=9.3 cm, 15 s/ projection). SPECT projections were reconstructed with the filtered back projections algorithm and Hamming filter (0.6 cut-off frequency). Each reconstructed matrix (80x80 pixels) was composed of 80 transverse images with a voxel size of 2.24x2.24x1.5 mm. Further, after SPECT acquisition, CT scans were acquired at 50 kV (320 mA) with 1024 projections over 360° and an FOV of 91 mm. Each reconstructed matrix (512x512) was composed of 512 transverse images with a cubic voxel edge dimension of 0.15 mm.

Visualization and quantification of radioactivity with a β imager
After intravenous injections, resected consecutive 4 mm thick slices were stained with H&E and prepared for β imaging. H&E histological sections were examined under an Olympus DX51 microscope (Olympus, Paris, France). Quantification of necrotic index was performed with Image J software. Quantification of [ 99m Tc]HYNIC-RGD binding was performed on a high speed Autoradiography β imager (BioSpace Lab, Nesles la Vallée, France). Briefly, dried consecutive sections of tumors were placed in a sample holder inside the detection chamber of the β imager. The levels of bound activity in the tumor sections were directly determined by counting the number of βparticles emerging from the tissue sections. The M3 Vision program (BioSpace Lab) was used to measure the activities in the region of interest (i.e. whole tumor section or by discriminating tumor parenchyma of necrotic areas). The radioligand binding signal was expressed in counts per minute per square millimeter (cpm/mm²).

Data analysis
Reconstructed data from SPECT, CT and planar imaging were analyzed using OsiriX-64 imaging software (Pixmeo, Geneva, Switzerland). Quantifications of [ 99m Tc]-HYNIC-RGD uptake in tumors were performed using MATLAB software (MathWorks, Meudon, France), expressed in terms of standardized uptake values (SUVs) and tumor to muscle (T/M) ratios. The T/M ratio was defined as follows. To define the tumor region of interest (ROI), we used a volumetric ROI that encompassed the entire lesion. To define the muscle ROI, a circular ROI (diameter 7 mm) was placed in both limbs; the mean value between these two ROIs was used to compute T/M ratios. The SUV mean was computed according to the following formula: SUV = [tumor activity (Bq/mL)]/[injected activity (Bq)/animal weight (g)], assuming a density of 1 g/cm 3 . The SUV max was defined as the maximum voxel value. Table  60°C/20s, a step specific for UII mRNA (73°C/5s), and 95°C/1s using the primers specified in Table  S1. Minus-reverse transcription ("-RT") controls were systematically performed and quality of PCR products was evaluated by generating a melting curve, which was also used to verify the absence of PCR artifacts (primer dimers) or non-specific PCR products. Samples were amplified at least in triplicates (3 different culture flasks and 3 different RT) and relative mRNA copy levels were determined using the comparative ∆∆Ct method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or Ubiquitin C (UBC) transcript levels were used as a reference, to control mRNA levels and stability within each cell line. Results were analyzed by using the Quantstudio design and analysis software (Applied Biosystems) and are expressed as mean of gene of interest expression relative to GAPDH or UBC reference gene.

Statistical analyses
Data were expressed as mean ± SEM and GraphPad Prism (version 5; GraphPad Software, Inc, La Jolla, USA) was used for statistical analyses. Student t test was used for parametric comparisons between paired variables, the Mann-Whitney U test was used for nonparametric pairwise comparisons, multivariate analyses were done with ANOVA with post hoc Dunnett (in vitro analyses) or Bonferroni tests (e.g., in vivo studies) as appropriate, and survival curves were generated by the Kaplan-Meier method. All reported P values were two-sided and considered to be statistically significant at P < 0.05. Supplementary Table S1. The primer sequences used for amplification of human UTS2, UTS2D, UTS2R, MMP9, ITGAV mRNAs and the mRNA encoding the housekeeping gene GAPDH or UBC. UBC, Ubiquitin C gene, GAPDH, Glyceraldehyde 3-Phosphate DeHydrogenase.
Supplementary Figure S3. Effects of a high dose of urantide on tumor growth. A, Tumor growth of Swiss Nude mice transplanted with U87 glioblastoma treated as in Figure 5D with vehicle (10 µL, n=10), UII (0.29 ng/kg, n=10) or urantide (290 ng/kg, n=10). High dose of urantide induced a persistent inhibition of tumor growth. Statistical significance for tumor growth was given by using two-way ANOVA with Bonferonni post hoc test compared with vehicle. **, P < 0.01; ***, P < 0.001. B, Late tumor growth of Swiss Nude mice transplanted with U87 glioblastoma treated with vehicle (10 µL, n=7) or urantide (29 ng/kg, , 290 ng/kg, , n=10) when tumor volume reached 500 mm 3 . The higher dose of urantide induced inflexion of the tumor growth kinetic for one week duration. Statistical significance for tumor growth was given by using two-way ANOVA with Bonferonni post hoc test compared with vehicle.
Supplementary Figure S5. Impact of UII on MMP9 and ITGAV alphav integrin gene expression in GBM and hCMEC/D3 cells. Cells were treated with UII (10 -9 M, 24 h) in the absence of FBS, and mRNAs were extracted for RT-qPCR analysis. MMP9 and ITGAV mRNA expressions were presented as ∆∆Ct, related to the UBC gene expression in GBM and hCMEC/D3 cells. Data were expressed as mean ± SEM of three independent cultures, and normalized to the housekeeping gene UBC. Statistical significance of treatments vs control condition was assessed with Mann-Whitney test. *, P<0.05.