All animal-involving procedures in this study were approved by the Faculty of Veterinary Medicine of Lisbon Ethics and Animal Welfare Committee. The generation of Dll4
(Dll4/LacZ) mice on CD1 background has been reported previously . The transgenic RIP1-Tag2 (RT2) mice of CD1 and C57/BL6 backgrounds, used for breeding with the Dll4
line and in experimental drug trials, respectively, were provided by Dr. Oriol Casanovas. Dll4 conditional knockout mice (Dll4
) were generated as previously described  and crossed with VE-cadherin-Cre-ERT2 mice, a kind gift from Dr. Ralph Adams, to produce a tamoxifen-inducible endothelial-specific Dll4 loss-of-function line (Dll4
Cre+). The animals were housed in well ventilated propylene cages with sawdust as bedding, in a room with controlled temperature between 22°C and 25°C and a 12-hours-light/12-hours-dark cycle. The mice were fed with standard laboratory diet and water ad libitum. From 12 weeks of age, all RT2 mice received 5% sugar in their water to relieve the hypoglycemia induced by the insulin-secreting tumors.
Experimental design, tumor burden analyses and therapeutic trials
To study the effects of impaired Dll4/Notch signaling on RT2 insulinoma growth, RT2 Dll4
and RT2 Dll4
littermates (CD1 background, n = 8 for each group) were sacrificed for tumor measurement, histological analysis of vascular morphology and gene expression analysis at 13.5 weeks of age. The pancreas glands were dissected and the macroscopic tumors (≥1 × 1 mm) were excised. Tumor volume was calculated using the formula V = 0.52 × a × b
2 where a and b equal the longer and shorter diameter of the tumor, respectively. The volumes of all tumors from each mouse were added to give the overall tumor burden per animal.
The effect of Dll4 allelic deletion in combination with Ephrin-B2/EphB4 signaling inhibition on the growth of the RT2 insulinoma was assessed by the administration of the soluble extracellular domain of EphB4 fused with albumin (sEphB4-Alb), which was produced as previously described . Both RT2 Dll4+/+ and RT2 Dll4+/- mice (CD1 background, n = 12 for each group) were separated in equal subgroups, treated intraperitoneally (i.p.) with vehicle (PBS) or sEphB4-Alb (10 mg/kg) 3×/wk for 3.5 weeks beginning at the age of 10 weeks and finally sacrificed for tumor measurement and histological analysis.
In the therapeutical trials. we assessed the efficacy of a systemically administered Dll4/Notch-inhibitor, soluble Dll4 extracellular domain fused to Fc (sDll4), both alone and in combination with sEphB4-Alb. sDll4 was produced as previously described . Vehicle (PBS, i.p. 3×/wk), sDll4 (10 mg/kg/day, i.p., 3×/wk), sEphB4-Alb (10 mg/kg/day, i.p., 3×/wk), and the combination of sDll4 (10 mg/kg/day, i.p., 3×/wk) with sEphB4-Alb (10 mg/kg/day, i.p., 3×/wk) treatments were started when RT2 mice (C57BL6 background) reached the age of 10 weeks and continued until mice were 13.5 week-old. Two independent experiments involved 6 animals per treatment group.
To evaluate the effect of Dll4 allelic deletion in combination with Ephrin-B2/EphB4 signaling inhibition on longevity, the RT2 Dll4
and RT2 Dll4
mice were separated in two equal groups (n = 10 for each group), treated i.p. with vehicle (PBS) or sEphB4-Alb (5 mg/kg, 3×/wk), beginning at the age of 10 weeks and continuously monitored for signs of hypoglycemic shock. The mice were sacrificed if found moribund or if body weight loss exceeded 15%. Survival rate was calculated as the percentage of live mice at the end of each week relative to the initial number of animals in the experimental group.
Assessment of toxicity
Heart, lung, liver, brain, kidney and intestines were collected from sDll4 and sEphB4-Alb treated mice used in the therapeutical trials, fixed in 10% formalin solution for 48 h, dehydrated in alcohol, cleared in xylene, embedded in paraffin, sectioned at 10 μm and stained with hematoxylin (Fluka AG Buchs SG Switzerland) and eosin Y (Sigma Chemicals, St. Louis, MO) to study eventual histopathological alterations. To assess the side effects that might arise from total (100%) inhibition of endothelial-specific Dll4 signaling, 8 week-old Dll4
Cre+ (n = 10) were treated with tamoxifen (50 mg/kg daily for 5 days) to produce endothelial-specific Dll4 null individuals, while a group of 8 week-old Dll4
Cre+ (n = 10) were left uninduced (control mice with constitutive Dll4 expression). Ten weeks later, the mice were sacrificed and heart, lung, liver, brain, kidney and intestines were collected, processed and examined as described above. Since Dll4 endothelial loss of function has been associated with hepatic lesions, we decided to determine the potential toxicological effect of a combination of Dll4/Notch and Ephrin-B2/EphB4 targeted therapy. Therefore another group of 8-week-old Dll4
Cre+ (n = 10) were treated with tamoxifen (50 mg/kg daily) for 5 days, subsequently divided in two equal subgroups that were injected with vehicle (PBS) or sEphB4-Alb (10 mg/kg) for ten weeks and then sacrificed. Liver samples were processed and examined as described above.
RT2 insulinomas obtained from tumor burden studies and therapeutical trials were fixed in a 4% paraformaldehyde (PFA) solution at 4°C for 1 h, cryoprotected in 15% sucrose, embedded in 7.5% gelatin, snap frozen in liquid nitrogen and cryosectioned at 10 and 20 μm. Double fluorescent immunostaining to the platelet endothelial cell adhesion molecule (PECAM) and the peri-vascular cell marker alpha smooth muscle actin (α-SMA) was performed on tissue sections to examine tumor vascular density and vessel maturity while double fluorescent immunostaining to PECAM and the pericyte marker neurogenin 2 chondroitin sulfate proteoglycan (NG2) was used to visualize pericyte recruitment. Rat monoclonal anti-mouse PECAM (BD Pharmingen, San Jose, CA), and rabbit polyclonal anti-mouse α-SMA (Abcam, Cambridge, UK) or rabbit polyclonal anti-mouse NG2 (Millipore, Billerica, MA) were used as primary antibodies. Species-specific secondary antibodies conjugated with Alexa Fluor 488 and 555 were from Invitrogen (Carlsbad, CA). Tissue sections were incubated with primary antibody overnight at 4°C and with secondary antibody for 1 hour at room temperature. Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Molecular Probes, Eugene, OR). Fluorescent immunostained sections were examined under a Leica DMRA2 fluorescence microscope with Leica HC PL Fluotar 10 and 20X/0.5 NA dry objective, captured using Photometrics CoolSNAP HQ, (Photometrics, Friedland, Denmark), and processed with Metamorph 4.6-5 (Molecular Devices, Sunnyvale, CA). Morphometric analyses were performed using the NIH ImageJ 1.37 v program. Vessel density corresponds to the percentage of each tumor section field occupied by a PECAM-positive signal. As a measure of vascular maturity, vessel wall assembly was assessed by quantifying the percentage of PECAM-positive structures lined by α-SMA-positive coverage while pericyte recruitment was assessed by quantifying the percentage of PECAM-positive structures lined by NG2-positive coverage.
Vessel perfusion study
To mark vessel perfusion, mice were anesthetized and biotin-conjugated lectin from Lycopersicon esculentum (100 μg in 100 μl of PBS; Sigma, St. Luis, MO) was injected via caudal vein and allowed to circulate for 5 minutes before the vasculature was transcardially perfused with 4% PFA in PBS for 3 minutes. Tumor samples were collected and processed as described above. Endothelial cells were stained with PECAM antibody and perfused vessels were visualised by streptavidin-Alexa 488 (Invitrogen, Carlsbad, CA), which binds to biotinylated lectin. The images were obtained and processed as described above. Tumor perfusion was quantified by determining the percentage of PECAM-positive structures that were colocalized with Alexa 488 signals.
Global gene expression and quantitative transcriptional analysis
Tumors from sEphB4-Alb or PBS treated RT2 Dll4
mice were harvested at week 13.5. RNA was then isolated and used for global gene expression analysis with Illumina MouseRef-8 v2.0 Expression BeadChip (Illumina, San Diego, CA). The genearray data were deposited to NCBI-GEO database http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24603. Genes with expression change between two groups higher than 2 fold and P value smaller than 0.05 were selected and the changes were validated by quantitative RT-PCR.
Using a SuperScript III FirstStrand Synthesis Supermix (Invitrogen, Carlsbad, CA), first-strand cDNA was synthesized from total RT2 Dll4
and RT2 Dll4
insulinoma RNA. Real-time PCR analysis was performed as described  using specific primers for β-actin, GAPDH, PECAM, Dll4, Hey2, VEGF-A, VEGFR1, VEGFR2, VEGF-C, VEGFR3, PDGF-β, Ephrin-B2, and Tie2. Primer pair sequences are available on request. Gene expression was normalized to β-actin and GAPDH.
Data processing was carried out using the Statistical Package for the Social Sciences version 15.0 (SPSS v. 15.0; Chicago, IL). Kaplan-Meier product-limit estimation with Breslow generalized Wilcoxon test was used for survival analyses. All other statistical analyses were performed using the Mann-Whitney-Wilcoxon test. All results are presented as mean ± SEM or mean ± SD when more appropriate. P-values < 0.05 and <0.01 were considered significant (indicated in the figures with *) and highly significant (indicated with **), respectively.