DC101 treatment increased mural cell coverage of tumor vessels, which could render the endothelium resistant to anti-angiogenic treatment. without affecting vessel regression and compromised the survival benefit of VEGFR2 inhibition by increasing vascular permeability. VEGFR2 inhibition normalized tumor vasculature while ectopic expression of Ang-2 diminished the beneficial effects of VEGFR2 blockade by inhibiting vessel normalization. Conclusion Cancer treatment regimens combining anti-VEGF and anti-Ang-2 agents may be an effective strategy to improve the efficacy of current anti-VEGF therapies. with a far-red lipophilic fluorescent dye (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate, DiD, Invitrogen, Carlsbad, CA), allowing observation deep inside the tissue via MPLSM. The labeled RBCs were mixed with the endogenous mouse blood via systemic injection at a ratio of 3C5 labeled RBCs per 100. Line scanning was performed using MPLSM to determine RBC velocity. All image analysis was completed using in-house algorithm (MATLAB, Mathworks, Natick, MA)(13). For more details of the data analysis, see Supplementary Methods. Magnetic Resonance Imaging (MRI) All MR images were acquired using a 9.4 Tesla MRI scanner (Bruker Biospin, Billerica, MA). Animals were anesthetized with a 50:50 mixture of O2 and medical air plus 1.5% isofluorane and placed prone in a home-built cradle. A custom-built transmit-receive birdcage mouse-head coil was used to acquire the images. T2 relaxation maps were generated from multi-echo spin-echo images and used to assess tumor edema. Acquisition parameters were: TE = 10 ms, 10 echoes, TR = 2500 ms, 11 image slices, 0.5 mm slice thickness, 150 m in-plane resolution, NA = 2. Voxelwise exponential fitting of the image signal intensity as a function of echo-time was performed using a MATLAB program written in-house to determine T2 relaxation time maps. Immunohistochemistry and Western blot analysis To label the perfused blood vessels, 100 g of biotinylated Lycopersicon Esculentum (Tomato) Lectin (Vector Laboratory, Burlingame, CA) was intravenously injected into mice, followed by perfusion fixation with 4% formaldehyde. 20m Tyk2-IN-3 thick frozen tissue sections were Tyk2-IN-3 blocked in 5% non-fat milk in PBS with 0.1% Triton X-100 and stained with Alexa 647-conjugated streptavidin (1:200, “type”:”entrez-protein”,”attrs”:”text”:”S21374″,”term_id”:”99986″,”term_text”:”pirS21374, Invitrogen, Carlsbad, CA). Pericytes were stained with Cy3-conjugated anti- smooth muscle actin (SMA) antibody (1:200, C6198, Sigma, St. Louis, MO) or anti-NG2 antibody (1:200, AB5320, Millipore, Billerica, MA). Ang-2 staining was done using Tyk2-IN-3 polyclonal anti-Ang-2 antibody (1:100, AF623, R&D Systems, Minneapolis, MN) after heat retrieval at 95 C for 5min in target retrieval solution (S1699, DAKO, Carpinteria, CA). Images of four different fields per tumor section were collected with Olympus laser scanning microscope using 20X objective lens. Quantification of the stained area was performed using our in-house segmentation algorithm (MATLAB, Mathworks, Natick, MA). Image Analysis was performed as previously described (3). Images were processed using Adobe Photoshop CS3 software (Adobe Systems Inc., San Jose, CA). Soluble Tie-2 expression was determined by Western blot analysis. Plasma samples (3 l each) from mice were separated on 8% denaturing polyacrylamide gel and transferred to PVDF membrane. The membrane was incubated with polyclonal anti-Tie-2 antibody (1:1000, AF313, R&D Systems, Minneapolis, MN), followed by horseradish peroxidase (HRP) conjugated donkey anti-rabbit IgG (1:5000, NA934V, Mouse monoclonal to KSHV ORF26 GE Healthcare, Piscataway, NJ). The membrane was incubated in enhanced chemiluminescence (ECL) plus detection reagent (RPN2132, GE Healthcare, Piscataway, NJ) and exposed to Kodak ML film. Data collection and Statistical analysis Data are expressed as mean SEM. Students t-test (two tailed with unequal variance) was performed for statistical analysis using Microsoft excel software. The KaplanCMeier method was used for survival studies. We considered a value of less than 0. 05 to be statistically significant. For survival, permeability and MRI studies, all data from multiple experiments were combined, analyzed and presented. Other studies were repeated at least three times and representative data were presented. Results Anti-VEGFR2 treatment transiently reduces Ang-2 expression We first determined the kinetics of Ang-2 expression during anti-VEGFR2 treatment. Ang-2 was predominantly expressed in tumor blood vessels of glioma xenografts (Fig. 1A) similar to patterns seen in autopsy samples from patients (14). Since Ang-2 was mainly expressed in vascular endothelial cells (ECs), we normalized the expression level of Ang-2 with respect to the EC specific marker, VE-Cadherin. As shown in Fig. 1B, DC101 treatment significantly, but transiently, reduced Ang-2 levels.