Animals and treatment.
Spontaneous pancreatic islet cell carcinomas in RIP-Tag2 mice (C57BL/6 background; ref. 65
) from our colony at UCSF were studied at 10–12 weeks of age (6
). A 1-mm3
piece of Lewis lung carcinoma from a donor mouse was implanted under the dorsal skin of wild-type 7- to 8-week-old C57BL/6 mice for 4 days (6
). All experimental procedures were approved by the UCSF Institutional Animal Care and Use Committee.
AG-013736, a potent small molecule inhibitor of VEGFR and related receptor tyrosine kinases (6
), was suspended at a concentration of 5 mg/ml in 0.5% carboxymethylcellulose and administered by gavage. In some experiments, AG-013736 was dissolved in 3 parts PEG-400 and 7 parts acidified water (pH 2–3) and injected i.p. In both cases, AG-013736 was administered at a dose of 25 mg/kg body weight in a volume of 5 μl/g, bid. Complementary studies used the selective VEGF receptor tyrosine kinase inhibitor AG-028262 (28
), which was prepared at a concentration of 21.92 mg/ml by suspension in 0.5% carboxymethylcellulose and administered by gavage (40 mg/kg in a volume of 5 μl/g, bid). The effect of metalloproteinase inhibition on regrowth of tumor vessels was assessed by treating mice with AG3340 (29
). AG3340, prepared in acidified water, pH 2.2–2.3, was administered by gavage (200 mg/kg in a volume of 5 μl/g, bid). AG-013736, AG-028262, and AG3340 were supplied by Pfizer Global Research and Development.
The basement membrane of regressing tumor vessels was targeted by treating mice with monoclonal antibody HUIV26, which recognizes cryptic sites on denatured type IV collagen (30
). HUIV26 at a concentration of 1.15 mg/ml was injected i.p. at a dose of 100 μg (volume, 87 μl) every other day (on days 0, 2, 4, 6 in all mice; days 8 and 10 for 11-day experiments; and days 8, 10, and 12 for 14-day experiments). HUIV26 was supplied by Cell Matrix Inc., a subsidiary of CancerVax.
Vascular regrowth after AG-013736 treatment was studied in groups of mice that received 1 of 4 treatments: (a) vehicle; (b) AG-013736 for 7 days; (c) AG-013736 for 7 days followed by a period without treatment lasting 1, 2, 4, 7, or 14 days; or (d) AG-013736 for 7 days followed by no treatment for 7 days and then a second 7-day round of AG-013736. Previous studies showed that vehicles for AG-013736 (carboxymethylcellulose or PEG-400 in acidified water) had no detectable effect on RIP-Tag2 tumors (6
). AG-028262 was studied in groups similar to a, b, and c. Group c had a 7-day period without treatment.
Effects of AG3340 on regrowth of tumor vessels were studied in groups of mice that received 1 of 4 treatments: (a) vehicle; (b) AG-013736 plus AG3340 for 7 days; (c) AG-013736 plus AG3340 for 7 days, then no treatment for 7 days; or (d) AG-013736 plus AG3340 for 7 days, then AG3340 alone for 7 days.
Effects of HUIV26 were examined in groups of mice that received 1 of 5 treatments: (a) vehicle; (b) AG-028262 for 7 days; (c) AG-028262 plus HUIV26 for 7 days; (d) AG-028262 for 7 days, then no treatment for 4 or 7 days; or (e) AG-028262 plus HUIV26 for 7 days, then HUIV26 alone for 4 or 7 days.
Lectin injection and fixation by vascular perfusion.
After the treatment and withdrawal periods, mice were anesthetized with ketamine (100 mg/kg i.p.) plus xylazine (10 mg/kg i.p.). Patency of individual tumor vessels was assessed in some mice by injection of 100 μl of FITC-labeled L. esculentum lectin (1 mg/ml in 0.9% NaCl; Vector Laboratories) into a femoral vein 2 minutes before the perfusion. The chest was opened rapidly, and the vasculature was perfused for 2 minutes at a pressure of 120 mmHg with fixative (1% paraformaldehyde in PBS, pH 7.4; Sigma-Aldrich) from an 18-gauge cannula inserted into the aorta via an incision in the left ventricle. Blood and fixative exited through an opening in the right atrium. Fixed tissues were removed, immersed in fixative for 1 hour at 4°C, rinsed several times with PBS, infiltrated with 30% sucrose, frozen in OCT compound, and processed for immunohistochemistry.
Cryostat sections of tissues were cut at a thickness of 80 μm unless otherwise indicated. Sections dried on Superfrost Plus slides (Fisher Scientific) were permeabilized with PBS containing 0.3% Triton X-100 (LabChem Inc.) and then incubated in a solution containing 5% normal serum (Jackson ImmunoResearch Laboratories Inc.), PBS-plus (PBS with 0.3% Triton X-100, 0.2% BSA; Sigma-Aldrich), and 0.01% thimerosal (Sigma-Aldrich) for 1 hour at room temperature to block nonspecific antibody binding (6
Sections on slides were incubated for 12–15 hours with primary antibodies diluted in 5% normal serum in PBS-plus at room temperature. Endothelial cells, pericytes, and vascular basement membrane of tumor vessels were identified by staining with combinations of 2 or 3 antibodies. Endothelial cells were labeled with rat monoclonal anti-CD31 (PECAM-1, clone MEC 13.3; 1:500; BD Biosciences — Pharmingen) or rabbit polyclonal anti–VEGFR-2 (antibody T014; 1:2,000; gift from R. Brekken and P. Thorpe, University of Texas Southwestern Medical Center, Dallas, Texas, USA). Pericytes were labeled with Cy3-conjugated mouse monoclonal anti–α-SMA (clone 1A4; 1:1,000; Sigma-Aldrich) and/or rat monoclonal anti–PDGFR-β (clone APB5; 1:2,000; gift from A. Uemura, Kyoto University, Kyoto, Japan). Vascular basement membrane was examined with rabbit polyclonal anti–type IV collagen antibody (1:10,000; Cosmo Bio Co.). VEGF was stained with goat polyclonal anti-VEGF antibody (1:400; R&D Systems).
After incubation with primary antibodies, sections were rinsed with PBS containing 0.3% Triton X-100 and incubated for 4–6 hours at room temperature with secondary antibodies diluted in 5% normal serum in PBS-plus. Secondary antibodies were FITC- or Cy3-labeled goat anti-rat IgG for rat anti-CD31 and anti–PDGFR-β antibodies; Cy3-, Cy5-, or FITC-labeled goat anti-rabbit IgG for rabbit anti-type IV collagen and anti-VEGFR-2 antibodies; and Cy3-labeled donkey anti-goat IgG for the goat anti-VEGF antibody (1:400; Jackson ImmunoResearch Laboratories Inc.). Sections were rinsed with PBS containing 0.3% Triton X-100, postfixed in 4% paraformaldehyde, rinsed again with PBS, and mounted in Vectashield (Vector Laboratories).
Tissue sections were examined with a Zeiss Axiophot fluorescence microscope equipped with single, dual, and triple fluorescence filters and a low-light, externally cooled, 3-chip charge-coupled device (CCD) camera (480 × 640–pixel RGB color images; CoolCam; SciMeasure) and with a Zeiss LSM 510 confocal microscope with Argon, Helium-Neon, and UV lasers (512 × 512– or 1024 × 1024–pixel RGB color images; Zeiss).
The anti-CD31 antibody uniformly stained the entire thickness of 80-μm sections of pancreas and tumors of RIP-Tag2 mice. The anti–type IV collagen antibody consistently stained the uppermost 50 μm of sections, but staining of deeper regions was variable, perhaps because the dilute antibody (1:10,000) was depleted during penetration. As a result, CD31-positive vessels in the lowermost part of some sections appeared unaccompanied by type IV collagen. This problem was circumvented by obtaining confocal microscopic images from the uppermost 50 μm of the sections.
Endothelial cells, pericytes, and basement membrane of blood vessels in tumors were quantified by measuring the proportion of sectional area (area density) occupied by fluorescence of specific immunohistochemical markers. Tumor vessel patency was assessed by measuring the extent of lectin staining. Digital fluorescence microscopic images, each representing a region measuring 960 × 1280 μm (×10 objective, ×1 Optovar), were captured from sections of at least 4 RIP-Tag2 tumors or 2 regions of Lewis lung carcinoma in each mouse. Images were analyzed using ImageJ software (http://rsb.info.nih.gov/ij) (6
). Based on an analysis of pixel fluorescence intensities, which ranged from 0 to 255, specific staining was distinguished from background by empirically using a threshold value of 45 or 50 (6
). Area densities of structures stained with lectin, CD31, α-SMA, PDGFR-β, or type IV collagen were calculated as the proportion of pixels having a fluorescence intensity value equal to or greater than the threshold (6
). Because the fluorescence threshold was set to provide the greatest signal-to-noise ratio for measuring specific immunoreactivity, some of the faintest immunoreactivity may not have been included. However, because thresholds were applied uniformly, all experimental groups were similarly affected.
Intensity of VEGFR-2 immunofluorescence.
VEGFR-2 immunofluorescence of RIP-Tag2 tumors was measured in 6 steps (6
): (a) Cryostat sections were cut at a thickness of 20 μm and stained for VEGFR-2 immunoreactivity or, as a control, with Cy3-labeled secondary antibody without a primary antibody. (b) Camera gain was calibrated on sections stained with the secondary antibody, which by definition had no foreground (specific) fluorescence. Camera gain was set so background fluorescence was barely visible in the digital images (×20 objective, ×1 Optovar; tissue region 480 × 640 μm). Analysis by ImageJ showed that 97% of pixels had a fluorescence intensity of less than 15 (intensity range, 0–255). The lower limit of specific fluorescence (threshold) was thus established as a fluorescence intensity of 15. (c) Digital images were then obtained from sections stained for VEGFR-2 immunoreactivity (Cy3-conjugated secondary antibody) with camera gain set as for the calibration sections. Camera gain was adjusted as needed from section to section to maintain the background fluorescence at the calibration level. Brightness of foreground fluorescence was ignored in this step. (d) RGB images were converted to 8-bit grayscale images with ImageJ. (e) Fluorescence intensity was determined for each pixel using ImageJ. (f) Mean fluorescence intensity of these pixels was calculated as the sum of the number of pixels at each intensity of at least 15, times the intensity, divided by the total number of pixels with intensity of at least 15.
The mean value for VEGFR-2 fluorescence intensity in RIP-Tag2 tumors in each mouse was calculated from at least 4 images. Group means were expressed as a percentage of the corresponding values for blood vessels in vehicle-treated tumors (n = 4–6 mice per treatment group). Tests of reproducibility confirmed that AG-013736 treatment reduced the intensity of VEGFR-2 immunofluorescence of RIP-Tag2 tumors by approximately 50% in 3 independent experiments.
Colocalization of pericyte markers.
As an index of pericyte phenotype, the amount of colocalization of 2 pericyte markers (α-SMA and PDGFR-β) was measured on images of immunohistochemically stained 20-μm sections of RIP-Tag2 tumors. Digital images of the red (Cy3-labeled α-SMA primary antibody) and green (PDGFR-β labeled with FITC-conjugated secondary antibody) channels of the same field were captured separately with the CCD camera on the fluorescence microscope (×10 objective, ×1 Optovar). The Colocalization plug-in function of ImageJ was used to identify pixels that had a fluorescence intensity equal to or greater than the threshold values (30 to 45) in both the red and green channels. The amount of colocalization of the 2 pericyte markers, expressed as the percentage of pixels with above-threshold PDGFR-β immunoreactivity that colocalized with above-threshold α-SMA staining, was calculated as the number of colocalized pixels divided by the number of above-threshold pixels in the PDGFR-β image.
The significance of differences among groups was assessed using ANOVA followed by the Bonferroni-Dunn or Fisher’s test for multiple comparisons. Values are expressed as mean ± SEM (n = 4–5 mice per group). P values less than 0.05 were considered significant, except where lower values were indicated in the Bonferroni-Dunn tests.