Figure 1.
The Miles assay measures vascular permeability changes in the CAM.
A. Bright field images of CAM vasculature following injection of Evan's blue dye subsequent to the systemic administration of PBS (left panel) or VEGF (right panel). Arrows indicate areas of visible vascular leak. B. When VEGF or PEP is injected intravenously distal to the site of analysis, a significant level of vascular permeability is observed in the CAM (left). Topically administered VEGF but not PEP induces a significant level of vascular permeability (right). C. Vascular permeability changes in the CAM were evaluated in the presence of human tumor xenografts. Increased vascular permeability was observed at the tumor site, particularly in HEp3 tumors. Systemically administered PEP (0.1 nM) further increases vascular permeability. Data are presented as Mean +/− SEM, n>15 for each group. * indicates statistical significance, p<0.05, ** p<0.01.
Figure 2.
Intravital imaging assesses real-time changes in vascular permeability induced by VEGF and PEP.
A. A series of representative images from intravital imaging experiments is shown. An accumulation of fluorescence outside the vasculature over time is seen in those embryos treated with VEGF or PEP compared to PBS. B. Images were captured and quantified every 15 minutes over a period of 3 hours to evaluate the extent of vascular leak. Vascular leak values were generated by subtracting time 0 values from subsequent time points. Asterisks indicate significant leak of dextran from the vasculature (2-way ANOVA, p<0.05 followed by Bonferroni post-tests, (p<0.05)) comparing either PBS vs VEGF, or PBS vs PEP at each time point.
Figure 3.
Assessment of regional permeability and vascular integrity in human tumors.
A. Representative fluorescence micrographs from two human HEp3 tumors displaying peri-tumoral (i) and tumor core (ii) vascular leak are shown with the 2000 kDa FITC-dextran (green) and 158 kDa TRITC-dextran (red). The normalized images were generated by subtracting the 0 hour image from the 3 hour image, and represent the net vascular leak. Tumor induced vascular leak is localized primarily to the tumor and especially to the central, necrotic core of the tumor. B. Areas utilized for regional vascular leak analyses are delineated. The solid circle represents an area of non-tumor tissue; the dashed circle denotes tumor and the dotted line indicates the avascular necrotic core. C. Quantitation of leak of large (green) and small (red) dextrans is shown for non-tumor tissue, the entire tumor and the core of the tumor. The relative leak of both dextrans was normalized to time zero; n = 6 for each analysis. Two-way ANOVA, (p<0.05) followed by Bonferroni post-tests, (p<0.05) was used to assess significant leak of the TRITC-dextran of either tumor versus non-tumour tissue, and necrotic core versus non-tumour tissue at each timepoint. Timepoints that demonstrated significance are indicated by an asterisk.
Figure 4.
Increasing vascular permeability enhances the accumulation of doxorubicin into the tumor.
Doxorubicin was injected intravenously subsequent to administration of PBS or VEGF and its uptake at the tumor site in real time was estimated using its natural fluorescence. A–B. Representative images of doxorubicin uptake over time, tumors (green) and doxorubicin uptake (red) are shown. C. Heat map of doxorubicin uptake after 3 hours in control and VEGF-treated tumors. D. Graph showing relative uptake of doxorubicin in the tumor in the presence or absence of systemic VEGF treatment. E. Graph showing relative uptake of doxorubicin in the normal tissues distal to the tumor in the presence or absence of systemic VEGF treatment. N = 4 per treatment; data were analyzed by 2 way ANOVA.