Effect of pazopanib on tumor microenvironment and liposome delivery

Abstract

Pathologic angiogenesis creates an abnormal microenvironment in solid tumors, characterized by elevated interstitial fluid pressure (IFP) and hypoxia. Emerging theories suggest that judicious downregulation of proangiogenic signaling pathways may transiently "normalize" the vascular bed, making it more suitable for drug delivery and radiotherapy. In this work, we investigate the role of pazopanib, a small-molecule inhibitor of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors, on tumor IFP, angiogenesis, hypoxia, and liposomal drug delivery. Nude mice bearing A549 human non-small cell lung cancer xenografts were treated with 100 mg/kg pazopanib (n = 20) or vehicle (n = 20) through oral gavage for 8 days, followed by a one-time intravenous dose of 10 mg/kg Doxil (liposomal doxorubicin). Pazopanib treatment resulted in significant reduction of tumor IFP and decreased vessel density, assessed by CD31 staining. Despite these trends toward normalization, high-performance liquid chromatography revealed no differences in doxorubicin concentration between pazopanib-treated and control tumors, with Doxil penetration from microvessels being significantly reduced in the pazopanib group. Additionally, tumor hypoxia, evaluated by CA-IX immunostaining and confirmed in a second study by EF5 expression (n = 4, 100 mg/kg pazopanib; n = 4, vehicle), was increased in pazopanib-treated tumors. Our results suggest that the classic definition of tumor "normalization" may undermine the crucial role of vessel permeability and oncotic pressure gradients in liposomal drug delivery, and that functional measures of normalization, such as reduced IFP and hypoxia, may not occur in parallel temporal windows.

Figure 1

Tumor volume response to pazopanib. Nude mice bearing A549 human non-small cell lung cancer xenografts (>350mm3) were randomized to receive daily oral administration of pazopanib 100mg/kg (n=18) or vehicle control (n=20), starting on day 0. Data presented as mean ± SEM. *, P < 0.05, One-way ANOVA.

Figure 2

Representative tri-color composites of Doxil demonstrating the distribution of Doxil (magenta) in a control tumor, in relation to CD-31-stained blood vessels (green) and hypoxia (CA-IX yellow). A, Whole tumor section, 10x objective. B, Magnified view demonstrating perivascular accumulation of Doxil, magnification 100%.

Figure 3

Effect of pazopanib on vasculature and vessel pericyte coverage. A, Representative composite image of vessels (CD-31, green) and pericytes (α-SMA, red). B, Mean vessel density (MVD) determined by CD-31 staining. C, Vessel pericyte coverage determined by co-localization of CD-31 and α-SMA staining. D, Vessel pericyte coverage determined by co-localization of CD-31 and NG2 staining. Columns and bars represent mean and SEM *, P < 0.05, ***, P < 0.001, Wilcoxon test.

Figure 4

Comparison of IFP and hypoxia markers in pazopanib and control tumors. A, Comparison of normalized tumor IFP measured at study end. Normalized tumor IFP = Tumor IFP/normal muscle IFP. Horizontal bars represent means. B, Correlation of normalized tumor IFP versus MVD. ρ, Spearman’s correlation coefficient. C, Percentage of tumor section area staining positive for CA-IX. B, Percentage of tumor section area staining positive for EF5. Columns and bars represent mean and SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001, Wilcoxon test.

Figure 5

Effect of pazopanib on Doxil delivery. Animals were treated with pazopanib 100mg/kg or vehicle control for 8 days, followed by a one-time dose of intravenous Doxil 10mg/kg. A, HPLC analysis of tumor doxorubicin concentration (ng of doxorubicin/mg of tumor tissue). Columns and bars represent mean and SEM. B, Average fluorescence intensity of Doxil versus distance from nearest microvessel. C, Penetration distance of Doxil from CD-31 stained blood vessels. Data represent distance from nearest microvessel at which Doxil fluorescence drops by 50% of its maximum value. *, P < 0.05, Wilcoxon test.

Figure 6

Total and phosphorylated levels of VEGF-R2 and PDGFR-ß. A plate-based antibody capture assay (Meso Scale Discovery platform) was used to quantify total receptor levels in vehicle-treated control tumors and pazopanib-treated tumors. Phosphorylated receptor levels were determined via quantitative Western blotting. A, Relative intensity values for total and phosphorylated VEGF-R2, and “activation status,” expressed as the ratio of phosphorylated/total VEGF-R2. B, Relative intensity values for total PDGFR-ß, phopsphorylated PDGFR-ß, and “activation status.” Columns and bars represent mean and SEM. *, P < 0.05, **, P < 0.01, Wilcoxon test.