Scheduling of radiation with angiogenesis inhibitors anginex and Avastin improves therapeutic outcome via vessel normalization

Abstract

Purpose: To test whether a direct antiangiogenic peptide (anginex) and a vascular endothelial growth factor antibody (bevacizumab, Avastin) can transiently normalize vasculature within tumors to improve oxygen delivery, alleviate hypoxia, and increase the effect of radiation therapy.

Experimental design: Tumor oxygenation levels, microvessel density and pericyte coverage were monitored in three different solid tumor models (xenograft human ovarian carcinoma MA148, murine melanoma B16F10, and murine breast carcinoma SCK) in mice. Multiple treatment schedules were tested in these models to assess the influence on the effect of radiation therapy.

Results: In all three tumor models, we found that tumor oxygenation levels, monitored daily in real time, were increased during the first 4 days of treatment with both anginex and bevacizumab. From treatment day 5 onward, tumor oxygenation in treated mice decreased significantly to below that in control mice. This "tumor oxygenation window" occurred in all three tumor models varying in origin and growth rate. Moreover, during the treatment period, tumor microvessel density decreased and pericyte coverage of vessels increased, supporting the idea of vessel normalization. We also found that the transient modulation of tumor physiology caused by either antiangiogenic therapy improved the effect of radiation treatment. Tumor growth delay was enhanced when single dose or fractionated radiotherapy was initiated within the tumor oxygenation window as compared with other treatment schedules.

Conclusions: The results are of immediate translational importance because the clinical benefits of bevacizumab therapy might be increased by more precise treatment scheduling to ensure radiation is given during periods of peak radiosensitivity. The oxygen elevation in tumors by non-growth factor-mediated peptide anginex suggests that vessel normalization might be a general phenomenon of agents directed at disrupting the tumor vasculature by a variety of mechanisms.

Figures

Fig. 1

The effect of anginex and Avastin (bevacizumab) on global tumor pO2 over time. A, global tumor pO2 is transiently increased by anginex and Avastin in MA148, B16F10, and SCK tumors as measured by Eppendorf pO2 histograph. -■-, control; -●-, anginex; -▲-, Avastin. Points, average median pO2 value derived from, on average, 10 values per track, 4 tracks per mouse and 3 to 7 mice per treatment group per day (n = 120-700); bars, SE. B, typical pimonidazole stainings of control, anginex, and Avastin treatment of SCK tumors on days 2 and 7. Representative images of the means for the amount of staining across each section. Original magnification, ×200; bar, 50 μm. C, quantification of pimonidazole stainings of SCK tumors on days 2 and 7 by morphometric analysis. Following binarization of images, hypoxia was estimated by scoring the total number of white pixels per field. Columns, mean black pixel count per image; bars, SE.

Fig. 2

Histologic analysis of pericytes and microvessel density after treatment with anginex and Avastin. A, quantification of immunohistochemical staining for pericytes and microvessel density in MA148, B16F10, and SCK tumors by morphometric analysis. *, P < 0.01, experimental group compared with control (Student's t test). B, typical colocalization staining of pericytes (green) and microvessel density (red) for control, anginex-treated, and Avastin-treated SCK tumors on days 2 and 7. Representative images of the means for the amount of staining. Original magnification, ×200; bar, 50 μm.

Fig. 3

Radiation treatment enhancement by rational scheduling of angiogenesis inhibitors. A, MA148 tumor volumes after anginex, radiation, or combination treatment. Tumor-bearing animals were treated with control (-■-), anginex (-●-; i.p. injections of 10 mg/kg Ax on days 0 and 1), radiation (-▲-; 5 Gy, day 2), RtAx combination (-◆-; 5 Gy on day 2 followed byAx on days 2 and 3), and AxRt combination (-▼-; Ax on days 0 and 1 followed by 5 Gy on day 2). B, B16F10 tumor volumes after anginex, radiation, or combination treatment. Tumor-bearing animals were treated with control (-■-), anginex (-●-; i.p. injections of 10 mg/kg Ax on days 0-2), radiation (-▲-; 5 Gy, days 2 and 3), and AxRt combination (-▼-; Ax on days 0-2 followed by 5 Gy on days 2 and 3). C, B16F10 tumor volumes after anginex, radiation, or combination treatment.Tumor-bearing animals were treated with control (-■-), anginex (-●-; i.p. injections of 10 mg/kg Ax on days 0-5), radiation (-▲-; 5 Gy, days 5 and 6), and AxRt combination (-▼-; Ax on days 0-5 followed by 5 Gy on days 5 and 6). Points, mean tumor volume (n = 5-8 animals per group); bars, SE.

Fig. 4

Anginex specifically targets and enhances the antiproliferative activity of radiation on endothelial cells. A, anginex radiosensitizes endothelial cells. Clonogenicity of human umbilical vein endothelial cells (HUVEC) is reduced to 50% by anginex exposure (20 μmol/L for 4 h) or to 17% by 5-Gy radiation treatment. Combining anginex before radiation caused an enhanced decrease in clonogenicity (4.6% P < 0.006) compared with either monotherapy alone. B, anginex has no effect on colony formation of B16F10 cells. The survival fraction was reduced by 70% by exposure to 5 Gy alone, but was not further decreased when anginex (20 μmol/L for 4 h) was combined before or after radiation.