Monitoring response to anti-angiogenic mTOR inhibitor therapy in vivo using 111In-bevacizumab

Abstract

Background: The ability to image vascular endothelial growth factor (VEGF) could enable prospective, non-invasive monitoring of patients receiving anti-angiogenic therapy. This study investigates the specificity and pharmacokinetics of ¹¹¹In-bevacizumab binding to VEGF and its use for assessing response to anti-angiogenic therapy with rapamycin. Specificity of ¹¹¹In-bevacizumab binding to VEGF was tested in vitro with unmodified radiolabelled bevacizumab in competitive inhibition assays. Uptake of ¹¹¹In-bevacizumab in BALB/c nude mice bearing tumours with different amounts of VEGF expression was compared to that of isotype-matched control antibody (¹¹¹In-IgG1κ) with an excess of unlabelled bevacizumab. Intratumoural VEGF was evaluated using ELISA and Western blot analysis. The effect of anti-angiogenesis therapy was tested by measuring tumour uptake of ¹¹¹In-bevacizumab in comparison to ¹¹¹In-IgG1κ following administration of rapamycin to mice bearing FaDu xenografts. Uptake was measured using gamma counting of ex vivo tumours and effect on vasculature by using anti-CD31 microscopy.

Results: Specific uptake of ¹¹¹In-bevacizumab in VEGF-expressing tumours was observed. Rapamycin led to tumour growth delay associated with increased relative vessel size (8.5 to 10.3, P = 0.045) and decreased mean relative vessel density (0.27 to 0.22, P = 0.0015). Rapamycin treatment increased tumour uptake of ¹¹¹In-bevacizumab (68%) but not ¹¹¹In-IgGκ and corresponded with increased intratumoural VEGF₁₆₅.

Conclusions: ¹¹¹In-bevacizumab accumulates specifically in VEGF-expressing tumours, and changes after rapamycin therapy reflect changes in VEGF expression. Antagonism of mTOR may increase VEGF in vivo, and this new finding provides the basis to consider combination studies blocking both pathways and a way to monitor effects.

Keywords: Angiogenesis; Bevacizumab; Cancer; Radionuclide; Rapamycin.

Fig. 1

An example of vessel analysis from CD31 immunohistochemistry images. a Original image. b The binary line image. c Orientation image indicating the angulation of vessels (scale represents degrees from 0 to 180). d Scale image representing the size of individual vessels (scale represents pixels)

Fig. 2

Distribution of ¹¹¹In-bevacizumab after 5 days in mice bearing different tumour xenografts. Black = FaDu, hatched = LS 174T, white = 786-O. Data are presented as mean ± SEM, n = minimum of 4 mice/group

Fig. 3

a Specificity of uptake of ¹¹¹In-bevacizumab. Uptake of ¹¹¹In-bevacizumab (black); ¹¹¹In-bevacizumab + excess of unlabelled bevacizumab (hatched) and ¹¹¹In-IgG1κ (white) in different tumour models. Data are presented as mean ± SEM, *P < 0.05; ****P < 0.0001, n = minimum of 4 mice/group. b SPECT-CT transaxial image of FaDu xenograft-bearing mice injected with either ¹¹¹In-bevacizumab or ¹¹¹In-IgG1κ (tumours in circles)

Fig. 4

Comparison of the distribution of VEGF, bevacizumab, and ¹¹¹In in FaDu xenograft sections. The rows are examples of two sections from different FaDu xenografts injected with ¹¹¹In-bevacizumab and an excess of unlabelled bevacizumab at 5 days. The left panels (green) are immunofluorescence images of VEGF staining, the centre panels (red) are immunofluorescence images of bevacizumab staining, and the right panels (grayscale) are autoradiographs of ¹¹¹In distribution

Fig. 5

The effect of rapamycin on FaDu xenografts. a Changes in tumour volume with rapamycin therapy. On day 10, tumour volumes are significantly different (P = 0.0004). b Select images of CD31 confocal microscopy of tumours from mice treated with rapamycin. c Changes in relative mean vessel size with rapamycin therapy. d Changes of relative mean vessel density of rapamycin. e Uptake changes of ¹¹¹In-bevacizumab and ¹¹¹In-IgGκ in FaDu xenografts treated with rapamycin (20 mg/kg). The control groups were treated with vehicle injections. Data are presented as mean ± SEM, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 6–7/group

Fig. 6

The effect of rapamycin therapy on VEGF levels in FaDu xenografts. a Changes in VEGF levels as measured by ELISA. Differences in levels were not statistically significant. b Changes in VEGF isoforms assessed by Western blot in FaDu xenografts treated with either rapamycin (20 mg/kg/day) or vehicle control for 10 days. c Changes in VEGF isoforms were measured using densitometry in the corresponding blots. Data are presented as mean ± SEM, **P < 0.01, n = 11/group