Acute tumour response to a bispecific Ang-2-VEGF-A antibody: insights from multiparametric MRI and gene expression profiling

Abstract

Background: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A).

Methods: Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg(-1) dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 10(6) mm(2) s(-1)), vascular perfusion/permeability (K(trans), min(-1)) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed.

Results: Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour K(trans) (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis.

Conclusions: Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.

Figure 1

Effects of vanucizumab on tumour progression. (A) Transverse T₂-weighted MR images of the central tumour slice acquired from paired mice bearing Colo205 xenografts before and 5 days after treatment, as indicated. (B) Summary of tumour volume response, determined by T₂-weighted MRI, for each treatment cohort before and 5 days after treatment with vanucizumab, bevacizumab, LC06 or omalizumab (control). Data points are mean±1 s.e.m., n⩾6 per treatment, **P<0.01, ***P<0.001, two-way ANOVA with Bonferroni correction.

Figure 2

Effects of vanucizumab on tumour vascular perfusion and permeability assessed by dynamic contrast-enhanced MRI. (A) Parametric K^trans maps acquired from paired Colo205 xenografts before and 5 days after treatment with vanucizumab, bevacizumab, LC06 or omalizumab (control), as indicated. (B) Estimates of the transfer constant (K^trans) and the initial area under the gadolinium uptake curve to 60 s (IAUGC₆₀) determined from each Colo205 xenograft before and 5 days after treatment with vanucizumab, bevacizumab, LC06 or omalizumab. (C) Summary of the percentage change in K^trans determined across all four treatment groups. Data points are mean±1 s.e.m, n⩾6 per treatment, *P<0.05, Wilcoxon matched pairs signed-rank test. There was no significant difference in the change in K^trans 5 days post treatment with vanucizumab, bevacizumab or LC06 when compared with omalizumab (control) – two-way ANOVA with Bonferroni correction.

Figure 3

Effects of vanucizumab on tumour fractional blood volume assessed by susceptibility contrast MRI. (A) Parametric fBV maps acquired from the central slice of paired Colo205 xenografts before and 5 days after treatment with either vanucizumab or omalizumab (control) as indicated. (B) Summary of treatment-induced changes in tumour fBV, determined by susceptibility contrast MRI. Data points are mean±1 s.e.m., n⩾6 per treatment, *P<0.03, Wilcoxon matched pairs signed-rank test.

Figure 4

Histological assessment of the effects of vanucizumab on tumour vasculature, hypoxia and necrosis. (A) Composite fluorescence images of Hoechst 33342 uptake (blue, perfusion) and pimonidazole adduct formation (green, hypoxia) acquired from whole sections of Colo205 xenografts 5 days after treatment with vanucizumab, bevacizumab, LC06 or omalizumab (control). Fluorescence images (× 100 magnification) of CD31 immunohistochemistry used to assess tumour microvessel density (MVD) post treatment. High-magnification (× 200) images of α-SMA immunohistochemical staining used to assess pericyte coverage. Composite images of whole tumour sections stained with H&E used to assess necrosis. (B) Summary of treatment-induced changes in perfused tumour vessels, hypoxia, MVD, vascular maturation and necrosis. Data points are mean±1 s.e.m., n⩾5 tumours per treatment, *P<0.05, **P<0.01, one-way ANOVA with Dunnett's multiple comparison test.

Figure 5

Differential gene expression in vanucizumab-treated tumours. Comparison of vanucizumab-treated tumours with omalizumab (control (ctrl)) tumour samples 5 days (5dy) after treatment led to the identification of 60 significant (*P<0.05 and |log₂ ratio|>0.5) differentially expressed genes in the tumour stroma. The upper part of the Table shows a comparison with the respective results in the other treatment groups, whereas the colour code specifies if the gene was identified from cohort 1 (C1), cohort 2 (C2) or both. A total of 26 of all genes were previously described in the VEGF-dependent vasculature signature (Brauer et al, 2013). Where known, the particular biological process in which a gene is known to be involved with is indicated.