Br J Cancer. 2015 Feb 3;112(3):495-503.

Dual targeting of Angiopoetin-2 and VEGF potentiates effective vascular normalisation without inducing empty basement membrane sleeves in xenograft tumours

 

O Coutelle*,1, L M Schiffmann1, M Liwschitz1, M Brunold1, V Goede1,2, M Hallek1, H Kashkar3,4,5,7 and U T Hacker1,6,7

1Department I for Internal Medicine, University of Cologne, Kerpener Strasse 62, 50924 Cologne, Germany;

2Department for Geriatric Care, St Marien Hospital, Kunibertskloster 11-13, 50668 Cologne, Germany;

3Institute for Medical Microbiology, Immunology and Hygiene, Medical Faculty, University of Cologne, Goldenfelsstrasse 19-21, 50935 Cologne, Germany;

4Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Medical Faculty, University of Cologne, Robert-Koch-Strasse 39, 50931 Cologne, Germany;

5Center for Molecular Medicine Cologne (CMMC), Robert-Koch-Strasse 39, 50931 Cologne, Germany and 6University Cancer Center Leipzig, Liebigstrasse 20, 04103 Leipzig, Germany

 

Abstract:

Background: Effective vascular normalisation following vascular endothelial growth factor (VEGF) inhibition is associated with endothelial cell regression leaving empty basement membrane sleeves (BMS). These long-lived BMS permit the rapid regrowth of tumour vasculature upon treatment cessation and promote resistance to VEGF-targeting drugs. Previous attempts at removing BMS have failed. Angiopoietin-2 (Ang2) is a vascular destabilizing factor that antagonises normalisation. We hypothesised that Ang2 inhibition could permit vascular normalisation at significantly reduced doses of VEGF inhibition, avoiding excessive vessel regression and the formation of empty BMS.

Methods: Mice xenografted with human colorectal cancer cells (LS174T) were treated with low (0.5 mg kg-1) or high (5 mg kg-1) doses of the VEGF-targeting antibody bevacizumab with or without an Ang2 blocking peptibody L1-10. Tumour growth, BMS formation and normalisation parameters were examined including vessel density, pericyte coverage, adherence junctions, leakiness, perfusion, hypoxia and proliferation.

Results: Dual targeting of VEGF and Ang2 achieved effective normalisation at only one-tenth of the dose required with bevacizumab alone. Pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth.

Conclusions: Dual targeting of VEGF and Ang2 can potentiate the effectiveness of VEGF inhibitors and avoid the formation of empty BMS.

PMID: 25562438

 

Background:

The success of antiangiogenic drugs is only partially dependent on the reduction in the number of tumor vessels that supply oxygen and nutrients to the tumor. In fact, antiangiogenic agents like VEGF inhibitors can transiently “normalize” the abnormal structure and function of tumor vasculature to make it more efficient for oxygen and drug delivery. It is thought that the preferential elimination of ineffective, leaky blood vessels by antiangiogenic drugs, leads to a reduction in the interstitial fluid pressure inside the tumor and thus increases blood flow, reduces hypoxia and improves access of chemotherapy (Jain, 2001; Hurwitz et al., 2004).

 

However a problem with VEGF targeting drugs is the narrow therapeutic window. Too much inhibition results in excessive vessel pruning and inadequate perfusion reducing access of chemotherapy and leading to hypoxia induced rebound angiogenesis. On the other hand, too little VEGF inhibition results in insufficient vessel pruning and a high interstitial fluid pressure from leaky tumor vessel, which would also prevent access for chemotherapy. Furthermore, once the anti-angiogenic therapy is paused, the rapid regrowth of ECs is facilitated by durable tunnels of pericytes and basement membrane sleeves that remain intact after EC regression and also serve as storage sites for angiogenic growth factors (Kalluri, 2003). Previous attempts at destroying these structures have so far been unsuccessful (Mancuso et al., 2006).

 

Therefore, if vascular normalization could be achieved with doses of VEGF inhibition that were just sufficient to prevent the capacity for sprouting angiogenesis and eliminate the most leaky blood vessels, but not cause excessive vessel regression and hypoxia, then the perpetual cycles of vessel regression followed by hypoxia induced new vessel formation with ever-increasing deposition of basement membrane scaffolds and the development of resistance to antiangiogenic drugs could be halted and the therapeutic window could be extended.

 

Angiopoietin-2 (Ang-2) destabilizes the association of ECs and pericytes, thus keeping the vasculature in a state of plasticity that allows new vessels to form in response to angiogenic cues. Ang-2 acts as an opponent of the stabilizing effects of the ANG-1/TIE2 signaling axis and has also been shown to directly promote sprouting angiogenesis by binding to integrins expressed on migrating EC tip cells. Ang-2 is therefore an attractive target to prevent vascular remodeling. Dual inhibition of Ang-2 and VEGF could prevent new vessel formation by closing the plasticity window which would preventing further cycles of revascularization in response to hypoxia and therefore limit cancer growth.

 

Here, we focused on two strategies to achieve vascular normalization without inducing hypoxia induced rebound angiogenesis.

  1. Avoiding excessive VEGF inhibition to prevent undue vessel regression and hypoxia as a stimulus for rebound angiogenesis and basement membrane sleeves.
  2. Combining VEGF targeting with Ang-2 inhibition to enhance vascular normalization at low levels of VEGF inhibition by preventing Ang-2 mediated vascular destabilization and sprouting angiogenesis

 

In our study we treated mice xenografted with human colorectal cancer cells (LS174T) with the VEGF-targeting antibody bevacizumab with low (“B10”=0.5 mg/kg) or high (“B100”= 5 mg/kg) doses with or without an Ang-2 blocking peptibody L1-10 (L1). Besides assessing differences in the tumor growth rate and basement membrane sleeves, our study focused on parameters of vascular normalization including vessel density, pericyte coverage, endothelial cell adherence junctions, vessel leakiness, perfusion as well as tumor hypoxia and proliferation.

 

Key Findings:

We found that under conditions of low doses of VEGF inhibition, additional Ang-2 blockade (B10/L1) induced a further reduction in vessel density and tumor growth (MS-Figure 1). While Ang-2 is classically recognized as a vascular destabilizer, its effect on sprouting angiogenesis likely accounts for the additional reduction in vessel density (Felcht et al., 2012; Nasarre et al., 2009). Vessel regression primarily occurs in response to VEGF inhibition, leading to hypoxia and rebound angiogenesis.

 

Importantly, dual Ang-2/VEGF inhibition severely limits the tumor capacity to form new blood vessels preventing new vessel formation for example in response to hypoxia. This results in a shift to increased tumor cell death (MS-Figure 4A). Dual inhibition also provides increased stability and perfusion for the surviving tumor vessels. Dual inhibition clearly improved parameters of normalization including endothelial integrity, increased pericyte coverage, enhanced VE-cadherin tight junctions, leading to reduced permeability (MS-Figures 2 and 3). The number of leaky blood vessels was reduced from 66% in controls to just 22% in L1/B10 and 21% in L1/B100 tumors. Furthermore, the increased normalization upon dual inhibition increased the penetration depth, permitting better access for chemotherapy (MS-Figure 3C). Finally, whereas the massive tumor vessel turnover after bevacizumab-treatment left a dense network of basement membrane sleeves, these sleeves were almost absent after dual targeting, indicating that the angiogenic remodeling activity was suppressed (MS-Figure 4B).

 

Conclusions:

Dual targeting of VEGF and Ang2 achieved effective normalization of the tumor at only one-tenth of the dose required with bevacizumab alone. Specifically, pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth. These changes create a more supportive tumor microenvironment for immuno- or chemotherapy (Huang et al., 2012)  and permit better penetration of cytostatic drugs, but are also thought to reduce the frequency of metastatic dissemination (Holopainen et al., 2012).

 

OC FIG1

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