Abstract 1388: Inhibition of VEGF and angiopoietin-2 to reduce brain metastases of breast cancer burden
Authors: Bohn Kaci A. , Sechrest Emily R. , Adkins Chris E. , Mittapalli Rajendar K. , Nounou Mohamed I. , Terrell-Hall Tori B. , Mohammad Afroz S. , Lockman Paul R. 
Published: August 2015

Introduction: In cancer, vascular destabilization and angiogenesis enhance tumor growth, metastatic potential, and correlate with poor patient outcome. A primary driver of angiogenesis is vascular endothelial growth factor (VEGF), secreted by tumor cells in response to decreased vessel density and hypoxia; and is highly expressed in breast cancer. A secondary driver, angiopoietin-2 (Ang-2), is activated by hypoxia, and induces vessel destabilization upon Tie2 receptor binding. VEGF and Ang-2 have shown to independently and synergistically induce angiogenesis. Preclinical brain metastases models of breast cancer have shown administering bevacizumab results in reduced vascular permeability, and reduces overall metastatic burden in brain. Based upon these observations, we hypothesize that inhibition of both VEGF and angiopoetin-2 will further reduce metastatic burden in brain.

Methods: To determine if inhibition of VEGF and Ang-2 results in decreased metastatic burden, a human breast cancer (MDA-MB-231Br) cell line transfected to stably express eGFP was injected into the peripheral circulation via the left cardiac ventricle and CNS metastases grew for 4-6 weeks. As metastases seeded the brain, treatments were administered beginning on day 10. Bevacizumab (10 mg/kg, twice weekly) and L1-10 (Ang-2 inhibitor, 4 mg/kg twice weekly) were administered until day 32, when animals were euthanized and metastatic burden in brain was determined.

Results: To confirm that VEGF and Ang-2 synergistically induced angiogenesis in vitro with bevacizumab and L1-10, we used aortic ring and wound healing assays. We observed significant increase (p&lt; 0.01) in branching compared to control at 48 and 72 hours (211 ± 18.2 and 303 ± 47.9 branches) in wells treated with the combination of Ang-2 (100 ng/mL) and VEGF (10 ng/mL). Upon addition of bevacizumab (20 μg/mL), a significant decrease (p&lt; 0.05) in branching was seen at 24 hours (54 ± 10 branches) and 48 hours (161 ± 9.8) but not 72 hours. Similarly, L1-10 (6.5 μg/mL) administration reduced endothelial branching significantly (p&lt; 0.05) at all time points. We observed that nearly all lesions had evidence of hypoxia, VEGF, and Ang-2. We observed the number of metastatic lesions which developed using bevacizumab and L1-10 (10 ± 4) was significantly less (p&lt; 0.001) than bevacizumab treatment (31.6 ± 6) or untreated controls (72 ± 10). The percentage of large lesions (7.7 ± 5) in the combinatorial group was significantly less than bevacizumab treatment (14.6 ± 4; p&lt;0.05) and significantly less (p&lt; 0.05) than un-treated controls (34.6 ± 8).

Conclusion: In summary, we demonstrate an integral role of Ang-2 and VEGF in angiogenesis and brain metastases progression. Simultaneous inhibition of these two angiogenic factors may potentially reduce the formation of experimental brain metastases. Further work is required to determine if simultaneous inhibition of VEGF and Ang-2 may be effective to reduce brain metastasis formation.

Citation Format: Kaci A. Bohn, Emily R. Sechrest, Chris E. Adkins, Rajendar K. Mittapalli, Mohamed I. Nounou, Tori B. Terrell-Hall, Afroz S. Mohammad, Paul R. Lockman. Inhibition of VEGF and angiopoietin-2 to reduce brain metastases of breast cancer burden. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2015-1388