During breast cancer growth and development, angiogenesis is triggered by the interaction between vascular endothelial growth factor (VEGF) and its receptors VEGF-R1 and VEGF-R2. In breast cancer, alternative VEGF receptors, the neuropilins (Np1 and Np2), are often upregulated and serve to augment the effects of VEGF-R1/VEGF-R2 binding and provide alternative signalling pathways. Recently, a humanized antibody, Bevacizumab (Bz), which prevents VEGF binding to VEGF-R1/VEGF-R2, in combination with chemotherapy demonstrated initial efficacy (increased progression-free survival) in breast cancer phase III clinical trials. Eventually, however, the tumours evade treatment control. This may be because neuropilins are not blocked by Bz and provide an alternative VEGF signalling pathway in breast cancer. Therefore the present study aims to evaluate the potential of enhancing efficacy of Bz treatment by simultaneously blocking VEGF–neuropilin binding.
Western blot analysis of VEGF receptors in human endothelial cells (HUVEC and HuDMEC) and breast cancer cell lines (MDA-MB-436, MCF-7 and T47D) assessed relative expressions of VEGF-R1, VEGF-R2, Np1 and Np2 in these cells. Microvascular endothelial tubule formation assays were then performed in vitro where cells were incubated on Matrigel in the presence of VEGF for 24 hours, and then Bz (1 to 4 mg/ml) and/or anti-Np (50 to 100 ng/ml) antibody were added to the wells. The number of branch points were quantified in three fields of view/well in three separate experiments.
Western blot analysis revealed Np1 and Np2 expression in both breast cancer and endothelial cell lines, whereas VEGF-R1 is expressed in MCF-7, MDA-MB-436 and endothelial cells and VEGF-R2 is only expressed by endothelial cells. HuDMEC stimulated by VEGF formed tubules in vitro, and the number of branch points/field of view for VEGF-stimulated controls (43 ± 6) versus Bz (25 ± 3) versus anti-NP antibody (26 ± 4) versus Bz + anti-Np antibody (17 ± 3) demonstrated a significant (P < 0.001) reduction in tubule formation.
These data show that anti-Np antibodies increase the inhibitory effect of Bz and suggest that efficacy of breast cancer treatment with Bz may be enhanced by addition of a neuropilin blocking agent.