Macropinocytosis of Bevacizumab by Glioblastoma Cells in the Perivascular Niche Affects their Survival

Abstract

Purpose

Bevacizumab, a humanized monoclonal antibody to VEGF, is used routinely in the treatment of patients with recurrent glioblastoma (GBM). However, very little is known regarding the effects of bevacizumab on the cells in the perivascular space in tumors.

Experimental Design

Established orthotopic xenograft and syngeneic models of GBM were used to determine entry of monoclonal anti-VEGF-A into, and uptake by cells in, the perivascular space. Based on the results, we examined CD133 ⁺ cells derived from GBM tumors in vitro. Bevacizumab internalization, trafficking and effects on cell survival were analyzed using multi-label confocal microscopy, immunoblotting and cytotoxicity assays in the presence/absence of inhibitors.

Results

In the GBM mouse models, administered anti-mouse-VEGF-A entered the perivascular tumor niche and was internalized by Sox2 ⁺/CD44 ⁺-tumor cells. In the perivascular tumor cells, bevacizumab was detected in the recycling compartment or the lysosomes, and increased autophagy was found. Bevacizumab was internalized rapidly by CD133 ⁺/Sox2 ⁺-GBM cells in vitro through macropinocytosis with a fraction being trafficked to a recycling compartment, independent of FcRn, and a fraction to lysosomes. Bevacizumab treatment of CD133 ⁺ GBM cells depleted VEGF-A and induced autophagy thereby improving cell survival. An inhibitor of lysosomal acidification decreased bevacizumab-induced autophagy and increased cell death. Inhibition of macropinocytosis increased cell death, suggesting macropinocytosis of bevacizumab promotes CD133 ⁺ cell survival.

Conclusions

We demonstrate that bevacizumab is internalized by Sox2 ⁺/CD44 ⁺-GBM tumor cells residing in the perivascular tumor niche. Macropinocytosis of bevacizumab and trafficking to the lysosomes promotes CD133 ⁺ cell survival, as does the autophagy induced by bevacizumab depletion of VEGF-A.