SC-22 * GLIOMA STEM CELLS INTERNALIZE BEVACIZUMAB FOUND IN THE PERIVASCULAR NICHE AND TARGET IT FOR RECYCLING OR DEGRADATION

Abstract

Antiangiogenic therapy shows great promise for treatment of cancer. Bevacizumab is a humanized mAb that blocks VEGF-A, thereby inhibiting angiogenesis. It has received FDA approval for patients with recurrent glioblastoma (GBM); however, approximately 30% of patients are non-responsive and the underlying mechanism for the lack of response is not known. It has been assumed that Bevacizumab solely targets circulating VEGF-A; however, VEGF-A found in the perivascular space of GBM tumors could also be an important target. We hypothesized that Bevacizumab is transcytosed across brain endothelial cells (ECs), gains access to the perivascular niche containing glioma stem cells (GSCs), and that differences in GSC endocytosis and targeting of Bevacizumab for recycling or degradation determines a patients' response. We found that Bevacizumab is internalized by normal and tumor-isolated brain ECs in a time-dependent manner that is partially inhibited by amiloride and stimulated by EGF, suggesting Bevacizumab enters the cells by macropinocytosis. ECs transcytose 62% of Bevacizumab over two-hours. Importantly, we found that 95% of GSCs internalize Bevacizumab in 30 minutes. Internalized Bevacizumab in GSCs can be recycled to the extracellular space or degraded; at 5 minutes we found that Bevacizumab was localized to the Rab4+ fast recycling compartment (67%); whereas at 30 minutes only 7% localized to the Rab4+ compartment and 16% localized to the LAMP1+ compartment (late endosome/lysosome). Furthermore, in an orthotopic xenograft mouse model of GBM administered Bevacizumab, we found a gradient of Bevacizumab extending from the vessel into the tumor and it was readily detected within perivascular tumor cells. Our data indicate that the trafficking of Bevacizumab by perivascular GSCs in GBM may influence Bevacizumab effectiveness in patients. Understanding the mechanism of internalization and trafficking of Bevacizumab in GSCs from different molecular subtypes of GBM may help us understand which patients will be responsive to Bevacizumab therapy.