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.

Single‐Cell Intravital Microscopy of Trastuzumab Quantifies Heterogeneous in vivo Kinetics

“…Is phagocyte uptake of mAbs beneficial for efficacy? Based on prior studies, HER2‐independent phagocyte uptake may involve scavenging of interstitial Tzm, for instance via nonspecific macropinocytosis or Fc‐mediated uptake of unbound mAbs by macrophages . In contrast, HER2‐dependent enhancement in phagocyte uptake likely involves the interaction between HER2‐bound Tzm and highly expressed Fcγ receptors (FcγR) on phagocytes.…”

An Interplay between Senescence, Apoptosis and Autophagy in Glioblastoma Multiforme—Role in Pathogenesis and Therapeutic Perspective

“…However, emerging evidence suggests a beneficial effect of autophagy inhibition in bevacizumab-based therapy in cancer, including GBM [ 114 , 115 , 116 , 117 , 118 ]. Carmustine, another drug used in GBM therapy, is an alkylating agent similar to TMZ [ 119 ]. However, its mechanism of anticancer action is different from TMZ as it dialkylates DNA and induces interstrand cross-links, which are serious DNA damage preventing DNA strand separation required for cell cycle progression, replication, transcription and recombination [ 120 ].…”

Shedding of bevacizumab in tumour cells-derived extracellular vesicles as a new therapeutic escape mechanism in glioblastoma

“…2b ). This mechanism appears to be quite rapid as suggested by the detection of bevacizumab in EVs following 2 h treatment, which could happen via fast recycling endosomes as recently proposed [ 9 ].…”