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Figure S3 shows co-localization of different fractions of bevacizumab or human IgG (hIgG) with Rab4 or LAMP1 in CD133+ GBM cells in vitro, and of rat anti-mouse VEGF-A IgG in an established tumor in the immune competent mouse model of GBM in vivo, show similar trafficking patterns.

Figure S1 shows that bevacizumab gains access to the perivascular tumor space and is internalized by tumor cells when administered to an orthotopic xenograft model of GBM and confirmation of cancer stem-like cell markers in vivo.

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 multilabel 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. Clin Cancer Res; 23(22); 7059–71. ©2017 AACR.

Figure S7 shows bevacizumab is transcytosed across normal brain ECs and TECs.

Figure S5 shows co-localization of a fraction of the bevacizumab-VEGF complex with Rab4 and a fraction with LAMP1 in CD133+ GBM tumor cells.

Figure S6 shows bevacizumab treatment causes a reduction in phospho-VEGFR2 and downregulation of VEGF-A induces autophagy in CD133+ GBM cells.

Figure S4 shows FcRn staining largely colocalizes with the endothelial cell marker vWf in xenograft GBM tumors.

Additional details regarding the methods for cells, reagents, animal studies, immunofluorescence, transcytosis, and ELISA assays.

Figure S2 shows the internalization of bevacizumab by CD133-negative non-stem GBM tumor cells was not inhibited by EIPA.