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

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.

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

PDF file - 695K, Significantly increased mRNA Expression of TNF-R1 and TNF-R2 in GBM as compared to normal brain

PDF file - 658K, Characterization of the integrin receptors mediating the attachment of primary brain ECs to collagen, fibronectin and laminin.

PDF file - 481K, Dose-dependent TNF�\�{induced apoptosis of primary brain ECs.

PDF file - 1MB, Absence of cFLIP expression in human GBM cells and human GBM stem cells plated on laminin

PDF file - 72K, Increased Expression of TNFalpha, TNF-R1 and TNF-R2 in the ECs in Recurrent GBM

PDF file - 972K, Percent of ECs expressing integrin alpha 6 beta 1 versus integrin alpha 6 beta 4.

PDF file - 71K, Increased Expression of TNFalpha, TNF-R1 and TNF-R2 in the ECs in Untreated GBM

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM), which requires endothelial cell (EC) migration. Here, we show that angiogenesis can also be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and ECs. The direct interaction between CSCs and ECs was mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. Using in vitro assays, we found that this interaction increased network formation and migration-associated signaling events in ECs, including activation of integrin αvβ3, FAK, bone marrow tyrosine kinase on chromosome X (BMX), p130CAS, ERK and JNK. Comparison of the effects of co-culturing CSCs with ECs versus the effects of conditioned media from CSCs co-cultured with ECs indicated that upstream and downstream effector activation was not attributed to a secreted factor(s). Activation of αvβ3 and BMX was required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells (consistent with CSCs) in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results show that direct interactions between CSCs and ECs have potent effects on EC migration and reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting. Citation Format: Monica Burgett, Justin Lathia, Patrick Roth, Amy Nowacki, Elena Pugacheva, Ping Huang, Amit Vasanji, Li Meizhang, Tatiana Byzova, Tom Mikkelsen, Shideng Bao, Jeremy Rich, Michael Weller, Candece Gladson. Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3380.

Sprouting angiogenesis requires endothelial cell (EC) migration. In glioblastoma (GBM), cancer stem cells (CSC) in the perivascular niche secrete factors that promote EC migration. Here, we identify a direct interaction between the CSCs and ECs that is mediated by RGD-peptide-dependent binding of L1CAM on CSCs with integrin αvβ3 on ECs. Using in vitro assays, we found that this interaction increased migration-associated signaling events in ECs, including activation of integrin αvβ3, ERK, JNK and Akt, and VCAM-1 and E-selectin mRNA expression. Comparison of the effects of co-culturing CSCs with ECs versus the effects of conditioned media from CSCs co-cultured with ECs indicated that the effects of the direct interaction and soluble factors differed quantitatively and qualitatively. The direct interactions stimulated the migration of ECs and primed them to the migratory effects of soluble factors produced by the CSCs. The direct interaction also enhanced expression of pro-survival genes in CSCs and ECs. In GBM biopsies, Sox2-positive tumor cells were localized in close proximity to ECs and injection of β3-negative ECs or L1CAM-negative CSCs into brain slices in organotypic culture resulted in reduced interactions between ECs and CSCs. RGD-peptide treatment of mice with established intracerebral GBM xenografts increased the mean distance of Sox2-positive tumor cells from ECs, decreased integrin αvβ3 activation and decreased vessel surface area. These data show that direct interactions between CSCs and ECs have potent effects on EC migration and angiogenesis in GBM and have implications for the design of angiogenesis-targeted therapies. Citation Format: Monica E. Burgett, Justin D. Lathia, Patrick Roth, Amy S. Nowacki, Ping Huang, Amit Vasanji, Meizhang Li, Tatiana Byzova, Tom Mikkelsen, Shideng Bao, Jeremy Rich, Michael Weller, Candece L. Gladson. L1CAM and integrin αvβ3 mediate direct cell contact between cancer stem cells and endothelial cells: Promotion of endothelial cell migration and survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4178. doi:10.1158/1538-7445.AM2015-4178

TNFα induces cell death of primary human brain microvessel endothelial cells (MvEC) in a dose-dependent manner through TNF-R1 activation. To determine whether TNF-R1 was available to be therapeutically manipulated on tumor-associated endothelial cells in glioblastoma tumors, we evaluated its expression on the endothelial cells in 50 human glioblastoma and 38 normal brain samples by immunohistochemistry. A significantly increased expression of TNF-R1 and TNFα was detected on the endothelial cells in the tumor biopsies as compared to the normal brain samples. We then investigated whether integrins regulate TNF-R1 pro-apoptotic signaling in MvEC isolated from normal brain; these cells proliferate in culture and resemble reactive endothelial cells. TNFα stimulation readily induced cell death of the brain MvEC when plated on collagen or fibronectin (attachment mediated by integrins α2β1 and α5β1, respectively), but the cells were protected from this pro-death signal when plated on laminin (mediated by integrin α6β1). In the cells plated on collagen (not on laminin) a time-dependent increase in the phosphorylation of p38 MAP kinase was detected with TNFα stimulation and knockdown of p38 MAP kinase blocked the TNFα-induced cell death suggesting p38 MAP kinase is a downstream effector for TNFα-induced apoptosis. We then examined whether engagement of a particular integrin signaled for expression of an anti-apoptotic protein and found expression of cFLIP when the MvEC were plated on laminin, but not when the cells were plated on collagen. Furthermore, down-regulation of cFLIP using siRNA restored the pro-apoptotic signal of TNFα in MvEC plated on laminin. To substantiate the clinical relevance of these findings, we examined the effect of TNFα stimulation on endothelial cells isolated from two different glioblastoma tumors, and found that TNFα induced cell death was similarly regulated by the ECM or integrins. Our data suggest that TNF-R1 on tumor-associated endothelial cells in glioblastoma tumors is available to be therapeutically manipulated as part of an anti-angiogenic therapy, and that the effectiveness of such a therapy maybe in part dependent on the activity of specific integrins expressed on the tumor endothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4761. doi:10.1158/1538-7445.AM2011-4761