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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

Angiogenesis, the development of a neovasculature, is a key histologic feature of malignant glioma. It occurs through multiple mechanisms, including sprouting and migration of existing endothelial cells (ECs), and is driven by signals in the microenvironment. Glioma stem cells (GSC) inhabit a region near ECs (perivascular niche). Here we examined the hypothesis that GSCs interact directly with ECs and promote EC motility. In 4 of 5 glioblastoma biopsies we found that ∼40% of GSCs were localized within 25 μm of ECs, based on double-labeling for Sox2 and vWf. We then examined the effect of GSCs on EC motility when plated on a 2D laminin substrate, and found a significant increase in EC motility in the presence of GSCs as well as a direct interaction. In a 3D assay, EC migration was enhanced by GSC-conditioned media, but the physical presence and interaction between ECs and GSCs further increased EC migration through a laminin-coated, 3-μm pore filter, as compared to GSC-conditioned media. Integrin αvβ3 and L1CAM have been reported to be upregulated on ECs and GSCs, respectively, in GBM tumors, and integrin αvβ3 can bind the RGD-peptide in L1CAM. We examined the effect of blocking or downregulating integrin αvβ3 or L1CAM on EC interaction with GSCs. In a cell-cell binding assay, blocking or downregulating the integrin β3 subunit significantly inhibited EC-GSC direct contact, as did an RGD peptide and an antibody to L1CAM. Furthermore, downregulation of integrin β3 on ECs significantly reduced EC migration in the presence of GSCs in the transwell migration assay and reduced EC association with GSCs in mouse brain slices in organotypic culture, analyzed by 2-Photon-LSM. These data suggest GSCs promote angiogenesis in part by promoting EC motility, and that the EC-GSC direct interaction mediated by integrin αvβ3 on ECs and L1CAM on GSCs also stimulates EC motility. Citation Format: Monica E. Burgett, Ping Huang, Russell S. Tipps, Amit Vasanji, Justin D. Lathia, Meizhang Li, Shideng Bao, Jeremy N. Rich, Candece L. Gladson. Glioma stem cells promote brain endothelial cell motility: a new mechanism for the direct interaction of glioma stem cells with endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2013-3898

Angiogenesis is a prominent characteristic of malignant glioma. Glioma stem cells (GSCs) reside adjacent to blood vessels in the perivascular niche, and recent evidence suggests a role for GSCs in promoting angiogenesis. Here we examined the interplay between GSCs and endothelial cells (ECs) on a laminin substrate, and the potential for GSC promotion of EC motility. In an in vitro 2D motility assay, we found that GSCs dramatically stimulated EC migration, and ECs also stimulated GSC migration. An antibody to integrin α6 blocked in part the motility of both GSCs and ECs. We also found that GSCs directly interact with ECs; therefore, we investigated which cell-adhesion molecules mediated the interaction. In a cell-cell binding assay, blocking antibodies to both L1-cell adhesion molecule (L1CAM) and integrin αvβ3 inhibited in part the direct contact between GSCs and ECs. In the motility assay, the combination of antibodies to L1CAM and αvβ3 significantly inhibited the motility and interaction of both cell types. These data suggest GSCs can promote angiogenesis by promoting EC migration, and indicate a role for the cell-adhesion receptors L1CAM and αvβ3 in the direct interaction between GSCs and ECs that may promote EC motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5288. doi:1538-7445.AM2012-5288