Matrigel (data not shown) suggesting that CXCL12 is not required for this process in vitro. HBMECs infected with lentivirus encoding scrambled shRNA induced a nearly twenty-fold increase in GBM cell number compared to GBM mono-culture. Despite consistent lattice formation, reduced expression of CXCL12 in HBMECs completely abrogated the trophic effect of endothelial cells on primary GBM cell growth (Figure 6B). Concordantly, culturing of U87 GBM cells with HUVECs did not enhance tumor cell growth (data not shown). Together, these data indicated that endothelial cell-derived CXCL12 mediates a direct trophic effect of endothelial cells on GBMs cells. To determine whether CXCL12 effects were mediated by CXCR4, we treated cultures with AMD3100 and measured tumor cell growth by bioluminescence imaging. The trophic effect of endothelial cells on primary GBM cells was inhibited by the specific CXCR4 antagonist, AMD3100 (Figure S5A). Consistent with prior studies in which AMD3100 and a second specific CXCR4 antagonist AMD3465 only blocked CXCL12-induced U87 cell growth [14,31], AMD3100 only inhibited primary GBM cell growth when the GBM cells were co-cultured with HBMECs, but not when they were in mono-culture. These data suggest that endothelial cells generated CXCL12 is responsible for the CXCR4 mediated trophic effect on GBM cells. In accordance with this conclusion, and consistent with published in vivo effects [14], AMD3100 treatment increased GBM cell apoptosis as measured by TUNEL staining (Figure S5B). Recently, a second CXCL12 receptor, CXCR7, has been reported to mediate survival effects in certain glioma cell lines [34]. To determine whether CXCR7 mediates GBM cell survival in response to endothelial cell-derived CXCL12 we evaluated CXCR7 expression in U87-HBMEC co-cultures by immunohis- tochemistry. Consistent with prior reports [48,49], HBMECs demonstrated a membranous pattern of CXCR7 expression cells (Figure S7A). In contrast, U87 cells exhibited little to no CXCR7 expression. To evaluate whether CXCR7 might also contribute to the trophic effect of endothelial cells we compared the effects of antagonizing both CXCL12 receptors CXCR4 and CXCR7 on U87 cell growth. Inhibition of CXCR4 by AMD3100 completely blocked HBMEC-induced enhanced growth. In contrast, the effects of the selective CXCR7 antagonists CCX733 and CCX771 were indistinguishable from the inactive control compound CCX704 (Figure S7B). Together these data suggest that in our co-culture models, endothelial cell-derived CXCL12 exerts a chemo-localization and a trophic effect on GBM cells through its primary signaling receptor CXCR4. Discussion There is an urgent need to understand the biology of microvascular endothelial cell-GBM interactions since they appear to support tumor growth, spread and resistance to treatment [6,7,16,39]. Practical experimental systems will be critical for establishing the basic biology and for high throughput screening for drugs capable of disrupting this interaction. The co-cultures described here are a tractable and relevant experimental system, as they recapitulate the chemo-localization and trophic effects of human brain endothelial cells on primary GBM cells. This simplified system is optimally suited for the detailed quantification of tumor cell migration as well as the trophic effects resulting from specific cell-cell interactions. In addition, uniform oxygenation in the co-cultures allows experimental separation of the trophic functions of endothelial cells away from their vascular functions such as oxygenation. Further, the absence of vasculogenesis in the co-cultures successfully isolates the contributions of CXCL12/ CXCL12 Mediated GBM-Endothelial Cell Interactions CXCR4 to growth regulation from their role in blood vessel formation. The ability to distinguish between these functions will be critical in interpreting and translating findings such as the correlation between increased CXCL12 signaling and increased vasculogenesis in an irradiated model of GBM xenografts [33]. Using the co-culture system, we identified CXCL12 as a mediator of endothelial cell trophic functions. Similar to its effects within germinal niches like the bone marrow [19,50,51,52] and external granule cell layer of the developing cerebellum [22], CXCL12 chemo-attracts brain tumor cells to the peri-endothelial cell space and stimulates their growth within that domain. The ability to target these endothelial cell functions could have significant therapeutic implications. The peri-endothelial cell space has been proposed to be a specialized brain tumor stem- like cell niche [6,53] as well as a route for GBM invasion [3,12,16,39]. While the existence of a distinct stem-like cell sub- population in GBM is controversial [54], tumor-derived cells with enhanced tumor-initiating activity have been repeatedly described (reviewed in [55,56]. Among the properties of these specialized cells appears to be enhanced resistance to radiation and chemotherapy [7,9,57]. If resistance to radiation therapy and chemotherapy are dependent upon localization to the peri- endothelial cell space, then mobilization of brain tumor cells with CXCR4 antagonists could be used in combination with radiation or standard chemotherapy to greater effect. This mechanism could in fact be the basis for the synergy observed between AMD3100 and BCNU in the treatment of intracranial U87 xenografts [58]. Similar observations were recently reported for acute myelogenous leukemia in which antagonism of CXCL12 resulted in leukemic blast mobilization from the bone marrow and enhanced anti- leukemia effect of tyrosine kinase inhibitors [59]. The combination of AMD3100 as a mobilizing agent for leukemic blasts and standard chemotherapy with mitoxanthrone, etoposide and cytarabine for the treatment of patients with relapsed or refractory AML is currently being evaluated in clinical trials. Further, it has been shown that CXCR4 mediates the perivascular migration of GBM cells [16,39]. The finding that tumor-associated endothelial cells express high levels of CXCL12 [13,14,39] supported the hypothesis that this characteristic feature of GBM would involve CXCR4. The present findings advance this hypothesis and provide additional rationale for pursuing CXCR4 antagonism in the treatment of GBM. Of particular importance may be the combination of CXCR4 antagonism and anti- angiogenic therapy. Anti-angiogenic therapy may decrease the number of perivascular sites capable of supporting and protecting brain tumor cells. Thus, combination therapy could provide both a structural and functional disruption of the perivasclar niche. In summary, primary GBM and HBMEC co-culture has identified an important role for CXCL12 in cell-cell interactions between GBM cells and endothelial cells. These data suggest that endothelial cells have ‘‘extra-vascular’’ functions that can directly drive tumor growth. Whether endothelial cell-derived molecules like CXCL12 create a specialized niche in the perivascular space, and how these molecules relate to the phenotype of stem-like cells remains to be determined. What is clear is that targeting both angiogenesis and endothelial cell function may represent a novel and powerful approach in the treatment of malignant brain tumors. Supporting Information Figure S1 Primary HBMECs Form Capillary-Like Net- works in Matrigel. (A) Primary HBMECs grow as a monolayer culture on uncoated coverslips (or plates) in endothelial cell growth PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33005