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In glioblastoma (GBM), tumor-associated macrophages (TAM) represent up to one half of the cells of the tumor mass, including both infiltrating macrophages and resident brain microglia. In an effort to delineate the temporal and spatial dynamics of TAM composition during gliomagenesis, we used genetically engineered and GL261-induced mouse models in combination with CX3CR1GFP/WT;CCR2RFP/WT double knock-in mice. Using this approach, we demonstrated that CX3CR1LoCCR2Hi monocytes were recruited to the GBM, where they transitioned to CX3CR1HiCCR2Lo macrophages and CX3CR1HiCCR2− microglia-like cells. Infiltrating macrophages/monocytes constituted approximately 85% of the total TAM population, with resident microglia accounting for the approximately 15% remaining. Bone marrow–derived infiltrating macrophages/monocytes were recruited to the tumor early during GBM initiation, where they localized preferentially to perivascular areas. In contrast, resident microglia were localized mainly to peritumoral regions. RNA-sequencing analyses revealed differential gene expression patterns unique to infiltrating and resident cells, suggesting unique functions for each TAM population. Notably, limiting monocyte infiltration via genetic Ccl2 reduction prolonged the survival of tumor-bearing mice. Our findings illuminate the unique composition and functions of infiltrating and resident myeloid cells in GBM, establishing a rationale to target infiltrating cells in this neoplasm. Cancer Res; 77(9); 2266–78. ©2017 AACR.

APLNR expression is upregulated in the infiltrating tumor zone in glioblastoma.

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

Dynamic modulation of CCR2 and CX3CR1 in myeloid cells isolated from tumors. With the decrease of Ly6C intensity, the cells present increased CX3CR1-GFP intensity but decreased CCR2-RFP intensity.

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.

Supplementary Figure S3. PROS1 is located in GSCs.

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 S1. Circulating Ly6CHi inflammatory monocytes diminish at terminal illness. Figure S2. Enhanced BM-derived cell infiltration in Cx3cr1GFP/GFP;Ccr2RFP/WT mice. Figure S3. RFP+ cells are only observed in the meninges in healthy naïve Cx3cr1GFP/WT;Ccr2RFP/WT mice. Figure S4. Bone marrow reconstitution efficiency as examined by flow cytometry. Figure S5. Significant up-regulated (red) and down-regulated (green) genes based on the pairwise comparisons. Figure S6. Loss of Ccl2 in stroma does not prolong survival of tumor-bearing mice. Figure S7. Immunohistochemistry staining and quantification of phosphor-histone 3 proliferating cells. Figure S8. Immunohistochemistry staining and quantification of Ki67+ proliferating cells. Figure S9. TUNEL staining of apoptotic cells. Figure S10. Survival curve of PDGF-driven tumor in WT, Ccl2+/- and Ccl2-/- mice. Supplementary Table S1. Combined Cx3cr1 and Ccr2 heterozygous knockout does not affect tumor development or survival. Supp. Table S2. Significantly enriched functional pathways in tumor-associated microglia. Supp. Table S3. Significantly enriched functional pathways in tumor-associated microglia and tumor-associate macrophages. Supp. Table S4. Significantly enriched functional pathways in tumor-associated macrophages.

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

Apelin is expressed in tumor vessels.

Supplementary Figure S1. AXL expression has inverse correlation with PDGFRα expression in GSCs.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1–AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors.Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002–13. ©2018 AACR.

Anti-invasive role of apelin in orthotopic models of proneural and classical glioblastoma.

APLN co-expressed genes are enriched for angiogenesis in glioblastoma subtypes

Supplementary figure legends and materials and methods

Supplementary Figure S5. BGB324 treatment decreases PD-L1 expression in tumors.

APLNR is expressed in glioblastoma cells and receptor internalization is activated by apelin peptides specifically.

Antiangiogenic therapy of glioblastoma (GBM) with bevacizumab, a VEGFA-blocking antibody, may accelerate tumor cell invasion and induce alternative angiogenic pathways. Here we investigate the roles of the proangiogenic apelin receptor APLNR and its cognate ligand apelin in VEGFA/VEGFR2 antiangiogenic therapy against distinct subtypes of GBM. In proneural GBM, apelin levels were downregulated by VEGFA or VEGFR2 blockade. A central role for apelin/APLNR in controlling GBM vascularization was corroborated in a serial implantation model of the angiogenic switch that occurs in human GBM. Apelin and APLNR are broadly expressed in human GBM, and knockdown or knockout of APLN in orthotopic models of proneural or classical GBM subtypes significantly reduced GBM vascularization compared with controls. However, reduction in apelin expression led to accelerated GBM cell invasion. Analysis of stereotactic GBM biopsies from patients as well as from in vitro and in vivo experiments revealed increased dissemination of APLNR-positive tumor cells when apelin levels were reduced. Application of apelin-F13A, a mutant APLNR ligand, blocked tumor angiogenesis and GBM cell invasion. Furthermore, cotargeting VEGFR2 and APLNR synergistically improved survival of mice bearing proneural GBM. In summary, we show that apelin/APLNR signaling controls GBM angiogenesis and invasion and that both pathologic features are blunted by apelin-F13A. We suggest that apelin-F13A can improve the efficiency and reduce the side effects of established antiangiogenic treatments for distinct GBM subtypes.Significance:Pharmacologic targeting of the APLNR acts synergistically with established antiangiogenic treatments in glioblastoma and blunts therapy resistance to current strategies for antiangiogenesis.See related commentary by Amoozgar et al., p. 2104

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

Supplementary Figure S2. CD11b(+) cells were depleted by BLZ945.

Glioblastoma cell- and host-derived apelin controls tumor angiogenesis.

Apelin expression is correlated with angiogenesis in human glioblastoma.

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

Supplementary Figure S6. BGB324 inhibits mouse tumor cells.

Supplementary Figure S4. Identification of T cells subpopulation in GBM dissociated tumor via CyTOF.

Supplementary Methods and Materials from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 6 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 4 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 1 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure 2 from Tuberous Sclerosis Complex Suppression in Cerebellar Development and Medulloblastoma: Separate Regulation of Mammalian Target of Rapamycin Activity and p27Kip1 Localization

Supplementary Figure 9 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 1 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 1 from Tuberous Sclerosis Complex Suppression in Cerebellar Development and Medulloblastoma: Separate Regulation of Mammalian Target of Rapamycin Activity and p27Kip1 Localization

Supplementary Figure 3 from Tuberous Sclerosis Complex Suppression in Cerebellar Development and Medulloblastoma: Separate Regulation of Mammalian Target of Rapamycin Activity and p27Kip1 Localization

Supplementary Figure Legends 1-4 from Tuberous Sclerosis Complex Suppression in Cerebellar Development and Medulloblastoma: Separate Regulation of Mammalian Target of Rapamycin Activity and p27Kip1 Localization

Gli signaling is critical for central nervous system development and is implicated in tumorigenesis. To monitor Gli signaling in gliomas in vivo, we created platelet-derived growth factor–induced gliomas in a Gli-luciferase reporter mouse. We find that Gli activation is found in gliomas and correlates with grade. In addition, we find that sonic hedgehog (SHH) is expressed in these tumors and also correlates with grade. We identify microvascular proliferation and pseudopalisades, elements that define high-grade gliomas as SHH-producing microenvironments. We describe two populations of SHH-producing stromal cells that reside in perivascular niche (PVN), namely low-cycling astrocytes and endothelial cells. Using the Ptc-LacZ knock-in mouse as a second Gli responsive reporter, we show β-galactosidase activity in the PVN and in some tumors diffusely throughout the tumor. Lastly, we observe that SHH is similarly expressed in human gliomas and note that an intact tumor microenvironment or neurosphere conditions in vitro are required for Gli activity. [Cancer Res 2008;68(7):2241–49]

Supplementary Figure 2 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure Legends 1-4 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure 8 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Methods from Tuberous Sclerosis Complex Suppression in Cerebellar Development and Medulloblastoma: Separate Regulation of Mammalian Target of Rapamycin Activity and p27Kip1 Localization

Supplementary Figure 3 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure 7 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 4 from Preclinical Evaluation of Radiation and Perifosine in a Genetically and Histologically Accurate Model of Brainstem Glioma

Supplementary Figure 4 from Tuberous Sclerosis Complex Suppression in Cerebellar Development and Medulloblastoma: Separate Regulation of Mammalian Target of Rapamycin Activity and p27Kip1 Localization

Supplementary Figure 3 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 2 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Supplementary Figure 10 from Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas