RAGE expression in tumor-associated macrophages promotes angiogenesis in glioma

Abstract

Interaction of RAGE (the receptor for advanced glycation endproducts) with its ligands can promote tumor progression, invasion, and angiogenesis. Although blocking RAGE signaling has been proposed as a potential anticancer strategy, functional contributions of RAGE expression in the tumor microenvironment (TME) have not been investigated in detail. Here, we evaluated the effect of genetic depletion of RAGE in TME on the growth of gliomas. In both invasive and noninvasive glioma models, animal survival was prolonged in RAGE knockout (Ager(-/-)) mice. However, the improvement in survival in Ager(-/-) mice was not due to changes in tumor growth rate but rather to a reduction in tumor-associated inflammation. Furthermore, RAGE ablation in the TME abrogated angiogenesis by downregulating the expression of proangiogenic factors, which prevented normal vessel formation, thereby generating a leaky vasculature. These alterations were most prominent in noninvasive gliomas, in which the expression of VEGF and proinflammatory cytokines were also lower in tumor-associated macrophages (TAM) in Ager(-/-) mice. Interestingly, reconstitution of Ager(-/-) TAM with wild-type microglia or macrophages normalized tumor vascularity. Our results establish that RAGE signaling in glioma-associated microglia and TAM drives angiogenesis, underscoring the complex role of RAGE and its ligands in gliomagenesis.

©2014 American Association for Cancer Research.

Figures

Figure 1

Impact of RAGE ablation in tumor microenvironment on glioma growth. A, Kaplan-Meier plots (top panel) and tumor luciferase activity (lower panel) in wild-type (WT) and RAGE knockout (Ager−/−) mice implanted with either intracranial GL261 or KLuc gliomas (n=7-8 mice/group, MS: median survival). B, Despite improvement in survival, tumor size (two weeks after implantation) was similar in WT and Ager−/− mice. Cross sections through the largest tumor area were used for size calculations (n=4 mice/group ± SD). C, Except for increased central necrosis in the GL261 tumors in Ager−/− mice, tumor histology and invasion pattern was similar in both strains. Experimental results are representative of three separate experiments.

Figure 2

Expression of RAGE and its ligands in gliomas. A, PCR (top panel) and immunostaining (lower panel) confirming lower RAGE expression in the lungs and brains of non-tumor bearing Ager−/− mice. B, Western analysis demonstrating in vitro expression of full-length (FL)-RAGE by each cell line prior to implantation. C, FL-RAGE Western blot and D, immunostaining of intracranial GL261 and K-Luc gliomas two weeks after implantation into either WT or Ager−/− mice (n=3 mice/group ± SD). E, Western analysis comparing the expression of RAGE ligands by each tumor type two weeks after intracranial implantation. Experimental results are representative of two separate experiments.

Figure 3

Impact of RAGE ablation on tumor inflammatory response. A, Infiltration of CD11b+ cells (microglia and macrophages) into GL261 and K-Luc gliomas two weeks after intracranial implantation into WT or Ager−/− CX3CR1GFP mice. B, Representative flow cytometry contour plots (top) and quantification (bottom) of tumor-associated leukocytes in two-week old intracranial GL261 and K-Luc tumors in WT or Ager−/− mice. (Lymphocytes: CD45+, CD11b-, microglia: CD45intermediate, CD11bhigh, macrophages: CD45+, CD11bhigh. n=3 mice/group ± SD). C, qPCR of pro-inflammatory cytokines in normal brain (NB), and intracranial GL261 and K-Luc tumors two weeks after implantation. (n=4 mice/group ± SD), *: p<0.05. Experimental results are representative of three separate experiments.

Figure 4

RAGE ablation abrogates tumor angiogenesis. A, qPCR of normal brain (NB) and intracranial GL261 and K-Luc gliomas demonstrating lower expression of tumor proangiogenic factors in Ager−/− mice (n=3 mice/group ± SD). B, Morphology (top panel) and quantification (lower panel) of GL261 and K-Luc blood vessels in WT and Ager−/− mice (n=4 mice/group ± SD). C, Tumor hypoxia (green staining) was measured by injecting glioma-bearing mice with pimonidazole two hours prior to tissue examination. Intracranial tumors were harvested two weeks after implantation. D, Effect of RAGE ablation on vascular permeability. Mice bearing two-week old intracranial GL261 or KLuc were injected with TRITC-dextran 150 two hours prior to image analysis. Vascular leakage is evident as dextran extravasation into perivascular space (arrows). E, Tumor vascular permeability was also assessed with Evans blue assay. Tumor-bearing mice were injected with Evans blue dye 30 minutes prior to sample collection. The dye was extracted and quantified with a spectrophotometer. (n=4 mice/group ± SD). *: p<0.05, ***: p<0.001. Experimental results are representative of two separate experiments.

Figure 5

RAGE expression in human glioblastomas. A, Representative immunohistochemistry of a glioblastoma tumor sample (left panel) and tumor edge (right panel) from the same patient demonstrating RAGE expression in tumor and not peritumoral white matter. B, RAGE expression in another glioblastoma tissue sample demonstrating nuclear (1), membranous (2) and cytoplasmic (3) RAGE expression in tumor cells (T). C, Expression of RAGE in tumor-associated vessels (CD34+, arrows) and macrophages (CD163+, arrows) in a representative glioblastoma sample. Nearly half of the tumor-associated macrophages expressed RAGE in every tumor.

Figure 6

Effect of RAGE ablation in tumor-associated macrophages (TAMs). A, Representative immunohistochemistry (left panel) and flow cytometry (right panel) of intracranial GL261 tumor in WT mice confirming RAGE expression in TAMs (left: CD11b+, arrows; right: red events). Nearly 20% of tumor macrophages CD45high CD11bhigh) and 50% of tumor microglia (CD45intermediate CD11bhigh) expressed RAGE. B, TAM expression of IL6 and VEGFα was suppressed in Ager−/− GL261 gliomas. TAMs were isolated by Percoll gradient from intracranial GL261 gliomas two weeks after implantation (n=4 mice/group ± SD). *: p<0.05, **: p<0.01. C, Expression of MMP9 was lower in Ager−/− primary bone marrow monocytes (BMM) after incubation with conditioned medium (CM) from GL261 cells. *: p<0.05, ***: p<0.001 D, Cathepsin S expression was modestly lower when Ager−/− BMM were incubated with IFNγ and GL261 CM, but not K-Luc CM. *: p<0.05. E, RAGE promoter activity was measured in WT and Ager−/− BMM after incubation with CM from GL261 and K-Luc cells. Although Ager promotor activity was lower in Ager−/− BMM under both conditions, its activity was not completely abolished when cells were exposed to K-Luc CM (n= 4 + SD). **: p<0.01, ***: p<0.001, ns: not significant as compared to cells transfected with control vector. Representative data from two separate experiments is shown.

Figure 7

RAGE ablation in TAMs abrogates angiogenesis in GL261 gliomas. A, To demonstrate the feasibility of generating chimeric mice, bone marrow (BM) cross transplant experiments between wild-type (WT) and CX3CR GFP1 mice were performed. After recovery, mice were implanted with GL261 tumors and analyzed by histochemistry and flow cytometry. Glioma macrophages were identified as CD45high CD11bhigh cells (green cells and green events in dot plot) and appeared to infiltrate throughout the tumors (left panel). In the reverse transplantation experiments (right panel) where recipient mice were CX3CR GFP1, tumor microglia were identified as CD45intermediate CD11bhigh cells (green events and cells) that mostly remained within the margin of the tumor. B, qPCR demonstrating suppression of VEGFα expression in GL261 tumors that were implanted into WT mice transplanted with Ager−/− BM. C, Tumor vessel characteristics in GL261 gliomas implanted into chimeric mice demonstrating normalization of dialated vessels in mice with either WT microglia or WT macrophages. Representative data from two separate experiments is shown. (n= 3 ± SD). *: p<0.05.