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Review
. 2016 Feb;23(2):122-33.
doi: 10.1111/micc.12251.

NG2 Proteoglycan-Dependent Contributions of Pericytes and Macrophages to Brain Tumor Vascularization and Progression

William B Stallcup  1 , Weon-Kyoo You  1   2 , Karolina Kucharova  1 , Pilar Cejudo-Martin  1 , Fusanori Yotsumoto  1   3
Affiliations
Review

NG2 Proteoglycan-Dependent Contributions of Pericytes and Macrophages to Brain Tumor Vascularization and Progression

William B Stallcup et al. Microcirculation. 2016 Feb.

Abstract

The NG2 proteoglycan promotes tumor growth as a component of both tumor and stromal cells. Using intracranial, NG2-negative B16F10 melanomas, we have investigated the importance of PC and Mac NG2 in brain tumor progression. Reduced melanoma growth in Mac-NG2ko and PC-NG2ko mice demonstrates the importance of NG2 in both stromal compartments. In each genotype, the loss of PC-endothelial cell interaction diminishes the formation of endothelial junctions and assembly of the basal lamina. Tumor vessels in Mac-NG2ko mice have smaller diameters, reduced patency, and increased leakiness compared to PC-NG2ko mice, thus decreasing tumor blood supply and increasing hypoxia. While the reduced PC interaction with endothelial cells in PC-NG2ko mice results from the loss of PC activation of β1 integrin signaling in endothelial cells, reduced PC-endothelial cell interaction in Mac-NG2ko mice results from 90% reduced Mac recruitment. The absence of Mac-derived signals in Mac-NG2ko mice causes the loss of PC association with endothelial cells. Reduced Mac recruitment may be due to diminished activation of integrins in the absence of NG2, causing decreased Mac interaction with endothelial adhesion molecules that are needed for extravasation. These results reflect the complex interplay that occurs between Mac, PC, and endothelial cells during tumor vascularization.

Keywords: macrophage recruitment; neuron-glia antigen 2 proteoglycan; pericyte-endothelial cell interaction; tumor growth; tumor microenvironment; tumor vascularization.

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Figures

Figure 1
Figure 1. Decreased brain tumor progression in myeloid- and pericyte-specific NG2 null mice
B16F10 tumors in control versus Mac-NG2ko mice (A) and in control versus PC-NG2ko mice (C) at 10 days after tumor initiation. Graphs quantify tumor volumes in control versus Mac-NG2ko mice (B) and in control versus PC-NG2ko mice (D). Data in B represent 21 control and 20 Mac-NG2ko mice. Data in D represent 22 control and 18 PC-NG2ko mice. *P<0.01 compared to controls. Figure taken from ref (90).
Figure 2
Figure 2. Myeloid-specific NG2 ablation impairs macrophage recruitment to tumors
Double immunostaining for CD11b (blue) and CD31 (green) in tumor sections from control (A-C) and Mac-NG2ko mice (D-F). Areas of overlap in z-stacks appear as pale blue (C,F). Quantification of macrophage abundance in tumors using the markers F4/80 (G) and CD11b (H). Data are plotted as the percentage of total tumor area occupied by marker pixels in 10-day tumors (G,H). Scale bars = 20 μm. *P<0.01 compared to controls. Figure taken from ref (90).
Figure 3
Figure 3. Loss of pericyte-endothelial cell interaction following myeloid-specific NG2 ablation
Double immunostaining for PDGFRβ (red) and CD31 (green) was used to quantify pericyte and endothelial cell abundance, along with pericyte ensheathment of endothelial cells, in tumor sections from control (A-C) and Mac-NG2ko mice (D-F). Z-stacks of confocal images were used to generate three-dimensional reconstructions for this purpose. 10-day tumors in Mac-NG2ko mice exhibit decreased CD31 pixel density (G) due to diminished vessel diameter (H). In addition, the percentage of pericytes lacking physical contact with endothelial cells (I; arrows in F) is increased, resulting in diminished pericyte ensheathment of endothelial cells (J). Scale bar = 20 μm. *P<0.01 compared to controls. Figure taken from ref (90).
Figure 4
Figure 4. NG2 down-regulation in pericytes decreases activation of β1 integrin in endothelial cells
(A) Three different in vitro culture formats were used to investigate interactions between human umbilical vein endothelial cells (HUVECs) and human brain vascular pericytes (HBVP). HUVECs and pericytes were cultured on opposite sides of a transwell membrane (in-contact); HUVECs alone were cultured on a transwell membrane (endothelium-only); HUVECs were cultured on a transwell membrane, and pericytes were cultured on the well bottom (non-contact). The in-contact model was used to examine pericyte-mediated activation of endothelial cell β1. Membranes were immunostained for CD31 (green) and activated β1 integrin (red), and confocal microscopy was used to examine the endothelial cell monolayer. CD31 labeling is similar in endothelial cell monolayers co-cultured with either control (B) or NG2 knockdown pericytes (D). However, activated β1 is more prominent and more closely co-localized with CD31 on the surfaces of HUVECs co-cultured with control pericytes (C) than on HUVECs co-cultured with NG2 knockdown pericytes (E, quantified in F). In the endothelium-only model, β1 integrin activation is increased by addition of soluble NG2 (sNG2)(G). In the non-contact model, activation of β1 integrin is not affected by treatment with control or NG2 siRNA (H). Levels of total β1 integrin are not affected in any of the three models (not shown). *P<0.05 compared to GAPDH siRNA (F). †P<0.05 compared to absence of sNG2 (G). Scale bar = 120 μm (B-E). Figure taken from ref (92).
Figure 5
Figure 5. NG2-dependent activation of β1 and β2 integrins in THP-1 macrophages
Human THP-1 monocytes were differentiated via PMA treatment and then transfected with control siRNA (siCT) or NG2 siRNA (siNG2). NG2 expression (A) was quantified by immunofluorescence with rabbit anti-NG2 antibody, revealing a 70% decrease in NG2 expression in NG2 knockdown cells. Activation of β1 integrin (B) was evaluated by immunolabeling with HUTS-21 monoclonal antibody, revealing a 50% decrease in activation in NG2 knockdown cells. Activation of β2 integrin (C) was investigated by immunolabeling with mAb-24 monoclonal antibody, revealing a 75% decrease in activation in NG2 knockdown cells. Labeling is quantified as pixels per cell. In B and C, levels of total β1 and β2 expression were not significantly altered by NG2 knockdown (not shown). Each data point is obtained from a separate culture well, and numerical values represent the number of pixels per cell averaged from 4 fields in each well. Thus in A, n = 17 wells, in B, n = 5 wells, and in C, n = 6 wells.
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