Cytomegalovirus promotes murine glioblastoma growth via pericyte recruitment and angiogenesis

Abstract

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV+ mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV+ mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV+ mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV+ mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

Keywords: Brain cancer; Mouse models; Oncology; Pericytes; Virology.

Figure 1. MCMV infection accelerates GBM growth in mice.

(A) Experimental overview. (B) Kaplan-Meier curves of GL261Luc2 tumor-bearing mice. Uninfected, n = 20; MCMV⁺, n = 19. P < 0.0001, log-rank test. Median survival is indicated on plot and shown in parentheses. (C) BLI and (D) MRI analysis of tumor-bearing MCMV⁺ and control animals 30 days after tumor implantation. (D) Tumor volume rendering from MRI images (left), tumor volume over time (right). n = 3. Box extends from the 25th to 75th percentile, and the median is indicated by a horizontal line. The whiskers represent the maximum and minimum values. Statistical analysis was performed by 2-way ANOVA with Bonferroni’s correction. *P < 0.05; ***P < 0.005. (E) H&E staining of GL261Luc2 tumors at end points. Scale bars: 1 mm (left panels); 50 μm (right panels).

Figure 2. MCMV infection accelerates GBM blood vessel formation in mice.

(A) Ki67 (green) immunofluorescence in brain sections taken from animals at the end point of survival studies. DAPI-stained nuclei are shown in blue. Scale bar: 50 μm. Graph shows Ki67-positive nuclei counts from 12 independent fields in 3 tumor samples. ***P < 0.005, Student’s t test. (B) CD31 (red) immunofluorescence in sections from GL261Luc2 tumors at survival end points (left panels). Scale bars: 100 μm. AngioTool analysis (right panels). Graphs show data for vascular parameters. Images are from 12 independent fields for each condition. n = 4. *P < 0.05; **P < 0.01 Student’s t test. (C) Kaplan-Meier survival curve of mice intracranially implanted with the murine CT-2A GBM cell line. Uninfected (n = 6) vs. MCMV⁺ (n = 6). P < 0.01, log-rank test. Median survival is indicated on plot and shown in parentheses. (D) Left panels show CD31 immunofluorescence staining (red) of tumor vasculature in 20 μm sections from CT-2A tumors in control and MCMV⁺ animals at survival end points. Scale bar: 100 μm. Graphs show representations of the data for vascular parameters based on analysis of angiogenesis and vessel morphology. Images are from 12 independent fields and 3 tumor specimens for each condition. *P < 0.05; ***P < 0.005, Student’s t test. (E) ASL-fMRI showing T2 coronal sections with ASL heatmap overlay. Quantitative analysis of both groups at corresponding ROIs. n = 3. Box extends from the 25th to 75th percentile, and the median is indicated by a horizontal line. Whiskers represent the maximum and minimum values. *P < 0.05, Student’s t test.

Figure 3. CMV associates with vascular pericytes in GBM.

(A) CMV (green, Virusys CA150-1 antibody; red, Virusys CA003-100 antibody) immunofluorescence staining in brain sections from animals at the end point of survival studies. DAPI-stained nuclei are shown in blue. Scale bar: 50 μm. (B) Real-time qPCR analysis of MCMV IE1/m123 mRNA levels in GL261Luc2 tumors from MCMV⁺ and naive mice. n = 3. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent maximum and minimum values. *P < 0.05; ***P < 0.005, Holm-Šídák test. (C) CMV (red, Virusys CA003-100), CD31 (green), and NG2 (green) immunofluorescence in tumor sections from MCMV⁺ mice. DAPI-stained nuclei are shown in blue. Scale bar: 50 μm. (D) CD31 (red), NG2 (green), and nuclei (blue) immunofluorescence in tumors from MCMV⁺ mice. Scale bar: 50 μm. (E) NG2 fluorescence intensity in 12 independent fields of view from murine GBMs. n = 3. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent maximum and minimum values. Scale bar: 50 μm. ***P < 0.005, Student’s t test. (F) CMV (red, Virusys CA003-100), CD31 (green), and NG2 (green) immunostaining immunofluorescence in human GBM. Scale bar: 50 μm. Pearson’s rank colocalization. n = 3. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent maximum and minimum values.

Figure 4. Conditioned medium from CMV-infected GBM cells increases pericyte migration and endothelial cell tube formation.

(A) Transwell migration of HBVPs and MBVPs. Data are presented as mean ± SD. ***P < 0.005, Holm-Šídák test. (B) HBMEC tube formation on Matrigel performed in the presence of conditioned media from HCMV-infected human GSCs or HBVPs. Data are presented as mean ± SD. ***P < 0.005, Holm-Šídák test. (C) Aortic ring assay performed using conditioned media from uninfected and CMV-infected GL261Luc2 cells or MBVPs. Graphical representation shows number of sprouting vessels. Data are presented as box and whisker plot. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent maximum and minimum values. ***P < 0.005, 2-way ANOVA. Scale bars: 50 μM (B); 1 mm for (C).

Figure 5. PDGF-D upregulation by CMV in GBM.

(A) qRT-PCR for PDGF-D in GSCs after HCMV infection. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent the maximum and minimum values. Outlier is shown by a green circle. (B) Western blot for PDGF-D in GSCs after HCMV infection. (C) qRT-PCR for PDGF-D in GL261Luc2 cells after MCMV infection. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent maximum and minimum values. (D) Western blot for PDGF-D in GL261Luc2 cells after MCMV infection. qRT-PCR data are represented as mean ± SD. *P < 0.05; ***P < 0.005, Student’s t test (A and C). (E and F) Transwell migration of HBVP and MBVP in the presence and absence of PDGF-D–neutralizing antibodies, or recombinant PDGF-D (rPDGF-D), as indicated. CMV-infected GBM cells and controls were grown on the bottom of a 12-well plate, as shown. Data are represented as mean ± SD. ***P < 0.005, Holm-Šídák test. (G) PDGF-D (red) and CMV (green) immunofluorescence showing areas of high CMV (lower right) and low CMV (lower left) in human GBM. DAPI-stained nuclei are shown in blue. Scale bar: 50 μm (upper images) 10 μm (lower images). (H) Increased phosphorylation of NF-κB p65 subunit RelA in G44 GSCs 24 hours after infection with HCMV Towne strain. (I) siRNA-mediated knockdown of RelA in G44 GSCs 48 hours after transfection. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent maximum and minimum values. (J) PDGF-D downregulation in response to HCMV infection after RelA knockdown in G44 GSCs.

Figure 6. PDGF-D mediates the effects of MCMV in murine GBM.

(A) Western blot for PDGF-D in GL261cas9-Pdgfd-KO cells. (B) Transwell migration assay of MBVPs toward GL261cas9-PDGFD-KO cells. Data are represented as mean ± SD. ***P < 0.005, 2-way ANOVA. (C) Kaplan-Meier curves of naive and MCMV⁺ mice intracranially implanted with GL261Cas9 (n = 4), GL261cas9-Pdgfd-KO (guide 3) (n = 4), or GL261cas9-Pdgfd-KO guide 5 (n = 4). Median survival is indicated on plot and shown in parentheses. (D) T2-weighted MRI at day 30. Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent the maximum and minimum values. *P < 0.05, 2-way ANOVA. (E) CD31 (red), NG2 (green), and nuclei (blue) immunofluorescence in GL261Pdgfd-KO tumors at end points. Scale bar: 50 μm. (F) Quantitative analysis of angiogenesis and vessel morphology in tumor sections. Data are represented as mean ± SD. **P < 0.01; ***P < 0.005, 2-way ANOVA.

Figure 7. Cidofovir reverses CMV-induced proangiogenic phenotype in MCMV⁺ GBM mice.

(A) Kaplan-Meier survival curve of naive and MCMV⁺ GL261Luc2 tumor-bearing mice treated with cidofovir (CDV). Median survival is indicated on plot and shown in parentheses. (B) CMV (green, Virusys CA150-1 antibody) and PDGF-D (magenta) immunofluorescence of tumor sections from CDV-treated mice. DAPI-stained nuclei are shown in blue. Scale bar: 100 μm. Box plot shows quantitation of PDGF-D fluorescence intensity (n = 3). Box extends from 25th to 75th percentile, and median is indicated by horizontal line. Whiskers represent maximum and minimum values. ***P < 0.005, Student’s t test. Error bars indicate SD. (C) Quantitative analysis of angiogenesis and vessel morphology after CDV treatment. Scale bar: 100 μm. ***P < 0.005, 2-way ANOVA. Error bars indicate SD. Scale bar: 50 μm. (D) CD31 (red), NG2 (green), and nuclei (blue) immunofluorescence in tumor sections at survival end points after CDV treatment.