gms | German Medical Science

Objective: Cytomegalovirus (CMV) is a ubiquitous member of the herpes virus family with a prevalence of between 50-100% in the human population and lifelong persistence. While normally clinically benign, severe cases are observed in neonates and immunocompromised patients. Over the last decade, CMV has been associated with glioblastoma (GBM) and other tumors. The aim of this study is to clarify the interrelationship between CMV and GBM, as to elucidate its contribution to GBM progression.

Methods: Pericytes, proneural (PN) and mesenchymal (MES) human GBM cell lines were assessed via flow cytometry for permissiveness to CMV (Towne Strain) infection. Cytokine expression pre- and post-infection was analyzed using Real-time-PCR. Transwell migration and endothelial tube formation were evaluated in co-culture with infected and uninfected GBM cell lines. C57BL/6 mice were infected with murine CMV (MCMV-Δm157) at P2 and syngeneic GL261 tumor cells were implanted at week 14 after latent infection was established. Murine as well as human brain sections were analyzed by immunofluorescence staining.

Results: CMV was found to closely co-localize with tumor vessels in patient derived GBM specimens. Interestingly in vitro studies confirmed a high permissiveness of pericytes for CMV infection. GBM cells, permissive for CMV, had a proneural, rather than a mesenchymal signature. In addition, upregulation of pro-angiogenic cytokines such as IL-6, TGF-beta and angiogenesis inducing receptor PAR-1 upon CMV infection was confirmed by RT-qPCR. Transwell assays revealed increased migration towards CMV infected cells after 48h. GBM cells towards infected pericytes: 1.5-fold (p=0.07); pericytes towards infected GBM cells: 1.48-fold (p=0.009). Further, co-culture of human brain microvascular endothelial cells (HBMEC) and CMV infected PN cells led to the establishment of larger (160%, p=<.0001) and more complex (number of junctions 9.5 (-) vs. 21 (+), p<.0001) tube formation on Matrigel compared to non-infected PN cells. In vivo, tumors formed by GL261 cells in MCMV infected mice (n=10) showed significantly higher numbers of infiltrating pericytes and were better vascularized compared to those of uninfected animals (n=10).

Conclusion: In this study, we show for the first time that pericytes are permissive for CMV infection. Infected GBM cells are characterized by a proneural signature. CMV induces a high cytokine production upon infection and establishes a pro-angiogenic microenvironment. Enhanced tube formation of HBMEC in vitro and the increased migration of GBM and pericytes towards infected cells, suggests a contribution of CMV to model the tumor microenvironment. Moreover, tumors show higher pericyte infiltration and increased angiogenesis in vivo. We conclude that CMV may cause GBM progression by increasing diffuse infiltration and angiogenesis via paracrine signaling.