Induction of CYR61 by Oncolytic HSV Infection of Glioma

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The Ohio State University

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Oncolytic viruses (OV) are engineered to replicate only within tumor cells, spreading within and destroying malignant gliomas without harming non-neoplastic cells. While clinical trials have confirmed its safety, the evidence for efficacy of OV therapy remains elusive. To enhance OV efficacy, a correlation linking the level of intratumoral OV over time and patient outcome/response is necessary. However, such a correlation is currently infeasible due to the lack of available biomarkers that faithfully reflect OV activity in tumors. An initial transcript profiling of human glioma cell lines in vitro treated with OV revealed significant and consistent cysteine-rich 61 protein (CYR61) induction that mirrored OV presence. CYR61 is a secreted heparin-binding protein that is over-expressed in a majority of tumors, including glioma, and is a potent angiogenic factor. Thus, we hypothesized that CYR61 mRNA and protein expression will be up-regulated upon OV infection in most glioma cell lines both in vitro and in vivo, thereby indicating its use as a reliable biomarker to evaluate the efficacy of ongoing OV infection and replication in tumors. CYR61 mRNA induction by OV infection in vitro was evaluated in 4 human glioma cell lines and 6 patient derived human tumor specimens using Quantitative Real Time PCR (QRT-PCR), and was confirmed in 9 out of 10 gliomas tested. We further confirmed the induction of CYR61 protein by western blot analysis of infected U343, U87, and U87ΔEGFR cells. To verify the induction of CYR61 in vivo, we implanted intracerebral tumors (human U87ΔEGFR cells) in athymic nude mice and treated the tumors with OV or PBS. The harvested tumor tissue samples were analyzed for CYR61 protein expression. Finally, we checked the induction of CYR61 protein in tumors derived from human gliomas and implanted in athymic nude mice by immunofluorescent staining and confocal fluorescent microscopy. The significant correlation between the levels of induced CYR61 and OV in tumor tissue indicates the potential usefulness of CYR61 as a biomarker for viral oncolysis in the tumor microenvironment. Our results indicate the importance of incorporating such measurements in future clinical trials using OV therapy to aid in the development of better therapeutic strategies.



oncolytic virus, brain tumor, CYR61, glioma, angiogenesis, HSV