Glioblastoma multiforme is one of the most devastating diseases of the central nervous system, leaving patients with a median survival of 12-15 months following standard of care treatment. Oncolytic herpes simplex viruses (OV) are genetically modified to selectively infect and kill cancer cells by lytic destruction, and have been increasingly recognized as effective therapies against gliomas, reducing tumor burden and enhancing animal survival in pre-clinical studies. The efficacy of this therapy, however, is limited by the ever-changing tumor microenvironment which helps confer resistance to subsequent virus infection. In this thesis document we take a closer look at some of the changes which occur within the tumor microenvironment following OV therapy, and use the insight gained to create more sophisticated oncolytic viruses to combat glioblastoma.
To reduce the increase in angiogenesis reported following OV therapy, we first describe the construction and testing of a novel oncolytic HSV-1 derived virus: 34.5ENVE (viral ICP34.5 is Expressed by Nestin promotor and Vstat120 Expressing). This virus showed significant glioma specific killing and anti-angiogenic effects in vitro and in vivo. Treatment of mice bearing subcutaneous and intracranial glioma with 34.5ENVE resulted in a significant increase in animal survival, with 100% (subcutaneous) and 75% (intracranial) of mice showing a complete response. Histology and dynamic contrast enhanced magnetic resonance imaging (DCE MRI) revealed reduced microvessel density and increased tumoral necrosis in tumors treated with 34.5ENVE compared to tumors treated with a control virus. Collectively, these results describe the enhanced therapeutic efficacy of a transcriptionally driven OV by way of exploiting its impact on the tumor microenvironment.
Next, we describe the role of Cysteine rich 61 (CCN1) in the tumor microenvironment on OV efficacy. CCN1 is a secreted extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. We examined a hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Expression is up-regulated in a variety of cancers, including glioma, resulting in a worse prognosis for these patients. As a significant induction of secreted CCN1 shortly following oncolytic viral therapy of glioma cells has been shown, we evaluated its role in the cellular response to viral infection. We found that exogenous CCN1 in glioma ECM orchestrates a cellular antiviral response that reduces viral replication and limits oncolytic virus efficacy. Gene expression profiling and real time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1. Using function blocking antibodies we discovered this effect was mediated by CCN1 binding the α6β1 integrin receptor, resulting in the rapid secretion of IFNα which was essential for this innate antiviral effect. Collectively, these results describe the novel role of a CCN1-integrin interaction in the activation of the type-I antiviral interferon response and ultimate inhibition of OV therapy.
Lastly, we investigate the relationship between CCN1 and macrophage-mediated oncolytic virus clearance in glioma. Using function-neutralizing antibodies, we show that inhibition of endogenously up-regulated CCN1 following OV therapy reduces OV mediated macrophage migration. Interestingly, CCN1’s coordinated increase in macrophage migration was found due both to its direct interaction with macrophages, as evidenced by an enhancement in macrophage migration with purified CCN1 protein, as well as to its direct interaction with glioma cells, which results in an increased production of chemokines. CCN1 enhanced the pro-inflammatory activation of macrophages following OV infection, which led to an increase in macrophage-mediated viral clearance in vitro. Though knock-down of the integrin α6β1 on glioma cells did reduce the type I IFN response presented above, it had no effect on CCN1’s relationship with macrophages. Examination of the cell surface integrin αMβ2, known to mediate the CCN1-macrophage interaction, revealed it to be the main effector for CCN1’s effect. In vivo, use of an anti-CCN1 antibody in mice bearing subcutaneous gliomas treated with oncolytic rHSVQ-IE4/5-Luc revealed enhanced luciferase activity along with reduced macrophage infiltration. Taken together, our findings indicate CCN1 not only has a role in the immediate activation of the type I IFN response, thereby inhibiting virus infection, but also in increasing macrophage infiltration and activation resulting in a macrophage mediated reduction in viral oncolysis. Further, this study warrants investigation of therapeutic strategies to reduce CCN1 following OV therapy, with the intention of creating a more suitable tumor microenvironment for virus therapy.
Collectively, the results presented in this doctoral thesis reveal very real limitations to sustainable OV therapy, brought on by the response of the tumor microenvironment to virus infection. We show these limitations may be reduced by harnessing the insight gained from a broader understanding of the tumor microenvironment to create more sophisticated OVs. Future studies will focus on the clinical relevance of 34.5ENVE for the treatment of patients suffering from glioblastoma multiforme. In addition we will continue to investigate the role for CCN1 and its inhibition in the improvement of OV therapy.