Changes in Extracellular Vesicles From Glioblastoma Cells Treated With Gene-Mediated Cytotoxic Immunotherapy.

Abstract

Glioblastoma (GBM) is an aggressive malignant brain cancer with rapid progression from diagnosis to death even with standard of care (SOC) therapy. Novel therapeutics are under investigation to help increase overall and progression free survival in patients. One area of investigation is the use of gene mediated cytotoxic immunotherapy (GMCI). With GMCI, the enzyme or protein for a therapeutic gene is inserted into the cell using an engineered virus. For GBM, the use of GMCI involves a non-replicating adenovirus (AdV) that expresses the herpes simplex virus thymidine kinase (HSV-tk) gene. In clinical trials, AdV-HSV-tk is injected into the resection cavity intraoperatively after resection of the GBM. The HSV-tk is required for activation of an anti-herpetic prodrug given systemically. The anti-herpetic prodrug acts as a toxic nucleotide that induces cell death by blocking DNA polymerase. The tumor antigens released from death of the cell promotes an immune response. As immunosuppression is thought to significantly contribute to the malignant nature of GBM, therapies that promote a robust anti-tumor immune response, such as GMCI and immunotherapy, hold great promise. GMCI combined with immunotherapy is now in a clinical trial for patients with GBM. Biomarkers that measure tumor responsiveness to treatment are under investigation. Extracellular vesicles (EVs) are mediators of intercellular communication through the transmission of biological information. Tumor derived EVs that are secreted into biological fluid, such as blood and CSF, can be isolated from patient blood and CSF samples. These EVs represent a good source of biomarkers. The purpose of this study is to identify changes in EVs secreted from virally infected cells compared to non-virally infected cells, and to assess the functional role of EVs in GMCI therapy. For cultured GBM cells infected with AdV-HSV-tk, the maximal expression of HSV-tk occurred at 24 hours after viral infection. Although no plateau of HSV-tk expression at increasing multiplicity of infection (MOI) was reached, a MOI of 240 was decided upon given the amount of viral stock available. Cell viability was assessed after infection with AdV-HSV-tk and treatment with the anti-viral drug ganciclovir (GCV). GCV had strong cytotoxicity at 96 hours with an IC50 of 0.1 uM. Differences in EV size was measured using the NanoSight particle analyzer. EVs from virally infected cells were significantly larger than EVs from non-virally infected cells. However, virally infected cells produced significantly less EVs than non-virally infected cells. EVs produced by virally infected cells contained HSV-tk mRNA. EVs from virally infected cells were able to merge with non-virally infected cells and transfer HSV-tk. When GCV was given to non-virally infected cells co-incubated with EVs from virally and non-virally infected cells, increased killing of the GBM cells incubated with EVs from virally infected cells occurred compared with GBM cells incubated with EVs from control cells. Our results suggest that GMCI therapy may alter the characteristics of EVs. EVs derived from virally infected GBM cells may serve as an important biomarker to monitor the response to treatment using GMCI. EVs may also play a functional role in the spread of viral mRNA to other cells in the local tumor microenvironment.