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BACKGROUND The dismal prognosis of glioblastoma (GBM) patients, with a median survival of less than 15 months despite maximal therapy urgently warrants new therapeutic approaches. Clinical trials employing oncolytic viruses (OVs) have shown encouraging results, however, in each OV clinical trial only a small subset of patients responded to treatment. As inter-tumoral heterogeneity has been the key challenge in treating GBM, we hypothesized that development of an in vitro co-culture model for assessment of viral replication and subsequent immune response might ultimately predict in vivo OV efficacy for individual GBM patients. METHODS 20 patient-derived GBM cell cultures were tested with dose-ranges of two clinically relevant OVs (DNX2401 and rQnestin34.5 V1) to determine the EC50 values (half-maximal effective concentration). To discriminate between responders and non-responders six of these cultures were infected using MOI 10 for DNX2401 and 0.25 for rQnestin34.5V1, and co-cultured with autologous PBMCs. OV-induced changes in gene and protein expression of immune associated genes were assessed using targeted gene expression (NanoString Technology) and ELISA. RESULTS DNX2401 EC50 values ranged from 0.35 to > 600 and from 0.04 to 1.77 for rQnestin34.5V1. Induction of pro-inflammatory cytokines and chemokines differed per virus and per patient. Enhanced OV infection or oncolysis efficiency did not lead to increased levels of IFNγ production. Importantly, longer exposure to dexamethasone prior to PBMC isolation correlated with suppressed IFNγ production. CONCLUSION We established an autologous GBM cells/PBMCs co-culture model which reflects inter-tumoral heterogeneity in terms of cytokine induction and virus-specific changes in gene and protein expression upon OV infection. Immune activation is not directly related to degree of infection or oncolysis and can be hampered by prolonged prior dexamethasone use. These first results support our hypothesis that improved prediction of response rates in oncolytic virotherapy for GBM may require a personalized approach with an in vitro test system.

Background: The brain tumor glioblastoma (GBM) is one of the most aggressive forms of cancer. The dismal prognosis of these patients, with a median survival of less than 15 months despite maximal therapy makes the need for new therapeutic approaches urgent. Clinical trials employing oncolytic viruses (OVs) have shown encouraging results, however, it appears that for each OV only a small group of patients responds to treatment. As inter- and intra-tumoral heterogeneity is a hallmark of GBM, we hypothesized that fresh patient-derived GBM cell cultures will reflect this inter-tumoral variability in response and allow identification of potential biomarkers of susceptibility to specific OVs. Furthermore, we established a co-culture system of primary GBM cultures with autologous peripheral blood mononuclear cells (PBMCs) to capture the degree of OV-induced oncolysis in conjunction with subsequent immune activation. Using these model systems, we attempt to develop tools which may guide future personalized trials of OV treatment for GBM. Methods: We tested the oncolytic potency of four OVs derived from different viral families (DNX2401, rQnestin34.5 V1, wild type Reovirus, lentogenic NDV-f0-GFP) on a panel of 19 molecularly characterized GBM cultures and calculated the half maximal effective concentration (EC50) for each virus on each cell culture. Quantitative PCR was performed to assess cytokine expression in tumor cells after infection with the 4 different OVs. OV-induced changes in the gene and protein expression of immune associated genes were assessed in co-cultures of GBM cells with PBMCs using Nanostring nCounter System and Elisa. Results: Screening of the 4 OVs on the panel of patient-derived GBM cell cultures revealed great inter-tumoral variability in oncolysis and cytokine response to the 4 different OVs with some degree of OV specific cytokine response profiles. Correlation analysis of transcriptome data with susceptibility to the four OVs shows that genes involved in distinct pathways are related to specific OV-sensitivity. In particular, cell cycle and immune related biological processes discriminate responders and non-responders. The co-culture of OV-infected glioma cells with PBMCs suggests that infection with different OVs leads to expression of distinct sets of genes and proteins in PBMCs; indicating that each OV mounts a specific immune response. Conclusion: Heterogeneity in OV sensitivity is demonstrated in primary GBM cultures, in terms of oncolysis, cytokine induction and in virus-specific changes in gene and protein expression in OV-infected tumor cells/PBMCs co-cultures. These results support the hypothesis that improving the response rates in oncolytic virotherapy for GBM may require a personalized approach. Citation Format: Eftychia Stavrakaki, Anne Kleijn, Wouter B. van den Bossche, Rutger K. Balvers, Lisette B. Vogelezang, Jie Ju, Andrew Stubbs, Yunlei Li, Dana Mustafa, Federica Fabro, Bernadette van den Hoogen, Rob Hoeben, William F. Goins, Hiroshi Nakashima, E. Antonio Chiocca, Clemens M. Dirven, Martine L. Lamfers. Towards personalized oncolytic virotherapy: Differential response of four oncolytic viruses in primary glioblastoma cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3560.