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Background Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). Methods Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age Results Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. Conclusion Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM. Graphical abstract

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.

Despite clinical intervention, glioblastoma (GBM) remains the deadliest brain tumor in adults. Its incurability is partly related to the establishment of drug resistance, both to standard and novel treatments. In fact, even though small kinase inhibitors have changed the standard clinical practice for several solid cancers, in GBM, they did not fulfill this promise. Drug resistance is thought to arise from the heterogeneity of GBM, which leads the development of several different mechanisms. A better understanding of the evolution and characteristics of drug resistance is of utmost importance to improve the current clinical practice. Therefore, the development of clinically relevant preclinical in vitro models which allow careful dissection of these processes is crucial to gain insights that can be translated to improved therapeutic approaches. In this review, we first discuss the heterogeneity of GBM, which is reflected in the development of several resistance mechanisms. In particular, we address the potential role of drug resistance mechanisms in the failure of small kinase inhibitors in clinical trials. Finally, we discuss strategies to overcome therapy resistance, particularly focusing on the importance of developing in vitro models, and the possible approaches that could be applied to the clinic to manage drug resistance.

INTRODUCTION The search for effective therapies for gliomas is progressively moving towards patient-specific medicine. In order to test patient-tailored therapies, it is vital to develop protocols for reliable establishment of patient-derived glioma cultures. We present a method for reliable culture establishment, with a 95% success rate in 114 consecutive high-grade samples. METHODS Cell cultures were established from either traditionally-resected tumor tissue or ultrasonic surgical aspirator (CUSA) derived tissue fragments, and expanded in serum-free culture, with selection of astrocytic populations if required. Cultures were started from single cells or small tumor fragments of 0.5-3mm (3D). Whole exome and RNA sequencing were carried out with the Illumina Novaseq and HiSeq platforms. Methylation profiling was performed with the Infinium MethylationEPIC array. Cultures and tumors were compared through analysis of single nucleotide polymorphisms and copy number profiles with the Infinium Global Screening Array. Intra-tumoral heterogeneity in cultures was investigated with single-cell transcriptomic sequencing (SORT-seq). We studied tumor-initiating potential by orthotopic injection of cultures in NOD-SCID mice. RESULTS Cultures started from single cells were established from CUSA material more efficiently (92%) than from traditional resection material (70%). 3D-derived cultures had a higher overall efficiency (95% for CUSA, 85% for traditional resection material). We confirmed high concordance in driver mutations, copy number and methylation profiles between tumors and derived cultures. Transcriptomics analysis, comparing tumors and derived cultures, revealed high consistency in gene expression distribution as demonstrated by correlation analysis (r=0.88). Singe-cell RNA-seq shows increased heterogeneity in CUSA derived-cultures, and decreased heterogeneity with passaging over time. Cultures faithfully produce tumors after orthotopic injection in NOD-SCID mice. CONCLUSION We present a highly successful method for the establishment of glioma cultures from patient material, with CUSA-derived cultures revealing greater heterogeneity. Cultures faithfully represent important molecular characteristics of parental tumors and can be used to test potential therapies in vitro.