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Background Molecular characterization based on genomic, transcriptomic and epigenetic profiling has led to a better delineation of various glioma subtypes and highlighted the individual paths of glioma evolution upon treatment and recurrence. However, due to cellular and molecular diversity of these tumors, the pharmacological treatment of gliomas, in particular of its most malignant subtype Glioblastoma (GBM), remains a major challenge. To address this challenge, we here apply a pharmacogenomics approach, modelling the disease in matched patient-derived preclinical models and profiling the differential drug response among individual patients and glioma subtypes Material and Methods We generated a cohort of 45 Patient-Derived Orthotopic Xenografts (PDOX) from a collection of over 400 glioma patients. We used a multi-parametric approach based on genetic, transcriptomic and longitudinal profiling of patients and their matched xenografts for a comprehensive subgrouping of our glioma cohort. Based on PDOX-derived 3D tumor organoids we carried out a targeted drug screen focused on epigenetic regulators. A high throughput drug screening using an unbiased large chemical library containing a unique collection of FDA approved compounds with high pharmacological diversity is currently ongoing. Results Our glioma cohort with matched PDOX and 3D tumor organoids represents diverse subgroups of glioma patients, including a unique collection of primary and relapsed tumors from the same patient. Our preliminary drug screen analysis on 3D organoids highlights selective susceptibility to certain epigenetic inhibitors in primary disease but not in the same patient’s relapse. Results of matching genomics and functional data will be presented. Conclusion An integrated personalized approach to profile gliomas at multiple genomic and functional levels allows for pharmacogenomic subgrouping of patients for personalized treatment strategies. This analysis will allow to link genotypes to functional phenotypes and hopefully identify therapeutic options for selected glioma sub-populations.

Background To date, glioblastoma (GBM) remains a fatal disease, with a median overall survival of roughly over a year. There is a crucial need of new treatment options, yet most clinical trials have failed partly due to the lack of predictive preclinical model systems. Currently, most patient-derived preclinical models suffer from the reduction or absence of immune system components, which represents a bottleneck for adequate immunotherapy testing. Humanized mice offer new opportunities here, since they rebuild an adaptive human immune system in a NSG mouse. Derivation of glioblastoma patient-derived orthotopic xenografts (PDOXs) in humanized mice appears thus as a promising tool for testing new treatment strategies targeting the tumor microenvironment (TME). Material and Methods We derived PDOXs through intracranial implantation of GBM primary organoids in different immunocompromised mouse strains (Nude, NOD/SCID, NSG). To introduce back the adaptive human immune system, GBM PDOXs were further derived in human CD34+ hematopoietic stem cell-engrafted NSG (HU-CD34+) mice. We applied single-cell RNA-sequencing, multicolor flow cytometry, immunohistochemical analyses and functional studies to examine the heterogeneous TME in a cohort of GBM PDOX models. We further interrogated the contribution and crosstalk between the human and mouse components constituting the brain TME in HU-CD34+ PDOXs. Results We show that glioma PDOXs can be derived in mice of different background including Nude, NOD-SCID, NSG and HU-CD34+ mice. Mouse-derived TME created in PDOX models contains tumor-associated macrophages (TAMs) known as major immuno-suppressive components of human GBM tumors. We further show that PDOXs derived in HU-CD34+ NSG mice present human CD45+ immune cells in the bone marrow and blood. Interestingly, we detect an influx of human immune cells in tumors developed in the mouse brain, which interact with the brain-derived immunosuppressive TME of mouse origin. Conclusion We here provide a thorough characterization of the heterogeneous brain TME created in GBM PDOX models. We show that human GBM can instruct mouse-derived brain cells towards immune-suppressive TME. The missing adaptive immune component can be introduced by derivation of GBM PDOXs in humanized mice. Such immunocompetent in vivo models will be important for testing novel therapies targeting different immune components in GBM.

Background Although immunotherapies represent an encouraging approach against cancer, to date none translated to the clinical benefit in Glioblastoma (GBM). One aspect contributing to this failure is the highly immunosuppressive GBM microenvironment. Our approach to overcome immunosuppression is to increase anti-tumor immune responses via adenovirus (AdV)-mediated delivery of the MHC-II Transactivator (CIITA) gene. CIITA-induced MHC-II expression is anticipated to convert GBM cells into surrogate antigen presenting cells able to prime T helper cells, therefore promoting CD4+ and CD8+ mediated immunity. Material and Methods We generated AdVs containing wild type CIITA (Ad-CIITA) using a replication-defective serotype5 adenoviral backbone. AdVs containing a mutated, non-functional version of CIITA (Ad-CIITA mutant) and an empty CMV promoter (Ad-null) were used as controls. AdV-mediated MHC-II expression was monitored at mRNA, protein and cell surface level. For the functional assessment of anti-tumor immune responses, we developed an advanced human GBM organoid model system consisting of tumor organoids co-cultured with either human peripheral blood mononuclear cells (PBMCs) or isolated CD3+ T cells. T cell mediated tumor cell killing was monitored over time via live cell imaging and flow cytometry. Results We successfully constructed and produced a CIITA-armed AdV that induces MHC-II expression in infected GBM cells, indicating the efficient expression of transcriptionally active CIITA for at least six days post infection. In immunocompetent human GBM organoids, Ad-CIITA infection of tumor cells led to prominent organoid disruption and tumor cell death, an effect that was not observed in the absence of PBMCs or CD3+ T cells. Tumor organoids infected with Ad-CIITA mutant remained intact, demonstrating the implication of cell surface MHC-II molecules in the observed phenotype. Conclusion Our results demonstrate that AdV-mediated delivery of CIITA is a promising strategy to increase T cell mediated immunity against glioblastoma.

Background A major contributing factor to Glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive tumor microenvironment (TME). GBM-associated myeloid cells, including resident microglia, macrophages and other peripheral immune cells are generally geared towards tumor-supportive roles. It is however unclear whether such recruited myeloid cells are phenotypically and functionally identical. Here, we aim to understand the heterogeneity of the GBM TME, using an unbiased, marker-free approach to systematically characterize cell type identities at the molecular and functional levels. Material and Methods We applied single-cell RNA-sequencing, multicolor flow cytometry, immunohistochemical analyses and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM patient-derived orthotopic xenografts (PDOXs) representing different tumor phenotypes were compared to glioma mouse GL261 model and patient tumors. Results We show that PDOX models recapitulate major components of the TME found in human GBM. Human GBM cells reciprocally interact with mouse cells to create a GBM-specific TME. The most prominent transcriptomic adaptations are found in tumor-associated macrophages (TAMs), which are largely of microglial origin. We reveal inter-patient heterogeneity of TAMs and identify key signatures of distinct phenotypic states within the microglia-derived TAMs across distinct GBM landscapes. GBM-educated microglia adapt expression of genes involved in immunosuppression, migration, phagocytosis and antigen presentation, indicating functional cross-talk with GBM cells. We identify novel phenotypic states with astrocytic and endothelial-like features. Identified gene signatures and phenotypic states are confirmed in GBM patient tumor tissue. Finally we show that temozolomide treatment leads to transcriptomic adaptation of not only the GBM tumor cells but also adjacent TME components. Conclusion Our data provide insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumor. We confirm a crucial role of microglia in supporting the immunosuppressive TME and show that PDOXs allow to monitor the highly plastic GBM ecosystem and its phenotypic adaptations upon treatment. This work further confirms the clinical relevance of PDOX avatars for testing novel therapeutics including modalities designed to target the myeloid compartment.

Intratumoral heterogeneity is one of the major barriers in the treatment of many tumor types. In glioblastoma (GBM) – the most aggressive brain tumor - survival of patients following standard therapy has stagnated to an average of 14 months, largely due to the inherent heterogeneity and phenotypic plasticity. Patient-derived orthotopic xenografts (PDOX) are important tools for preclinical cancer studies. We have shown that they recapitulate histological and molecular features of patient tumors, thus producing more clinically relevant study outcomes when compared to other preclinical models. To understand the impact of treatment on intratumoral heterogeneity and interactions of tumor cells with the microenvironment, we applied single-cell RNA-seq in GBM PDOX models generated from naive and treatment exposed patient tumors. Here we show that PDOX models recapitulate all the major cell types and transcriptional programs reported in GBM patient samples. We demonstrate that GBM tumor cells occur in distinct phenotypic states dictated by microenvironmental cues and treatment-induced pressure. Non-neoplastic cells present within xenografts undergo transcriptomic adaptation similar to the GBM microenvironment in patients. Analysis of treated PDOXs and longitudinal PDOX models derived from patients prior and after treatment allows us to investigate short-term and stable long-term transcriptomic adaptations. We aim to reveal key molecular regulators enhancing tumor cell plasticity and resistance, that could be targeted in combinatory treatments. Our GBM PDOX models provide a platform for high-quality pre-clinical research and an improved clinical relevance in the development and testing of novel therapeutics.

It is well recognized that long term cell cultures are poor models to study human cancer, largely because of loss of clonal heterogeneity, accumulation or loss of genomic alterations and adaptation to a highly artificial environment. Patient-derived orthotopic xenografts (PDOX) based on organotypic three-dimensional tumor spheroids from human glioma samples are proposed to represent a reliable and clinically-relevant animal model. We have generated a living biobank of PDOX models from 34 glioma patients (grade III and IV), including longitudinal patient samples with matched recurrent tumors. Using an efficient orthotopic xenografting procedure we obtain an overall tumor take-rate of close to 80%. We show that our glioma PDOX retain the genetic and epigenetic profiles of primary patient biopsies throughout several generations of xenotransplantation. In particular they not only faithfully recapitulate gene amplification and expression of EGFR and EGFRvIII variant in a reproducible manner, also amplification and expression of rarer patient-specific EGFR variants is maintained. Overall genome-wide transcriptomic profiles of PDOX remain very similar to patient biopsies and correlate better with the GBM cohort of TCGA (538 GBM samples) than conventional cell lines. Observed differences at the transcriptomic level are largely based on the replacement of human to mouse stromal cells, which impacts on the molecular sub-classification of GBM. We conclude that glioma PDOX models closely reflect patient heterogeneity and treatment response, and thus represent appropriate avatars for reproducible pre-clinical trials. Furthermore, by combining profiling of the somatic mutational landscape with large-scale drug screening, PDOX-derived tumor organoids can elucidate druggable targets and tumor response profiles in a personalized patient-specific manner.

BACKGROUND A major hallmark of glioblastoma (GBM) is its highly invasive capacity, contributing to its aggressive behaviour. Since invasive cells cannot be easily removed by surgery or irradiation, they are left behind and eventually result in lethal recurrence. Therefore, a better understanding of the invasion process and of the key molecular players underlying the invasive capacities of GBM may lead to the identification of new therapeutic targets for GBM patients. MATERIAL AND METHODS To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM sphere cultures. The phenotype of the most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cell lines was compared with overexpression in non-invasive cells. RNA sequencing of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion. RESULTS Through a whole genome shRNA screen, a zinc-finger containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong decrease in invasion capacity in two highly invasive GBM cell lines. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of either one or both zinc-finger motifs led to decreased invasion indicating that the two zinc-finger motifs are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus. CONCLUSION We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through a transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent a novel promising target to overcome invasive capacities in GBM.

Background Deregulated growth factor signaling is a major driving force in the initiation and progression of glioblastoma. The tumor suppressor and stem cell marker Lrig1 is a negative regulator of the epidermal growth factor receptor (EGFR) family. Here, we addressed the therapeutic potential of the soluble form of Lrig1 (sLrig1) in glioblastoma treatment and the mechanism of sLrig1-induced growth inhibition. Methods With use of encapsulated cells, recombinant sLrig1 was locally delivered in orthotopic glioblastoma xenografts generated from freshly isolated patient tumors. Tumor growth and mouse survival were evaluated. The efficacy of sLrig1 and the affected downstream signaling was studied in vitro and in vivo in glioma cells displaying variable expression of wild-type and/or a constitutively active EGFR mutant (EGFRvIII). Results Continuous interstitial delivery of sLrig1 in genetically diverse patient-derived glioma xenografts led to strong tumor growth inhibition. Glioma cell proliferation in vitro and tumor growth in vivo were potently inhibited by sLrig1, irrespective of EGFR expression levels. Of importance, tumor growth was also suppressed in EGFRvIII-driven glioma. sLrig1 induced cell cycle arrest without changing total receptor level or phosphorylation. Affected downstream effectors included MAP kinase but not AKT signaling. Of importance, local delivery of sLrig1 into established tumors led to a 32% survival advantage in treated mice. Conclusions To our knowledge, this is the first report demonstrating that sLrig1 is a potent inhibitor of glioblastoma growth in clinically relevant experimental glioma models and that this effect is largely independent of EGFR status. The potent anti-tumor effect of sLrig1, in combination with cell encapsulation technology for in situ delivery, holds promise for future treatment of glioblastoma.

Standard-of-care for glioblastoma (GBM) includes surgery, radiation and temozolomide. Nearly all tumors recur and 5-year survival is less than 3%. Unmethylated promoter status for O(6)-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for temozolomide-resistance. Second-line treatment with bevacizumab has not only failed to improve survival, but has also been shown to induce intratumor hypoxia, which is implicated in increased chemoresistance. VAL-083 is a bi-functional DNA-targeting agent that readily crosses the blood-brain barrier. VAL-083 targets N(7)-guanine, causing DNA double-strand breaks and cancer cell-death in GBM cancer stem cells (CSCs) and non-CSCs, independent of MGMT. We have previously shown that bevacizumab treatment upregulates expression of glucose transporters GLUT-1/GLUT-3 on GBM cells. We hypothesized that, based on its unique monosaccharide backbone structure, VAL-083 may benefit from bevacizumab-induced GLUT transporter upregulation leading to enhanced VAL-083 uptake and anti-tumor activity. Methods: To investigate the in vivo anti-tumor effect of VAL-083+bevacizumab, we used a orthotopic patient-derived xenograft GBM model. All mice carried MGMT-unmethylated, temozolomide-resistant recurrent T16 GBM tumors as detected by MRI 35 days post-implantation. Mice were grouped into control, bevacizumab, VAL-083, and VAL-083+bevacizmab. Tumor progression was measured by MRI on days 49 and 56, and tumor growth rate was calculated for the entire study (day 35 vs. 56) and for the last 7 days (day 49 vs. 56). Results: Tumors were significantly smaller in VAL-083-treated mice both compared to control (-83%, p

BACKGROUND: Glial tumors presenting a heterozygous point mutation in isocitrate dehydrogenase (IDH) accumulate the oncometabolite D-2-hydroxyglutarate (2-HG) leading to a hypermethylation phenotype through inhibition of α-ketoglutarate (α-KG) dependent demethylases. However the direct effect of the IDH mutation on metabolic dysregulation remains poorly understood. IDH mutant glioma cells are notoriously difficult to grow in vitro and only a handful of laboratories, including ours, have been able to generate patient derived xenograft models in vivo. Here we aimed to get insight into the major metabolic aberrations observed in human IDH mutant gliomas with a special emphasis on the energy metabolism, oxidative stress and glutamine/glutamate pathways. METHODS: In situ high resolution mass spectrometric imaging (MSI) was performed on IDH1 mutant xenograft sections derived from human oligodendroglial tumors with 1p19q deletion, providing a detailed metabolic profiling and anatomical localization in tumor sections. Key enzymes of the perturbed metabolic pathways were monitored by Western blot and immunohistochemistry. Liquid chromatography mass spectrometry (LC-MS) was applied to validate findings in clinical specimen. RESULTS: 2-HG was specifically localized in the tumor bed but not in the surrounding brain parenchyma, while α-KG levels were not significantly altered in IDH1 mutant tumors. We identified a large set of differential metabolites in IDH1-mutant tumors, including changes in lipid metabolism, in the methylation and the glutathione pathway. CONCLUSIONS: We provide a detailed metabolic mapping of human IDH mutant gliomas with high anatomical resolution in situ. Our data point to the importance of oxidative stress regulation and its possible link to epigenetic dysregulation in IDH mutant gliomas.

BACKGROUND: The oncometabolite D-2-hydroxyglutarate (2-HG) is strongly increased in glial tumors presenting a heterozygous point mutation in isocitrate dehydrogenase (IDH) and is a key indicator of aberrant metabolic circuitries in these tumors. IDH1 is a dimeric enzyme converting isocitrate to α-ketoglutarate (α-KG), a metabolite involved in energy production, macromolecule biosynthesis and an important enzyme co-factor. In IDH1-mutant tumors, α-KG is processed to 2-HG in a NADPH consuming reaction, leading to a hypermethylation phenotype through inhibition of α-KG-dependent demethylases. However the direct effect of the IDH mutation on metabolic dysregulation remains poorly understood. A limitation in the analysis of IDH mutant tumors is the lack of appropriate pre-clinical models. Indeed glioma cells carrying the IDH mutation are notoriously difficult to grow in vitro and only a handful of laboratories, including ours, have been able to generate patient derived xenograft models in vivo. Thus most current studies addressing the role of 2-HG are based on cell lines overexpressing mutant IDH1 in a wildtype background, with unknown consequences for the function of the oncogenic enzyme. Here we aimed to get insight into the major metabolic aberrations observed in human IDH mutant gliomas with a special emphasis on oxidative stress and glutamine/glutamate pathways. METHODS: In situ high resolution mass spectrometric imaging (MSI) was performed on IDH1 mutant xenografts derived from human oligodendroglial tumors with 1p19q deletion, providing a detailed metabolic profiling and anatomical localization in tumor sections. Key enzymes of the perturbed metabolic pathways were monitored by Western blot and immunohistochemistry. Liquid chromatography mass spectrometry (LC-MS) was applied to validate our findings in fresh clinical specimen. RESULTS: 2-HG was specifically localized in the tumor bed but not in the surrounding brain parenchyma, while α-KG levels were not significantly altered in IDH1 mutant tumors. We identified a large set of differentially present metabolites in IDH1-mutant tumors, including the anti-oxidant glutathione. Importantly we reveal significant differences in metabolite levels between human glioma samples with an endogenous IDH mutation compared to previously reported data on mutant IDH1 overexpressing cell lines. CONCLUSIONS: We provide for the first time a detailed metabolic profiling of human IDH mutant gliomas with high anatomical resolution in situ. Our data point to the importance of oxidative stress regulation and its possible link to epigenetic dysregulation in IDH mutant oligodendrogliomas. SECONDARY CATEGORY: Imaging.