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Background: Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. Methods: BTICs of different subtypes representing tumor heterogeneity were challenged with OXPHOS (oxidative phosphorylation) inhibition to assess the differential effects of metabolic intervention on key resistance features. Results: Whereas mesenchymal BTICs were more invasive, more glycolytic and less responsive to OXPHOS-inhibition, proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathways, and responded better to OXPHOS inhibition. Conclusion: Targeting glycolysis may be a promising approach to inhibit highly invasive tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors to overcome resistance to current treatments.

Glioblastoma (GBM) is a particularly devastating tumor with a median survival of about 16 months. Recent research has revealed novel insights into the outstanding heterogeneity of this type of brain cancer. However, all GBM subtypes share the hallmark feature of aggressive invasion into the surrounding tissue. Invasive glioblastoma cells escape surgery and focal therapies and thus represent a major obstacle for curative therapy. This review aims to provide a comprehensive understanding of glioma invasion mechanisms with respect to tumor-cell-intrinsic properties as well as cues provided by the microenvironment. We discuss genetic programs that may influence the dissemination and plasticity of GBM cells as well as their different invasion patterns. We also review how tumor cells shape their microenvironment and how, vice versa, components of the extracellular matrix and factors from non-neoplastic cells influence tumor cell motility. We further discuss different research platforms for modeling invasion. Finally, we highlight the importance of accounting for the complex interplay between tumor cell invasion and treatment resistance in glioblastoma when considering new therapeutic approaches.

Introduction: Glioblastoma (GBM) belongs to the most aggressive cancers. Main pathophysiological feature of GBM is fast and widespread invasion of the brain parenchyma by a subpopulation of progenitor tumour cells, rendering localized therapies ineffective. In addition, brain tumour initiating cells (BTIC) from GBM may repopulate the tumour site after initially effective treatments. Thus, the invasive progenitor tumour cell fraction is a strategic prime target for early and sustained anti-invasion therapies. To date, the dependency on and interaction with the microenvironment of progenitor cells and their adaptive development from the tumour cell fraction has not been thoroughly investigated. Methods: With organotypic brain slice cultures (OBSC), single cell RNA expression analysis and bioinformatics workup, we established a novel integrated approach to investigate the expression profile of invasion leading cells. A micromanipulator adapter was developed to isolate different three subpopulations within human GBM progenitor cells: leader, follower, and stationary, from the OBSCs. In comparison, we use long-term intravital imaging to analyse the behaviour and interaction of the three cellular fractions with each other and the microenvironment in vivo. Imaging through a cranial window in the brain of a living mouse enables us to visualize single tumour cells. Using a photoswitchable vector expressed in BTICs we specifically label and isolate leader, follower, and stationary cells separately to compare leader cell behaviour in vivo, in situ, and in the patient situation. Findings: The in situ OBSC model is a valuable tool to analyse migration and invasion in conditions simulating normal brain tissue including some microenvironmental influences. This allowed us to monitor human BTIC cell invasion in real time. Invasive cells emerge from the initial cell population, driving invasion into the surrounding tissue. Those leader cells differ in morphology and behaviour from stationary cells, which remain at the implantation site. A subsequent microarray analysis of single cells revealed that leader cells have a markedly distinct expression pattern in comparison to follower and stationary cells. This leader cell-specific expression signature is currently verified in fresh patient tumour biopsies, other BTICs analysed in the OBSC as model, and in the cranial window model. Updated results will be presented at the WFNOS meeting. Outlook: We showed that invasive leader cells have a specific functional and molecular profile. A thorough characterization of these cells in respect to their interaction with and response to the microenvironment that may be involved in the process of transforming cells into invasive leader cells is ongoing. We hypothesize that a deep characterization of these events will shed light on the chronology of one of the gliomagenesis milestones, namely invasion.

Tumor Treating Fields (TTFields) have shown to be effective in prolonging progression-free and overall-survival and increasing the rate of two- and five-year survivors of patients with primary glioblastoma. However, the two-year survival rate is still below 50%. A promising approach to enhance the efficiency of TTFields is the use of drugs which extend metaphase-anaphase transition and telophase. In a previous study we tested the efficacy of the combined treatment of TTFields and the Aurora B kinase inhibitor AZD1152 in different established glioma cell lines: U87-MG, U87-MGshP53 and U-251. We found that the combined treatment of TTFields and AZD1152 led to a significant reduction in the number of glioma cells in all three cell lines as compared to each treatment alone. In the present study we analyzed primary tumor-cell-lines to validate these data. In addition, we tested MLN8237, an Aurora A kinase inhibitor, to confirm that Aurora kinase inhibition is a valuable target for a combination therapy with TTFields. Primary tumor-cell lines were established from glioblastoma tissue taken intraoperatively. TTFields (1.6 V/cm RMS, 200 kHz) were applied for 72 hours using the inovitro system. AZD1152 was added to the media in concentrations of up to 100 nmol/l. Cell counts, cell cycle and clonogenic potential were determined at the end of treatment. Formation of multinuclear cells was determined using microscopic images of cells stained with crystal violet. MLN8237 was used in concentrations up to 50 nmol/l for the treatment of U87 MG cells. The combined treatment of TTFields and AZD1152 led to a significant reduction in the number of primary glioblastoma cells (Mann-Whitney-U-test, p The results presented in this work demonstrate that the combination of TTFields and aurora kinase inhibition can be an effective treatment against glioma cells. Based on the above, there is a strong rational to continue exploring the potential of combining TTFields and aurora kinase inhibition in early clinical trials. Citation Format: Paula Bartmann, Silvia Roosz, Achim Temme, Moshe Giladi, Eilon D. Kirson, Yoram Plati, Uri Weinberg, Adrian Kinzel, Verena Leidgens, Dietmar Krex. Aurora kinase inhibition to enhance Tumor Treating Fields efficacy in glioblastoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 239.

// Verena Leidgens 1 , Judith Proske 1, * , Lisa Rauer 1, * , Sylvia Moeckel 1 , Kathrin Renner 2 , Ulrich Bogdahn 1 , Markus J. Riemenschneider 3 , Martin Proescholdt 4 , Arabel Vollmann-Zwerenz 1 , Peter Hau 1 , Corinna Seliger 1 1 Department of Neurology and Wilhelm Sander-NeuroOncology Unit, University Hospital Regensburg, Regensburg, Germany 2 Department of Internal Medicine III, University Hospital Regensburg, Regensburg, Germany 3 Department of Neuropathology, Regensburg University Hospital, Regensburg, Germany 4 Department of Neurosurgery, University Hospital Regensburg, Regensburg, Germany * These authors have contributed equally to this work Correspondence to: Corinna Seliger, email: corinna.seliger@klinik.uni-regensburg.de Keywords: glioma, BTIC, STAT3, Stattic, metformin Received: August 20, 2016 Accepted: November 21, 2016 Published: December 24, 2016 ABSTRACT Glioblastoma (GBM) is the most common and malignant type of primary brain tumor and associated with a devastating prognosis. Signal transducer and activator of transcription number 3 (STAT3) is an important pathogenic factor in GBM and can be specifically inhibited with Stattic. Metformin inhibits GBM cell proliferation and migration. Evidence from other tumor models suggests that metformin inhibits STAT3, but there is no specific data on brain tumor initiating cells (BTICs). We explored proliferation and migration of 7 BTICs and their differentiated counterparts (TCs) after treatment with Stattic, metformin or the combination thereof. Invasion was measured in situ on organotypic brain slice cultures. Protein expression of phosphorylated and total STAT3, as well as AMPK and mTOR signaling were explored using Western blot. To determine functional relevance of STAT3 inhibition by Stattic and metformin, we performed a stable knock-in of STAT3 in selected BTICs. Inhibition of STAT3 with Stattic reduced proliferation in all BTICs, but only in 4 out of 7 TCs. Migration and invasion were equally inhibited in BTICs and TCs. Treatment with metformin reduced STAT3-phosphorylation in all investigated BTICs and TCs. Combined treatment with Stattic and metformin led to significant additive effects on BTIC proliferation, but not migration or invasion. No additive effects on TCs could be detected. Stable STAT3 knock-in partly attenuated the effects of Stattic and metformin on BTICs. In conclusion, metformin was found to inhibit STAT3-phosphorylation in BTICs and TCs. Combined specific and unspecific inhibition of STAT3 might represent a promising new strategy in the treatment of glioblastoma.

To this day, glioblastoma (GBM) remains an incurable brain tumor. Previous research has shown that metformin, an oral anti-diabetic drug, may decrease GBM cell proliferation and migration especially in brain tumor initiating cells (BTICs). As transforming growth factor β 2 (TGF-β2) has been reported to promote high-grade glioma and is inhibited by metformin in other tumors, we explored whether metformin directly interferes with TGF-β2-signaling. Functional investigation of proliferation and migration of primary BTICs after treatment with metformin+/−TGF-β2 revealed that metformin doses as low as 0.01 mM metformin thrice a day were able to inhibit proliferation of susceptible cell lines, whereas migration was impacted only at higher doses. Known cellular mechanisms of metformin, such as increased lactate secretion, reduced oxygen consumption and activated AMPK-signaling, could be confirmed. However, TGF-β2 and metformin did not act as functional antagonists, but both rather inhibited proliferation and/or migration, if significant effects were present. We did not observe a relevant influence of metformin on TGF-β2 mRNA expression (qRT-PCR), TGF-β2 protein expression (ELISA) or SMAD-signaling (Western blot). Therefore, it seems that metformin does not exert its inhibitory effects on GBM BTIC proliferation and migration by altering TGF-β2-signaling. Nonetheless, as low doses of metformin are able to reduce proliferation of certain GBM cells, further exploration of predictors of BTICs' susceptibility to metformin appears justified.

Glioblastomas (GBM) are characterized as highly malignant brain tumours, hallmarked by infiltrating tumour cells. Their pathogenesis may depend on Brain Tumour Initiating Cells (BTIC), according to recent publications. The subventricular zone and hippocampus are areas where BTICs may arise and migrate to distinct areas to initiate a tumour bulk via differentiation into the glial linage. In consequence, it is relevant to investigate the transdifferentiation, migratory and invasive potential of BTICs through the identification of molecules and pathways controlling the induction of invasion. Therefore, this study aimed to identify characteristics specific for invasion-driving GBM cells. Invasion of GBM cells was induced in an in situ model of organotypic brain slice cultures (OBSC), a valuable tool to visualize and analyse migration and invasion in a setting that simulates normal brain tissue conditions. Fluorescence tagged BTICs were inoculated in the hippocampal region of fresh brain slice cultures from new-born rats to monitor tumour cell invasion for three weeks. During this time, invasive cells emerge from the initial cell population, driving invasion into the surrounding tissue. Those invasive cells differ in morphology and behaviour from stationary cells remaining at the implantation site. An adapter was constructed to fit to a micromanipulator to isolate the different cell subpopulations from the OBSC. After cDNA library generation and subsequent microarray analysis, a detailed comparison has been made between both groups to compare gene expression profiles of genes associated with e.g. proliferation, invasion, cell-cell contacts, and stemness. Here, invasive cells showed a markedly distinct expression pattern in comparison to stationary cells. Identified transcription factors and their target gene expression will be verified in different BTIC subsets. Overall expressed genes specific for leader cells will be further characterized and regulated to manipulate the invasive behaviour and tumourigenicity of BTICs.

Versican is a large chondroitin sulphate proteoglycan produced by several tumor cell types, including high-grade gliomas. Increased expression of distinct versican isoforms in the extracellular matrix plays a role in tumor cell growth, adhesion and migration. We have recently shown that transforming growth factor (TGF-beta)2, an important modulator of glioma invasion, interacts with versican isoforms V0/V1 during malignant progression of glioma in vitro. However, the distinct subtype of versican that modulates these effects could not be specified. Here, we show that transient down-regulation of V1 by siRNA leads to a significant reduction of proliferation and migration in glioblastoma cell lines and glioblastoma progenitor cells, whereas tumor cell attachment stays unaffected. We conclude that V1 plays a predominant role in modulating central pathophysiological mechanisms as proliferation and migration in glioblastoma. Considering that TGF-beta is a master regulator of glioma pathophysiology, and that V0/1 is induced by TGF-beta2, therapeutic regulation of V1 may induce meaningful effects on glioma cell migration not only in vitro, but also in vivo.