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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.

Receptor tyrosine kinase (RTK) pathways are known to play an important role in tumor cell proliferation of glioblastoma (GBM). Cellular determinants of RTK-inhibitor sensitivity are important to optimize and tailor treatment strategies. The stress response gene activating transcription factor 4 (ATF4) is involved in homeostasis and cellular protection. However, little is known about its function in GBM. We found that the ATF4/p-eIF2α pathway is activated in response to Sunitinib in primary tumor initiating progenitor cell cultures (BTICs). Furthermore, lysosome entrapment of RTK-inhibitors (RTK-Is) leads to accumulation of autophagosomes. In case of Sunitinib treated cells, autophagy is additionally increased by ATF4 mediated upregulation of autophagy genes. Inhibition of ATF4 by small interfering RNA (siRNA) reduced autophagy and cell proliferation after Sunitinib treatment in a subset of BTIC cultures. Overall, this study suggests a pro-survival role of the ATF4/p-eIF2α pathway in a cell type and treatment specific manner.

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.

Glioblastoma remains a fatal diagnosis. Previous research has shown that metformin, which is an inhibitor of complex I of the respiratory chain, may inhibit some brain tumor initiating cells (BTICs), albeit at dosages that are too high for clinical use. Here, we explored whether a combined treatment of metformin and diclofenac, which is a non-steroidal anti-inflammatory drug (NSAID) shown to inhibit glycolysis by interfering with lactate efflux, may lead to additive or even synergistic effects on BTICs (BTIC-8, -11, -13 and -18) and tumor cell lines (TCs, U87, and HTZ349). Therefore, we investigated the functional effects, including proliferation and migration, metabolic effects including oxygen consumption and extracellular lactate levels, and effects on the protein level, including signaling pathways. Functional investigation revealed synergistic anti-migratory and anti-proliferative effects of the combined treatment with metformin and diclofenac on BTICs and TCs. Signaling pathways did not sufficiently explain synergistic effects. However, we observed that metformin inhibited cellular oxygen consumption and increased extracellular lactate levels, indicating glycolytic rescue mechanisms. Combined treatment inhibited metformin-induced lactate increase. The combination of metformin and diclofenac may represent a promising new strategy in the treatment of glioblastoma. Combined treatment may reduce the effective doses of the single agents and prevent metabolic rescue mechanisms. Further studies are needed in order to determine possible side effects in humans.

Aim: Recently, D,L-methadone has been put forward as adjuvant treatment in glioblastoma (GBM). Methods: We analyzed the μ-opioid receptor expression in a set of GBM cell lines and investigated the efficacy of D,L-methadone alone and in combination with temozolomide (TMZ). Results & conclusion: Expression of the μ-opioid receptor was similar in the tested cell lines. High concentrations of D,L-methadone induced apoptosis in all cell lines and showed treatment interaction with TMZ. However, in lower dosages, reflecting clinically attainable concentrations, D,L-methadone alone showed no efficacy, and induced even higher proliferation in one specific cell line. Also, no interaction with TMZ was observed. These results suggest caution to the premature use of D,L-methadone in the treatment of GBM patients.

The literature on TGF-ß in cancer including data on the expression or activation of TGF-ß pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-ß pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-ß isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-ß1, TGF-ß2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-ß isoform directed therapies. Of note, TGF-ß antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-β isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-ß directed antisense therapies based upon TGF-β immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.

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.

// 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.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 60-70% of all gliomas. Current median patient survival time is 14-16 months after diagnosis. Numerous efforts in therapy have not significantly altered the nearly uniform lethality of this malignancy. The Transforming Growth Factor beta (TGF-β) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-β pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-β inhibitor peptide, was analyzed. P144 decreased proliferation, migration, invasiveness, and tumorigenicity in vitro, whereas apoptosis and anoikis were significantly increased for GBM cell lines. SMAD2 phosphorylation was reduced, together with a downregulation of SKI and an upregulation of SMAD7 at both transcriptional and translational levels. Additionally, P144 was able to impair tumor growth and increase survival in an in vivo flank model. Our findings suggest a potential effect of P144 in vitro and in vivo that is mediated by regulation of transcriptional target genes of the TGF-β pathway, suggesting a therapeutic potential of P144 for GBM treatment.

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.

The combination of radiotherapy, temozolomide and valproic acid (VPA) has shown some promise in retrospective analyses of patients with glioblastoma, although their mechanisms of action remain unknown.We investigated the in vitro and in vivo effects of pretreating glioma cells with temozolomide and VPA as an immunization strategy to boost an adaptive immune response in a syngeneic mouse model.Temozolomide and VPA induced autophagy in GL261 glioma cells, and caused tumor antigen-specific T-cells to become activated effector T-cells. Mice with a pre-existing glioma showed no improvement in clinical outcome when immunized with temozolomide- and VPA-treated glioma cells.Although temozolomide and VPA treatment of glioma cells can boost the adaptive immune response, in the context of a vaccine therapy, additional factors are necessary to eradicate the tumor and improve survival.

BACKGROUND: The transcription factor C/EBPβ has been identified as a master regulator of the mesenchymal phenotype in malignant glioma. It is transcribed from an intronless gene, but can be translated into three isoforms (LAP, LAP*, LIP). C/EBPβ has been shown to be upregulated in mesenchymal GBMs and its expression correlates to a worse prognosis in patients. Downregulation of C/EBPβ reduces proliferation and migration in vitro and diminishes tumor growth in vivo. In addition, C/EBPβ protein levels are increased in hypoxic and necrotic areas in human GBM tissue samples. However, expression levels and impact of the different isoforms in glioma are unknown. Crosstalk of C/EBPβ and TFGβ has been shown to influence metastatic behavior of breast carcinoma cells. The aim of this project is therefore to investigate the impact of C/EBPβ isoforms LAP and LIP on phenotype, proliferation and migration of brain tumor initiating cells (BTICs) and their possible interaction with transforming growth factor beta (TGFβ). Methods: LAP and LIP isoforms were stably overexpressed in a set of proneural and mesenchymal BTIC lines. Marker expression was investigated by immunocytochemistry and Western blot. Proliferation was monitored by crystal violet staining and migration by spheroid assay. Genome wide transcriptome analysis was performed by next generation RNA sequencing on a HiSeq (Illumina). Results: Overexpression of LAP ­(full-length protein) induced proneural BTICs to switch towards a mesenchymal phenotype, whereas overexpression of LIP had no such effect. Increased LAP led to enhanced migration in the majority of cell lines irrespective of subtype, whereas LIP mostly decreased migration. Rather the reverse effect was observed regarding proliferation. RNASeq analysis revealed highly differential consequences of LAP and LIP overexpression regarding expressed genes and principal component analysis. TGFβ signaling proved to be one of the most significantly altered pathways both in LAP and LIP cells. However, TGFβ treatment had either no effect or inhibited proliferation or migration independent of C/EBPβ isoform levels. Conclusions: We could show for the first time that overexpression of C/EBPβ is sufficient to induce a mesenchymal phenotype in proneural BTICs and that the LAP isoform is responsible for this transdifferentiation. Independent of glioma subtype, LAP and LIP can elicit opposing effects. Nevertheless, both isoforms might contribute to tumor progression, since shifting the balance between LAP and LIP might add to BTICs capability to adapt to microenvironmental cues, e.g. by adopting a more migratory or proliferative phenotype. In contrast to the findings in breast CA, TGFβ acted independently of C/EBPβ isoforms. Ongoing experiments will provide further insight into downstream effector molecules and regulatory mechanisms for each isoform in GBM.

The immunological impact on antibody-based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD-scid IL2Rγ(null) mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor-cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T-cell maturation and tumor cell-specific T-cell activation. In addition, Natural-Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody-dependent cellular cytotoxicity-based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system-related target cancer therapy and resistance.

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.

Glioblastomas (GBM) are highly malignant brain tumors, hallmarked by infiltrating growth of tumor cells. Brain Tumor Initiating Cells (BTIC) may initiate GBM pathogenesis according to recent publications. In particular the subventricular zone and hippocampus are areas where BTICs may arise and migrate to distinct areas to initiate the tumor bulk via differentiation into the glial linage. As a valuable tool an in situ model of organotypic brain slice cultures is utilized to visualize and analyse migration and invasion in a setting that simulates normal brain tissue conditions. This method allows the inoculation of tagged BTIC in the hippocampal region of fresh brain slice cultures from newborn rats (P8-12), and to monitor tumor cell invasion for up to three weeks. During this process the original cell population divides into different subpopulations: a stationary that proliferates at the implantation site, and an infiltrating one. Stationary and infiltrating cells differ in morphology and behaviour. In order to reveal why an initial cell population separates into subpopulations, a method was developed to microdissect single cells from the slices after an inoculation time of up to three weeks. cDNA libraries were generated for subsequent microarray analysis to compare the subgroups regarding gene expression profiles of genes associated with e.g. proliferation, invasion, cell-cell contacts, and stemness. Besides the different phenotypic appearance of the subpopulations, the genetic pattern of cells from the subpopulations determined by microarray analysis should unveil genes driving tumor invasion. These newly identified targets will be further characterized and regulated to manipulate the behaviour of BTICs.

Metformin is one of the most widely prescribed oral antidiabetic drugs. In addition to its known hypoglycemic effects there is current evidence, that metformin may also reduce the risk and mortality of cancer in diabetic patients. The antineoplastic role of metformin has been extensively studied in breast-, prostate-, pancreatic-, lung- and colon cancer, however, the effect of metformin on glioblastomas, one of the most devastating forms of human tumors, has not been thoroughly investigated. We examined the effects of metformin on a variety of brain tumor initiating cells (BTIC) from fresh tumor resections of primary glioblastomas using proliferation assays (cell count, BRDU-assay) and migration assays (scratch and spheroid migration assay), and investigated differentiation of cells by means of immunocytochemistry. Tumor cell metabolism was investigated by measuring extracellular lactate levels, mitochondrial respiration by high resolution respirometry and determination of cellular oxygen consumption under cell culture conditions using the PreSens-assay. Important signalling molecules, such as AMPK, Akt and mTOR were investigated by means of Western Blot analysis. Functional assays were compared in BTIC and differentiated tumor cells from the same patients. In BTIC, we observed a markedly impaired proliferation and migration of cells after treatment with metformin and inhibition of complex I of the respiratory chain. In addition, we observed increased extracellular lactate levels and activation of the AMPK/mTOR signalling cascade. Metformin-treated BTIC showed reduced expression of stem cell markers. Differentiated tumor cells were distinctly less responsive to treatment with metformin. We hypothesize that differential sensitivity of brain tumor initiating cells and differentiated tumor cells to metformin is due to different use of and dependence on oxidative energy production and impaired glycolytic and glutaminolytic rescue pathways in BTIC. Further investigation of metformin as a possible antineoplastic agent in glioblastoma is warranted, especially in the light of a multitude of promising ongoing clinical studies on metformin and other tumor types.

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.

The prognostic significance of HER2 expression in human breast carcinomas is beyond dispute nowadays. The HER family of receptor tyrosine kinases comprises four members (HER1/ErbB1/EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4) that act in concert via transactivation and consequently compose a functional signaling unit. Besides HER2 overexpression, coexpression of other HER receptors has substantial impact on course of disease and potential therapeutic benefit. This observation is substantiated by numerous preclinical studies and retrospective studies done on patients with breast cancer. Against this background, the quantification of all HER receptor expressions at the same time would significantly extend the information content revealed by routine diagnosis of breast cancer tissues. Moreover, the knowledge of HER receptor coexpression profiles in primary tumor samples could provide the basis to design and develop highly specific antireceptor treatment strategies. Here, we report on a simultaneous flow cytometric detection of all four HER receptors on carcinoma cells isolated from primary breast cancer tissues and separated from nonepithelial cells by cytokeratin staining. Combined with DNA, i.e. ploidy quantification, the approach resulted in a six-parameter assay that could complement the diagnosis of a variety of diseases in which HER receptor expression has a pivotal impact on the degree of malignancy. © 2010 International Society for Advancement of Cytometry

Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.

Small molecule inhibitors have been investigated in a large set of clinical trials in malignant gliomas. Despite promising preclinical studies, results of pilot trials have been generally disappointing. A deeper understanding of the complex biology of malignant glioma cells, and their adaptation to targeted agents is therefore critical for further therapy development. We analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Geneset enrichment analysis (GSEA) revealed that some of the regulated patterns are associated to the amino acid metabolism. In gene ontology annotations, additional evidence was raised that ER-stress response signaling which is connected to the amino acid metabolism is induced by Sunitinib. Based on the observation of gene network analysis (STRING) we hypothesize that the central mediator of the integrated stress response ATF4 (activating transcription factor 4) plays an important role in the regulation of the Sunitinib induced expression profiles and probably in the adaptation to treatment conditions. First, we confirmed a concentration dependent induction of ATF4 protein expression in Sunitinib treated BTICs. For further evaluation, we analyzed the expression of ATF4 in paraffin embedded tissue blocks from high grade glioma patients treated with Sunitinib by immunohistochemistry. ATF4 significantly correlated with shorter overall survival from the beginning of Sunitinib treatment. Interestingly, expression data from high grade gliomas retrieved from a public platform (REMBRANDT) revealed that transcription of the ATF4-induced TRB3 pseudokinase is negatively correlated with overall survival independent of treatment. In summary our data suggest that ATF4 expression may be predictive for response to Sunitinib and might also be involved in general resistance mechanisms in high grade gliomas. Further studies are planned to elucidate the induction of apoptosis and autophagy in dependence of ATF4 expression and its contribution to therapy resistance.