Combined Modulation of Tumor Metabolism by Metformin and Diclofenac in Glioma

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

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo

Scaffold-Based (Matrigel™) 3D Culture Technique of Glioblastoma Recovers a Patient-like Immunosuppressive Phenotype

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance

Untersuchungen zum Einsatz von small interfering RNAs gegen epidermal growth factor receptor in humanen Gliomzellen

Humane maligne Gliome stellen die häufigste Form primärer Hirntumore des Erwachsenenalters dar. Ihre anatomische Lokalisation, die Infiltration des umgebenden gesunden Gehirngewebes und die Unterdrückung einer gegen den Tumor gerichteten Immunantwort tragen zu ihrem hochgradig aggressivem Phänotyp bei. Trotz intensiver Bemühungen, die neurochirurgischen, radiologischen und chemotherapeutischen Behandlungsmethoden zu verbessern, beträgt die mediane Überlebensdauer bei Patienten mit der aggressivsten Form, dem Glioblastoma multiforme (GBM) nur circa 18 Monate nach Diagnose. Maligne Gliome sind durch eine Reihe distinkter genetischer Veränderungen gekennzeichnet, wobei eine der häufigsten Aberrationen den epidermalen Wachstumsfaktor-Rezeptor (EGFR, ErbB1, HER-1) betrifft. Dieser ist in 40-50% der Gliome amplifiziert, außerdem liegt häufig eine transkriptionelle Überexpression und eine Aktivierung des Signalwegs über autokrine Rückkopplungsmechanismen vor. Zudem kann die Amplifikation mit einer aktivierenden Mutation (EGFRvIII) einhergehen. Der EGFR stellt den Prototyp von Rezeptortyrosinkinasen der Klasse I dar und gehört einer Familie von vier homologen Rezeptoren (ErbB1-ErbB4) an. Die Aktivierung des Rezeptors erfolgt durch die Bindung spezifischer Liganden (z.B. EGF, TGFalpha) mit anschließender Bildung von ErbB- Homo- oder Heterodimeren und Phosphorylierung bestimmter intrazellulärer Aminosäurereste. Dadurch rekrutierte Effektormoleküle vermitteln die Aktivierung verschiedener Signalkaskaden (z.B. MAPK, PI(3)K), die zu Proliferation, Migration und erhöhten Überlebensraten der Zellen führen können. Der EGFR ist das Ziel verschiedener therapeutischer Strategien. Während kleine synthetische Tyrosinkinase-Inhibitoren (TKIs) und monoklonale Antikörper die Aktivität des EGFR blockieren, inhibieren Antisense-Oligonukleotide und Ribozyme dessen Proteinsynthese. Einen alternativen Ansatz zu letztgenannten Methoden stellt die Verwendung von small interfering RNAs (siRNAs) dar. Diese 21 Nukleotide langen doppelsträngigen RNAs wurden als Bestandteile eines intrazellulären Mechanismus´ (RNA Interferenz, RNAi) identifiziert, der zur Abwehr von Viren und mobilen genetischen Elementen dient und RNA-Moleküle degradiert, die Sequenzhomologie zu den eingesetzten siRNAs aufweisen. Durch Selektion spezifischer Abschnitte der Ziel-mRNA und die Herstellung dazu homologer synthetischer siRNAs, kann die mRNA eines therapeutisch relevanten Gens, hier des EGFR, spezifisch abgebaut werden. Das Ziel der vorliegenden Arbeit war zunächst, die zu Beginn der Untersuchungen noch völlig neuartige Anwendung von siRNAs in humanen Gliomzellen zu etablieren. Im folgenden sollte die Expression des für die Progression dieser Tumore potentiell relevanten EGFR durch spezifische siRNAs reduziert und so ein weniger maligner Phänotyp in den verwendeten Zellinien induziert werden. Der Effekt der EGFR-Regulation auf die Genexpression wurde anhand von Mikroarray-Analysen untersucht

Tumor Cell Invasion in Glioblastoma

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

Effective silencing of EGFR with RNAi demonstrates non-EGFR dependent proliferation of glioma cells

The epidermal growth factor receptor (EGFR, ErbB1) is frequently dysregulated in a variety of solid human tumors, including malignant glioma. EGFR expression has been associated with disease progression, resistance to standard therapies and poor survival. The application of small interfering RNAs (siRNAs) has become an effective and highly specific tool to modulate gene expression, and a wide range of oncogenes have been silenced successfully. Here we show the siRNA-mediated down-regulation of EGFR in two established glioma cell lines with different EGFR expression levels (U373 MG, LN18). The expression of EGFR mRNA and protein was down-regulated by 70-90%. However, siRNA treatment had no inhibitory effect on cell proliferation, migration and activation status of EGFR-coupled signaling cascades. In accordance with these results, gene expression analysis with microarrays revealed only small, albeit specific changes in expression patterns. In conclusion, these data indicate that the specific down-regulation of EGFR might not be sufficient for a single agent therapeutic approach in malignant glioma

L’expérience infirmière en psychiatrie et santé mentale : enquête sur les dynamiques de professionnalisation et de construction du rapport au métier

This research confronts the concerns surrounding the professionalisation of psychiatric and mental health nurses. It takes place in a financed context through its implementation as a CIFRE programme (research and training agreement). As its sponsor, the mental health care facility of the Lille Metropolitan area has set up a plan called “professionalisation and tutoring” in order to complete nurses’ initial training, judged insufficient since the psychiatric nurses’ degree and its associated training were shut down in 1992. The plan’s significance is analysed through three levels and from a comprehensive approach. The first level regards its situation in the historical and cultural evolution of the professional group. The second, that which concerns the plan’s actors getting involved to make it work. The final level refers to the neo-nurses appropriation of the educative work instituted by the plan. It is shown that the plan expresses an intention of (re)professionalising the social group by establishing a relation between work and training. This third level is used as an analytical line allowing the study of how, in neo-nurses’ experience, the relationship with the craft is built. The theme of violence in psychiatry is also examined through the relation between work and training to study how it is taken into account in the nurses’ practices. Finally, the typicality of this research, financed by a sponsor’s funds, implies that it has to deal with a system of expectations composed of different levels. Here, the researcher is made to consider its involvement and give an interventionist dimension by training some actors of the field.Cette recherche traite des préoccupations de professionnalisation des infirmiers en psychiatrie et santé mentale. Elle s’inscrit dans un contexte commandité via sa mise en œuvre en convention industrielle de formation par la recherche (CIFRE). Commanditaire de ce travail, l’établissement public de santé mentale Lille-Métropole a développé un dispositif dit de « professionnalisation et tutorat » pour compléter une formation initiale jugée insuffisante après que le diplôme d’infirmier de secteur psychiatrique, et la formation y conduisant, aient été supprimés en 1992. La portée de ce dispositif est analysée à trois échelles, à partir d’une démarche compréhensive. D’abord, celle de son inscription dans l’évolution historico-culturelle du groupe professionnel. Ensuite, celle des acteurs mobilisés pour son fonctionnement. Enfin, celui de son appropriation par les néo-infirmiers destinataires de ce travail éducatif institué par le dispositif. Il est montré que le dispositif exprime une intention de (re)professionnalisation du groupe social par l’institution d’une relation entre travail et formation. Celle-ci est mobilisée comme axe analytique permettant d’examiner comment, dans l’expérience des néo-infirmiers, se construit le rapport au métier. La question de la violence en psychiatrie est aussi examinée à l’aune de cette relation pour étudier la manière dont elle est prise en compte dans les pratiques infirmières. Enfin, le caractère commandité de cette recherche la positionne dans un système d’attentes à plusieurs niveaux. Le chercheur est alors conduit à penser son implication et lui donner une dimension interventionniste, ceci par la formation de certains acteurs du terrain

P144, a Transforming Growth Factor beta inhibitor peptide, generates antitumoral effects and modifies SMAD7 and SKI levels in human glioblastoma cell lines

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-beta) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-beta pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-beta 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-beta pathway, suggesting a therapeutic potential of P144 for GBM treatment. (C) 2016 Elsevier Ireland Ltd. All rights reserved