Células-tronco mesenquimais (CTM) apresentam tropismo a tumores, sendo importantes componentes do estroma tumoral. No cérebro, o nicho perivascular é uma importante fonte de CTM, as quais podem contribuir direta e/ou indiretamente para o desenvolvimento de tumores, embora os mecanismos envolvidos sejam pouco conhecidos. No presente trabalho, investigou-se a influência de CTM sobre a proliferação, capacidade invasiva e tumorigenicidade de células de Glioblastoma (GBM) humano. Sabe-se que CTM produzem TGFB1, uma citocina multifuncional envolvida em imunomodulação, proliferação, migração e transição epitelial-mesenquimal de células tumorais. Experimentos in vitro, realizados com meios condicionados de CTM de cordão umbilical humano com silenciamento permanente do gene TGFB1, demonstraram que o TGFB1 secretado por CTM é capaz de aumentar significativamente a proliferação e viabilidade de células de GBM humano da linhagem U87FP635. Esses resultados revelam uma importante ação parácrina dessa citocina regulatória, quando produzida por outros tipos celulares contidos no microambiente tumoral. Entretanto, sob condições experimentais que melhor mimetizam o microambiente tumoral, detectou-se que CTM também afetam o comportamento de células tumorais por um mecanismo alternativo, dependente de contato celular, mas independente dos níveis de TGFB1 secretados pelas CTM. Sob condições de cocultivo celular, envolvendo contato físico entre CTM e células de GBM U87FP635, detectou-se um aumento significativo na quantidade de células tumorais viáveis. Quando cultivadas na forma de esferoides tumorais, o contato com CTM aumentou a capacidade invasiva das células U87FP635. Finalmente, em modelo in vivo ectópico de GBM, células U87FP635 geraram tumores mais desenvolvidos quando coinjetadas com CTM. Esses efeitos pró-tumorigênicos foram observados tanto em contato com CTM controles, quanto com CTM contendo o gene TGFB1 permanentemente silenciado. Assim, esses achados indicam que CTM podem exercer efeitos pró-tumorigênicos por dois mecanismos alternativos e independentes: ação parácrina de TGFB1 secretado por CTM e ação mediada por contato célula-célula. Nas condições experimentais testadas, o mecanismo dependente de contato célula-célula demonstrou ser predominante. O estudo proteômico do secretoma dessas células identificou 126 proteínas diferencialmente expressas além de 10 proteínas exclusivamente detectadas em meios condicionados de cocultivos de CTM com células de GBM U87FP635. Cerca de 80% dessas proteínas exclusivamente secretadas pelo contato célula-célula são componentes de exossomos e estão envolvidas em proliferação celular e desenvolvimento tecidual. Esses resultados apontam uma interação dinâmica de comunicação entre CTM e células tumorais, e revelam algumas proteínas interessantes potencialmente envolvidas em uma ação pró-tumorigênica de CTM mediada por contato celular / Mesenchymal stem cells (MSC) display tropism to tumors, being recruited to its microenvironment where they comprise the tumor stroma. In brain, perivascular niche is a substantial source of MSC. Although mechanisms involved are poorly understood, MSC may directly and/or indirectly contribute to tumor development. Herein, the influence of MSC on the proliferation, invasiveness and tumorigenicity of human glioblastoma cells (GBM) was investigated. Moreover, since MSC releases TGFB1, a multifunctional cytokine with roles in immunomodulation, proliferation, migration and epithelial-mesenchymal transition of tumor cells, we analyzed if MSC-secreted TGFB1 affects GBM behavior. In vitro studies performed in the presence of conditioned media from human umbilical cord MSC with a stable TGFB1 gene expression knockdown showed that MSC-secreted TGFB1 is able to significantly increase the proliferation and viability of a GBM cell line (U87FP635). These results revealed an important paracrine effect of this regulatory cytokine when secreted by other cell types in tumor microenvironment. However, under experimental conditions that better mimic the tumor microenvironment, it was found that MSC also affect tumor cell behavior by an alternative mechanism dependent on cell-cell contact, but independent of TGFB1 levels secreted by MSC. The cell-cell contact between MSC and GBM U87FP635 significantly enhaced tumor viable cells. Additionally, the spheroid tumor cell culture with MSC cell contact increased the invasiveness of U87FP635 cells. Finally, in vivo ectopic implantation model showed more developed tumors when GBM U87FP635 cells were coinjected with MSC. These pro-tumorigenic effects were found both in cell-cell contact with control MSC, as with MSC containing TGFB1 gene expression knockdown. Thus, these findings indicate that MSC can exert pro-tumorigenic effects by two alternative and independent mechanisms: paracrine action of TGFB1 secreted by MSC and action mediated by cell-cell contact. In the present experimental conditions, the cell-cell contact-dependent mechanism was predominant. The secretome proteomic study of those cells identified 126 differentially expressed proteins as well as 10 proteins exclusively detected in conditioned media from GBM U87FP635 cell cocultures with MSC. About 80% of proteins uniquely secreted by cell-cell contact are exosomes components and are involved in cell proliferation and tissue development. These results indicate a dynamic interaction of communication between MSC and tumor cells and reveal some interesting proteins potentially involved in a MSC pro-tumorigenic action mediated by cell contact

The World Health Organization classifies glioblastoma (GBM) as a type IV astrocytoma, making it one of the most fatal tumors that exists. Despite the advances in chemotherapy, surgery, and radiation treatments that improve a patients length of survival, the overall trajectory of the disease remains unchanged. It has been shown that GBM cells produce significant levels of prostaglandins, including prostaglandin D2 (PGD2). PGD2 possesses pro- and anti-tumorigenic properties. Hence, a more complete understanding of PGD2 activity in GBM could yield more effective treatments against GBM. Through techniques like RT-PCR, immunohistochemistry, and HPLC tandem mass spectrometry, we were able to confirm the presence of the PGD2 synthesis in GBM cell lines. We treated GBM cell lines with various concentrations of exogenous PGD2 over 72 hours and observed its effects on cell count, apoptosis, mitosis and viability. Our results suggest that PGD2 possesses contradictory functions in GBM depending on concentration (mM PGD2 vs. nM PGD2) and receptor activation. / A Organização Mundial de Saúde classifica glioblastoma (GBM) como um astrocitoma tipo IV, fazendo uns dos tumores mais fatais que existe. A pesar dos avanços em quimioterapia, cirurgia e radioterapia que melhoram a longevidade de sobrevivência, a trajetória geral da doença permanece imutável. Tem sido demonstrado que células de GBM produzem níveis significativos de prostaglandinas, incluindo prostaglandina D2 (PGD2). PGD2 possui propriedades pro- e anti-tumorigenicos. Então, um entendimento mais completo da atividade de PGD2 em GBM pode gerar tratamentos mais efetivos. Através de técnicas como RT-PCR, imunohistoquimicas e HPLC espectrometria de massa em tandem, conseguimos confirmar a presença da síntese de PGD2 em linhagens de GBM. Tratamos linhagens de GBM com concentrações variáveis de PGD2 exógeno durante 72 horas e observamos seus efeitos na contagem de células, apoptose, mitose e viabilidade. Nossos resultados sugerem que PGD2 possui funções opostas em GBM dependendo em concentração (mM PGD2 vs. nM PGD2) e ativação de receptores.

Das Glioblastom ist mit 70 % aller Gliome der häufigste humane Hirntumor mit sehr ungünstiger Prognose. Es konnte gezeigt werden, dass das natürlich vorkommende Dipeptid Carnosin (β-Alanyl-L-histidin) die Proliferation von Glioblastomzellen inhibiert. Diese Wirkung des Carnosins konnte ebenfalls in vivo nachgewiesen werden. Da Carnosin auch einen Einfluss auf den ATP-Haushalt der Glioblastomzellen besitzt, war das Ziel dieser Arbeit einen Wirkungsort von Carnosin zu identifizieren, womit die ATP mindernden und proliferationshemmenden Eigenschaften erklärt werden können. Es wurde untersucht, ob Carnosin den Energiemetabolismus der Glioblastome beeinflusst. Dabei konnte mithilfe zellbiochemischer Methoden gezeigt werden, dass die untersuchten Zelllinien nicht von der Energieversorgung durch die mitochondriale oxidative Phosphorylierung abhängen, da sich weder Hemmung (KCN) noch Entkopplung (DNP) der Elektronentransportkette auf den zellulären ATP-Gehalt auswirkten. Carnosin hingegen verringerte den ATP-Spiegel dieser Zellen. Die Hemmung der Glykolyse durch Oxamat (LDH-Hemmung), bewirkte einen starken Abfall des intrazellulären ATP-Spiegels, worauf Carnosin keinen zusätzlichen Effekt mehr besaß. Carnosin konnte eine Wirkung auf die glykolytische ATP-Synthese zugesprochen werden. Da ein direkter, molekularer Wirkungsort auf diesem Weg nicht identifiziert werden konnte, wurde parallel untersucht, ob sich über Proteomanalysen der Glioblastomzelllinie T98G ein Wirkungsort, bzw. -mechanismus bestimmen lässt. Anhand der Methode der zweidimensionalen Gelelektrophorese (2D-GE) konnten 31 signifikant differenziell exprimierte Proteine detektiert werden, von denen 6 Proteine (VBP-1, OLA-1, TALDO 1, UROD, BAG-2, GRPEL1) über MALDI-TOF-Analysen identifiziert wurden. In Western-Blot-Analysen konnte ein Protein (VBP-1), neben T98G, auch in primären Glioblastomzelllinien als differenziell exprimiert nachgewiesen werden. Anhand der zellbiologischen und proteinbiochemischen Untersuchungen konnte einerseits eine Verbindung des Carnosins zum HIF1α-Signalweg und andererseits zur generellen posttranslationalen Peptidprozessierung hergestellt werden. Der direkte Nachweis eines Einflusses von Carnosin auf HIF1α wurde aber bisher nicht erbracht.

Primary glioblastoma (GBM), IDH-wildtype, especially with multifocal appearance/growth (mGBM), is associated with very poor prognosis. Several clinical parameters have been identified to provide prognostic value in both unifocal GBM (uGBM) and mGBM, but information about the influence of radiological parameters on survival for mGBM cohorts is scarce. This study evaluated the prognostic value of several volumetric parameters derived from magnetic resonance imaging (MRI). Data from the Department of Neurosurgery, Leipzig University Hospital, were retrospectively analyzed. Patients treated between 2014 and 2019, aged older than 18 years and with adequate peri-operative MRI were included. Volumetric assessment was performed manually. One hundred and eighty-three patients were included. Survival of patients with mGBM was significantly shorter (p < 0.0001). Univariate analysis revealed extent of resection, adjuvant therapy regimen, residual tumor volume, tumor necrosis volume and ratio of tumor necrosis to initial volume as statistically significant for overall survival. In multivariate Cox regression, however, only EOR (for uGBM and the entire cohort) and adjuvant therapy were independently significant for survival. Decreased ratio of tumor necrosis to initial tumor volume and extent of resection were associated with prolonged survival in mGBM but failed to achieve statistical significance in multivariate analysis.

Das Glioblastoma multiforme gehört zu den ZNS-Tumoren neuroepithelialen Ursprungs. Es zeichnet sich durch ein multiformes Zellbild, einen geringen Differenzierungsgrad und eine schnelle Krankheitsprogression aus. Trotz mikrochirurgischer Entfernung und anschließender Radiochemotherapie entwickeln die Patienten im Durchschnitt nach 7 Monaten einen Rezidivtumor und haben eine mittlere Überlebenszeit von 14,6 Monaten. Die Rezidivneigung stellt somit ein großes Problem in der Behandlung von Glioblastompatienten dar. In früheren Arbeiten konnte nachgewiesen werden, dass die Rezidivtumore eine andere Zellzusammensetzung und auch ein aggressiveres Wachstumsverhalten als deren Primärformen aufweisen. Ziel dieser Arbeit war es, zu prüfen ob mittels 2D-Gelelektrophorese und anschließender MALDI-TOF-Massenspektrometrie Unterschiede im Proteinexpressionsmuster zwischen Gewebeproben vom Primärtumor eines Glioblastoms WHO Grad IV und dem korrespondierendem Rezidivtumor eines Patienten detektierbar sind. Hierbei wurden 43 Proteine als differentiell exprimiert erkannt, von denen mit Hilfe der MALDI-TOF-Massenspektrometrie sechs genauer charakterisiert wurden. Vier der sechs Proteine waren im Rezidivtumor erhöht: EnoylCoA-Hydratase, ATP-Synthase Untereinheit d, Tropomyosin alpha-3-Kette Isoform 2 und Cathepsin D. Die anderen zwei waren im Rezidivtumor niedriger ausgeprägt: Nukleosid-Diphosphatkinase A und L-3-Phosphoserin-Phosphatase. Eine weitere Untersuchung mittels Western-Blot-Analyse bestätigte, dass Cathepsin D (als eines der sechs charakterisierten Proteine) tatsächlich auch in den Rezidivtumoren dreier weiterer Patienten stärker exprimiert war als in den korrespondierenden primären Glioblastomen.

Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumor that mainly affects adults. GBM displays remarkable intra- and inter-tumoral heterogeneity and contains a subpopulation of cells named glioma stem cells that is believed to be responsible for tumor maintenance, progression and recurrence. We have established and characterized a biobank of 48 cell lines derived from GBM patients. The cells were explanted and maintained as adherent cultures in serum-free, defined neural stem cell medium. These GBM cells (GCs) displayed NSC marker expression in vitro, had orthotopic tumor initiating capability in vivo, harboured genomic alterations characteristic of GBM and represented all four TCGA molecular subtypes. Our newly established biobank is also connected with a database (www.hgcc.se) that provides all molecular and clinical data. This resource provides a valuable platform of valid in vitro and in vivo models for basic GBM research and drug discovery. By using RCAS/tv-a mouse models for glioma, we found that GBMs originating from a putative NSC origin caused more tumorigenic GCs that had higher self-renewal abilities than those originating from putative glial precursor cell origin. By transcriptome analysis a mouse cell origin (MCO) gene signature was generated to cluster human GCs and GBM tissue samples and a functional relationship between the differentiation state of the initially transformed cell and the phenotype of GCs was discovered, which provides the basis for a new predictive MCO-based patient classification. LGR5 was found to be highly expressed in the most malignant mouse GC lines of putative NSC origin and also enriched in proneural GBMs characterized by PDGFRA alterations and OLIG2 up-regulation. By overexpressing or depleting LGR5 we discovered that high LGR5 expression in proneural GC lines increased the tumorigenicity, self-renewal and invasive capacities of the cells and could potentiate WNT signalling through its ligand RSPO1. Through transcriptome analysis we identified the candidate genes CCND2, PDGFRA, OLIG2, DKK1 that were found to be regulated by LGR5. In the last study, we found that mouse OPCs could initiate both astrocytic and oligdendroglial gliomas, which indicated that oncogenic signalling is dominant to cell of origin in affecting the histology of gliomas.

<p>The most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.</p><p>Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both <italic>in vitro</italic> and <italic>in vivo</italic> using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials. </p><p>In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.</p> / Dissertation

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

High Mobility Group AT-hook protein 2 (HMGA2) is a non-histone chromatin binding protein expressed in stem cells, cancer cells but not in normal human somatic cells. The presence of HMGA2 in cancer correlates with advanced neoplastic disease and poor prognosis. HMGA2 plays important roles in Base Excision Repair (BER) and at replication forks. HMGA2 is present at mammalian metaphase telomeres and its loss induces chromosomal aberrations. However, the functional role of HMGA2 at telomeres remains elusive. We hypothesized a protective role of HMGA2 that guards telomeres and modulates DNA damage repair signaling pathways. Employing different HMGA2+ human tumor cell models, we investigated the HMGA2-mediated functions that contribute to chemoresistance in glioblastoma (GB). This study presents a novel interaction of HMGA2 with telomeric protein TRF2 (Telomere Repeat-Binding Factor 2). This interaction retains TRF2 at telomeres, thus capping the telomeres and reducing telomere-dysfunction induced foci despite induced telomere stress. Loss of HMGA2 coincides with increased phosphorylation of TRF2, decreased TRF2 retention at telomeres and increased formation of telomeric aggregates, anaphase bridges and micronuclei. These findings provide new evidence for a unique role of HMGA2 at telomeres as a novel contributor of telomeric integrity. We show that upon DNA damage, HMGA2 causes increased and sustained phosphorylation of Ataxia Telangiectasia and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1). Prolonged presence of pCHK1Ser296 coincides with prolonged G2/M block and increased tumor cell survival. The relationship between (ATR)-CHK1 DNA damage response pathway and HMGA2 identifies a novel mechanism by which HMGA2 can alter DNA repair function in cancer cells. We identified HMGA2 as a novel factor contributing to temozolomide (TMZ) resistance in GB. HMGA2 knockdown sensitizes the GB cells to TMZ. We propose a specific combination of FDA-approved drugs, TMZ and Dovitinib (DOV), to increase GB cell death. We show that DOV downregulates key BER proteins, attenuates pSTAT3-coordinated Lin28A and HMGA2 expression. Our results suggest that a sequential therapeutic strategy of pretreating GB cells with DOV followed by a sequence of TMZ and DOV diminishes TMZ resistance and enhances the ability of TMZ to induce GB cell death. Overall, we identified HMGA2 as a multifunctional survival factor in human cancer cells and showed that targeting HMGA2 is a valid strategy to combat HMGA2+ cancer cells. / February 2016

Da hochgradige Gliome nur eine geringe Tendenz zur Metastasierung aufweisen, beschränkte sich das klinische Wissen über diesen seltenen Krankheitsverlauf bisher im Wesentlichen auf die Erkenntnisse aus Einzelfallberichten und kleineren Fallserien. Eine detaillierte Analyse der beschriebenen Fälle war bisher nicht verfügbar. Die vorliegende Arbeit stellt eine systematische Auswertung der wissenschaftlichen Literatur über Patienten mit metastasierten Glioblastomen oder Gliosarkomen dar. Unser Ziel war es, sämtliche Publikationen zu berücksichtigen, welche bis April 2013 veröffentlicht worden sind. Mit Hilfe einer systematischen Literaturrecherche in den beiden Datenbanken PubMed und Web of Science konnten 215 Arbeiten identifiziert werden, welche insgesamt 357 Fallberichte enthielten. Die Prognose nach Diagnose einer Metastasierung ist infaust. In der untersuchten Patientenkohorte betrug die mediane Überlebenszeit lediglich 3.0 ± 0.4 Monate. Eine univariate Datenanalyse ergab, dass Geschlecht, Alter, der histologische Subtyp und das Zeitintervall zwischen der Diagnose des Primärtumors und der Metastasen die Überlebenszeit nicht beeinflussten. Im Gegensatz dazu war eine Metastasierung, die ausschließlich außerhalb des zentralen Nervensystems (ZNS) auftrat, mit längeren Überlebenszeiten verbunden. In den letzten Jahrzehnten wurden offenbar keine entscheidenden therapeutischen Fortschritte erzielt. Fälle, die in Publikationen bis zum Jahr 2000 Erwähnung fanden, wiesen keine schlechteren Überlebenszeiten auf als die nach der Jahrtausendwende publizierten Fälle. Aktuell gibt es keinen Datensatz, der geeignet wäre, die vielfältigen Therapieansätze systematisch auf ihre Wirksamkeit hin zu überprüfen. Wir sehen hier die Notwendigkeit, ein zentrales Register zu etablieren.