Mammalian neural stem cell (NSC) lines provide a useful experimental model for basic and applied research across stem cell and developmental biology, regenerative medicine and neuroscience. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with functional genetic analyses. However, targeted genetic manipulations have not been reported for mammalian NSC lines. Here, we deploy the CRISPR/Cas9 technology and demonstrate a variety of diverse targeted genetic modifications in both mouse and human NSC lines such as: targeted transgene insertion at safe harbour loci; biallelic knockout of neurodevelopmental genes; knock-in of epitope tags and fluorescent reporters; and engineering of glioma driver mutations at endogenous proto-oncogenes. Leveraging these new optimised methods, we explored gene editing to model the earliest stages of paediatric gliomagenesis in primary human NSCs. Our data indicate that oncogenic mutations in histone H3.3 play a role in NSC transformation and may operate through suppression of replication induced senescence. By comparing cellular responses of NSC cultures from different compartments of the developing brain, we also identify differences in susceptibility to distinct H3.3 mutations that mirror the disease etiology. Altogether, our findings indicate that CRISPR/Cas9-assisted genome editing in NSC lines is a versatile tool to explore gene function in CNS development and cancer biology. Our project resulted in the creation of valuable human cellular models of paediatric gliomagenesis, which will allow us to further our understanding of the disease and carry out cell based drug discovery projects.

Orientador: Valeria Valente / Banca: Ana Lucia Fachin Saltoratto / Banca: Cleverton Roberto de Andrade / Resumo: O câncer é um conjunto de alterações celulares que favorecem a proliferação descontrolada e a aquisição de propriedades metastáticas. A ativação de oncogenes e/ou a perda de genes supressores tumorais leva a desbalanços nos mecanismos de controle do ciclo celular e/ou na inativação das vias apoptóticas, contribuindo para a instabilidade genômica presente em todos os tipos tumorais. Relatos na literatura têm sugerido que o estresse replicativo oriundo desse aumento proliferativo exacerbado é um fator importante na formação e progressão de muitos tipos de câncer. Dentro dessa perspectiva, é plausível pensar que as células tumorais tenham desenvolvido certas competências que as permitam lidar com o estresse replicativo para continuar se propagando. Resultados prévios de nosso laboratório sugerem que a proteína centromérica HJURP (Holliday Junction Recognizing Protein) esteja envolvida nesse ganho de competência. Dessa forma, esse trabalho teve como objetivo a análise do papel dessa proteína frente ao estresse replicativo em linhagens de glioblastoma. Nossos resultados demonstraram que a superexpressão de HJURP confere um aumento na capacidade proliferativa celular da linhagem U87MG. Além disso, vimos que em baixas concentrações de camptotecina as células superexpressoras de HJURP possuíam um comportamento proliferativo muito semelhante à condição não tratada. Os ensaios de citotoxicidade revelaram uma maior capacidade de recuperação frente ao estresse replicativo exógeno nas sit... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cancer is characterized by a subset of features that supports uncontrolled cell proliferation and acquisition of metastatic properties. Oncogene activation and/ or the deactivation of tumor suppressor genes lead to disbalances in cell cycle control progression and/or inactivation of apoptotic pathways, followed by genomic instability present in all types of tumors. According to the literature, replicative DNA stress arising from uncontrolled cell growth is the major driver in the development and progression of many cancers. In this perspective, it is believed that cancer cells have developed some skills in order to deal with high levels of endogenous replicative stress for their continued proliferation. Previous results from our group suggest that the centromeric protein HJURP (Holliday Junction Recognizing Protein) is involved in this mechanism. Thus, in this work, we aimed to analyze the role of this protein in the cellular response to the replicative stress in glioblastoma U87MG cell line. Our data showed that cells who levels of HJURP were high, had an increase in cell growth. Besides that, we also observed that at lower camptothecin concentrations the curve proliferation of overexpressed HJURP cells had a similar behavior as the control cells. The cytotoxicity results demonstrated that cells overexpressing HJURP had a better replicative induced stress when compared to the control cells. Together, this data suggests... (Complete abstract click electronic access below) / Mestre

Dissertação (mestrado)—Fundação Universidade de Brasília, Programa de Pós-Graduação em Biologia Animal, 2018. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). / O câncer é o nome dado a um conjunto de doenças relacionadas responsáveis pelas maiores taxas de morte ao redor do mundo, tanto em países mais ou menos desenvolvidos. Dentre eles está o Glioblastoma Multiforme (GBM), originado nas células gliais e classificado como um astrocitoma de grau IV, caracteristicamente agressivo e invasivo. Sabendo da baixa expectativa de vida dos pacientes com esta condição, terapias alternativas têm sido incentivadas de forma a tentar combater o tumor com as mais diversas estratégias, visando atingir todas as células da massa tumoral. O uso de células mesenquimais (MSCs) como carreador de terapias à tumores já vem sendo investigado e tem apresentado bons resultados, entretanto, estabelecer um protocolo de administração efetivo é essencial. Uma das estratégias que se tem usado para entender esse potencial migratório é sua marcação com nanopartículas magnéticas. Por isso, este estudo teve como objetivo investigar e comparar a administração de MSCs por via intranasal e endovenosa em um modelo murino de glioma. Para tal, as MSCs foram isoladas de lipoaspirado humano e marcadas com nanopartículas magnéticas para visualização e quantificação in vivo. Foi feita a transdução lentiviral da linhagem tumoral U87MG com luciferase para facilitar o estabelecimento do modelo animal de glioma por meio de acompanhamento de sua bioluminescência. Posteriormente, as MSCs marcadas com nanopartículas foram administradas pelas vias intranasal e endovenosa em camundongos imunodeficientes previamente enxertados com células U87MG e, ao final, sua capacidade migratória foi avaliada por meio de analise histológica e determinação da biodistribuição de ferro por espectrometria de emissão óptica com plasma acoplado indutivamente. Os dados obtidos foram analisados conforme o teste estatístico apropriado e apresentados como média e erro padrão. Observamos por meio dos ensaios in vitro que a marcação das MSCs com nanopartículas foi eficiente, visto que foi possível evidenciar o ferro na célula e estas tiveram tendência migratória em direção ao campo magnético. O estabelecimento da linhagem tumoral modificada com luciferase foi efetivo, porém a transdução com GFP não foi efetiva, visto que poucas células se mostraram fluorescentes. Já nos ensaios in vivo, a administração das MSCs marcadas após o estabelecimento do modelo murino de glioblastoma demonstrou uma maior eficiência da via intranasal, pois teve um maior acúmulo de ferro no cérebro e menor concentração nos pulmões, em comparação com a via endovenosa. Dessa forma, a via intranasal se mostrou mais eficaz e deve ser priorizada quando o objetivo é atingir o cérebro e reduzir possíveis efeitos associados ao acúmulo do tratamento nos pulmões. / Cancer is the name given to a set of related diseases responsible for highest death rates around the world, both in underdeveloped or developed countries. Among them, is Glioblastoma Multiforme (GBM), which originates in glial cells and is classified as a grade IV astrocytoma, characteristically aggressive and invasive. Knowing low life expectancy of patients with this condition, alternative therapies have been encouraged in order to try to fight tumor with most diverse strategies, aiming to reach all cells of the tumor mass. The use of mesenchymal stromal cells (MSCs) as a carrier of tumor therapies has been investigated and has shown good results, however, establishing an effective administration protocol is essential. One of the strategies that has been used to understand this migratory potential is labbeling the cells with magnetic nanoparticles. Therefore, this study aimed to investigate and compare intranasal and intravenous administrations routes for MSCs in a murine model of glioma. For this, human MSCs were isolated from liposuction and labeled with magnetic nanoparticles for visualization and quantification in vivo. Transduction of the U87MG tumor cell line with luciferase lentiviral particles was performed to facilitate establishment of the animal model of glioma by monitoring bioluminescence. Subsequently, MSCs labeled with magnetic nanoparticles were injected intravenously and intranasally in immunodeficient glioblastoma mice model with U87MG cells and, ultimately, their migratory capacity was evaluated by both histological analysis and biodistribution of iron analised by optical emission spectrometry with inductively coupled plasma. Data were analyzed by using appropriate statistical tests and reported as mean and standard error. We observed through in vitro assays that labelling and further tracking of MSCs with nanoparticles was efficient, since it was possible to evidence the iron into the cell and these had a migratory tendency towards the magnetic field. Establishment of luciferase-expressing tumor line was effective, but the GFP transduction was not effective, since few cells were fluorescent. In the in vivo assays, after established of glioblastoma murine model, we observed a higher efficiency of intranasal administration route when comparing to endovenous route. Higher accumulation of iron in brain and a lower concentration in lungs were found in intranasal group, in comparison with intravenous route group. Thus, intranasal route has proven to be more effective and should be prioritized when the goal is to reach brain and reduce possible effects associated with accumulation of treatment in lungs.

Glioblastoma (GBM) is the most common and aggressive type of brain cancer, with an average life expectancy of 15 months. The standard of care for GBM, surgery accompanied by radiation and chemotherapy (temozolomide-TMZ), has not changed in over 10 years illustrating the need for new and efficacious treatments. Therefore, it is imperative to improve our knowledge of GBM physiology to understand the mechanisms driving recurrence and chemoresistance so that more effective therapeutic options can be developed. Mitochondria-cell communication is key to monitor and maintain both mitochondrial and cellular health, and signaling events on the outer mitochondrial membrane (OMM) have emerged as a crucial signal integration site for cellular responses. Consequently, proteins on the OMM are crucial to determining cellular survival and dictating organelle physiology. Thus, the goal of our current study is to evaluate OMM proteins to determine how alterations in organelle regulation may impact CNS tumor biology. We first measured the concentrations of Bcl-2 family proteins on mitochondria from ten continuous GBM cell lines and correlated the protein levels to IC50values of genotoxic agents TMZ and irinotecan. We found that Bcl-2 levels corresponded to chemoresistance, while increased Bim concentrations promoted chemosensitivity. In contrast to our studies in gynecological cancers, the concentrations of the pro-dysfunction OMM scaffold protein Sab had no impact on chemosensitivity of the GBM cell lines, despite diminished Sab expression in GBM patients. However, we identified a novel truncated variant of Sab in the GBM cell lines. We found that GBM cells expressing only full-length Sab had a slower proliferation rate than those with the variant, which could be attributed to increased glycolysis in GBM cells expressing the Sab variant. To determine if the lack of Sab-mediated apoptosis was consistent across CNS tumors, we analyzed publicly-available patient data and found that Sab expression is down-regulated in neuroblastoma patients, a pediatric malignancy responsible for 12% of childhood cancer deaths. We found that that inhibiting Sab-mediated signaling in human neuroblastoma (SH-SY5Y cells) enhanced oxidative phosphorylation in a pyruvate dehydrogenase-dependent manner, increased BCl-2 levels (pro-survival), decreased Bim concentrations (pro-apoptotic), and promoted chemoresistance. Furthermore, examination of additional neuroblastoma cells derived from CNS tumors revealed that Sab levels correspond to proliferation rate, metabolic phenotype, and chemosensitivity. Our studies demonstrate the importance of OMM signaling in CNS tumor physiology and emphasizes the importance of cellular context to the outcomes of OMM signaling events.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Ein LOH (loss of heterozygosity, Heterozygotieverlust) im Bereich des kurzen Arms des Chromosoms 10 tritt sehr häufig in hochgradigen Gliomen auf. Vornehmlich konzentriert sich der allele Verlust auf die Region 10p14-p15. Die chromosomale Instabilität dieser Region deutet auf die Existenz eines oder mehrerer Tumorsuppressorgene hin. Bis jetzt konnte der Krüppel-like Transkriptionsfaktor 6 (KLF6) als mögliche Zielstruktur eines LOH in 10p15 identifiziert werden. Das Gen des regulatorischen Glykolyseenzyms PFKFB3 ist im Bereich 10p15.1 lokalisiert. Die Spleißvariante 4 (UBI2K4) des PFKFB3-Gens hat eine hemmende Wirkung auf das dreidimensionale Wachstum von U87 Glioblastomzellen. Folglich könnte der PFKFB3 Genlocus Zielstruktur eines LOH in Glioblastomen sein. In dieser Arbeit wurden 40 Glioblastome mit der Mikrosatellitenanalyse basierend auf PCR-Technik untersucht. Bei 55 % (22/40) der Glioblastome zeigte sich ein LOH des PFKFB3 Gens. Der LOH des KLF6 Locus, 2,5 cM telomerisch zur PFKFB3 lokalisiert, wurde in 60 % (21/35) der Glioblastome festgestellt und korrelierte nicht mit dem PFKFB3-LOH. Die Deletion eines Allels des PFKFB3-Gens führt zu einer signifikanten Abnahme der PFKFB3 mRNA- und Proteinkonzentration. Die Expression der das Glioblastomzellwachstum hemmenden Spleißvariante UBI2K4 war in der Gruppe der LOH PFKFB (+) Glioblastome deutlich erniedrigt im Vergleich zur PFKFB3-LOH (-) Gruppe und korrelierte mit der totalen PFKFB3 Expression. Die Prognose der Glioblastom-Patienten verschlechtert sich durch den PFKFB3-LOH und die daraus resultierende niedrigere UBI2K4 Expression. Die Ergebnisse der Untersuchungen zeigen, dass der für Glioblastome typische LOH der Region 10p14-p15 auf die PFKFB3 und speziell auf die Spleißvariante UBI2K4 als Tumorsuppressorprotein abzielt.

<p>Transforming growth factor–β (TGF–β) signaling regulates a range of processes in a variety of cell types. Consequently, TGF–β plays a complex role in the progression of several types of cancers; it acts as a tumor suppressor in normal cells and early in tumor progression, yet it can promote tumor progression in later stages of cancer.</p><p>Among the cancers that TGF–β has been implicated in is glioblastoma multiforme (GBM), the most common primary brain neoplasm and one of the most lethal types of cancer. Because of its high mortality rate and the lack of effective treatments, discovering the molecular mechanisms that underlie GBM formation and growth is of great clinical interest. To this end, we investigated the function of a TGF–β target gene — the putative tumor suppressor N‐Myc downstream‐regulated gene 4 (NDRG4) — in GBM cell viability, proliferation and tumor formation. Contrary to the established roles of other NDRG family members, we found that NDRG4 expression is elevated in GBM and that NDRG4 is required for the survival of established GBM cell lines and primary GBM xenograft cells enriched for highly tumorigenic GBM cancer stem cells. Knockdown of NDRG4 expression results in G₁ cell cycle arrest followed by apoptosis that is associated with a decrease in the expression of XIAP and survivin. Finally, knockdown of NDRG4 expression in established GBM cell lines and GBM cancer stem cells results in decreased tumorigenicity following intracranial implantation of these cells into immunocompromised mice. Collectively, these data indicate that NDRG4 does not function as a tumor suppressor like other NDRG family members, but rather it is essential for GBM tumorigenicity and may represent a potential therapeutic target for this devastating disease.</p><p>In the second portion of this dissertation, we examine the TGF–β cytostatic signaling pathway in B lymphocytes. TGF–β–induced growth inhibition is the most extensively studied biological response to a TGF–β signal. Although in most cell types this response is mediated by Smad3– dependent regulation of c–Myc, p15<super>Ink4B</super>, and p21<super>Cip1</super> transcription, studies from Smad3 null mice suggest that TGF–β–induced growth inhibition in B lymphocytes occurs regardless of Smad3 status. We prove that this response does indeed occur independently of Smad3 in purified primary B lymphocytes and WEHI–231 cells. Consistent with this, p15<super>Ink4B</super> and p21<super>Cip1</super> are not noticeably induced by TGF–β in these cells, whereas Id3 and cyclin G2 are induced in a Smad3–independent manner. Finally, unlike the MAPK pathways we tested, the BMP–specific Smads 1 and 5 are activated in response to TGF–β in these cells, and this activation is dependent on ALK5 kinase activity. Collectively, these data indicate that TGF–β induces growth inhibition in B lymphocytes through a novel signaling pathway, and Smads 1 and 5 may help mediate this response.</p> / Dissertation

<p>Cytosolic cytochrome c promotes apoptosis by triggering caspase activation. In healthy cells cytochrome c localizes to mitochondria, where it participates in the electron transport chain. Apoptotic stimuli induce permeabilization of the outer mitochondrial membrane and release of cytochrome c. Once cytosolic, cytochrome c binds Apaf-1, inducing the formation of a protein complex that recruits and activates caspases, which serve to dismantle the dying cell. Although the steps of this signaling pathway have been described, many of the regulatory mechanisms influencing the cellular response to cytosolic cytochrome c remain unclear. Using apoptosis assays and microinjection techniques, we investigated the response of several cell-types to cytosolic cytochrome c.</p> <p>First, we demonstrate that cytosolic cytochrome c kills brain tumor cells but not normal brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in brain tumors compared with negligible Apaf-1 in brain tissue. These differences in Apaf-1 abundance correlate with differences in E2F1, a previously identified activator of Apaf-1 transcription. Chromatin immunoprecipitation assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to Apaf-1 expression in brain tumors. These results demonstrate an unexpected sensitivity of brain tumors to cytochrome c and raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancers.</p> <p>Secondly, we develop a method for monitoring caspase activity in Xenopus laevis oocytes and early embryos. The approach, utilizing microinjection of a near-infrared dye that emits fluorescence only after its cleavage by active caspases, has enabled the elucidation of subtleties in the apoptotic program. We demonstrate that brief caspase activation is sufficient to cause death. We illustrate the presence of a cytochrome c dose threshold, which is lowered by neutralization of inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that eventual death of these oocytes is caspase-independent. Imaging caspase activity in the embryo suggests that apoptosis in early development is not cell-autonomous. Finally, we believe this method presents a useful screening modality for identifying novel apoptotic regulators as well as pro-apoptotic small-molecules that could be useful in treating brain tumors.</p> / Dissertation

Glioblastoma multiforme (GBM), a grade IV malignant astrocytic tumor according to WHO classification, is one of the most common and malignant brain tumor. Temozolomide (TMZ) is the current standard treatment for GBM. Nevertheless, resistance to chemotherapy in GBM is common and therefore a major obstacle to successful treatment. Adducin 3 (ADD3), a cytoskeletal protein, has been found to be associated with chemoresistance in osteosarcoma, but its potential role in glioblastoma is unclear. A TMZ-resistant model was established by chronically exposing the glioma cells (D54 cell line) to an increasing dose of TMZ. A resistant subclone (D54-R) was successfully generated. ADD3 expression level was found to be upregulated in the D54-R when compared to the parental D54 cells (D54-C). CD133 is a putative cancer stem cell marker. Its expression level was found also to be higher in D54-R when compared to D54-C cells. Among the D54-R cells, a subgroup of cells was found to express ADD3 intensely. The proportion of these spherical cells was higher in D54-R than D54-C. Moreover, these cells were spherical in morphology and expressed putative cancer stem cell markers: CD133, NANOG and OCT-3/-4. Therefore, ADD3 is associated with cancer stem cells in human glioma. The upregulation of ADD3 expression is associated with TMZ-resistance in GBM. / published_or_final_version / Surgery / Master / Master of Research in Medicine

<p>The histologic subtypes of malignant glial neoplasms range from anaplastic astrocytoma to the most deadly World Health Organization (WHO) Grade IV glioblastoma (GBM), the most common primary brain tumor in adults. Over the past 40 years, only modest advancements in the treatment of GBM tumors have been reached. Current therapies are predominantly for palliative endpoints rather than curative, although some treatment modalities have been shown to extend survival in particular cases. Patients undergoing current standard of care therapy, including surgical resection, radiation therapy, and chemotherapy, have a median survival of 12-15 months, with less than 25% of patients surviving up to two years and fewer than 10% surviving up to five years. A variety of factors contribute to standard treatment failure, including highly invasive tumor grade at the time of diagnosis, the intrinsic resistance of glioma cells to radiation therapy, the frequent impracticality of maximal tumor resection of eloquent cortical structures, and the fragile intolerance of healthy brain for cytotoxic therapies. Treatment with immunotherapy is a potential answer to the aforementioned problems, as the immune system can be harnessed and educated to license rather potent antitumor responses in a highly specific and safe fashion. One of the most promising vehicles for immunotherapy is the use of dendritic cells, which are professional antigen-presenting cells that are highly effective in the processing of foreign antigens and the education of soon-to-be activated T cells against established tumors. The work outlined in this dissertation encompasses the potential of dendritic cell therapy, the current limitations of reaching full efficacy with this platform, and the recent efforts employed to overcome such barriers. This work spans the characterization and preclinical testing of utilizing protein antigens such as tetanus-diphtheria toxoid to pre-condition the injection site prior to dendritic cell vaccination against established tumors expressing tumor-specific antigens. </p><p>Chapter 1 comprises an overview of the current standard therapies for malignant brain tumors. Chapters 2 and 3 provide a review of immunotherapy for malignant gliomas in the setting of preclinical animal models and discuss issues relevant to the efficacy of dendritic cell vaccines for targeting of GBM. Chapters 4 provides the rationale, methodology, and results of research to improve the lymph node homing and immunogenicity of tumor antigen-specific dendritic cell vaccines in mouse models and in patients with newly diagnosed GBM. Chapter 5 delineates the interactions discovered through efforts in Chapter 4 that comprise protein antigen-specific CD4+ T cell responses to induced chemokines and how these interactions result in increased dendritic cell migration and antitumor responses. Lastly, Chapter 6 discusses the future utility of migration of DC vaccines as a surrogate for antitumor responses and clinical outcomes. </p><p>This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix. </p><p>In summary, this work 1) identifies inefficient lymph node homing of peripherally administered dendritic cells as one of the glaring barriers to effective dendritic cell immunotherapy, 2) provides answers to overcome this limitation with the use of readily available pre-conditioning recall antigens, 3) has opened up a new line of investigation for interaction between recall responses and host chemokines to activate immune responses against a separate antigen, and 4) provides future prospects of utilizing chemokines as adjuvants for additional immunotherapies targeting aggressive tumors. Together, these studies hold great promise to improve the responses in patients with GBM.</p> / Dissertation