by Chong Yin Yue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 146-161). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract (English/Chinese) --- p.ii / Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.xi / Chapter I. --- INTRODUCTION --- p.1 / Chapter I.1. --- Central Nervous System Tumors --- p.1 / Chapter I.2. --- Histopathological Classification of Gliomas --- p.4 / Chapter I.2.1. --- Astrocytic Tumors --- p.4 / Chapter I.2.1.1. --- Diffuse Astrocytomas --- p.4 / Chapter I.2.1.1.1. --- Low Grade Diffuse Astrocytomas --- p.5 / Chapter I.2.1.1.2. --- Anaplastic Astrocytomas --- p.5 / Chapter I.2.1.1.3. --- Glioblastomas --- p.6 / Chapter I.2.1.2. --- Pilocytic Astrocytomas --- p.9 / Chapter I.2.1.3. --- Pleomorphic Xanthoastrocytomas --- p.10 / Chapter I.2.2. --- Non-Astrocytic Tumors --- p.10 / Chapter I.2.2.1. --- Oligodendroglial Tumors --- p.10 / Chapter I.2.2.2. --- Ependymal Tumors --- p.16 / Chapter I.2.2.3. --- Dysembryoplastic Neuroepithelial Tumors --- p.20 / Chapter I.3. --- Molecular Genetics in Gliomas --- p.21 / Chapter I.4. --- Telomeres and Telomerase --- p.29 / Chapter I.4.1. --- History --- p.29 / Chapter I.4.2. --- Telomeres --- p.30 / Chapter I.4.3. --- End Replication Problem --- p.32 / Chapter I.4.4. --- Telomerase --- p.34 / Chapter I.4.5. --- Telomerase Components --- p.36 / Chapter I.4.5.1. --- RNA Component --- p.36 / Chapter I.4.5.2. --- Protein Component --- p.39 / Chapter I.4.5.3. --- Catalytic Subunit --- p.40 / Chapter I.4.5.4. --- Telomeric Repeat Binding Factor 1 --- p.41 / Chapter I.4.6. --- Cellular Immortality and Aging --- p.42 / Chapter I.4.7. --- Detection of Telomerase Activity --- p.44 / Chapter I.4.7.1. --- Telomeric Repeat Amplification Protocol Assay --- p.44 / Chapter I.4.7.2. --- Other Detection Methods --- p.45 / Chapter I.4.8. --- An Overview of Telomerase Activity --- p.48 / Chapter I.4.9. --- Alternative Lengthening of Telomeres --- p.50 / Chapter I.4.10. --- Telomeres Suppressor Genes --- p.51 / Chapter I.4.11. --- Telomerase and pl6/pRb Pathway --- p.52 / Chapter I.5. --- Telomeres and Telomerase Studies in Brain Tumors --- p.54 / Chapter I.5.1. --- Telomerase Activity in Brain Tumors --- p.54 / Chapter I.5.2. --- Telomere Length in Brain Tumors --- p.57 / Chapter I.5.3. --- Telomerase RNA Component Expression in Brain Tumors --- p.57 / Chapter II. --- OBJECTIVES of STUDY --- p.59 / Chapter III. --- MATERIALS AND METHODS --- p.61 / Chapter III.l. --- Telomerase Activity Study --- p.61 / Chapter III.1.1. --- Specimens --- p.61 / Chapter III.l.2. --- Telomerase Extraction --- p.63 / Chapter III.1.3. --- Protein Concentration Measurement --- p.63 / Chapter III.1.4. --- RNase-treated Samples --- p.64 / Chapter III.l.5. --- TRAP Assay --- p.64 / Chapter III.1.5.1. --- TS Primer Labelling --- p.65 / Chapter III.1.5.2. --- TS Primer Extension And PCR Amplification --- p.65 / Chapter III. 1.6. --- Non-Denaturing Polyacrylamide Gel Electrophoresis --- p.66 / Chapter III.1.7. --- Data Analysis --- p.67 / Chapter III.1.8. --- Statistical Analysis --- p.67 / Chapter III.2. --- Telomere Length Study --- p.68 / Chapter III.2.1. --- Specimens --- p.68 / Chapter III.2.2. --- DNA Extraction --- p.70 / Chapter III.2.3. --- DNA Concentration Measurement --- p.71 / Chapter III.2.4. --- Analysis of Telomere Length by Southern Hybridization --- p.71 / Chapter III.2.4.1. --- DNA Digestion --- p.73 / Chapter III.2.4.2. --- Southern Blotting --- p.73 / Chapter III.2.5. --- Data Analysis --- p.76 / Chapter III.2.6. --- Statistical Analysis --- p.77 / Chapter III.3. --- hTERT and hTEP1 mRNA Expression Study --- p.78 / Chapter III.3.1. --- Specimens --- p.78 / Chapter III.3.2. --- RNA Extraction and DNase Treatment --- p.78 / Chapter III.3.3. --- Reverse Transcription-Polymerase Chain Reaction --- p.80 / Chapter III.3.3.1. --- Primer Design --- p.81 / Chapter III.3.3.2. --- Standard Protocol --- p.81 / Chapter III.3.4. --- Statistical Analysis --- p.84 / Chapter III.4. --- p16 and pRb Immunostaining --- p.85 / Chapter III.4.1. --- Specimens --- p.85 / Chapter III.4.2. --- Slide Preparation --- p.85 / Chapter III.4.3. --- Immunohistochemistry --- p.87 / Chapter III.4.4. --- Data Analysis --- p.88 / Chapter III.4.5. --- Statistical Analysis --- p.89 / Chapter IV. --- RESULTS --- p.90 / Chapter IV.1. --- Telomerase Activity Study --- p.90 / Chapter IV.2. --- Telomere Length Study --- p.96 / Chapter IV.3. --- hTERT and hTEPl mRNA Expression Study --- p.104 / Chapter IV.4. --- p16 and pRb Protein Expression --- p.109 / Chapter V. --- DISCUSSION --- p.115 / Chapter V. 1. --- Telomerase Activity in Gliomas --- p.115 / Chapter V.2. --- Telomere Length in Oligodendroglial and Ependymal Tumors --- p.122 / Chapter V.3. --- hTERT and hTEPl mRNA Expression in Oligodendroglial and Ependymal Tumors --- p.128 / Chapter V.4. --- Protein Expression ofpl6 and pRb in Oligodendroglial and Ependymal Tumors --- p.132 / Chapter V.5. --- Discussion on Telomerase Activity-Related Factors --- p.135 / Chapter V.6. --- Significance of Study and Clinical Application --- p.137 / Chapter V.7. --- Future Direction --- p.141 / Chapter VI. --- CONCLUSION --- p.143 / Chapter VII. --- REFERENCES --- p.146

The prognosis for patients diagnosed with glioma has changed little over the past two decades. Many therapies that appeared promising in preclinical studies have been unsuccessful in the clinic. In an attempt to address this problem I developed a method for the efficient derivation of glioma primary cultures from fresh human brain tumours. These cultures are enriched for putative cancer stem-like cells that are thought to be responsible for glioma initiation, therapy resistance and recurrence. This mechanism of tumour development is a departure from the traditional multistep model of cancer. It is hoped that preclinical models incorporating glioma stem-like cells will more effectively recapitulate the biology of human disease and so better predict the likely clinical efficacy of inhibitor compounds tested in vitro and in the preclinical setting. In contrast to the majority of the existing literature, I identified two distinct tumourderived glioma stem-like cell phenotypes in my primary cultures that I have called ‘branched’ and ‘flat.’ The branched cells had similarities to the radial glia-like cells previously described in glioma stem-like cultures. In contrast, the flat cells had mesenchymal-like features. I discuss the implications of these observations for understanding glioma cell biology. I describe the development of high content phenotypic assays that incorporate these putative glioma stem-like cells. I screened inhibitor compounds of the PI3 kinase pathway, which is important in glioma cell behaviour, and identified that PIK75, a drug that targets the p110α catalytic subunit of PI3 kinase, inhibited growth of all the primary cells tested. I examined PIK75 activity in some detail. In vivo models of glioma are used to validate the findings of in vitro compound screening, so I describe my attempt to develop a novel genetically engineered mouse model designed to initiate glioma formation from the glioma stem-like cell. Surprisingly, these mice actually developed malignant peripheral nerve sheath tumours and that gave me a novel insight into the pathogenesis of this rare disease. This also informed future work on my long-term goal of generating a genetic model of glioma that recapitulates human disease.

Introduction: Low-grade gliomas are a sub-group of primary brain tumours that typically affect young adults and which present specific challenges to conventional diagnostic imaging. They demonstrate a pattern of growth whereby tumour cells infiltrate healthy brain tissue without distortion of the surrounding brain or blood-brain barrier integrity. These features limit the capacity of conventional neuro-imaging strategies to effectively delineate the tumour extent or characterise the degree of 'malignancy'. One solution is to apply multiple imaging modalities to image different aspects of the tumour behaviour, analogous to histological classification based upon changes in mitotic activity, cellular atypia, microvascular proliferation and necrosis. Published information regarding how imaging techniques that address these parameters correlate within the tumour volume is limited. This reflects the technical challenges in acquiring and processing data at an adequate spatial resolution to characterise small but heterogenous tumours. In this thesis, following a series of experiments seeking to optimise the sensitivity and reproducibility of PET analysis in gliomas, a prospective multi-modal neuro-imaging study is presented addressing this need. Methods: Retrospective [11C]-methionine PET (MET PET) data made available through a collaboration with the Max-Planck Institute for Neurological Research in Cologne was carried out first to address the optimal method of analysis of PET data in gliomas. A normal methionine uptake map was created and its use in the analysis of patient scans validated against a conventional approach. Automated methods for delineating the extent of abnormal methionine uptake and identifying the region of peak uptake were developed and evaluated to optimise the reproducibility of the approach. High-resolution MET PET and a comprehensive MRI brain tumour protocol were then acquired prospectively in 20 subjects in Manchester. Detailed analysis of the peak uptake and extent of abnormal tissue defined using PET and MRI modalities including structural, diffusion- and perfusion-weighted techniques was performed. Results: Evaluation of methionine uptake with respect to population normal data, the 'RatioMap' technique, yielded peak uptake measurements that correlated closely with a conventional approach (r = 0.97) but with improved reproducibility. The constrained 3D region-growing algorithm designed to delineate the abnormal region was shown to be reproducible and to generate volumes that correlated with tumour grade. High-resolution multi-modal data in suspected low-grade gliomas demonstrated consistent correlation between peak methionine uptake ratio and peak regional cerebral blood volume (r = 0.85) but with disparity between the location of the maximal uptake regions (mean distance = 11.2mm). Significant correlation was seen between multi-modal MRI and PET ‘tumour’ volumes (r = 0.91) but with substantially larger MRI defined abnormal volumes (ratio = 2.0) including small regions identified as abnormal by multiple MRI parameters but normal on PET imaging. Conclusion: A novel method to enhance the reproducibility of analysis of MET PET images in gliomas has been presented and validated but there remains no single imaging modality capable of fully characterising glioma extent and 'malignancy' non-invasively. Considerable correlation between PET and MRI tumour biomarkers has been demonstrated but there are significant differences between the regions identified as the 'most malignant' for biopsy targeting and the extent of potentially tumour bearing tissue. Combined use of diffusion- and perfusion-weighted MRI parameters can provide results very closely correlated to the PET findings but cannot yet completely replace the use of nuclear medicine techniques. The use of multi-modal approaches to tumour characterisation as demonstrated in this study provides the most effective currently available approach to fully characterise a suspected glioma.

Background: In a previous study, it was shown that melanoma patients have a greater incidence of glioma as compared to the general population in United States. Because glioma and melanoma do not have common environmental risk factors, this observation suggests a common genetic predisposition shared by glioma and melanoma that maybe used to lead future research of the specific genes and drug targets for both malignancies. However, this observation has to be confirmed in other populations. The aim of this study was to investigate the association between melanoma and glioma in Taiwanese population. Methods: We used claim data of Taiwan’s National Health Insurance Research Database (NHIRD) from year 1998 to 2010. The study population included 1,000,000 randomly selected men and women ages 20 and older from the NHIRD database. Glioma was defined by ICD-9-CM codes 191, 192.0-192.3, 192.8, 192.9, 225 and 237.5. Melanoma was defined by ICD-9-CM codes 172, 173, 190.0, 190.9, 192.1, 216.X (X=0, 3-7, 9), 224, 223.2, 235.1, 235.2, 237.6, 238.2, 238.3, 238.8. We excluded participants under ages 20 at 1998 and unknown gender (n=324,879). Cox's proportional hazard regression analysis was conducted to estimate the association between the history of melanoma on glioma risk. Main results: The hazard ratio of developing glioma was significantly higher in patients with melanoma than in those without melanoma (hazard ratio (HR) = 6.18; 95% confidence interval (CI) = 5.57-6.85). The hazard ratio for developing glioma was lower in male patients than in female patients, with the hazard ratio of 0.77 (95% CI = 0.71-0.84), adjusted for melanoma and age. The hazard ratio increased with age peaking at age group age from 60 to 69 and decrease after 70 years and older. Conclusion: The present study showed that Taiwanese patients with melanoma are at a higher risk of developing glioma. The exact underlying etiologies require further investigation.

Chan Yung. / Publication date from spine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 80-95). / Acknowledgement --- p.3 / Abstract --- p.4 / Chapter Chapter 1 --- Introduction --- p.7 / Chapter "1,1" --- Current status in treatment of gliomas --- p.7 / Chapter 1.1.1 --- Surgery of gliomas --- p.7 / Chapter 1.1.2 --- Radiation therapy of gliomas --- p.9 / Chapter 1.1.3 --- Chemotherapy for gliomas --- p.10 / Chapter 1.1.4 --- New modalities --- p.11 / Chapter 1.2 --- Basic principle of photodynamic therapy --- p.13 / Chapter 1.2.1 --- Photosensitizer --- p.13 / Chapter 1.2.2 --- Photosensitising effect of HpD --- p.13 / Chapter 1.2.3 --- Mechanism of selectivity in PDT --- p.15 / Chapter 1.2.4 --- Selective localization and the blood brain barrier --- p.16 / Chapter 1.2.5 --- Cytotoxic effect of PDT --- p.18 / Chapter 1.2.6 --- PDT on normal brain --- p.19 / Chapter 1.3 --- Photodynamic therapy in clinical neurosurgery --- p.21 / Chapter 1.4 --- The aims of the study --- p.26 / Chapter Chapter 2 --- Materials and methods --- p.28 / Chapter 2.1 --- Patient population --- p.28 / Chapter 2.2 --- Photosensitizer --- p.28 / Chapter 2.3 --- Administration of HpD --- p.29 / Chapter 2.4 --- Measurement of HpD --- p.30 / Chapter 2.5 --- Fluorescence microscopic examination --- p.32 / Chapter 2.6 --- Confocal laser scanning microscopic examination --- p.33 / Chapter Chapter 3 --- Results --- p.38 / Chapter 3.1 --- HpD measurement --- p.38 / Chapter 3.2 --- Fluorescence microscope examination --- p.40 / Chapter 3.3 --- Confocal laser scanning microscope (CLSM) examination --- p.41 / Chapter Chapter 4 --- Discussion --- p.56 / Chapter 4.1 --- Quantifying HpD level --- p.58 / Chapter 4.2 --- Quantifying HpD in the intravenous administration group --- p.59 / Chapter 4.3 --- Quantifying HpD in the intratumoural injection group --- p.63 / Chapter 4.4 --- Flourescence microscopic study of HpD distribution of malignant gliomas --- p.67 / Chapter 4.5 --- Confocal laser scanning microscopy (CLSM) study of HpD localization of malignant gliomas --- p.72 / Chapter 4.6 --- Conclusion --- p.74 / Chapter Chapter 5 --- Summary --- p.76 / Chapter Chapter 6 --- References --- p.80

Glioblastomas Multiformes (GBM) são tumores do Sistema Nervoso Central com altas taxas de invasibilidade e grande resistência a quimio e radioterapias, cuja origem foi inicialmente atribuída a células tronco neurais (NSCs). Mais recentemente, trabalhos de rastreamento de linhagem celular (lineage tracing) revelaram que a célula de origem em GBM, ao menos para certos subtipos, é na verdade a célula precursora de oligodendrócitos (OPC). A identificação da origem do tumor pode auxiliar na compreensão da doença e no desenvolvimento de terapias mais eficazes. OPCs são células com grande capacidade migratória e constituem a população de células cerebrais mais proliferativamente ativa, características compatíveis com a biologia de glioblastomas. Neste trabalho criamos uma meta-assinatura de OPCs que, quando aplicada a amostras populacionais e de céulas únicas de GBMs humanos, indica a presença de características de OPCs em virtualmente todos os tumores, principalmente dentre os do subtipo Proneural. Apesar disso, o cultivo de GBMs in vitro tem sido tradicionalmente realizado em meio próprio de NSCs, como forma de preservar as características originais do tumor. Entretanto, no caso de OPC ser a verdadeira célula de origem, o ideal seria a utilização de meio próprio para esta célula. Utilizamos meio padrão para NSCs e meio para OPCs em linhagens de camundongo bem como em biópsias humanas. Meio de NSCs provoca alterações morfológicas e em marcadores, enquanto meio de OPCs mantém as células mais similares ao tumor que lhes deu origem. Principalmente, meio de NSCs reduz o potencial tumorigênico das células in vivo, e faz com que alvos errôneos sejam identificados na resposta a drogas, devido à expressão de marcadores nãoautênticos pra célula de origem. A análise de gliomas humanos indica que a população proliferativamente ativa expressa marcadores de OPCs, independentemente do subtipo em que o tumor foi classificado. Assim, concluímos que o papel de OPCs no desenvolvimento de GBMs é mais importante do que se imaginava, e que a utilização de meio de cultivo baseado na célula de origem é fundamental para a correta identificação de alvos terapêuticos. / Glioblastoma Multiformes (GBM) are Central Nervous System tumors that present high invasibility rates and great resistance to chemo- and radiotherapies, whose origin was initially accredited to Neural Stem Cells (NSCs). More recently, papers employing lineage tracing revealed that the cell of origin in GBM, at least for certain subtypes, is in fact an Oligodendrocyte Precursor Cell (OPC). The elucidation about the origin of a tumor can help in the disease comprehension and in the development of more efficient therapies. OPCs are naturally migratory cells and constitute the most actively proliferating cell population in the brain, characteristics that are compatible with glioblastoma biology. In this work we created an OPC meta-signature that, once applied to populational and single-cell data in GBM, reveals the presence of OPC features in virtually every tumor, mainly from the Proneural subtype. Moreover, GBM in vitro culture has traditionally been done in NSC media, as an attempt to preserve original characteristics from the tumor. However, if OPC is the real cell of origin, it would be better to grow GBM samples in OPC media. Here, we used NSC media and OPC media in mice lines as well as in human byopsies. NSC media induces morphological and marker changes, while OPC media maintains the cells more similar to the tumor from where they were originated. Mainly, NSC media reduces the tumorigenic potential of the cells in vivo, and causes false targets to be identified in response to drugs due to the expression of non-authentic markers to the cell of origin. Human glioma analysis indicates that the actively proliferating population expresses OPC markers, regardless of the subtype in which the tumor was classified. Therefore, we conclude that the role of OPCs in GBM development is more importante than originally thought, and that the employment of culture media based on cell of origin is of fundamental importance for the correct identification of therapeutical targets.

Gliomas são tumores do sistema nervoso central caracterizados por alta invasibilidade e mortalidade. Inúmeros esforços foram feitos nas últimas décadas para melhorar a sobrevida dos pacientes, porém o último marco no tratamento se deu pela implementação da temozolomida (TMZ) combinada a ressecção cirúrgica e a radioterapia (RTX) em 2005. O projeto do atlas do genoma humano do câncer (TCGA) sequenciou tumores de mais de 500 pacientes com diagnóstico de glioblastoma (GBM) e categorizou os tumores em quatro subtipos moleculares, baseados na expressão, mutações e deleções de genes. Essas alterações genéticas já foram correlacionadas à melhora na sobrevida e à sensibilidade a terapia, porém, até o momento estudos prospectivos falham em direcionar o tratamento dos pacientes baseados nas características moleculares. Em busca de melhorar o entendimento sobre a correlação entre a sensibilidade a terapia in vitro e características genotípicas e fenotípicas, nós realizamos culturas primárias de células derivadas de tumores do sistema nervoso central de 23 pacientes (24 biopsias diferentes). As células foram crescidas em DMEM/F12 suplementado com soro fetal bovino e expostas aos tratamentos em doses e tempos semelhantes aos encontrados na clínica. Foram realizadas análises de viabilidade celular sete dias após o início do tratamento para 11 culturas primárias, ou as culturas foram tratadas por cinco dias em combinação, ou não, com RTX (sem RTX: 16 culturas; com RTX: 9 culturas) seguidos de sete dias em meio livre de droga, ao fim dos quais as células remanescentes foram contadas. A radioterapia e fármacos que agem sobre o citoesqueleto (vincristina, vimblastina, paclitaxel e mebendazole) sozinhos foram os tratamentos mais eficientes em reduzir a população celular. Uma segunda rodada de tratamento com TMZ, paclitaxel e a combinação de procarbazina, CCNU e vincristina (PCV) sugere que a resistência não é estável, e sucessivas exposições ao mesmo, ou a um fármaco diferente, podem ter seus efeitos somados na diminuição do crescimento populacional ou na massa tumoral final. Ao administrar PCV e paclitaxel nas células em cultura observamos um aumento nos níveis de autofagia que correlaciona com o declínio da população. Combinações desses fármacos com concentrações plasmáticas de um bloqueador da autofagia (cloroquina) não são capazes alterar o crescimento populacional. Resultados de análises do banco disponibilizado pelo TCGA mostram que alguns pacientes não apresentam aumento na sobrevida após tratamento com TMZ, e que essa falha no tratamento correlaciona com a baixa expressão de alguns genes nos seus tumores, por exemplo, aqueles com baixa expressão de FGFR3 e AKT2. As culturas primárias e linhagens celulares com menor expressão desses genes foram sensíveis in vitro a combinação de TMZ com dois fármacos que agem sobre o citoesqueleto: vimblastina e mebendazole (TVM). Essa associação retarda o crescimento de linhagens e culturas primárias resistentes a TMZ, além de induzir parada no ciclo celular, senescência e aumento da expressão de Notch3. Devido a falha na terapia padrão, ao baixo custo e aos resultados promissores essa associação de TVM poderia ser testada em pacientes cujos tumores apresentam baixos níveis de AKT2 e FGFR3. / Gliomas are tumors of the central nervous system with high invasiveness and mortality. Efforts have been done in last decades to improve patients overall survival, but the last treatment gain was given by the introduction of temozolomide (TMZ) combined with surgical resection and radiotherapy (RTX), in 2005. The cancer genome atlas (TCGA) consortion sequenced tumors from 500 patients with glioblastoma diagnosis and clustered tumors into four molecular subtypes, based on gene expression, mutations and deletions. Theses genetic alterations are associated with improved overal survival and sensitivity to therapy, but up to date, prospective studies have failed to address patients’ treatment based on molecular characteristics. In order to better understand the correlation among sensitivity in vitro and genotypic and phenotypic characteristics, we performed patient-derived cell cultures of central nervous system tumors from 23 patients (24 biopsies). Cells were grown in DMEM/F12 supplemented with fetal bovine serum and treated at doses and times similars to those administered in patients. Cell viability analyses were performed seven days after start of treatment for 11 cultures or cultures were treated for five days plus RTX (without RTX: in 16 cultures; with RTX: in 9 cultures) followed by seven day in drug-free medium, at the end, the remaining amount of cells were counted. RTX and drugs acting on cytoskeleton (vincristine, vinblastine, paclitaxel and mebendazole) alone were the most efficient treatments to reduce the population. A second round of treatment with TMZ, paclitaxel and the combination of procarbazine, CCNU and vincristine (PCV) suggests that the resistance is not stable, and repeated exposures to the same, or to another drug, could have additional effects in population or tumor mass reduction. PCV and paclitaxel treated cells showed an increase in autophagy levels correlated with reduction in population. Combining these drugs with plasmatic concentrations of an autophagy inhibitor (chloroquine) did not change population growth. Results from TCGA databank showed that some patients did not benefit in overal survival after TMZ treatment and this correlates with expression of some genes, for example, those harboring tumors with FGFR3 and AKT2 low expression. Patient-derived cultures and cell lines with low expression of these genes were sensitive in vitro to the combination of TMZ with two drugs that act on cytoskeleton: vinblastine and mebendazole (TVM). This association slowed the growth of patient-derived and cell lines tolerant to TMZ, besides inducing cell cycle arrest, senescence and increased Notch3 expression. In the case of failure of standard therapy, low cost and promising results, TVM association could be tested in patients harboring FGFR3Low/AKT2Low tumors.

Gliomas malignos compreendem o subtipo mais comum e devastador de tumores primários do sistema nervoso central (SNC), sendo o Glioblastoma Multiforme (GBM) a forma mais agressiva e mortífera. Pacientes com este tipo de tumor apresentam um prognóstico de sobrevida que não ultrapassa os 18 meses, após o diagnóstico. Isso é decorrente do fato de que os GBMs possuem elevada resistência aos tratamentos de quimio e radioterapia, além de serem altamente infiltrativos e neurologicamente destrutivos. Um importante fator que contribui para essa resistência é a superexpressão da proteína XIAP (X-linked Inhibitor of Apoptosis), o mais potente inibidor de apoptose da família das IAPs. Este atua inibindo diretamente a ativação das Caspases 3, 7 e 9. P53 é considerada uma das mais potentes proteínas supressoras tumorais, uma vez que esta atua como reguladora central em diversas vias de sinalização de mecanismos celulares distintos, como a via de controle do ciclo celular, reparo ao DNA, apoptose, senescência, autofagia, entre outras. Isto justifica o fato de p53 estar frequentemente mutada, nos mais variados tipos de cânceres. Portanto, o objetivo do presente trabalho foi estudar os efeitos resultantes da interação entre a superexpressão da proteína supressora tumoral p53 e o silenciamento da proteína anti-apoptótica XIAP na proliferação, na sobrevivência de GBMs. Quando p53 foi superexpressa (P+) na linhagem de glioma U87 silenciada para XIAP (Xi), através de RNAi, a taxa de proliferação celular reduziu significativamente em relação às células com as intervenções isoladas (U87Xi e U87wtP+) ou em relação ao controle selvagem U87wt, ao longo de dez dias. O ensaio de citotoxicidade LDH revelou um aumento na desestabilização da membrana citoplasmática nas células com a manipulação gênica combinada (U87XiP+) em relação os controles U87Xi e U87wtP+. O ensaio com AnexinaV/PI demonstrou uma elevação na marcação de células U87XiP+ com os dois marcadores, indicando uma maior indução de morte celular em relação aos controles. Além disso, como consequência da modulação combinada de p53 e XIAP, os níveis das proteínas pró-apoptóticas Bax e PUMA aumentaram tanto na linhagem U87wt, quanto na linhagem U87Xi, ao superexpressar p53. Além disso, os níveis de p21 e da proteína anti-apoptótica Bcl-xL foram reduzidos na linhagem U87Xi em relação ao controle U87wt. Outra constatação importante foi o aumento dos níveis de caspase-3 na linhagem U87Xi em relação ao controle U87wt e um aumento ainda maior nas células com a intervenção combinada (U87XiP+). Todos estes dados convergem para o fato de que a combinação do silenciamento de XIAP e superexpressão de p53 é mais efetiva na redução da proliferação e indução de morte na linhagem de glioma U87, do que as manipulações gênicas realizadas de forma isoladas, além de sugerir uma possível via de integração entre XIAP e p53, tendo como ponto de conexão o controle dos níveis citoplasmáticos da proteína p21 tanto por p53, quanto por XIAP, mostrando que a modulação combinada de proteínas da via apoptótica é capaz de potencializar os efeitos produzidos pelas intervenções de forma isoladas, representando uma nova e promissora estratégia no desenvolvimento de novas terapias para o tratamento de Gliomas.

Glioblastomas são os tumores mais agressivos do Sistema Nervoso Central (SNC). Caracterizam-se por sua alta invasibilidade, proliferação, altos índices de recorrência e morte, assim como quimio e radiorresistência. Tumores sólidos apresentam uma organização hierárquica, em que há uma pequena população de células tronco tumorais (CSCs) ou células iniciadoras de tumor (CICs). Essas células são capazes de repopular o tumor, que leva à recorrência. CSCs caracterizam-se por realizar também a mitose assimétrica, na qual parte das células continuam como CSCs e a outra parte sofre diferenciação. Essas células diferenciadas são incapazes de repopular o tumor, uma vez que perderam a sua capacidade tronco. CSCs mostraram ser relativamente resistentes as terapias anticâncer tradicionais como quimio e/ou radioterapia. Doxorrubicina (Doxo) é um agente anticâncer usado em diversos tipos de tumor e atua formando adutos no DNA e também inibindo a ação da topoisomerase II. Dependendo da concentração utilizada de Doxo, diferentes efeitos são observados; por exemplo, baixas doses (100 nM) levam à senescência celular, enquanto que altas doses (10 μM) levam à apoptose. Temozolomida (Tmz) é um anti-tumoral utilizado na terapia de diversos tumores, inclusive gliomas. Resveratrol (Rsv) é um polifenol encontrado em diversas plantas, como a uva, e possui efeitos como: neuroproteção, antiinflamatório, anti-oxidante, proteção cardíaca, entre outros. Nosso objetivo foi avaliar a influência do efeito dessas três drogas nas CSCs derivadas de glioblastoma humano. Realizou-se o ensaio de formação de esferas, um indicador da presença de CSCs, citometria de fluxo para Oct4 e Nanog e ensaio de senescência. Nossos ensaios utilizaram a linhagem U87 e dois tipos diferentes de meio de cultura. O primeiro continha DMEM Low Glucose com SFB e o segundo, um meio de cultura para células tronco (SCM), contendo DMEM F12 suplementado com FGF (fator de crescimento fibroblástico), EGF (fator de crescimento epidermal), LIF (fator inibidor de leucemia) e B27. Os tratamentos realizados utilizaram Doxo 1 e 10 nM; Tmz 5 μM e Rsv 1, 10 e 30 μM. O número de esferas formadas foi reduzido tanto em Doxo 1 quanto em 10 nM quando SFB foi utilizado. Doxo 1 não alterou o número de células Oct4 e Nanog positivas enquanto que Doxo 10 nM reduziu. Ambas as doses induziram senescência. Tmz reduziu o número de esferas formadas e também a porcentagem de células Oct4 e Nanog positivas. Usando SCM, observamos que Doxo e Tmz reduziram o número de esferas e a porcentagem de células Nanog e Oct4 positivas. Rsv 10 μM reduziu o número de esferas formadas enquanto que Rsv 30 μM reduziu o número de células positivas para CD133 e Oct4 com meio com SFB. Nossos resultados sugerem que Doxo 10 nM possui um melhor efeito nas CSCs do que Doxo 1 nM. Doxo 10 nM, além de reduzir o número de esferas, reduziu a porcentagem dos marcadores de CSCs e também induziu senescência celular na presença de SFB. Tmz e Rsv apresentaram resultados semelhantes à Doxo 10 nM. / Glioblastomas are the most aggressive tumors of the central nervous system (CNS). They are characterized by their high invasiveness, proliferation, high rates of recurrence and death, as well as chemo and radioresistance. Solid tumors have a hierarchical organization, in which there is a small tumor stem cell population (CSCs) or tumor initiator cells (CICs). These cells are able to repopulate the tumor, which leads to recurrence. CSCs are characterized by also performing asymmetric mitosis, in which the cells remain as CSCs and the other part undergoes differentiation. These differentiated cells are unable to repopulate the tumor, since they have lost their stem cell capacity. CSCs showed to be relatively resistant to traditional anti-cancer therapies such as chemo and / or radiotherapy. Doxorubicin (Doxo) is an anticancer agent used in several tumor types and acts by forming DNA adducts as well as inhibiting the action of topoisomerase II. Depending on the concentration of Doxo used, different effects was observed, for example, low doses (100 nM) lead to cellular senescence, whereas high doses (10 μM) lead to apoptosis. Temozolomide (Tmz) is an anti-tumor therapy used in many tumors, including gliomas. Resveratrol (Rsv) is a polyphenol found in various plants, such as grapes, and has effects such as neuroprotective, antiinflammatory, anti-oxidant, cardiac protection, among others. Our objective was to evaluate the effects of these three drugs on CSCs derived from human glioblastoma. We performed the sphere formation assay, an indicator of the presence of CSCs, flow cytometry for Oct4 and Nanog and senescence assay. Our experiments used the cell line U87 and two different types of culture medium. The first contained DMEM Low Glucose with FBS and the second, a culture medium for stem cells (SCM), containing DMEM F12 supplemented with FGF (fibroblastic growth factor), EGF (epidermal growth factor), LIF (leukemia inhibitor factor) and B27. The tests were performed using Doxo 1 and 10 nM, Tmz 5 μM and Rsv 10 and 30 μM. The number of spheres formed was reduced in both doses of Doxo when FBS was used. Doxo 1 nM did not alter the number of Oct4 and Nanog positive cells while Doxo 10 nM reduced. Both doses induced senescence. Tmz reduced the number of spheres formed and also the percentage of Nanog and Oct4 positive cells. Using SCM, we saw that Doxo and Tmz reduced the number of spheres and the percentage of Nanog and Oct4 positive cells. Rsv 10 μM reduced the number of spheres formed while Rsv 30 μM reduced the number of CD133 and Oct4 positive cells with medium supplemented with FBS. Our results suggest that Doxo 10 nM has a better effect in the CSCs than Doxo 1 nM. Doxo 10 nM besides reducing the number of spheres also decreased the percentage of CSCs markers, and induced cellular senescence in the presence of FBS. Tmz and Rsv had similar effects as Doxo 10 nM.

Patients diagnosed with malignant glioma tumours have median survivals of 1.6 yrs and 5 months, respectively, highlighting the deadly nature of this disease. Despite aggressive multimodal treatment, patients with malignant glioma often present with secondary brain tumours at sites distal to the primary tumour mass. These secondary tumours are a consequence of renegade neoplastic cells that infiltrate the surrounding normal brain, a hallmark feature of malignant glioma. Brain fatty acid-binding protein (FABP7), which binds omega-3 docosahexaenoic acid (DHA) and omega-6 arachidonic acid (AA), is overexpressed and associated with a poor prognosis in patients with malignant glioma compared with normal brain. These data suggest that FABP7 plays an important role in gliomagenesis; however, the mechanism(s) underlying a role for FABP7 in malignant glioma has, until now, been unexplored. Here, we demonstrate that expression of FABP7 in malignant glioma cells is accompanied by increased cell migration. Consistent with our in vitro results, we show that expression of FABP7 in astrocytoma tumours is associated with sites of tumour infiltration and tumour recurrence. Furthermore, we demonstrate that the fatty-acid ligands of FABP7 affect cell migration in an FABP7-dependent manner. More specifically, DHA inhibits migration, whereas AA stimulates cell migration. Finally, we reveal that uptake and incorporation of DHA and AA in the phospholipids of malignant glioma cells is enhanced by FABP7 expression, suggesting a mechanism by which DHA and AA may affect cell migration by altering signal transduction at the cell membrane. We propose that the inherent ability of malignant glioma cells to express the radial glial marker FABP7 underlies their infiltrative capacity, allowing tumour cells to migrate long distances from the main tumour mass. We propose a model whereby FABP7 expression and relative levels of DHA and AA determine tumour infiltrative potential. Our findings provide insight into the role of FABP7 and its fatty acid ligands in controlling the migration of malignant glioma cells and point to the potential use of DHA as a natural anti-infiltrative agent in the treatment of malignant glioma. We believe that targeting FABP7-expressing cells may make a significant impact on the treatment of high grade astrocytomas. / Experimental Oncology