Células tumorais têm sua proliferação e mobilidade modificada por diversos fatores de crescimento, citocinas e mediadores inflamatórios, dentre os quais a amilóide sérica A (SAA). Estudos prévios do nosso grupo mostraram o efeito direto da SAA em processos de proliferação, migração e invasão de células de glioblastoma multiforme (GBM), A172 e T98G. Neste estudo nós complementamos resultados prévios de migração e invasão; avaliamos SAA como possível indutora de moléculas importantes para a invasividade do tumor, como as MMP-2 e -9 e ROS; realizamos ensaio clonogênico com a intenção de investigar uma possível contribuição da rSAA no estágio inicial de desenvolvimento do tumor; avaliamos o impacto da hipóxia na expressão dos diferente genes da SAA; estimulamos as células com indutores hepáticos clássicos da SAA e analisamos a possibilidade destes induzirem os diferentes genes da SAA em células tumorais; avaliamos possíveis receptores e vias de sinalizações envolvidas nos processos de proliferação, migração e invasão. Construímos knockdowns (KDs) dos genes da SAA de fase aguda (SAA1 e 2) e constitutiva (SAA4) e avaliamos a função de cada um deles para a morfologia e para os processos de proliferação, migração e invasão de GBM. Por fim investigamos SAA como possível biomarcadora de gliomas em amostras clínicas. Nossos resultados sugerem que rSAA afetou a atividade das MMP-2 e -9 e a produção de ROS em ambos GBM, mas não se mostrou clonogênica. As citocinas IL-6, TNF-α e IL-1β, mas não a hipóxia, foram capazes de induzir os diferentes genes da SAA. A adição de rSAA às culturas celulares estimulou a transrição dos diferentes genes da SAA, sugerindo a ativação de mecanismos intracelulares retroalimentados. Efeitos pró-tumorais da rSAA parecem ser viabilizados via RAGE, enquanto efeitos anti-tumorais parecem ser induzidos via TLR-4. Pela primeira vez mostramos que SAA induz aumento de RAGE. KDs da SAA inibiram proliferação, migração e invasão, sugerindo que SAA seja um produto tumoral importante para a manutenção do fenótipo invasivo de GBM. A adição de SAA exógena reverteu grande parte dos efeitos nas células T98G KD, enquanto células A172 KD responderam parcialmente à rSAA. KDs da SAA sugerem a mesma como mantenedora da morfologia das células de GBM. De maneira inédita mostramos que o gene SAA4 até então descrito como um gene constitutivo de função desconhecida é importante para a proliferação, migração e invasão de GBM. Nós especulamos que os efeitos diferenciados induzidos por rSAA nos GBM estejam associados à natureza multiligante da SAA e às diferenças genéticas dos GBM. Pacientes com GBM apresentaram aumento significativo na transcrição e expressão de SAA1 no tecido tumoral, bem como aumento sérico de SAA. A correlação na expressão de SAA1 com moléculas importantes para progressão tumoral, como CXCR4, CXCR7, CD163 e HIF-1α também a identificam como uma proteína associada à malignidade. / Tumor cells have their proliferation and migration modified by several growth factors, cytokines and inflammatory mediators, such as serum amyloid A (SAA). Previous studies from our group showed the direct effect of SAA on proliferation, migration and invasion of glioblastoma multiforme (GBM) cells, A172 and T98G. In this study we complemented previous migration and invasion data; evaluated SAA as possible inducer of MMP-2, -9 and ROS; performed clonogenic assay to investigate a possible contribution of rSAA in the early stage of tumor development; evaluated the impact of hypoxia on the expression of different genes of SAA; stimulated the cells with classics inducers of hepatic SAA and analyzed the possibility of these different genes to induce SAA in tumor cells; evaluated possible receptors and signaling pathways involved in proliferation, migration and invasion processes. SAA knockdowns (KDs) were made for acute phase (SAA1 and 2) and constitutive protein (SAA4) and evaluated their role in cell proliferation, migration, morfology and invasion. Finally it was investigated SAA as a possible biomarker of glioma grade in clinical samples. Our results suggest that rSAA affects MMP-2 and -9 activity and ROS production in both GBM, but did not affect clonogenicity. IL-6, TNF-α and IL-1β, but not hypoxia, were able to induce SAA expression. rSAA addition to cell cultures stimulated transcription of the three different SAA genes, suggesting the activation of intracellular feedback mechanisms. Pro-tumor effects of rSAA seem to occur via RAGE and anti-tumor effects appear to be induced via TLR-4. This was de first time that induction of RAGE triggered by rSAA was shown. Proliferation, migration and invasion were inhibited in SAA KDs, suggesting that SAA is an important tumoral product for the maintenance of the invasive phenotype of GBM. The addition of exogenous SAA largely reversed the effects on SAA KDs T98G cells, whereas SAA KDs A172 cells partially responded to the rSAA. The findings with SAA KDs suggest that SAA affect cell morphology. Another new contribution from our study was that SAA4, a constitutive gene with unknown function, was important for the proliferation, migration and invasion of GBM and it can be induced by rSAA, IL-6, TNF-α and IL-1β. We speculate that the different effects induced by rSAA in GBM are associated with the affinity of SAA to different receptors and the different genetic backgrounds of GBM. Patients with GBM showed a significant increase in the transcription and expression of SAA1 in tumor tissue as well as increased serum SAA. The correlation between the expression of SAA1 with important molecules for tumor progression, such as CXCR4, CXCR7, CD163 and HIF-1α also identified SAA as a protein associated with malignancy.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

La caractérisation moléculaire multidimensionnelle des tumeurs et des tumeurs gliales en particulier est une étape importante pour l’identification de biomarqueurs (diagnostique, pronostique, théranostique et/ou de prédisposition), pour l’identification de cibles thérapeutiques et pour une meilleure compréhension de l’oncogénèse moléculaire.Nos travaux ont permis de confirmer et de consolider certaines données de la littérature comme par exemple : (i) la valeur pronostique favorable de la codélétion 1p/19q, (ii) la valeur pronostique favorable de la mutation IDH, (iii) le caractère mutuellement exclusive des mutations TP53 et de la codélétion 1p/19q et (iv) la rareté des altérations génétiques du PDGFRA dans les gliomes de bas grade (GDBG). De manière plus originale, nous avons identifié plusieurs sous-groupes génomiques de GDBG pertinents sur le plan clinico-biologique, notamment au sein des GDBG non 1p/19q codélétés : (i) 19q-délété ; (ii) 11p-délété, (iii) 7-gagné, (iv) 19-gagné et (v) inclassés. La perte du bras chromosomique 19q annule la valeur pronostique favorable de la mutation IDH dans les GDBG non 1p/19q codélétés. Nous avons également identifié des mutations géniques originales dans les GDBG (i.e. mutation TEP1 et RNF40) qui renforcent le rôle des télomères et du remodelage de la chromatine au sein des GDBG.Enfin, nous nous sommes concentrés sur la caractérisation des GDBG 11p-délétés qui sont de phénotype majoritairement astrocytaire et de moins bon pronostic. Ces GDBG surexpriment des gènes des cellules immunitaires (les GIM -Glioma infiltrating microglia-, les macrophages de type 1, les macrophages de type 2) et sont infiltrés par des cellules macrophagiques et microgliales. Ce microenvironnement dérégulé peut constituer une cible thérapeutique au sein des GDBG 11p-délétés. En conclusion, nos travaux participent à la dissection clinico-moléculaire des GDBG et à préciser la biologie d’un sous-type de GDBG caractérisé la perte du bras chromosomique 11p. / Multildimensional molecular characterization of tumors and more specifically of gliomas is of pivotal importance to identify: (i) new biomarkers (i.e. diagnostic, prognostic, theranostic or predisposing), (ii) new therapeutic targets and (iii) to improve our understanding of molecular oncogenesis.Our work has confirmed and consolidated previous data published in the literature, for example that: (i) 1p/19q co-deletion is associated with better prognosis, (ii) IDH mutation is associated with better prognosis, (iii) TP53 mutations and 1p/19q codeletion are mutually exclusive and (iv) PDGFRA is rarely altered, at genomic level, in low-grade gliomas (LGG).More originally, we have identified several genomic groups, with clinical and biological relevances, in LGG and more specifically in LGG without 1p/19q co-deletion: (i) 19q-deleted, (ii) 11p-deleted, (iii) 7-gained, (iv) 19-gained and (v) unclassified. Interestingly, 19q deletion abrogates the positive prognostic value of IDH mutation in LGG without 1p/19q codeletion.We have also identified new recurrent somatic gene mutations in LGG (i.e. TEP1 and RNF40 mutations), supporting the critical role of telomeres and chromatin remodelling in LGG.Finally, we have characterized further 11p-deleted LGG that exhibit mostly astrocytic phenotype and poor prognosis. This subgroup includes LGG overexpressing genes of inflammatory/immune cells (GIM -Glioma infiltrating microglia-, M1 macrophages and M2 macrophages) and infiltrated by macrophagic/microglial cells. This peculiar microenvironment detected in 11p-deleted LGG might be used as a therapeutic target. In conclusion, our work participates to characterize clinico-biological portrait of LGG and to describe a singular genomic subgroup of LGG characterized by 11p loss.

Gliomas are brain tumors that present with neurological symptoms including seizures and cognitive deficits. Starting at early stages of tumor development glioma cells diffusely infiltrate brain tissue where they interact with non-neoplastic cells including neurons and can perturb normal brain function. While the clinical consequences of glioma induced cortical dysfunction are well established, the neuronal alterations that underlie cortical dysfunction in glioma are unknown. We hypothesize that glioma cells infiltrate surrounding brain tissue and induce alterations in neurons that may contribute to the neurological symptoms associated with gliomas. Due to intermingling of glioma cells and neurons it has been challenging to isolate and characterize neurons from glioma brain tissue while preserving complex neuronal morphology. To address this issue we developed a new mouse glioma model that allowed us to obtain a neuron specific gene expression profile, otherwise obscured by the predominantly large population of glioma cells within the tumor. In this thesis I use this model to test the hypothesis that infiltrating glioma cells induce phenotypic alterations in neurons that contribute to the neurological symptoms associated with glioma. The Camk2a-Ribotag mouse glioma model enabled us to isolate neuron specific transcripts from glioma brain tissue. The Ribotag mouse has a conditional HA-tagged ribosomal protein (Rpl22) that can be expressed upon Cre-recombination. Camk2a is specifically expressed in excitatory neurons, the Camk2a-Cre mouse induces Cre-recombination in the Ribotag mouse so that Camk2a+ neurons selectively express HA-tagged Rpl22. We used the Camk2a-Ribotag glioma model to isolate neuron specific ribosome bound transcripts to characterize neuronal alterations in glioma. In chapter 2 of this thesis I describe how we developed and characterized the Camk2a-Ribotag mouse glioma model. We first obtained mouse glioma cells that have p53 deletion and overexpress PDGFRa, then we injected these cells in the Camk2a-Ribotag mouse and use this as our glioma model to extract neuron specific ribosome bound transcripts. This method is referred to as translating ribosome affinity purification (TRAP) which is used to obtain cell type specific translational profiles. Using this approach we identified alterations in neuronal gene expression, specifically we show that there is an upregulation of actin binding genes associated with dendritic spine morphology and a downregulation of synaptic genes associated synaptic regulation. We demonstrate that drebrin, an actin binding protein in dendritic spines, is upregulated in tumor brain synaptosomes, we also show a downregulation of dendritic spine density in HA-tagged neurons which suggests that these neuronal alterations contribute to synaptic dysfunction in our glioma model. Dendritic spines are dynamic structures that regulate synaptic function in response to diverse stimuli. mTOR signaling can regulate brain specific functions such as synaptic plasticity. Alterations in mTOR signaling can result in cognitive deficits, epilepsy and brain abnormalities that are associated with neurological disease. We hypothesized that mTOR regulates the neuronal alterations we identified in our glioma model. In chapter 3 of this thesis I describe how we tested this hypothesis by acutely inhibiting mTOR signaling with the ATP competitive inhibitor AZD8055 in the Camk2a-Ribotag mouse glioma model. Using TRAP we show that acute mTOR inhibition reverses many neuron specific alterations that occurs in the glioma infiltrated cortex, actin binding genes that were upregulated in tumor brains were downregulated after mTOR inhibition and synaptic genes that were downregulated in tumor brains were upregulated after mTOR inhibition. These results suggest that key neuron specific alterations are regulated by mTOR signaling in our glioma model. In chapter 4 of this thesis I describe how we used ribosome profiling to identify translational alterations in our Camk2a-Ribotag mouse glioma model. Ribosome profiling in an RNA sequencing based method that is used to measure translation efficiency by calculating the number of ribosomes per transcript. Using this approach we identified an upregulation in the translation of DNA methylation and demethylation gene ontologies. These results suggest that alterations in specific DNA methylation and demethylation gene ontologies are regulated at the level of translation and warrant further analysis of cell type specific translational alterations using ribosome profiling. The work described in this thesis demonstrates 1) use of the Camk2a-Ribotag mouse glioma model for the identification of neuron specific alterations, 2) neuron specific alterations include the upregulation of dendritic spine genes, downregulation of synaptic genes and downregulation of dendritic spine density, 3) acute mTOR inhibition reverses many of these neuronal alterations, 4) ribosome profiling revealed the translational upregulation of epigenetic genes in our mouse glioma model. The findings described in this thesis provide the first characterization of neuron specific transcriptional and translational alterations in glioma infiltrated cortex that and provide new insights into the mechanisms that underlie the devastating neurological symptoms in glioma patients.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Novel strategies for the treatment of malignant gliomas are urgently needed. They are characterised by an inherent resistance to both chemo- and radiotherapeutics resulting in unrelenting tumour progression. While the exact mechanisms of treatment resistance remain undefined, it is now recognized that multiple components within the apoptotic pathway are heavily dysregulated in glioma cells and that the over-expression of anti-apoptotic proteins in patient samples correlates with inferior patient survival. The Inhibitor of Apoptosis Proteins (lAPs) represent the final molecular blockade preventing cellular apoptosis and have been identified as a potential rational therapeutic target in gliomas. The work described herein was focused on the development of novel therapeutic strategies that target the lAPs in malignant gliomas, that are readily translatable to the clinic, and that have the potential to improve patient outcomes. The first series of studies examined the hypothesis that targeting the lAPs in conjunction with other conventional and targeted therapies would overcome treatment resistance, and enhance anti-tumour activity. The novel, small molecule, lAP inhibitor LBW242 was shown to successfully target the lAPs in glioma cells and inhibit their ability to bind to and inactivate caspases. However when tested as a single agent in vitro, no stand alone anti-glioma activity of LBW242 was demonstrated. A screen of the activity of LBW242 in combination other pro-apoptotic compounds led to the discovery that lAP inhibition applied in combination with receptor tyrosine kinase (RTK) inhibition led to enhanced caspase activation and induction of apoptosis with a subsequent synergistic anti-glioma effect. The most profound effect was demonstrated with the specific combination of PDGFR and lAP inhibition both in vitro and in vivo as well as in primary patient derived glioma tumourspheres. While multiple RTKs have previously been validated as rational therapeutic targets, the clinical failure of RTK inhibitors in glioma patients has to date remained unexplained. The results in this thesis provide a novel explanation for the resistance of glioma cells to these targeted therapies, and more importantly offer a clinically tractable strategy of overcoming that resistance and improving patient outcomes. The second series of studies investigated the mechanism of synergy between lAP and RTK inhibition. The results showed that PDGFR inhibition does not stimulate apoptosis in glioma cells by previously described pathways. A screen of the entire apoptotic pathway revealed that treatment with imatinib modulates the expression of the anti-apoptotic protein NOL3/ARC. The results showed that imatinib treatment leads to down-regulation of NOL3 and that this effect is critical to the synergy between lAP and PDGFR inhibition. Further analysis suggested a critical role for NOL3 in gliomagenesis and treatment resistance NOL3 was found to be highly expressed in malignant gliomas and with expression levels that are inversely correlated with patient outcomes. A role for NOL3 has not previously been described in malignant gliomas. Finally, a series of studies were undertaken that tested the use of LBW242 in combination with the standard-of-care therapies of irradiation and temozolomide. In vitro assays demonstrated that LBW242 enhanced the pro-apoptotic activity of radiotherapy, and clonogenic assays showed that the combination therapy led to a synergistic anti-glioma effect in multiple glioma cell lines. Athymic mice bearing established human malignant glioma tumour xenografts treated with LBW242 plus radiation and temozolomide demonstrated a profound and synergistic suppression of tumour growth. Neurosphere assays revealed that the combination of radiation and LBW242 led to a pro-apoptotic effect in highly resistant glioma stem cells with a corresponding inhibition of tumour growth. The results indicate a potentially powerful strategy to enhance the therapeutic activity of standard-of-care therapies in glioma patients. Collectively, the findings of the studies in this thesis contribute to a better understanding of the mechanisms of treatment resistance in malignant gliomas, and demonstrate that the pro-apoptotic and anti-glioma effects of radiotherapy, chemotherapy and specific targeted therapies can be enhanced by the addition of a novel, small molecule lAP inhibitor. These results are readily translatable to clinical trial, and offer the potential for improved treatment outcomes for glioma patients.

Thesis (Ph.D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Additional advisors: Louis B. Nabors, James M. Markert, Donald B. Twieg. Description based on contents viewed July 8, 2009; title from PDF t.p. Includes bibliographical references (p. 73-79).

Made available in DSpace on 2016-05-10T12:03:03Z (GMT). No. of bitstreams: 1 000478477-Texto+Completo-0.pdf: 5818535 bytes, checksum: 5b089589ca820b17117fd0c8e9d1cc90 (MD5) Previous issue date: 2016 / Gliomas represent the most common class of malignant tumors of the central nervous system being the most aggressive, and lethal brain tumors in primary brain tumors. Among the treatments, radiation is one of the most used therapies, but the intrinsic radioresistance of these tumors remains a critical problem in the management of these patients. Currently it is known that the effect of radiation extends beyond the directly cytotoxicity caused in tumor cells. Radiation therapy appears to induce an immunogenic cell death that among the features is ATP release. The ATP can cause cytotoxicity via P2X7 receptor and also acts as a sign of damage activating the immune system. The ATP can be hydrolyzed by enzymes of the purinergic system, among them the ectonucleotidase CD39, to adenosine, which has an opposite effect to ATP, causing immunosuppression. The radiation-induced ATP release and the ability of this nucleotide in modulate immune responses raised the hypothesis about the purinergic signaling participation in the tumor and immune cells response to radiation. Therefore, this study investigated: i) the role of ectonucleotidase CD39/NTPDase1 in the radiation-induced immune response in gliomas, ii) the importance of ATP-P2X7 receptor in the gliomas response to radiotherapy. Using knockout mice for CD39/NTPDase1, we observed that the deletion of this enzyme combined with radiotherapy significantly reduced the immunosuppressive cells Tregs in the tumor and spleen, attenuated the infiltration of myeloid derived suppressor cells caused by radiation and increased CCR7 expression in splenic dendritic cells and macrophages, indicating the presence of freshly mobilized antigen presenting cells available to differentiate in immune-effector cells that sustain a more prolonged antigen-specific T-cell–mediated immune response. Thereby, showing that blocking the activity of CD39/NTPDase1 can control immunosuppressive mechanisms generated by the tumor and promises to improve the radiotherapy response. Furthermore, in this study we observed that radiation actives the P2X7 receptor and by silencing this receptor on the GL261 glioma cell line, we have shown that radiotherapy is less efficient in vivo when compared with mice injected with GL261 WT cells, which constitutively express the P2X7 receptor. We also showed that patients with glioma that overexpress the P2X7 receptor, showed a better response to radiotherapy, revealing the importance of the expression of this receptor on glioma cells as a useful marker to analyze the tumor sensitivity to radiation and a successful radiotherapy response. In summary, our data shed light on the purinergic signaling for modulating the radiotherapy response in gliomas. / Os gliomas representam a classe mais comum de tumores malignos do sistema nervoso central, sendo o tumor cerebral mais agressivo e letal entre os tumores cerebrais primários. Dentre os tratamentos, a radiação é uma das terapias mais utilizadas, porém a radiorresistência intrínseca destes tumores continua a ser um problema crítico na gestão de destes pacientes. Atualmente, sabe-se que o efeito da radiação se estende além da citotoxicidade direta causada nas células tumorais. A radioterapia parece induzir uma morte celular imunogênica, que entre as características está a liberação de ATP. O ATP pode causar citotoxicidade através do receptor P2X7 e também atua como um sinal de dano celular ativando o sistema imune. O ATP pode ser hidrolisado por enzimas do sistema purinérgico, dentre elas a ectonucleotidase CD39/NTPDase1, à adenosina, que tem um efeito aposto ao ATP, causando imunossupressão. A secreção de ATP induzida pela radioterapia e a capacidade deste nucleotídeo em modular a resposta imune, levantou a hipótese da participação da sinalização purinérgica na resposta de células tumorais à radiação e na resposta imune induzida pela radioterapia. Portanto, neste estudo visou-se investigar: i) o papel da ectonucleotidase CD39/NTPDase1 na resposta imune induzida pela radioterapia em gliomas, ii) a importância da via ATP-receptor P2X7 na resposta de gliomas à radioterapia. Através de camundongos knockout para a enzima CD39/NTPDase1, observamos que a deleção desta enzima combinada com a radioterapia reduziu significativamente as células imunossupressoras Tregs no tumor e no baço, atenuou a infiltração de células mieloides supressoras causada pela radiação, e aumentou a expressão de CCR7 em células dentríticas e macrófagos localizados no baço, indicando a presença células apresentadoras de antígeno recémmobilizadas e disponíveis para se diferenciarem em células imunes efetoras que sustentam uma resposta imune mais prolongada mediada por células T antígeno específicas. Deste modo, mostrou-se que o bloqueio da atividade da CD39/NTPDase1 pode controlar mecanismos imunossupressores gerados pelo tumor e promete melhorar a resposta à radioterapia. Além disso, neste estudo observou-se que a radioterapia ativa o receptor P2X7 e através do silenciamento deste receptor na linhagem de glioma GL261, demonstramos que a radioterapia foi pouco eficiente in vivo, quando comparado com camundongos injetados com a GL261 WT, que expressa constitutivamente o receptor P2X7. Também demonstramos que pacientes com glioma que expressaram mais o receptor P2X7, apresentaram uma melhor resposta a radioterapia, revelando a importância da expressão deste receptor em células de glioma como um marcador útil para analisar a sensibilidade tumoral à radioterapia e para uma resposta bem-sucedida à radioterapia. Em suma, nossos dados lançam luz sobre a sinalização purinérgica para a modulação da resposta à radioterapia em gliomas.

Gliomas constitute 36% of all primary brain tumors and 81% of all primary malignant brain tumors. The overall prognosis in patients with gliomas depends mainly on the location and histologic grade of the tumor.<p>The World Health Organization classification of gliomas is the primary basis for guiding therapy and assessing overall prognosis in gliomas. This classification system, based on histological features, often falls short of predicting therapeutic response of individual tumors within the same histological grade. Yet, it still remains the grading method for both research and clinical prospects.<p>Unlike any other organ the brain has multiple protective layers such as the skull that ensure a homeostatic environment. The resulting reduced access to the brain and the absence of plasmatic brain tumor markers bring neuroimaging in a central position in diagnosis and management of brain tumors. Moreover, neuroimaging has evolved from a purely morphologic investigation into a diagnostic tool that allows characterization of particular physical alterations within brain tissue. Understanding the relationship between the physical characteristics of tumor tissue, studied by MR imaging, and biological characteristics of the tumor is therefore important for the appropriate integration of neuroimaging in brain tumor management. The general objective of this work is to define the relationship between physiologybased MR imaging and biological features of glial tumors. Diffusion and perfusionweighted imaging, physiologybased MR techniques provide the data based on physical characteristics of the tissues. Diffusion weighted imaging (DWI) allows the measurement of water molecules diffusivity within the brain tissue by means of apparent diffusion coefficient (ADC) measurements. Perfusion weighted imaging (PWI) is based on changes of MR signal during the passage of contrast material through the intravascular space and allows hemodynamic measurements such as those of cerebral blood volume (CBV)within the brain tissue.<p>Highgrade diffuse gliomas are currently differentiated from low grade diffuse gliomas by increased cellularity with nuclear atypia, mitotic activity, endothelial proliferation and necrosis. Components of the extracellular matrix and angiogenesis constitute some other features of gliomas, which have established links with oncogenic processes that influence the proliferative and infiltrative potentials of these tumors. We have specifically targeted these features in our comparative studies with the working hypothesis that physiologybased MR measurements, obtained in vivo, might provide information that is pertinent in terms of tumor malignancy.<p>We chose to approach the biology of brain tumors in two ways: in vivo, by means of metabolic imaging techniques such as positron emission tomography (PET) and ex vivo, by means of histological and immunohistochemical analyses of tumor specimens.<p>Many studies have investigated the relation between ADC values and cellularity in gliomas. The underlining theory is based on the premise that water diffusivity within the 9 extracellular compartment is inversely related to the content and attenuation of the constituents of the intracellular space. Therefore when cellularity increases, intracellular space volume increases with a relative reduction of the extracellular space, leading to restricted diffusion of water molecules. However other factors may affect the value of ADC in gliomas such as the extracellular matrix which contains various amounts of hydrophilic macromolecules susceptible to change water molecules diffusivity. Hyaluronic acid is one highly hydrophilic component of the extracellular matrix of gliomas and its level of expression changes significantly during the progression to anaplasia in gliomas. Our hypothesis was that hyaluronan may influence ADC values in high and low grade gliomas.<p>We demonstrated a positive correlation between ADC values and the immunohistochemical level of hyaluronan in glial tumors.<p>Previous studies have confirmed the utility of positron emission tomography using C11 Methionine (PETMET) as a prognostic tool in patients with gliomas. Higher MET uptake is associated with greater proliferative potential and a higher level of malignancy in gliomas.<p>The increased aminoacid uptake in gliomas seems to reflect increased transport mediated by aminoacid carriers located in the endothelial cell membrane. Our hypothesis was that CBV measurements, index of tumor vascularity, may be related to tumor aminoacid metabolism.<p>We found a positive correlation between maximum CBV values and maximum MET uptake values in gliomas.<p>A limitation to these preliminary studies was lack of direct correlation between MRbased measurements and histologic and metabolic data. Indeed, glial tumors are known for their remarkable tissue heterogeneity across different grades, within the same grade, and even within a single given tumor. Therefore we used image coregistration and stereotactic biopsies to further assess the relationship between MRbased imaging data and both metabolic and histologic analysis.<p>The second part of our studies was based on measurements at the exact same localization on both MR and PET images where biopsy specimens were performed. We found a local relationship between CBV and MET uptake values. Both measurements correlated with mitotic activity and endothelial proliferation; two features of tumor aggressiveness.<p>In order to quantify tumor cellularity and tumor angiogenesis, we respectively measured cell density and vessel density using immunohistochemical markers to identify vessels. We found a regional relationship between CBV and cell density, as well as vessel density in gliomas whereas no correlation was found regionally between ADC and cell density.<p>We concluded that CBV measurements may be used locally as indices of angiogenesis and cellularity in gliomas; whereas local ADC measurements are more variable and may not be used as a marker of cellularity in heterogeneous tumors such as gliomas. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished