Players in glioma progression: Genomic exploration of glioma cell compartments

Diffuse gliomas are malignant tumors of the central nervous system (CNS), characterized by infiltrative growth, high heterogeneity and poor prognosis. Characterization of the genetic alterations in these tumors is important for correct diagnosis, therapeutic approaches and overall patient survival. Based on genetic mutations, gliobastomas (GMB) can be divided into proneural, classical and mesenchymal subtypes. In this thesis, the potential value of a family of ‘inhibitor of differentiation’ proteins to differentiate between astrocytic and oligodendrocytic gliomas was determined. Glioma tumorigenicity is not only controlled by genetic alterations, interaction between tumor cells and cells of the non-tumor tissue are critical for glioma growth and aggressiveness. Non-tumor cells that are important for GBM progression are immune cells that enter the brain tumor and microglia. Microglia are the local immune cells of the CNS, essential for brain development, and the first cells to detect and respond to damage or danger. In this thesis, microglia from normal and tumor CNS tissue are characterized. Microglia were isolated from the CNS autopsy samples and gliomas, and their gene expression profiles were determined. Human microglia are very similar to mouse microglia but the effect of aging was very different. Microglia in diffuse gliomas are immune-suppressed and tumor-growth supportive, especially in tumors with poor clinical outcome. This data is the first extensive human microglia gene expression profile, from both physiological and pathological conditions. These results provide insight in glioma heterogeneity and the tumor-supportive properties of the non-tumor microenvironment, potentially leading to the identification of new therapeutic targets

Molecular associations of ID4 in diffusely infiltrative astrocytomas

O inibidor da ligação do DNA 4 (ID4) é membro da família hélice-alça-hélice de fatores de transcrição, presente durante o desenvolvimento embrionário Sistema Nervoso Central, e que tem sido associado à mutações no gene TP53 e a ativação de SOX2. Juntamente com outros fatores de transcrição, ID4 está envolvido no processo de tumorigênese dos astrocitomas, contribuindo para a de-diferenciação celular, a proliferação e a quimiorresistência. Nesse estudo, nosso objetivo foi caracterizar o padrão de expressão de ID4 em astrocitomas humanos difusamente infiltrativos de graus II a IV de malignidade, classificados de acordo com a Organização Mundial de Saúde (OMS); correlacionar os níveis de expressão de ID4 com os de SOX2, SOX4, OCT-4 e NANOG, juntamente com o status mutacional de TP53; e correlacionar os resultados com os dados de sobrevida dos pacientes com GBM. PCR quantitativo em tempo real (qRT-PCR) foi realizado em 130 amostras de astrocitomas para obter a expressa relativa de cada gene, mostrando hiperexpressão de todos os fatores de transcrição analisados nas amostras tumorais. Correlação positiva foi encontrada comparando a expressão relativa de ID4 em astrocitomas infiltrativos com a expressão de SOX2 (r=0.50; p<0.005), SOX4 (r=0.43; p<0.005) e OCT-4 (r=0.39; p<0.05). Os resultados da análise mutacional de TP53 permitiu comparações entre os grupos mutado e não-mutado apenas nos casos de astrocitomas de baixo grau (AGII), que demonstraram altos níveis de ID4, SOX2 e SOX4 nos casos mutados (p<0.05). Esse padrão foi mantido em amostras de glioblastoma (GBM) secundário e foi confirmado por imunohistoquímica. A combinação da hiperexpressão de ID4, SOX4 e OCT-4 conferiu uma menor sobrevida (mediana de 6 meses) muito menor aos pacientes de GBM do que a hipoexpressão (mediana de 18 meses). Uma vez que ID4 isoladamente e SOX4 e OCT-4 como um complexo ativam a transcrição de SOX2, é possível que múltiplas vias de ativação de SOX2 prejudiquem o prognóstico dos pacientes de GBM. Esses resultados sugerem uma via comum para esses quatro alvos na tumorigênese dos astrocitomas, com ID4 surgindo como um novo alvo para estudos e terapias futurasInhibitor of DNA Binding 4 (ID4) is a member of the helix-loop-helix ID family of transcription factors, mostly present in the central nervous system during embryonic development, that has been associated with TP53 mutation and activation of SOX2. Along with other transcription factors, ID4 has been implicated in the tumorigenic process of astrocytomas, contributing to cell dedifferentiation, proliferation and chemoresistance. In this study, we aimed to characterize the ID4 expression pattern in human diffusely infiltrative astrocytomas of World Health Organization (WHO) grades II to IV of malignancy (AGII-AGIV); to correlate its expression level to that of SOX2, SOX4, OCT-4 and NANOG, along with TP53 mutational status; and to correlate the results with the clinical end-point of overall survival among glioblastoma patients. Quantitative real time PCR (qRT-PCR) was performed in 130 samples of astrocytomas for relative expression, showing up-regulation of all transcription factors in tumor cases. Positive correlation was found when comparing ID4 relative expression of infiltrative astrocytomas with SOX2 (r=0.50; p<0.005), SOX4 (r=0.43; p<0.005) and OCT-4 (r=0.39; p<0.05). The results from TP53 coding exon analysis allowed comparisons between wild-type and mutated status only in AGII cases, demonstrating significantly higher levels of ID4, SOX2 and SOX4 in mutated cases (p<0.05). This pattern was maintained in secondary GBM and further confirmed by immunohistochemistry. Combined hyperexpression of ID4, SOX4 and OCT-4 conferred a much lower (6 months) median survival than did hypoexpression (18 months). Because both ID4 alone and a complex of SOX4 and OCT-4 activate SOX2 transcription, it is possible that multiple activation pathways of SOX2 impair the prognosis of GBM patients. These results imply a similar pathway for these four targets in astrocytoma tumorigenesis, with ID4 appearing to be a new target for further studies and therapie

Human microglia expression profile and its alterations related to glioma

A micróglia é essencial para a homeostase do Sistema Nervoso Central (SNC), função neuro-imune inata, e exerce papel importante na neurodegeneração, envelhecimento cerebral e tumorigênese. Gliomas difusos são tumores cerebrais primários caracterizados por crescimento infiltrativo e altas taxas de heterogeneidade, o que torna a doença praticamente incurável. Avanços em análises genéticas caracterizaram alterações moleculares relacionadas ao tempo de sobrevida e à resposta clínica desses pacientes, especialmente em glioblastomas (GBM). No entanto, a tumorigenicidade dos gliomas não é controlada unicamente por suas alterações genéticas. As interações entre as células tumorais, a micróglia residente e os macrófagos/monócitos infiltrados desempenham um papel crucial na modulação do crescimento e agressividade do glioma. Neste estudo, analisamos o fenótipo de ativação da micróglia/macrófagos em gliomas, incluindo astrocitomas e oligodendroglimas de diferentes graus de malignidade, apresentamos o perfil de expressão gênica da população pura de micróglia cortical e do tecido cerebral total correspondente. Usando sequenciamento de DNA de alta performance, classificamos as amostras de GBM em Proneural, Clássico e Mesenquimal. Em seguida, avaliamos os status de ativação da micróglia/macrófagos dessas amostras. Apesar do alto grau de heterogeneidade, pudemos observar níveis mais altos dos marcadores mielóides (IBA1, CD11b and CD68) em tumores astrocíticos comparados a tumores de origem oligodendrocítica e ao tecido não-neoplásico. Marcadores de anti-inflamação, como CD163, foram mais abundantes em astrocitomas, bem como em GBMs do subtipo Mesenquimal e Clássico; enquanto que marcadores de pró-inflamação, como IL1-beta, mostraram uma expressão mais heterogênea entre as amostras. Em seguida, micróglia foi isolada de 25 amostras de córtex parietal provenientes de autópsia de indivíduos cognitivamente preservados e foi feito o RNA-seq. Os resultados foram comparados à micróglia de camundongo e a outras células mielóides. Boa parte dos genes expressos pela micróglia humana foram similares àqueles expressos pela micróglia murina, como CX3CR1, P2YR12 e ITGAM. Porém, foram identificados genes de característica imune, abundantemente expressos na micróglia humana e não identificados na micróglia de camundongos, como TLR, Fcy, receptores do tipo SIGLEC, e fatores de transcrição NLRC5 e CIITA. A comparação dos dados de expressão gênica da micróglia com monócitos e macrófagos identificou novos marcadores que distinguem a micróglia humana de outras células mielóides. Nossos dados sobre a micróglia em gliomas sugerem características de imunossupressão e de pró-crescimento em tumores de pior prognóstico, ligado a um fenótipo específico de ativação das células mielóides. Este é o primeiro estudo a identificar o transcriptoma da micróglia humana pura, demonstrando que ela é claramente diferente da micróglia murina e de outras células mielóides. Esses resultados abrem portas para estudos de populações específicas de células mielóides em gliomasMicroglia are essential for central nervous system (CNS) homeostasis and innate neuroimmune function, and play important roles in neurodegeneration, brain aging and tumorigenesis. Diffuse gliomas are primary brain tumors characterized by infiltrative growth and high heterogeneity, which renders the disease mostly incurable. Advances in genetic analysis have characterized molecular alterations leading to impact on patients\' overall survival and clinical outcome, particularly in glioblastoma (GBM). However, glioma tumorigenicity is not controlled uniquely by its genetic alterations. The crosstalk between tumor cells, resident microglia and infiltrating monocytes/macrophages plays a crucial role in modulating glioma growth and aggressiveness. Here, we assess the activation status of microglia/macrophages in gliomas,including astrocytomas and oligodendrogliomas of different grades of malignancy, and present the gene expression profile of pure cortical human microglia and corresponding unsorted brain tissue. Using high-throughput DNA sequencing, we have classified GBM samples in Proneural, classical and mesenchymal. Next, we evaluated the activation status of microglia/macrophages within these samples. Despite the great heterogeneity, we observed higher levels of myeloid markers (IBA1, CD11b and CD68) in astrocytic tumors compared to oligodendrocytic ones and to non-neoplastic (NN) tissue. Anti-inflammation markers, such as CD163, are also more abundant in astrocytomas, as well as in the mesenchymal and classical GBM subtypes, while pro-inflammation markers, such as IL1-beta, show a more widespread expression throughout samples. Next, microglia were isolated from the parietal cortex of 25 autopsy samples of cognitively preserved humans and RNA sequenced. Overall, genes expressed by human microglia are similar to mouse microglia, such as CX3CR1, P2YR12, and ITGAM. Interestingly, a number of immune genes, not identified as mouse microglia signature genes, were abundantly expressed in human microglia, such as TLR, Fcy and SIGLEC receptors and NLRC5 and CIITA transcription factors. Comparison of microglia to monocyte and macrophage expression data underscored the CNS-specific functions of microglia and new markers were identified that distinguish human microglia from other myeloid cells. Our glioma-related data suggests an immune-suppressive and growth supportive characteristic for tumors with worse clinical outcome, linked to an activation profile of myeloid cells. This data is the first comprehensive pure human microglia gene expression profile; human microglia clearly differ from mouse microglia and other myeloid cells. These results will help further studies focusing on pure myeloid cells populations in gliom

Plasmatic membrane toll-like receptor expressions in human astrocytomas

<div><p>Toll-like receptors (TLRs) are the first to identify disturbances in the immune system, recognizing pathogens such as bacteria, fungi, and viruses. Since the inflammation process plays an important role in several diseases, TLRs have been considered potential therapeutic targets, including treatment for cancer. However, TLRs’ role in cancer remains ambiguous. This study aims to analyze the expression levels of plasmatic cell membrane TLRs (TLR1, TLR2, TLR4, TLR5, and TLR6) in human astrocytomas the most prevalent tumors of CNS different grades (II-IV). We demonstrated that TLR expressions were higher in astrocytoma samples compared to non-neoplastic brain tissue. The gene and protein expressions were observed in GBM cell lines U87MG and A172, proving their presence in the tumor cells. Associated expressions between the known heterodimers TLR1-TLR2 were found in all astrocytoma grades. In GBMs, the mesenchymal subtype showed higher levels of TLR expressions in relation to classical and proneural subtypes. A strong association of TLRs with the activation of cell cycle process and signaling through canonical, inflammasome and ripoptosome pathways was observed by <i>in silico</i> analysis, further highlighting TLRs as interesting targets for cancer treatment.</p></div

Differential Expression of <i>ID4</i> and Its Association with <i>TP53</i> Mutation, <i>SOX2</i>, <i>SOX4</i> and <i>OCT-4</i> Expression Levels

<div><p>Inhibitor of DNA Binding 4 (ID4) is a member of the helix-loop-helix ID family of transcription factors, mostly present in the central nervous system during embryonic development, that has been associated with <i>TP53</i> mutation and activation of <i>SOX2</i>. Along with other transcription factors, <i>ID4</i> has been implicated in the tumorigenic process of astrocytomas, contributing to cell dedifferentiation, proliferation and chemoresistance. In this study, we aimed to characterize the <i>ID4</i> expression pattern in human diffusely infiltrative astrocytomas of World Health Organization (WHO) grades II to IV of malignancy (AGII-AGIV); to correlate its expression level to that of <i>SOX2</i>, <i>SOX4</i>, <i>OCT-4</i> and <i>NANOG</i>, along with <i>TP53</i> mutational status; and to correlate the results with the clinical end-point of overall survival among glioblastoma patients. Quantitative real time PCR (qRT-PCR) was performed in 130 samples of astrocytomas for relative expression, showing up-regulation of all transcription factors in tumor cases. Positive correlation was found when comparing <i>ID4</i> relative expression of infiltrative astrocytomas with <i>SOX2</i> (<i>r</i> = 0.50; <i>p</i><0.005), <i>SOX4</i> (<i>r</i> = 0.43; <i>p</i><0.005) and <i>OCT-4</i> (<i>r</i> = 0.39; <i>p</i><0.05). The results from <i>TP53</i> coding exon analysis allowed comparisons between wild-type and mutated status only in AGII cases, demonstrating significantly higher levels of <i>ID4</i>, <i>SOX2</i> and <i>SOX4</i> in mutated cases (<i>p</i><0.05). This pattern was maintained in secondary GBM and further confirmed by immunohistochemistry, suggesting a role for <i>ID4</i>, <i>SOX2</i> and <i>SOX4</i> in early astrocytoma tumorigenesis. Combined hyperexpression of <i>ID4</i>, <i>SOX4</i> and <i>OCT-4</i> conferred a much lower (6 months) median survival than did hypoexpression (18 months). Because both ID4 alone and a complex of SOX4 and OCT-4 activate <i>SOX2</i> transcription, it is possible that multiple activation of <i>SOX2</i> impair the prognosis of GBM patients. These observational results of associated expression of <i>ID4</i> with <i>SOX4</i> and <i>OCT-4</i> may be used as a predictive factor of prognosis upon further confirmation in a larger GBM series.</p> </div

Immunofluorescence of TLR1, TLR2, TLR4, TLR5, and TLR6 in GBM cell lines.

<p>A172 (A) and U87MG (B). TLR1, TLR4, and TLR6 are stained in red, TLR2 and TLR5 in green, and nuclei in blue by DAPI. The presence of all five TLRs was detected in both cell lines. Expression of TLR5 was more intense in A172 compared to U87MG. TLR4 and TLR5 positivity were detected in both tumor lineage cells nuclei. Magnification of 400x.</p

Used primer sequences.

<p>Used primer sequences.</p

Heatmap with major genes of the TLR signaling pathways from the TCGA dataset.

<p>RPKM gene expression levels are normalized by z-scores, and comparatively up-regulated RNA expression values are presented in red and down-regulated values in blue. Mean values are in white. TLRs downstream signaling pathways: canonical, ripoptosome, and inflammasome pathways are activated in mesenchymal GBM subtype. Genes of unrelated pathways were added to show their randomic expression levels, including a microglia marker.</p

<i>TLR1</i>, <i>TLR2</i>, <i>TLR4</i>, <i>TLR</i>5, and <i>TLR</i>6 expression levels in astrocytomas of different malignant grades.

<p>(A) The analyzed samples consisted of 22 non-neoplastic (NN) cases, 26 astrocytoma grade II (AGII) cases, 18 astrocytoma grade III (AGIII) cases, and 96 glioblastoma (GBM) cases. Data are represented by box and whisker plots, with the median represented by the line in the middle of the boxes, and top and bottom boxes represent the first and third quartiles. qRT-PCR values are normalized by three housekeeping genes (<i>HPRT</i>, <i>GUSB</i>, <i>TBP</i>). For statistical analysis, Kruskal-Wallis and Dunn’s tests were applied, wherein (*) <i>p</i> < 0.05 when compared to NN cases and (†) <i>p</i> < 0.05 when compared to AGII (Dunn test), all the genes present <i>p</i><0.01 (Kruskal-Wallis). (B) Correlation between <i>TLR2</i>-<i>TLR1</i>, <i>TLR2</i>-<i>TLR6</i>, <i>TLR2</i>-<i>TLR4</i>, <i>TLR2</i>-<i>TLR5</i>, <i>TLR4</i>-<i>TLR1</i>, <i>TLR4</i>-<i>TLR5</i>, and <i>TLR4</i>-<i>TLR6</i> are demonstrated in GBM cases. Statistical analysis was made by the Spearman-rho correlation, and <i>p</i><0.05 were considered significative.</p