Endothelial cell ferroptosis influences IDH wild-type glioblastoma growth in recurrent glioblastoma multiforme patients

Glioblastomas are known for their poor clinical prognosis, with recurrent tumors often exhibiting greater invasiveness and faster growth rates compared to primary tumors. To understand the intratumoral changes driving this phenomenon, we employed single-cell sequencing to analyze the differences between two pairs of primary and recurrent glioblastomas. Our findings revealed an upregulation of ferroptosis in endothelial cells within recurrent tumors, identified by the significant overexpression of the NOX4 gene. Further analysis indicated that knocking down NOX4 in endothelial cells reduced the activity of the ferroptosis pathway. Utilizing conditioned media from endothelial cells with lower ferroptosis activity, we observed a decrease in the growth rate of glioblastoma cells. These results highlighted the complex role of ferroptosis within tumors and suggested that targeting ferroptosis in the treatment of glioblastomas requires careful consideration of its effects on endothelial cells, as it may otherwise produce counterproductive outcomes.

The 2021 World Health Organization classification of gliomas: an imaging approach / Nova classificação das neoplasias gliais segundo a Organização Mundial da Saúde 2021, com enfoque radiológico

Abstract The purpose of this pictorial essay is to describe the recommendations of the 2021 World Health Organization classification for adult-type and pediatric-type gliomas and to discuss the main modifications in relation to the previous (2016) classification, exemplified by imaging, histological, and molecular findings in nine patients followed at our institutions. In recent years, molecular biomarkers have gained importance in the diagnosis and classification of gliomas, mainly because they have been shown to correlate with the biological behavior and prognosis of such tumors. It is important for neuroradiologists to familiarize themselves with this new classification of central nervous system tumors, so that they can use this knowledge in evaluating and reporting the imaging examinations of patients with glioma.

Resumo O propósito deste ensaio iconográfico é descrever e discutir as novas recomendações da Organização Mundial da Saúde de 2021, referente aos gliomas dos tipos adulto e infantil, e suas principais diferenças com a classificação anterior (2016), exemplificadas com imagens de nove casos de pacientes atendidos nas nossas instituições. Recentemente, há uma crescente significância dos marcadores moleculares no diagnóstico e classificação dos gliomas e tumores do sistema nervoso central, principalmente pela correlação com o comportamento biológico e o prognóstico. É importante que os neurorradiologistas estejam familiarizados com a nova classificação dos tumores do sistema nervoso central para a prática clínica, na avaliação e emissão de laudos e opiniões nas imagens dos pacientes com gliomas.

Neuroimmunomodulatory Properties of Flavonoids and Derivates: A Potential Action as Adjuvants for the Treatment of Glioblastoma.

Glioblastomas (GBMs) are tumors that have a high ability to migrate, invade and proliferate in the healthy tissue, what greatly impairs their treatment. These characteristics are associated with the complex microenvironment, formed by the perivascular niche, which is also composed of several stromal cells including astrocytes, microglia, fibroblasts, pericytes and endothelial cells, supporting tumor progression. Further microglia and macrophages associated with GBMs infiltrate the tumor. These innate immune cells are meant to participate in tumor surveillance and eradication, but they become compromised by GBM cells and exploited in the process. In this review we discuss the context of the GBM microenvironment together with the actions of flavonoids, which have attracted scientific attention due to their pharmacological properties as possible anti-tumor agents. Flavonoids act on a variety of signaling pathways, counteracting the invasion process. Luteolin and rutin inhibit NFκB activation, reducing IL-6 production. Fisetin promotes tumor apoptosis, while inhibiting ADAM expression, reducing invasion. Naringenin reduces tumor invasion by down-regulating metalloproteinases expression. Apigenin and rutin induce apoptosis in C6 cells increasing TNFα, while decreasing IL-10 production, denoting a shift from the immunosuppressive Th2 to the Th1 profile. Overall, flavonoids should be further exploited for glioma therapy.

Expression analysis of progesterone­regulated miRNAs in cells derived from human glioblastoma.

Glioblastomas (GBMs) are the most frequent and malignant type of brain tumor. It has been reported that progesterone (P4) regulates the progression of GBMs by modifying the expression of genes that promote cell proliferation, migration and invasion; however, it is not fully understood how these processes are regulated. It is possible that P4 mediates some of these effects through changes in the microRNA (miRNA) expression profile in GBM cells. The present study investigated the effects of P4 on miRNAs expression profile in U­251MG cells derived from a human GBM. U­251MG cells were treated for 6 h with P4, RU486 (an antagonist of the intracellular progesterone receptor), the combined treatment (P4+RU486) and cyclodextrin (vehicle) and then a miRNA microarray analysis conducted. The expression analysis revealed a set of 190 miRNAs with differential expression in the treatments of P4, RU486 and P4+RU486 in respect to the vehicle and P4 in respect to P4+RU486, of which only 16 were exclusively regulated by P4. The possible mRNA targets of the miRNAs regulated by P4 could participate in the regulation of proliferation, cell cycle progression and cell migration of GBMs. The present study provided insight for understanding epigenetic modifications regulated by sex hormones involved in GBM progression, and for identifying potential therapeutic strategies for these brain tumors.

Enrichment and Transcriptional Characterization of Stem Cells Isolated from Human Glioblastoma Cell Lines.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.

Testosterone Promotes Glioblastoma Cell Proliferation, Migration, and Invasion Through Androgen Receptor Activation.

Glioblastomas (GBM) are the most frequent and aggressive human brain tumors due to their high capacity to migrate and invade normal brain tissue. Epidemiological data report that GBM occur in a greater proportion in men than in women (3:2), suggesting the participation of sex hormones in the development of these tumors. It has been reported an increase in testosterone (T) levels in patients with GBM. In addition, androgen receptor (AR) is overexpressed in human GBM, and genetic silencing of AR, and its pharmacological inhibition, induce GBM cell death in vivo and in vitro. However, the role of T in proliferation, migration and invasion in human GBM cell lines has not been evaluated. We observed that T increased the number of U87, U251, and D54 cells derived from human GBM due to an increase in cell proliferation. This induction was blocked with flutamide, an antagonist of AR. T also induced migration and invasion of GBM cells that flutamide partially blocked. These data suggest that T through AR contributes to the progression of GBM by promoting proliferation, migration, and invasion.

Expression of the Annexin A1 and its correlation with matrix metalloproteinases and the receptor for formylated peptide-2 in diffuse astrocytic tumors.

Astrocytomas represent the majority of cerebral gliomas. Studies show that the anti-inflammatory protein Annexin-A1 (ANXA1) is associated with the tumor invasion process and that its actions can be mediated by the receptor for formylated peptides (FPR). Therefore, we evaluated the expression of ANXA1, the receptor FPR2 and matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in brain astrocytomas. Detection of proteins was performed in sections of diffuse astrocytomas (grade II), anaplastic astrocytomas (grade III) and glioblastomas (GBM, grade IV) and quantifications were made by densitometry. Our analyses showed increased expression of ANXA1 in astrocytomas of all grades, but especially in GBM. The expression of FPR2 is similar to that found for ANXA1, being higher in GBM. Immunostaining for MMPs is also stronger as the degree of malignancy increases, especially with respect to MMP-9. The positive correlation between ANXA1/FPR2 and ANXA1/MMP-9 was observed in all tumors studied. The data indicate the possible action of ANXA1 and FPR2 on the development and progression of astrocytomas, related to increased expression of MMP-9. Thereby, ANXA1 and FPR2 are involved in the biology and malignancy of diffuse astrocytic tumors.

Allopregnanolone Alters the Gene Expression Profile of Human Glioblastoma Cells.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors. In these malignancies, progesterone (P4) promotes proliferation, migration, and invasion. The P4 metabolite allopregnanolone (3α-THP) similarly promotes cell proliferation in the U87 human GBM cell line. Here, we evaluated global changes in gene expression of U87 cells treated with 3α-THP, P4, and the 5α-reductase inhibitor, finasteride (F). 3α-THP modified the expression of 137 genes, while F changed 90. Besides, both steroids regulated the expression of 69 genes. After performing an over-representation analysis of gene ontology terms, we selected 10 genes whose products are cytoskeleton components, transcription factors, and proteins involved in the maintenance of DNA stability and replication to validate their expression changes by RT-qPCR. 3α-THP up-regulated six genes, two of them were also up-regulated by F. Two genes were up-regulated by P4 alone, however, such an effect was blocked by F when cells were treated with both steroids. The remaining genes were regulated by the combined treatments of 3α-THP + F or P4 + F. An in-silico analysis revealed that promoters of the six up-regulated genes by 3α-THP possess cyclic adenosine monophosphate (cAMP) responsive elements along with CCAAT/Enhancer binding protein alpha (CEBPα) binding sites. These findings suggest that P4 and 3α-THP regulate different sets of genes that participate in the growth of GBMs.

Allopregnanolone promotes proliferation and differential gene expression in human glioblastoma cells.

Allopregnanolone (3α-THP) is one of the main reduced progesterone (P4) metabolites that is recognized as a neuroprotective and myelinating agent. 3α-THP also induces proliferation of different neural cells. It has been shown that P4 favors the progression of glioblastomas (GBM), the most common and aggressive primary brain tumors. However, the role of 3α-THP in the growth of GBMs is unknown. Here, we studied the effects of 3α-THP on the number of cells, proliferation and gene expression in U87 cell line derived from a human GBM. 3α-THP (10, 100nM and 1µM) increased the number of U87 cells, and at 10nM exerted a similar increase in both the number of total and proliferative U87 cells as compared with P4 (10nM). Interestingly, finasteride (F; 100nM), an inhibitor of 5α-reductase (5αR), an enzyme necessary to metabolize P4 and produce 3α-THP, blocked the increase in the number of U87 cells induced by P4. By using RT-qPCR, we determined that U87 cells express 5α-R isoenzymes 1 and 2 (5αR1 and 5αR2), being 5αR1 the predominant one in these cells. 3α-THP (10nM) increased the expression of TGFß1, EGFR, VEGF and cyclin D1 genes. P4 increased TGFß1 and EGFR expression, and this effect was blocked by F. These data provide evidence that P4, through its metabolite 3α-THP, can promote in part cell proliferation of human GBM cells by changing the expression of genes involved in tumor progression.

Copy Number Profiling of Brazilian Astrocytomas.

Copy number alterations (CNA) are one of the driving mechanisms of glioma tumorigenesis, and are currently used as important biomarkers in the routine setting. Therefore, we performed CNA profiling of 65 astrocytomas of distinct malignant grades (WHO grade I-IV) of Brazilian origin, using array-CGH and microsatellite instability analysis (MSI), and investigated their correlation with TERT and IDH1 mutational status and clinico-pathological features. Furthermore, in silico analysis using the Oncomine database was performed to validate our findings and extend the findings to gene expression level. We found that the number of genomic alterations increases in accordance with glioma grade. In glioblastomas (GBM), the most common alterations were gene amplifications (PDGFRA, KIT, KDR, EGFR, and MET) and deletions (CDKN2A and PTEN) Log-rank analysis correlated EGFR amplification and/or chr7 gain with better survival of the patients. MSI was observed in 11% of GBMs. A total of 69% of GBMs presented TERT mutation, whereas IDH1 mutation was most frequent in diffuse (85.7%) and anaplastic (100%) astrocytomas. The combination of 1p19q deletion and TERT and IDH1 mutational status separated tumor groups that showed distinct age of diagnosis and outcome. In silico validation pointed to less explored genes that may be worthy of future investigation, such as CDK2, DMRTA1, and MTAP Herein, using an extensive integrated analysis, we indicated potentially important genes, not extensively studied in gliomas, that could be further explored to assess their biological and clinical impact in astrocytomas.

Expression and hormonal regulation of membrane progesterone receptors in human astrocytoma cells.

Progesterone (P) participates in the regulation of the growth of several tumors, including astrocytomas, the most common and malignant human brain tumors. It has been reported that P induces astrocytomas growth in part by its interaction with its intracellular receptors (PR). Recently, it has been reported that membrane progesterone receptors (mPRs) are expressed in ovarian and breast cancer cells, and that P could exert some actions through these receptors, however, it is unknown whether mPRs are expressed in astrocytomas. In this work, U251 and U87 cell lines derived from human astrocytomas grade IV were used to study the expression, localization and hormonal regulation of three mPRs subtypes. Using RT-qPCR and Western blot techniques, we found that mPRα and mPRß are clearly expressed at mRNA and protein levels in astrocytoma cells whereas mPRγ was barely expressed in these cells. Immunofluorescence staining showed that mPRα and mPRß were mainly located in the cell surface. Flow cytometry assays demonstrated that in U251 and U87 cells, mPRß is expressed by a higher percentage of both permeabilized and non-permeabilized cells as compared with mPRα. The percentage of cells expressing mPRγ was very low. P and estradiol (E) (10, 100 nM and 1 µM) decreased mPRα protein content at 12 h. In contrast, both P (100 nM and 1 µM) and E (10 and 100 nM) increased mPRß content. Finally, by in silico analysis, we identified that mPRα, mPRß and mPRγ promoters contain several progesterone and estrogen response elements. Our results indicate that mPRs are expressed in human astrocytoma cells, exhibiting a differential regulation by E and P. These data suggest that some P actions in astrocytoma cells may be mediated by mPRs.

Detecção da proteína PLP2 em glioblastomas / Detection of PLP2 protein in glioblastomas

Os astrocitomas correspondem á grande maioria dos gliomas, e são classificados segundo a organização mundial de saúde ( OMS) em quatro subgrupos: astrocitoma policítico ( grau I), astrocitoma de baixo grau (grau II), astrocitoma anaplástico ( grau III) e glioblastoma multiforme ( grau IV)...

Astrocytomas account of the large majority of gliomas, and are classified according to World Health Organization ( WHO) into four groups: pilocytic astrocytoma ( grade I), low-grade astrocytoma (grade II), anaplastic astrocytoma ( grade III) and glioblastoma multiforme ( grade IV)...

Neuropareidolia: diagnostic clues apropos of visual illusions / Neuropareidolia: pista diagnóstica a partir de uma ilusão visual

Diagnosis in neuroimaging involves the recognition of specific patterns indicative of particular diseases. Pareidolia, the misperception of vague or obscure stimuli being perceived as something clear and distinct, is somewhat beneficial for the physician in the pursuit of diagnostic strategies. Animals may be pareidolically recognized in neuroimages according to the presence of specific diseases. By associating a given radiological aspect with an animal, doctors improve their diagnostic skills and reinforce mnemonic strategies in radiology practice. The most important pareidolical perceptions of animals in neuroimaging are the hummingbird sign in progressive supranuclear palsy, the panda sign in Wilson's disease, the panda sign in sarcoidosis, the butterfly sign in glioblastomas, the butterfly sign in progressive scoliosis and horizontal gaze palsy, the elephant sign in Alzheimer's disease and the eye-of-the-tiger sign in pantothenate kinase-associated neurodegenerative disease.

O diagnóstico em neuroimagem envolve o reconhecimento de padrões específicos indicativos de doenças particulares. Pareidolia, é a perceção equivocada de algo claro e distinto a partir de um estímulo vago e obscuro, por vezes benéfico a quem interpreta exames de imagem na procura do diagnóstico. A este propósito, alguns animais podem pareidolicamente ser reconhecidos em neuroimagens associadas a determinadas doenças específicas, promovendo mais rapidez na habilidade diagnóstica e naturalmente reforçando estratégias mnemônicas individuais na prática do diagnóstico neuroradiológico. Alguns dos sinais de neuroimagens relacionados a percepções pareidolicas de animais são: o sinal do beja-flor na paralisia supra nuclear progressiva; o sinal do panda na doença de Wilson; o sinal do panda na sarcoisdose; o sinal da borboleta no glioblastoma; o sinal da borboleta no escoliose progressiva e paralisia do olhar horizontal; o sinal do elefante na doença de Alzheimeir; e o sinal do olho de tigre na doença degenerativa ligada a pantothenato kinase.

Investigação do efeito do glutamato sobre o conteúdo de rEGF em linhagens celulares derivadas de glioblastomas humanos estabelecidas e caracterizadas

Os gliomas são as neoplasias cerebrais primárias que mais freqüentemente acometem o Sistema Nervoso Central (SNC). Cerca de 50% dos casos de gliomas são neoplasias com fenótipo maligno, incluindo os glioblastomas. Os glioblastomas possuem grande capacidade de expansão e invasão do tecido cerebral adjacente, o que contribui para o seu prognóstico sombrio. O glutamato é o principal neurotransmissor excitatório do SNC, mas sob condições patológicas a ativação excessiva do sistema glutamatérgico provoca danos citotóxicos às células neurais. A toxicidade glutamatérgica está associada a uma série de doenças agudas e crônico-degenerativas que acometem o SNC. Os glioblastomas humanos são capazes de liberar quantidades tóxicas de glutamato sobre os tecidos adjacentes e esta liberação parece favorecer o crescimento e a expansão tumoral. A fim de avaliarmos se a progressão tumoral mediada pelo glutamato é desencadeada por elementos das vias de sinalização, investigamos o efeito do glutamato sobre o conteúdo e expressão do receptor do fator de crescimento epitelial (rEGF). Desta forma, os cultivos primários Gli1, Gli2 e Gli3, assim como a linhagem estabelecida U87MG, foram tratados com doses crescentes de glutamato (5-200 mM) por 48 horas e após o tratamento a viabilidade celular, os conteúdos de rEGF e de Akt foram avaliados. Os cultivos analisados apresentaram suscetibilidades distintas aos efeitos citotóxicos do glutamato, porém em todos os casos as doses efetivas foram muito superiores (valores de ICso de 45mM a 100mM) às concentrações tóxicas descritas para células neurais (valores de ICso de100uM a 1mM). A expressão protéica de rEGF e o conteúdo de Akt fosforilado aumentaram após o tratamento com 5mM de glutamato, sugerindo que a ativação dos receptores de EGF possam exercer função na via de sinalização desencadeada pelo glutamato. Após o término dos experimentos com glutamato dois dos três cultivos primários utilizados (Gli1 e Gli2) se mantiveram viáveis em cultivo por mais de 50 passagens, o que sugere estabilidade das alterações moleculares e a seleção de uma subpopulação especifica de células que pode ser denominada linhagem celular. Assim, em os à caracterização destas duas novas linhagens celulares derivadas de glioblastomas - Gli1 e Gli2. Para tal avaliamos as suas taxas de proliferação, morfologia celular, o perfil de cariótipo de ambas e os conteúdos de rEGF, Hsp70, 6, p53 e MMP2, marcadores relacionados à progressão tumoral. Em suma, os cultivos Gli1 e Gli2 demonstraram alterações cromossômicas e expressão de marcadores compatíveis com as anomalias descritas para a manifestação do fenótipo de glioblastomas. Igualmente, comparamos o crescimento destas linhagens glioblastomas cultivadas como monocamada com o cultivo tridimensional em esferóides multicelulares. Os resultados obtidos revelaram padrões de crescimento e conteúdos dos marcadores diferentes entre as condições de cultivo

The gliomas are the primary brain tumors that more frequntIy accomplished the Central Nervous System (CNS). About 50% of the glioma cases presented malignant phenotype, including glioblastomas. Glioblastoma are tumors with high capacity of proliferation and invasion through the adjacent healthy nervous tissue, which largely contributes for its poor prognosis. The glutamate is the main excitatory neurotransmitter of the CNS, but under pathological conditions the extreme activation of the glutamatergic system induces cytotoxic damages to the neural cells. The glutamatergic citotoxicity is associated with severaI acute and chronic-degenerative diseases of the CNS. Human glioblastomas are capable of release toxic amounts of glutamate on adjacent brain tissue and this release seems to favor tumoral growth and expansion. In order to evaluate if the tumoral progression mediated by glutamato is a product of the activation of elements 6f1he signaling pathways, we investigated the glutamate effect on the content and expression of EGFi . For this reason, the primary culture cells Gli1, Gli2 and Gli3, as well as the established cell line U-87MG, were treated with increasing doses of glutamate (5-200 mM) for 48 hours and after the treatment the cell viability, EGFr and Akt contents were evaluated. The studied cells presented distinct susceptibilities to the cytotoxic effects of glutamate, however in all the cell cultures the toxicity was revealed only with very high glutamate doses (values of ICso range from 45mM to 100mM) when compared to the described toxic concentrations for neural cells (ICso values range from 100uM to 1 mM). The EGFr protein expression and the phosphor-Akt content increased after the treatment with 5mM of glutamate, suggesting that the activation of the EGF receptors can have a role in the glutamate signaling pathways. After the conclusion of these set of experiments, twoof out three tested primary cultures remained viable in cell culture for more than 50 passages, which suggests stability of the molecular profile and the selection of a specific ce11 subpopulation that can be named by 0011 line. Thus, in order to characterize these two new glioblastoma cell lines (Gli1 and Gli2), we carry out a series of experiments. The experiments had included the evaluation of the cellular growth rate, the cellular morphology, the kariotype profile and the identification of molecular markers related to tumor progression (EGF, Hsp70, Ki76, p53 e MMP2). 80th glioblastoma cell lines had demonstrated chromosomic alterations and expression of molecular markers similar to those described for glioblastoma phenotype. Equally, we compared the growth of these cells in monolayer cultures to three-dimensional multicellular spheroids cultures. Accordingly the results demonstrated different standarts of growth and molecular markers between the culture conditions

Glioblastoma de células gigantes: Reporte de un caso / Giant cell glioblastoma: Case Report

Los glioblastomas multiformes (OMS grado IV) son las neoplasias más frecuentes y malignas del sistema nervioso central, una variante histológica de éstos, denominada, de células gigantes representa aproximadamente 1% de todos los tumores cerebrales y 5% de todos los glioblastomas. Presentamos el caso de un paciente de sexo femenino a quien se le diagnosticó y trató un glioblastoma de células gigantes de localización intra y paraventricular derecho. Se enfatiza la importancia de las características histológicas de este tumor en relación a su pronóstico.

Glioblastomas (WorldHealth Organization, (WHO), grade IV) are the most frequent and malignant neoplasms of the human nervous system, Giant cells glioblastomas, a subtype of these, account for less than 1% of all brain toumors and up to 5% of glioblastomas. We present the case of a female who was diagnosed and treated for a right intra and paraventricular giant cell glioblastoma. We enfatize the importance of histological features of this toumor related to its prognosis.