Case report: Regression of Glioblastoma after flavivirus infection.

Glioblastoma is the most frequent and aggressive primary brain cancer. In preclinical studies, Zika virus, a flavivirus that triggers the death of glioblastoma stem-like cells. However, the flavivirus oncolytic activity has not been demonstrated in human patients. Here we report a glioblastoma patient who received the standard of care therapy, including surgical resection, radiotherapy and temozolomide. However, shortly after the tumor mass resection, the patient was clinically diagnosed with a typical arbovirus-like infection, during a Zika virus outbreak in Brazil. Following the infection resolution, the glioblastoma regressed, and no recurrence was observed. This clinical response continues 6 years after the glioblastoma initial diagnosis.

Obstacles to Glioblastoma Treatment Two Decades after Temozolomide.

Glioblastomas are considered the most common and aggressive primary brain tumor in adults, with an average of 15 months' survival rate. The treatment is surgery resection, followed by chemotherapy with temozolomide, and/or radiotherapy. Glioblastoma must have wild-type IDH gene and some characteristics, such as TERT promoter mutation, EGFR gene amplification, microvascular proliferation, among others. Glioblastomas have great heterogeneity at cellular and molecular levels, presenting distinct phenotypes and diversified molecular signatures in each tumor mass, making it difficult to define a specific therapeutic target. It is believed that the main responsibility for the emerge of these distinct patterns lies in subcellular populations of tumor stem cells, capable of tumor initiation and asymmetric division. Studies are now focused on understanding molecular mechanisms of chemoresistance, the tumor microenvironment, due to hypoxic and necrotic areas, cytoskeleton and extracellular matrix remodeling, and in controlling blood brain barrier permeabilization to improve drug delivery. Another promising therapeutic approach is the use of oncolytic viruses that are able to destroy specifically glioblastoma cells, preserving the neural tissue around the tumor. In this review, we summarize the main biological characteristics of glioblastoma and the cutting-edge therapeutic targets that are currently under study for promising new clinical trials.

GBM-Derived Wnt3a Induces M2-Like Phenotype in Microglial Cells Through Wnt/ß-Catenin Signaling.

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/ß-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial ß-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1ß gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/ß-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.

p53 expression and subcellular survivin localization improve the diagnosis and prognosis of patients with diffuse astrocytic tumors.

PURPOSE: Diffuse astrocytic tumors are the most frequently occurring primary central nervous system (CNS) tumors. Their histological sub-classification into diffuse astrocytoma (DA), anaplastic astrocytoma (AA) and glioblastoma (GB) is challenging and the available prognostic factors are limited to age and tumor subtype. Biomarkers that may improve the histological sub-classification and/or serve as prognostic factors are, therefore, urgently needed. The relationship between survivin and p53 in diffuse astrocytic tumor progression and survival is currently unclear. Here, we aimed to assess the relevance of these proteins in the accuracy of the histological sub-classification of these tumors and their respective treatment responses. METHODS: One hundred and thirty-three formalin-fixed paraffin-embedded diffuse astrocytic tumor samples were included. The tumor samples were histologically reviewed and subsequently assessed for p53 and survivin expression and the presence of the IDH R132H mutation by immunohistochemistry. p53 expression levels and survivin subcellular localization patterns were correlated with histological classification and clinical outcome. RESULTS: We found that age and histological subtype were the only features with a prognostic impact. In addition, we found that high p53 expression levels and a nuclear survivin localization correlated with the AA subtype, whereas cytoplasmic survivin localization correlated with the GB subtype. We also found that patients carrying tumors with a high cytoplasmic survivin expression, a high nuclear survivin expression or a high p53 expression, and who did not receive radiotherapy, exhibited poorer short-term and long-term overall survival rates. CONCLUSIONS: Our data suggest that subcellular survivin localization and p53 expression may be employed as valuable tools to improve the accuracy of the histological sub-classification of diffuse astrocytic tumors. Patients whose tumors overexpress these proteins may benefit from radiotherapy, irrespective age and/or histological classification.

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells.

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition.

PURPOSE: Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells. METHODS: The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of ß3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry. RESULTS: We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of ß3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling. CONCLUSION: From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro.

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFß, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.

Gliomas and the vascular fragility of the blood brain barrier.

Astrocytes, members of the glial family, interact through the exchange of soluble factors or by directly contacting neurons and other brain cells, such as microglia and endothelial cells. Astrocytic projections interact with vessels and act as additional elements of the Blood Brain Barrier (BBB). By mechanisms not fully understood, astrocytes can undergo oncogenic transformation and give rise to gliomas. The tumors take advantage of the BBB to ensure survival and continuous growth. A glioma can develop into a very aggressive tumor, the glioblastoma (GBM), characterized by a highly heterogeneous cell population (including tumor stem cells), extensive proliferation and migration. Nevertheless, gliomas can also give rise to slow growing tumors and in both cases, the afflux of blood, via BBB is crucial. Glioma cells migrate to different regions of the brain guided by the extension of blood vessels, colonizing the healthy adjacent tissue. In the clinical context, GBM can lead to tumor-derived seizures, which represent a challenge to patients and clinicians, since drugs used for its treatment must be able to cross the BBB. Uncontrolled and fast growth also leads to the disruption of the chimeric and fragile vessels in the tumor mass resulting in peritumoral edema. Although hormonal therapy is currently used to control the edema, it is not always efficient. In this review we comment the points cited above, considering the importance of the BBB and the concerns that arise when this barrier is affected.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells.

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma.

Glioblastomas (GBMs) are considered to be one of the deadliest human cancers, characterized by a high proliferative rate, aggressive invasiveness and insensitivity to radio- and chemotherapy, as well as a short patient survival period. Moreover, GBMs are among the most vascularized and invasive cancers in humans. Angiogenesis in GBMs is correlated with the grade of malignancy and is inversely correlated with patient survival. One of the first steps in tumor invasions is migration. GBM cells have the ability to infiltrate and disrupt physical barriers such as basement membranes, extracellular matrix and cell junctions. The invasion process includes the overexpression of several members of a super-family of zinc-based proteinases, the Metzincin, in particular a sub-group, metalloproteinases. Another interesting aspect is that, inside the GBM tissue, there are up to 30% of microglia or macrophages. However, little is known about the immune performance and interactions of the microglia with GBMs. These singular properties of GBMs will be described here. A sub-population of cells with stem-like properties may be the source of tumors since, apparently, GBM stem cells (GSCs) are highly resistant to current cancer treatments. These cancer therapies, while killing the majority of tumor cells, ultimately fail in GBM treatment because they do not eliminate GSCs, which survive to regenerate new tumors. Finally, GBM patient prognostic has shown little improvement in decades. In this context, we will discuss how the membrane-acting toxins called cytolysins can be a potential new tool for GBM treatment.