Reproducibility of the NanoString 22-gene molecular subgroup assay for improved prognostic prediction of medulloblastoma.

Medulloblastoma is the most frequent malignant brain tumor in children. Four medulloblastoma molecular subgroups, MBSHH , MBWNT , MBGRP3 and MBGRP4 , have been identified by integrated high-throughput platforms. Recently, a 22-gene panel NanoString-based assay was developed for medulloblastoma molecular subgrouping, but the robustness of this assay has not been widely evaluated. Mutations in the gene for human telomerase reverse transcriptase (hTERT) have been found in medulloblastomas and are associated with distinct molecular subtypes. This study aimed to implement the 22-gene panel in a Brazilian context, and to associate the molecular profile with patients' clinical-pathological features. Formalin-fixed, paraffin-embedded (FFPE) medulloblastoma samples (n = 104) from three Brazilian centers were evaluated. Expression profiling of the 22-gene panel was performed by NanoString and a Canadian series (n = 240) was applied for training phase. hTERT mutations were analyzed by PCR followed by direct Sanger sequencing and the molecular profile was associated with patients' clinicopathological features. Overall, 65% of the patients were male, average age at diagnosis was 18 years and 7% of the patients presented metastasis at diagnosis. The molecular classification was attained in 100% of the cases, with the following frequencies: MBSHH (n = 51), MBWNT (n = 19), MBGRP4 (n = 19) and MBGRP3 (n = 15). The MBSHH and MBGRP3 subgroups were associated with older and younger patients, respectively. The MBGRP4 subgroup exhibited the lowest 5-year cancer-specific overall survival (OS), yet in the multivariate analysis, only metastasis at diagnosis and surgical resection were associated with OS. hTERT mutations were detected in 29% of the cases and were associated with older patients, increased hTERT expression and MBSHH subgroup. The 22-gene panel provides a reproducible assay for molecular subgrouping of medulloblastoma FFPE samples in a routine setting and is well-suited for future clinical trials.

Quality of life and physical limitations in primary brain tumor patients.

OBJECTIVE: To correlate quality of life (QoL) and physical limitations in histological proven primary brain tumor patients using a battery of generic, disease-specific and symptom questionnaires. METHODS: Thirty patients with primary brain tumors were selected from a neuro-oncology outpatient clinic. The FACT-Br Functional Assessment of Cancer Therapy-Brain (specific quality of life questionnaire for patients with brain tumors), SF-36 (generic quality of life questionnaire), HADS (anxiety and depression), and Barthel Index (functionality scale) were answered by the patients at the same interview. RESULTS: The Barthel index did not demonstrate correlation with any subscale of the FACT-Br questionnaire. The HADS had a negative correlation with all FACT-Br subscales and its total score. The SF-36 had a significant weak to moderate correlation with the FACT-Br questionnaire. CONCLUSION: Considering that the FACT-Br is a quick specific questionnaire, it can be a valuable and simple option in evaluating QoL in brain tumor patients with good functional capacity.

ASPM gene expression in medulloblastoma.

PURPOSE: Medulloblastomas are the most common malignant tumors of the central nervous system in childhood. The incidence is about 19-20% between children younger than 16 years old with peak incidence between 4 and 7 years. Despite its sensibility to no specific therapeutic means like chemotherapy and radiotherapy, the treatment is very aggressive and frequently results in regression, growth deficit, and endocrine dysfunction. From this point of view, new treatment approaches are needed such as molecular targeted therapies. Studies in glioblastoma demonstrated that ASPM gene was overexpressed when compared to normal brain and ASPM inhibition by siRNA-mediated inhibits tumor cell proliferation and neural stem cell proliferation, supporting ASPM gene as a potential molecular target in glioblastoma. The aim of this work was to evaluate ASPM expression in medulloblastoma fragment samples, and to compare the results with the patient clinical features. METHODS: Analysis of gene expression was performed by quantitative PCR real time using SYBR Green system in tumor samples from 37 children. The t test was used to analyze the gene expression, and Mann-Whitney test was performed to analyze the relationship between gene expressions and clinical characteristics. Kaplan-Meier test evaluated curve survival. RESULTS: All samples overexpressed ASPM gene more than 40-fold. However, we did not find any association between the overexpressed samples and the clinical parameters. CONCLUSION: ASPM overexpression may modify the ability of stem cells to differentiate during the development of the central nervous system, contributing to the development of medulloblastoma, a tumor of embryonic origin from cerebellar progenitor cells.

Expression of GNAS, TP53, and PTEN Improves the Patient Prognostication in Sonic Hedgehog (SHH) Medulloblastoma Subgroup.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is currently classified in four main molecular subgroups with different clinical outcomes: sonic hedgehog, wingless, group 3, and group 4 (MBSHH, MBWNT, MBGRP3, or MBGRP4). Presently, a 22-gene expression panel has been efficiently applied for molecular subgrouping using nCounter technology. In this study, formalin-fixed, paraffin-embedded samples from 164 Brazilian medulloblastomas were evaluated, applying the 22-gene panel, and subclassified into the low and high expression of nine key medulloblastoma-related genes. In addition, TP53 mutation status was assessed using TruSight Tumor 15 Panel, and its correlation with expression and prognostic impact was evaluated. Samples from 149 of 164 patients (90%) were classified into MBSHH (47.7%), MBWNT (16.1%), MBGRP3 (15.4%), and MBGRP4 (20.8%). GNAS presented the highest expression levels, with higher expression in MBSHH. TP53, MYCN, SOX2, and MET were also up-regulated in MBSHH, whereas PTEN was up-regulated in MBGRP4. GNAS, TP53, and PTEN low expression was associated with the unfavorable patient outcome only for MBSHH (P = 0.04, P = 0.01, and P = 0.02, respectively). TP53 mutations were detected in 28.57% of MBSHH cases and exhibited association with lower expression and worse clinical outcome, although not statistically significant. The 22-gene panel for molecular classification of medulloblastoma associated with the expression of GNAS, TP53, and PTEN improves the patient prognostication in MBSHH subgroup and can be easily incorporated in the 22-gene panel without any additional costs.

Lack of KBTBD4 Mutations in Molecularly Classified Brazilian Medulloblastomas.

Medulloblastoma is the most frequent malignant brain tumor in children, representing 20% of all childhood brain tumors. Currently, medulloblastomas are molecularly classified in 4 subgroups that are associated with distinctive clinicopathological features. KBTBD4 mutations were recently described in a subset of MBGRP3 and MBGRP4 medulloblastomas subgroups. However, no other studies reported KBTBD4 mutations in medulloblastomas. Thus, our aim was to investigate KBTBD4 mutations in a Brazilian series of medulloblastoma. We evaluated 128 medulloblastoma patients molecularly classified from 4 Brazilian reference centers. DNA from formalin-fixed, paraffin-embedded samples was screened for KBTBD4 hotspot mutations by Sanger sequencing. Most of the patients were male, average age was 16.5 years old and average overall survival was 55.9 months. The predominant histological subtype was the classic subtype, followed by nodular/desmoplastic, and the predominant medulloblastoma molecular subtype was the MBSHH subgroup (46%), followed by MBGRP3 and MBGRP4 (19%/each), and MBWNT (16%). Among the 128 samples, 111 were successfully sequenced. No KBTBD4 mutations were identified in 111 samples. Our findings suggest that KBTBD4 mutations are uncommon in Brazilian MBGRP3 and MBGRP4 medulloblastomas subgroups. Further studies in a larger series of MBGRP3 and MBGRP4 medulloblastomas are warranted to better assess role of KBTBD4 mutations.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X.

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo.

BACKGROUND: Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. METHODS/RESULTS: We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. CONCLUSIONS: Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology.

BACKGROUND: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. RESULTS: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. CONCLUSIONS: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. METHODS: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Performance of adjuvant treatment correlates with survival in reoperated glioblastomas.

OBJECTIVE: To analyze cases of recurrent glioblastoma subjected to reoperation at a Brazilian public healthcare service. METHODS: A total of 39 patients subjected to reoperation for recurrent glioblastoma at the Department of Neurosurgery, São Paulo Hospital, Federal University of São Paulo, from January 2000 to December 2013 were retrospectively analyzed. RESULTS: The median overall survival was 20 months (95% confidence interval - CI = 14.9-25.2), and the median survival after reoperation was 9.1 months (95%CI: 2.8-15.4). The performance of adjuvant treatment after the first operation was the single factor associated with overall survival on multivariate analysis (relative risk - RR = 0.3; 95%CI = 0.2-0.7); p = 0.005). CONCLUSION: The length of survival of patients subjected to reoperation for glioblastoma at a Brazilian public healthcare service was similar to the length reported in the literature. Reoperation should be considered as a therapeutic option for selected patients.

Mesenchymal stem cell-like properties of CD133+ glioblastoma initiating cells.

Glioblastoma is composed of dividing tumor cells, stromal cells and tumor initiating CD133+ cells. Recent reports have discussed the origin of the glioblastoma CD133+ cells and their function in the tumor microenvironment. The present work sought to investigate the multipotent and mesenchymal properties of primary highly purified human CD133+ glioblastoma-initiating cells. To accomplish this aim, we used the following approaches: i) generation of tumor subspheres of CD133+ selected cells from primary cell cultures of glioblastoma; ii) analysis of the expression of pluripotency stem cell markers and mesenchymal stem cell (MSC) markers in the CD133+ glioblastoma-initiating cells; iii) side-by-side ultrastructural characterization of the CD133+ glioblastoma cells, MSC and CD133+ hematopoietic stem cells isolated from human umbilical cord blood (UCB); iv) assessment of adipogenic differentiation of CD133+ glioblastoma cells to test their MSC-like in vitro differentiation ability; and v) use of an orthotopic glioblastoma xenograft model in the absence of immune suppression. We found that the CD133+ glioblastoma cells expressed both the pluripotency stem cell markers (Nanog, Mush-1 and SSEA-3) and MSC markers. In addition, the CD133+ cells were able to differentiate into adipocyte-like cells. Transmission electron microscopy (TEM) demonstrated that the CD133+ glioblastoma-initiating cells had ultrastructural features similar to those of undifferentiated MSCs. In addition, when administered in vivo to non-immunocompromised animals, the CD133+ cells were also able to mimic the phenotype of the original patient's tumor. In summary, we showed that the CD133+ glioblastoma cells express molecular signatures of MSCs, neural stem cells and pluripotent stem cells, thus possibly enabling differentiation into both neural and mesodermal cell types.

MR cerebral blood volume maps correlated with vascular endothelial growth factor expression and tumor grade in nonenhancing gliomas.

BACKGROUND AND PURPOSE: Relative cerebral blood volume (rCBV) measurements derived from perfusion-weighted imaging (PWI) may be useful to evaluate angiogenesis and preoperatively estimate the grade of a glioma. We hypothesized that rCBV is correlated with vascular endothelial growth factor (VEGF) expression as marker of the angiogenic stimulus in presumed supratentorial low-grade gliomas (LGGs). METHODS: From February 2001 to February 2004, we examined 20 adults (16 men, four women; mean age 36 years; range, 23-60 years) with suspected (nonenhancing) supratentorial LGG on conventional MR imaging. Preoperative MR imaging used a dynamic first-pass gadolinium-enhanced, spin-echo echo-planar PWI. In heterogeneous tumors, we performed stereotactic biopsy in the high-perfusion areas before surgical resection. Semiquantitative grading of VEGF immunoreactivity was applied. RESULTS: Nine patients had diffuse astrocytomas (World Health Organization grade II), and 11 had other LGG and anaplastic gliomas. In patients with heterogeneous tumors on PWI, the high-rCBV focus had areas of oligodendroglioma or anaplastic astrocytoma on stereotactic biopsy, whereas the surgical specimens were predominantly astrocytomas. Anaplastic gliomas had high rCBV ratios and positive VEGF immunoreactivity. Diffuse astrocytomas had negative VEGF expression and mean rCBV values significantly lower than those of the other two groups. Three diffuse astrocytomas had positive VEGF immunoreactivity and high rCBV values. CONCLUSION: Our results confirmed the correlation among rCBV measurements, VEGF expression, and histopathologic grade in nonenhancing gliomas. PWI may add useful data to the preoperative assessment of nonenhancing gliomas. Its contribution in predicting tumor behavior and patient prognosis remains to be determined.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Galectin-3 as an immunohistochemical tool to distinguish pilocytic astrocytomas from diffuse astrocytomas, and glioblastomas from anaplastic oligodendrogliomas.

The distinction of astrocytomas and oligodendrogliomas, mainly pilocytic astrocytomas (PILOs) from infiltrating astrocytomas and oligodendrogliomas (ODs), and high-grade oligodendrogliomas from glioblastomas (GBMs), poses a serious clinical problem. There is no useful immunohistochemical (IHC) marker to differentiate these gliomas, and sometimes the differential diagnosis between them is arbitrary. We identified galectin-3 (Gal-3) as a possible tool to differentiate them based on gene expression profiles of GBMs. We confirmed the differential expression in 45 surgical samples (thirteen GBMs; seven PILOs; 5 grade II ODs; 5 anaplastic oligodendrogliomas [AODs], including 2 Oligo-astrocytomas; 8 diffuse astrocytomas [ASTs], and 7 non-neoplastic samples) by quantification of Gal-3 gene expression by real-time quantitative PCR (rt-PCR). Higher expression of Gal-3 was observed in GBMs and PILOs than in OD, AODs and ASTs. The IHC expression of Gal-3 was evaluated in 90 specimens (fifteen PlLOs, fourteen ASTs, 10 anaplastic astrocytomas, fifteen GBMs, eleven ODs, fifteen AODs, and 10 dysembryoplastic neuroepithelial tumors). The mean labeling score for Gal-3 determined according to the percentage of labeled cells in the tumor bulk was significantly different in GBMs versus AODs and in PILOs versus ASTs. Hence, Gal-3 is differentially expressed in central nervous system tumors, making IHC detection of Gal-3 a useful tool in distinguishing between these gliomas.

Stereotactic biopsy guidance in adults with supratentorial nonenhancing gliomas: role of perfusion-weighted magnetic resonance imaging.

OBJECT: The. diagnosis of low-grade glioma (LGG) cannot be based exclusively on conventional magnetic resonance (MR) imaging studies, and target selection for stereotactic biopsy is a crucial issue given the high risk of sampling errors. The authors hypothesized that perfusion-weighted imaging could provide information on the microcirculation in presumed supratentorial LGGs. METHODS: All adult patients with suspected (nonenhancing) supratentorial LGGs on conventional MR imaging between February 2001 and February 2004 were included in this study. Preoperative MR imaging was performed using a dynamic first-pass gadopentate dimeglumine-enhanced spin echo-echo planar perfusion-weighted sequence, and the tumors' relative cerebral blood volume (rCBV) measurements were expressed in relation to the values observed in contralateral white matter. In patients with heterogeneous tumors a stereotactic biopsy was performed in the higher perfusion areas before resection. Among 21 patients (16 men and five women with a mean age of 36 years, range 23-60 years), 10 had diffuse astrocytomas (World Health Organization Grade II) and 11 had other LGGs and anaplastic gliomas. On perfusion-weighted images demonstrating heterogeneous tumors, areas of higher rCBV focus were found to be oligodendrogliomas or anaplastic astrocytomas on stereotactic biopsy; during tumor resection, however, specimens were characterized predominantly as astrocytomas. Diffuse astrocytomas were associated with significantly lower mean rCBV values compared with those in the other two lesion groups (p < 0.01). The rCBV ratio cutoff value that permitted better discrimination between diffuse astrocytomas and the other lesion groups was 1.2 (80% sensitivity and 100% specificity). CONCLUSIONS: Perfusion-weighted imaging is a feasible method of reducing the sampling error in the histopathological diagnosis of a presumed LGG, particularly by improving the selection of targets for stereotactic biopsy.

Neurologic complications after heart transplantation.

OBJECTIVE: Neurologic complications are known as important cause of morbidity and mortality in orthotopic heart transplantation. Our aim was to identify the frequency and outcome of neurologic complications after heart transplantation in a prospective observational study. METHOD: From September 93 to September 99, as part of our routine heart transplantation protocol all patients with end-stage cardiac failure were evaluated by the same neurologist before and at the time of any neurologic event (symptom or complaint) after transplantation. RESULTS: Out of 120 candidates evaluated, 62 were successfully transplanted (53 male; median age 45.5 years, median follow-up 26.8 months). Fifteen patients (24%) had ischemic, 22 (35%) idiopathic, 24 (39%) Chagas' disease and 1 (2%) had congenital cardiomyopathy. Neurologic complications occurred in 19 patients (31%): tremor, severe headache, transient encephalopathy and seizures related to drug toxicity or metabolic changes in 13; peripheral neuropathy in 4; and spinal cord compression in two (metastatic prostate cancer and epidural abscess). No symptomatic postoperative stroke was observed. CONCLUSIONS: Although frequent, neurologic complications were seldom related to persistent neurologic disability or death. Most of the complications resulted from immunosuppression, however, CNS infection was rare. The absence of symptomatic stroke in our series may be related to the lower frequency of ischemic cardiomyopathy.

[Arteriovenous malformation-glioma association: study of four cases]. / Associação de malforma o vascular e gliomas: estudo de quatro casos.

We reviewed the clinical presentation, imaging and histopathologic findings in 4 patients with the diagnosis of arteriovenous malformation associated with glioma that were operated on from 1991 to 2000 in our institution. Four patients (2 males; age between 15 and 52 years) presented with progressive headache with clinical evidence of intracranial hypertension (in 3) and partial seizures (in 1). CT scan showed a brain tumor without any detectable pathologic vessels. Histologic examination revealed astrocytic tumors associated with arteriovenous malformation. No patient presented the vascular component intermixed with the tumor. The arteriovenous-glioma association is rare and must be identified by a clear demarcation between the malformation and the tumor.

In vitro Analysis of Neurospheres Derived from Glioblastoma Primary Culture: A Novel Methodology Paradigm.

Glioblastomas are the most lethal primary brain tumor that frequently relapse or progress as focal masses after radiation, suggesting that a fraction of tumor cells are responsible for the tumor regrowth. The identification of a brain tumor cell subpopulation with potent tumorigenic activity supports the cancer stem cell hypothesis in solid tumors. The goal of this study is to determine a methodology for the establishment of primary human glioblastoma cell lines. Our aim is achieved by taking the following approaches: (i) the establishment of primary glioblastoma cell culture; (ii) isolation of neurospheres derived from glioblastoma primary cultures; (iii) selection of CD133 cells from neurospheres, (iv) formation of subspheres in the CD133-positive population, (v) study of the expression level of GFAP, CD133, Nestin, Nanog, CD34, Sox2, CD44, and CD90 markers on tumor subspheres. Hence, we described a successful method for isolation of CD133-positive cell population and establishment of glioblastoma neurospheres from this primary culture, which are more robust than the ones derived straight from the tumor. Pointed out that the neurospheres derived from glioblastoma primary culture showed 29% more cells expressing CD133 then the ones straight tumor-derived, denoting a higher concentration of CD133-positive cells in the neurospheres derived from glioblastoma primary culture. These CD133-positive fractions were able to further generate subspheres. The subspheres derived from glioblastoma primary culture presented a well-defined morphology while the ones derived from the fresh tumor were sparce and less robust. And the negative fraction of CD133 cells was unable to generate subspheres. The tumor subspheres expressed GFAP, CD133, Nestin, Nanog, CD44, and CD90. Also, the present study describes an optimization of neurospheres/subspheres isolation from glioblastoma primary culture by selection of CD133-positive adherent stem cell.

Fas, FasL, and cleaved caspases 8 and 3 in glioblastomas: a tissue microarray-based study.

This investigation analyzed the immunoexpression of FasL, Fas, cleaved caspase-8, and cleaved caspase-3 in glioblastomas. Formalin-fixed and paraffin-embedded glioblastoma tissues and control brain tissues from 97 patients were analyzed by tissue microarrays and immunohistochemistry. Patients with glioblastomas that were negative or weakly stained (<50% of cells positive) for cleaved caspase-8 had worse cancer-specific overall survival (median=8.5 months) than did patients with tumors that highly expressed cleaved caspase-8 (median=11.7 months; P=0.0325), independent of clinical variables. There was no association of other markers with survival, treatment, sex, age, tumor size, and primary site. Among the tumors, there were reasonable to good positive correlations between the expression of FasL and Fas (r=0.47) and between Fas and cleaved caspase-8 (r=0.41), and there were poor positive correlations between Fas and cleaved caspase-3 (r=0.26), FasL and cleaved caspase-8 (r=0.22), and cleaved caspase-8 and -3 (r=0.31). Our results suggest that Fas-Fas-ligand signal transduction could be inhibited, especially at the stage of caspase-8 activation, thereby establishing a major mechanism for evasion of apoptosis by these tumors. The absence or low expression of cleaved caspase-8 in the tumors was a negative prognostic indicator for patient survival.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo

Background Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. Methods/results We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. Conclusions Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.