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Background: Glioblastoma multiforme (GBM) is an incurable tumor, with a median survival rate of only 14–15 months. Along with heterogeneity and unregulated growth, a central matter in dealing with GBMs is cell invasiveness. Thus, improving prognosis requires finding new agents to inhibit key multiple pathways, even simultaneously. A subset of GBM stem-like cells (GSCs) may account for tumorigenicity, representing, through their pathways, the proper cellular target in the therapeutics of glioblastomas. GSCs cells are routinely enriched and expanded due to continuous exposure to specific growth factors, which might alter some of their intrinsic characteristic and hide therapeutically relevant traits. Methods: By removing exogenous growth factors stimulation, here we isolated and characterized a subset of GSCs with a “mitogen-independent” phenotype (I-GSCs) from patient’s tumor specimens. Differential side-by-side comparative functional and molecular analyses were performed either in vitro or in vivo on these cells versus their classical growth factor (GF)-dependent counterpart (D-GSCs) as well as their tissue of origin. This was performed to pinpoint the inherent GSCs’ critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential. Transcriptomic fingerprints were pointed out by ANOVA with Benjamini-Hochberg False Discovery Rate (FDR) and association of copy number alterations or somatic mutations was determined by comparing each subgroup with a two-tailed Fisher’s exact test. The combined effects of interacting in vitro and in vivo with two emerging GSCs’ key regulators, such as Wnt5a and EphA2, were then predicted under in vivo experimental settings that are conducive to clinical applications. In vivo comparisons were carried out in mouse-human xenografts GBM model by a hierarchical linear model for repeated measurements and Dunnett’s multiple comparison test with the distribution of survival compared by Kaplan–Meier method. R

Introduction: Posterior fossa syndrome (PFS) is a set of debilitating complications that can occur after surgery for posterior fossa tumors. This study aimed to assess the preoperative radiological and surgical risk factors for the onset of PFS in a histologically homogeneous population of children with medulloblastoma and compare it to a similar population of young adults. Methods: Included patients underwent posterior fossa surgery for medulloblastoma at 11 Italian neurosurgical wards (2003–2019) and were referred to Fondazione IRCCS Istituto Nazionale dei Tumori in Milan (INT) for postoperative treatments. We collected patients’ pre- and post-operative clinical, surgical and radiological data from the INT charts. To compare the distribution of variables, we used the Mann–Whitney and Fisher tests for continuous and categorical variables, respectively. Results: 136 patients (109 children and 27 young adults) were included in the study. Among children, 29 (27%) developed PFS, and all of them had tumors at midline site with invasion of the fourth ventricle. Radiological evidence of involvement of the right superior (39% versus 12%; p = 0.011) or middle cerebellar peduncles (52% versus 18%; p = 0.002) seemed more common in children who developed PFS. Young adults showed an expected lower incidence of PFS (4 out of 27; 15%), that may be due to anatomical, physiological and oncological elements. Conclusions: This study confirmed some factors known to be associated with PFS onset and shed light on other debated issues. Our findings enhance an already hypothesized role of cerebellar language lateralization. The analysis of a population of young adults may shed more light on the often-neglected existence of PFS in non-pediatric patients.

Background: The aim of this study was to achieve a consensus on the minimum set of outcome measures and predictors to be used in the neurosurgical practice and on the timing of outcome assessment. Methods: A consensus building approach was employed. All neurosurgical departments in Lombardy (Italy) were invited to participate by the Carlo Besta Neurologic Institute IRCCS Foundation. Three workshops were organized during which a multidisciplinary group called Neurosurgical Outcome Network (NEON) was created and the methodology to select outcome measures, predictors, and timing of outcome assessment was established. Eight working groups were created for the different neurosurgical diseases (neuro-oncological, skull base, vascular, traumatic, spinal, peripheral nervous system, malformation, functional) and 8 workshops were organized to identify the outcome measures and predictors specific for each of the neurosurgical diseases based on the experts' clinical practice and the existing literature. Results: A total of 20 neurosurgical departments participated in this study. Specific outcome measures, predictors and the timing of outcome assessment were identified for each of the 8 neurosurgical diseases. Moreover, a list of variables common to all pathologies were identified by the NEON group as further data to be collected. Conclusions: A consensus on the minimum set of outcome measures and predictors and the timing of outcome assessments for 8 neurosurgical diseases was achieved by a group of neurosurgeons of the Lombardy region, called NEON. These sets could be used in future studies for a more homogeneous data collection and as a starting point to reach further agreement also at national and international level.

Background: In the aftermath of COVID-19 outbreak, there is a strong need to find strategies to monitor SARSCoV-2 transmission. While the application of screening techniques plays a major role to this end, there is evidence challenging the real significance of seroconversion. We reported a case of COVID-19 reactivation associated with a neurosurgical operation with early neuropsychiatric involvement presumably promoted by olfactory and gustatory impairment in the first infection. Case Descriptio: A 57-year-old man was referred for a 2-month history of progressive development of imbalance, dizziness, and vomiting. Magnetic resonance imaging showed two bilateral hemispheric cerebellar lesions. In line with our triage protocol, the patient underwent a nasopharyngeal swab for RNA of SARS-CoV-2 detection, which resulted positive. Of note, the patient had reported in the previous month hyposmia and hypogeusia. After a period of 14 days, three new swabs were performed with negative results, leading the way to surgery. In the early post-operative period, the patient manifested acute onset of psychotic symptoms with hyperactive delirium, followed by fever and acute respiratory failure. A chest computed tomography revealed a specific pattern of ground-glass opacities in the lower lobes bilaterally, suggesting a viral pneumonia. Serological tests demonstrated the seroconversion and a new nasopharyngeal swab confirmed SARS-CoV-2 infection. Conclusion: Our report highlights the importance of comprehensive screening assessments in sensitive cases highly susceptible to COVID-19 recurrence.

Background. The radio- and chemo-resistance of glioblastoma stem-like cells (GSCs), together with their innate tumor-initiating aptitude, make this cell population a crucial target for effective therapies. However, targeting GSCs is hardly difficult and complex, due to the presence of the blood-brain barrier (BBB) and the infiltrative nature of GSCs arousing their dispersion within the brain parenchyma. Methods. Liposomes (LIPs), surface-decorated with an Apolipoprotein E-modified peptide (mApoE) to enable BBB crossing, were loaded with doxorubicin (DOXO), as paradigm of cytotoxic drug triggering immunogenic cell death (ICD). Patient-derived xenografts (PDXs) obtained by GSC intracranial injection were treated with mApoE-DOXOLIPs alone or concomitantly with radiation. Results. Our results indicated that mApoE, through the engagement of the low-density lipoprotein receptor (LDLR), promotes mApoE-DOXO-LIPs transcytosis across the BBB and confers target specificity towards GSCs. Irradiation enhanced LDLR expression on both BBB and GSCs, thus further promoting LIP diffusion and specificity. When administered in combination with radiations, mApoE-DOXO-LIPs caused a significant reduction of in vivo tumor growth due to GSC apoptosis. GSC apoptosis prompted microglia/macrophage phagocytic activity, together with the activation of the antigen-presenting machinery crucially required for anti-tumor adaptive immune response. Conclusions. Our results advocate for radiotherapy and adjuvant administration of drug-loaded, mApoE-targeted nanovectors as an effective strategy to deliver cytotoxic molecules to GSCs at the surgical tumor margins, the forefront of glioblastoma (GBM) recurrence, circumventing BBB hurdles. DOXO encapsulation proved in situ immune response activation within GBM microenvironment.

Purpose: Despite recent advances, the prognosis of glioblastoma (GBM) remains poor. The aim of this study was to assess the efficacy and tolerability of multiple daily fraction radiotherapy performed with multiple temozolomide (TMZ) administrations in newly diagnosed patients with GBM. Methods: This trial was a prospective, open-label, monocentric, nonrandomized, single arm, phase II study. The primary endpoint was the proportion of progression-free patients at 12 months, and the secondary endpoints were overall survival (OS) and toxicity. Thirty-five patients underwent two radiotherapy courses concomitant with TMZ after surgery. At each course, radiation was delivered 3 times daily, 2 Gy/fraction, for 5 consecutive days, and the total dose was 60 Gy; concurrent TMZ was administered in a total dose of 150-200 mg/m2/day. Results: The primary endpoint failed to be applied; Macdonald criteria could be used in 16 (46%) patients with local or intracerebral recurrence (group A). In 12 patients, due to suspicion of radiation necrosis vs recurrence, Macdonald criteria were not applied (group B). The OS was 22 months, and OS probabilities at 12, 18, and 24 months were 82%, 59%, and 44%, respectively. Hematologic toxicities generally did not exceed grade 2. The quality of life and cognitive functioning did not significantly change between baseline and the first follow-up. In the multivariate analysis, necrosis and pseudoprogression were significant prognostic factors of OS. Conclusions: To improve local control and OS, a more aggressive treatment schedule should be explored. The related higher necrosis risk and its implications regarding local control deserve further investigation.

BACKGROUND: Concern about radiation-induced optic neuropathy (RION) has governed recent thinking about the role of radiation therapy in the treatment of meningiomas involving the anterior optic pathways. Despite this concern, during the last few years, the use of radiosurgery for such lesions has increased steadily. OBJECTIVE: To define both the tumor control rate and the risk of RION over a long-term follow-up period in a large cohort of patients treated with multisession radiosurgery. METHODS: The local control and visual outcome of 143 patients who underwent multisession radiosurgery (mRS) were evaluated. Neurological outcome was also analyzed. The data for the present study were obtained from a prospectively maintained database. RESULTS: The mean follow-up was 44 months (range, 12-113 months). All patients underwent mRS. The median prescription dose was 25 Gy delivered in 3 to 5 fractions. The prescription isodose, which typically encompassed at least 95% of the tumor, ranged from 65% to 86% (median, 80%). The mean tumor volume was 11.0 cm 3 (range, 0.1-126.3 cm 3; median, 8 cm 3). The progression-free survival at 3, 5, and 8 years was 100%, 93%, and 90%, respectively. Compared with baseline, visual function improved in 36% of patients, whereas 7.4% experienced a worsening in visual function (5.1% excluding the patients with progressive disease). CONCLUSION: Good local control rate and a low risk of RION indicate that mRS is a safe and effective treatment option in cases of large meningiomas. ABBREVIATIONS: AOP, anterior optic pathway AVP, anterior visual pathway mRS, multisession radiosurgery PD, progressive disease RION, radiation-induced optic neuropathy sRS, stereotactic radiosurgery.

Despite various treatment strategies being available, recurrent high-grade gliomas (r-HGG) are difficult to manage. To obtain local control, radiosurgery (SRS) reirradiation has been considered as potential treatment. In the present study, a retrospective analysis was performed on r-HGG patients treated with salvage single- (s-SRS) or multi-fraction SRS (m-SRS). The aim of this study was to evaluate the effectiveness of salvage SRS in terms of overall survival (OS); toxicity was analyzed as well. Between 2004 May and 2011 December, 128 r-HGG patients (161 lesions) treated with CyberKnife® SRS reirradiation were retrospectively analyzed. Toxicity was graded according to Radiation Therapy Oncology Group and by Common Terminology Criteria for Adverse Events v.3 criteria. OS from the diagnosis date and OS from reirradiation were estimated using the Kaplan–Meier method. Median follow-up was 9 months (range 15 days–82 months). All patients completed SRS without high-grade toxicity. Radiation necrosis was observed in seven patients (6 %) with large volume lesions. The median survival from initial diagnosis was 32 months. The 1-, 2-, and 3-years survival rates from diagnosis were 95, 62, and 45 % respectively. Median survival following SRS was 11.5 months. The 1-, 2-, and 3-years survival rate following SRS was 48, 20, and 17 % respectively. On multivariate analysis, age <40 years, salvage surgery before SRS, and other post-SRS therapies (second-line chemotherapy and/or surgery) were found to significantly improve survival (p = 0.03). SRS represents a safe and feasible option to treat r-HGG patients with low complication rates and potential survival benefit.

Despite various treatment strategies being available, recurrent high-grade gliomas (r-HGG) are difficult to manage. To obtain local control, radiosurgery (SRS) reirradiation has been considered as potential treatment. In the present study, a retrospective analysis was performed on r-HGG patients treated with salvage single- (s-SRS) or multi-fraction SRS (m-SRS). The aim of this study was to evaluate the effectiveness of salvage SRS in terms of overall survival (OS); toxicity was analyzed as well. Between 2004 May and 2011 December, 128 r-HGG patients (161 lesions) treated with CyberKnife(®) SRS reirradiation were retrospectively analyzed. Toxicity was graded according to Radiation Therapy Oncology Group and by Common Terminology Criteria for Adverse Events v.3 criteria. OS from the diagnosis date and OS from reirradiation were estimated using the Kaplan-Meier method. Median follow-up was 9 months (range 15 days-82 months). All patients completed SRS without high-grade toxicity. Radiation necrosis was observed in seven patients (6 %) with large volume lesions. The median survival from initial diagnosis was 32 months. The 1-, 2-, and 3-years survival rates from diagnosis were 95, 62, and 45 % respectively. Median survival following SRS was 11.5 months. The 1-, 2-, and 3-years survival rate following SRS was 48, 20, and 17 % respectively. On multivariate analysis, age <40 years, salvage surgery before SRS, and other post-SRS therapies (second-line chemotherapy and/or surgery) were found to significantly improve survival (p = 0.03). SRS represents a safe and feasible option to treat r-HGG patients with low complication rates and potential survival benefit

Despite various treatment strategies being available, recurrent high-grade gliomas (r-HGG) are difficult to manage. To obtain local control, radiosurgery (SRS) reirradiation has been considered as potential treatment. In the present study, a retrospective analysis was performed on r-HGG patients treated with salvage single- (s-SRS) or multi-fraction SRS (m-SRS). The aim of this study was to evaluate the effectiveness of salvage SRS in terms of overall survival (OS); toxicity was analyzed as well. Between 2004 May and 2011 December, 128 r-HGG patients (161 lesions) treated with CyberKnife(®) SRS reirradiation were retrospectively analyzed. Toxicity was graded according to Radiation Therapy Oncology Group and by Common Terminology Criteria for Adverse Events v.3 criteria. OS from the diagnosis date and OS from reirradiation were estimated using the Kaplan-Meier method. Median follow-up was 9 months (range 15 days-82 months). All patients completed SRS without high-grade toxicity. Radiation necrosis was observed in seven patients (6 %) with large volume lesions. The median survival from initial diagnosis was 32 months. The 1-, 2-, and 3-years survival rates from diagnosis were 95, 62, and 45 % respectively. Median survival following SRS was 11.5 months. The 1-, 2-, and 3-years survival rate following SRS was 48, 20, and 17 % respectively. On multivariate analysis, age <40 years, salvage surgery before SRS, and other post-SRS therapies (second-line chemotherapy and/or surgery) were found to significantly improve survival (p = 0.03). SRS represents a safe and feasible option to treat r-HGG patients with low complication rates and potential survival benefit

Despite various treatment strategies being available, recurrent high-grade gliomas (r-HGG) are difficult to manage. To obtain local control, radiosurgery (SRS) reirradiation has been considered as potential treatment. In the present study, a retrospective analysis was performed on r-HGG patients treated with salvage single- (s-SRS) or multi-fraction SRS (m-SRS). The aim of this study was to evaluate the effectiveness of salvage SRS in terms of overall survival (OS); toxicity was analyzed as well. Between 2004 May and 2011 December, 128 r-HGG patients (161 lesions) treated with CyberKnife® SRS reirradiation were retrospectively analyzed. Toxicity was graded according to Radiation Therapy Oncology Group and by Common Terminology Criteria for Adverse Events v.3 criteria. OS from the diagnosis date and OS from reirradiation were estimated using the Kaplan–Meier method. Median follow-up was 9 months (range 15 days–82 months). All patients completed SRS without high-grade toxicity. Radiation necrosis was observed in seven patients (6 %) with large volume lesions. The median survival from initial diagnosis was 32 months. The 1-, 2-, and 3-years survival rates from diagnosis were 95, 62, and 45 % respectively. Median survival following SRS was 11.5 months. The 1-, 2-, and 3-years survival rate following SRS was 48, 20, and 17 % respectively. On multivariate analysis, age <40 years, salvage surgery before SRS, and other post-SRS therapies (second-line chemotherapy and/or surgery) were found to significantly improve survival (p = 0.03). SRS represents a safe and feasible option to treat r-HGG patients with low complication rates and potential survival benefit.

In a two-stage phase II study, 24 patients with first diagnosis of glioblastoma (GBM) were treated with dendritic cell (DC) immunotherapy associated to standard radiochemotherapy with temozolomide (TMZ) followed by adjuvant TMZ. Three intradermal injections of mature DC loaded with autologous GBM lysate were administered before adjuvant TMZ, while 4 injections were performed during adjuvant TMZ. According to a two-stage Simon design, to proceed to the second stage progression-free survival (PFS) 12 months after surgery was expected in at least 8 cases enrolled in the first stage. Evidence of immune response and interaction with chemotherapy were investigated. After a median follow up of 17.4 months, 9 patients reached PFS12. In these patients (responders, 37.5%), DC vaccination induced a significant, persistent activation of NK cells, whose increased response was significantly associated with prolonged survival. CD8+T cells underwent rapid expansion and priming but, after the first administration of adjuvant TMZ, failed to generate a memory status. Resistance to TMZ was associated with robust expression of the multidrug resistance protein ABCC3 in NK but not CD8+T cells. The negative effect of TMZ on the formation of T cell-associated antitumor memory deserves consideration in future clinical trials including immunotherapy

Object: Incident reporting systems are universally recognized as important tools for quality improvement in all complex adaptive systems, including the operating room. Nevertheless, introducing a safety culture among neurosurgeons is a slow process, and few studies are available in the literature regarding the implementation of an incident reporting system within a neurosurgical department. The authors describe the institution of an aviation model of incident reporting and investigation in neurosurgery, focusing on the method they have used and presenting some preliminary results.Methods: In 2010, the Inpatient Safety On-Board project was developed through cooperation between a team of human factor and safety specialists with aviation backgrounds (DgSky team) and the general manager of the Fondazione IstitutoNeurologico Carlo Besta. In 2011, after specific training in safety culture, the authors implemented an aviation-derived prototype of incident reporting within the Department of Neurosurgery. They then developed an experimental protocol to track, analyze, and categorize any near misses that happened in the operating room. This project officially started in January 2012, when a dedicated team of assessors was established. All members of the neurosurgical department were asked to report near misses on a voluntary, confidential, and protected form (Patient Incident Reporting System form, Besta Safety Management Programme). Reports were entered into an online database and analyzed by a dedicated team of assessors with the help of a facilitator, and an aviation-derived root cause analysis was performed.Results: Since January 2012, 14 near misses were analyzed and classified. The near-miss contributing factors were mainly related to human factors (9 of 14 cases), technology (1 of 14 cases), organizational factors (3 of 14 cases), or procedural factors (1 of 14 cases).Conclusions: Implementing an incident reporting system is quite demanding; the process should involve

One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However, the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs, we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here, we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably, RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth, both in vitro and in vivo. Thus, our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment, suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment. ©2011 AACR.

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within 2 years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. Lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma. © 2010 American Chemical Society.

Brain metastases (BMs) are the most common malignancy of the central nervous system. Recently it has been demonstrated that plasminogen activator inhibitor serpins promote brain metastatic colonization, suggesting that mutations in serpins or other members of the coagulation cascade can provide critical advantages during BM formation. We performed whole-exome sequencing on matched samples of breast cancer and BMs and found mutations in the coagulation pathway genes in 5 out of 10 BM samples. We then investigated the mutational status of 33 genes belonging to the coagulation cascade in a panel of 29 BMs and we identified 56 Single Nucleotide Variants (SNVs). The frequency of gene mutations of the pathway was significantly higher in BMs than in primary tumours, and SERPINI1 was the most frequently mutated gene in BMs. These findings provide direction in the development of new strategies for the treatment of BMs.

Glioblastomas (GBMs) contain transformed, self-maintaining, multipotent, tumour-initiating cancer stem cells, whose identification has radically changed our perspective on the physiology of these tumours. Currently, it is unknown whether multiple types of transformed precursors, which display alternative sets of the complement of properties of true cancer stem cells, can be found in a GBM. If different subsets of such cancer stem-like cells (CSCs) do exist, they might represent distinct cell targets, with a differential therapeutic importance, also depending on their characteristics and lineage relationship. Here, we report the presence of two types of CSCs within different regions of the same human GBM. Cytogenetic and molecular analysis shows that the two types of CSCs bear quite diverse tumorigenic potential and distinct genetic anomalies, and, yet, derive from common ancestor cells. This provides critical information to unravel the development of CSCs and the key molecular/genetic components underpinning tumorigenicity in human GBMs. © 2009 Macmillan Publishers Limited. All rights reserved.

Human glioblastomas (hGBMs) have been shown to embody a relatively small subset of cells which bear the defining features of somatic stem cells and the ability to establish, expand and perpetuate these tumors. They are defined cancer stem cell or tumor propagating cells (TPCs). This has caused a paradigmatic shift in the way we interpret hGBM physiology, for it identifies TPCs as a major culprit to be tackled for the development of novel therapeutics. Hence, we inferred that the study of regulatory mechanisms of normal neurogenesis may lead us to identify specific inhibitors of TPCs. METHODS: TPCs from human GBMs were used to determine the role of a neural stem cell regulator, the EphA2 receptor, in modulating self-renewal and tumorigenicity. We used a combined series of in vitro experiments which include primary tissue cultures, FACS analysis, next generation sequencing screening and in vivo, orthotopic xenografting experiments entailing the intracerebral delivery of putative therapeutic agents. RESULTS: We determined the over-expression of the Ephrin receptor type A2 (EphA2) in hGBM TPCs. EphA2 upregulation causally underlies self-renewal and expansion of the TPCs pool. Most important, both the EphA2 cognate ligand ephrinA1-Fc causes EphA2 downregulation in TPCs and suppress TPC self-renewal and intracranial tumorigenicity. This points to EphA2 as a key effector in TPCs self-renewal and tumorigenicity. Notably, intracranial administration of ephrinA1-Fc causes both down-regulation of EphA2 in hGBMs orthotopic xenografts pre-established in mice, whose growth and expansion throughout the brain parenchyma is significantly hindered in the absence of cytotoxic effects. CONCLUSIONS: We identify TPCs as a major target of EphA2 overexpression in hGBM, that drives their self-renewal and tumorigenicity. We propose TPCs as a specific cellular target in which enforced down-regulation of a molecular target such as EphA2 may be exploited for therapeutic purposes, under therapy

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor and is associated with poor survival. Recently, stem-like cell populations have been identified in numerous malignancies including GBM. To identify genes whose expression is changed with differentiation, we compared transcript profiles from a GBM oncosphere line before and after differentiation. Bioinformatic analysis of the gene expression profiles identified podocalyxin-like protein (PODXL), a protein highly expressed in human embryonic stem cells, as a potential marker of undifferentiated GBM stem-like cells. The loss of PODXL expression upon differentiation of GBM stem-like cell lines was confirmed by quantitative real-time PCR and flow cytometry. Analytical flow cytometry of numerous GBM oncosphere lines demonstrated PODXL expression in all lines examined. Knockdown studies and flow cytometric cell sorting experiments demonstrated that PODXL is involved in GBM stem-like cell proliferation and oncosphere formation. Compared to PODXL-negative cells, PODXL-positive cells had increased expression of the progenitor/stem cell markers Musashi1, SOX2, and BMI1. Finally, PODXL expression directly correlated with increasing glioma grade and was a marker for poor outcome in patients with GBM. In summary, we have demonstrated that PODXL is expressed in GBM stem-like cells and is involved in cell proliferation and oncosphere formation. Moreover, high PODXL expression correlates with increasing glioma grade and decreased overall survival in patients with GBM. © 2013 Binder et al.

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Although CD90 has been identified as a marker for various kinds of stem cells including liver cancer stem cells (CSCs) that are responsible for tumorigenesis, the potential role of CD90 as a marker for CSCs in gliomas has not been characterized. To address the issue, we investigated the expression of CD90 in tissue microarrays containing 15 glioblastoma multiformes (GBMs), 19 WHO grade III astrocytomas, 13 WHO grade II astrocytomas, 3 WHO grade I astrocytomas and 8 normal brain tissues. Immunohistochemical analysis showed that CD90 was expressed at a medium to high level in all tested high-grade gliomas (grade III and GBM) whereas it was barely detectable in low-grade gliomas (grade I and grade II) and normal brains. Double immunofluorescence staining for CD90 and CD133 in GBM tissues revealed that CD133+ CSCs are a subpopulation of CD90+ cells in GBMs in vivo. Flow cytometry analysis of the expression of CD90 and CD133 in GBM-derived stem-like neurospheres further confirmed the conclusion in vitro. The expression levels of both CD90 and CD133 were reduced along with the loss of stem cells after differentiation. Furthermore, the limiting dilution assay demonstrated that the sphere formation ability was comparable between the CD90+/CD133+ and the CD90+/CD133- populations of GBM neurospheres, which is much higher than that of the CD90-/CD133- population. We also performed double staining for CD90 and a vascular endothelial cell marker CD31 in tissue microarrays which revealed that the CD90+ cells were clustered around the tumor vasculatures in high-grade glioma tissues. These findings suggest that CD90 is not only a potential prognostic marker for high-grade gliomas but also a marker for CSCs within gliomas, and it resides within endothelial niche and may also play a critical role in the generation of tumor vasculatures via differentiation into endothelial cells

In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications. © 2012 Elsevier Inc.

In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2(High) and EphA2(Low) populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation, and differentiation. Here we applied a multilectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSRGBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells that may be useful in stem-cell therapy of glioblastoma. © 2011 American Chemical Society.

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by gamma-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.

Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem-like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone-modifying agents to elucidate mechanisms by which the phenotype and tumor-promoting capacity of GBM-derived neoplastic stem-like cells are regulated. Using established GBM-derived neurosphere lines and low passage primary GBM-derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch-like epidermal growth factor-related receptor (DNER), inhibited the growth of GBM-derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor-promoting effects of canonical Notch signaling in brain cancer stem-like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem-like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies.

Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.

Transformed stem cells have been isolated from some human cancers. We report that, unlike other brain cancers, the lethal glioblastoma multiforme contains neural precursors endowed with all of the critical features expected from neural stem cells. Similar, yet not identical, to their normal neural stem cell counterpart, these precursors emerge as unipotent (astroglial) in vivo and multipotent (neuronal-astroglial-oligodendroglial) in culture. More importantly, these cells can act as tumor-founding cells down to the clonal level and can establish tumors that closely resemble the main histologic, cytologic, and architectural features of the human disease, even when challenged through serial transplantation. Thus, cells possessing all of the characteristics expected from tumor neural stem cells seem to be involved in the growth and recurrence of adult human glioblastomas multiforme.