Molecular signature of stem-like glioma cells (SLGCs) from human glioblastoma and gliosarcoma.

Glioblastoma multiforme (GBM) and the GBM variant gliosarcoma (GS) are among the tumors with the highest morbidity and mortality, providing only palliation. Stem-like glioma cells (SLGCs) are involved in tumor initiation, progression, therapy resistance, and relapse. The identification of general features of SLGCs could contribute to the development of more efficient therapies. Commercially available protein arrays were used to determine the cell surface signature of eight SLGC lines from GBMs, one SLGC line obtained from a xenotransplanted GBM-derived SLGC line, and three SLGC lines from GSs. By means of non-negative matrix factorization expression metaprofiles were calculated. Using the cophenetic correlation coefficient (CCC) five metaprofiles (MPs) were identified, which are characterized by specific combinations of 7-12 factors. Furthermore, the expression of several factors, that are associated with GBM prognosis, GBM subtypes, SLGC differentiation stages, or neural identity was evaluated. The investigation encompassed 24 distinct SLGC lines, four of which were derived from xenotransplanted SLGCs, and included the SLGC lines characterized by the metaprofiles. It turned out that all SLGC lines expressed the epidermal growth factor EGFR and EGFR ligands, often in the presence of additional receptor tyrosine kinases. Moreover, all SLGC lines displayed a neural signature and the IDH1 wildtype, but differed in their p53 and PTEN status. Pearson Correlation analysis identified a positive association between the pluripotency factor Sox2 and the expression of FABP7, Musashi, CD133, GFAP, but not with MGMT or Hif1α. Spherical growth, however, was positively correlated with high levels of Hif1α, CDK4, PTEN, and PDGFRß, whereas correlations with stemness factors or MGMT (MGMT expression and promoter methylation) were low or missing. Factors highly expressed by all SLGC lines, irrespective of their degree of stemness and growth behavior, are Cathepsin-D, CD99, EMMPRIN/CD147, Intß1, the Galectins 3 and 3b, and N-Cadherin.

Captopril inhibits Matrix Metalloproteinase-2 and extends survival as a temozolomide adjuvant in an intracranial gliosarcoma model.

BACKGROUND: Captopril is a well-characterized, FDA-approved drug that has demonstrated promise as a repurposed oncology therapeutic. Captopril's known anti-cancer effects include inhibition of Matrix Metalloproteinase-2 (MMP-2), an endopeptidase which selectively breaks down the extracellular matrix to promote cell migration. MMP-2 is a known therapeutic target in gliomas, tumors with significant clinical need. Using an aggressive gliosarcoma model, we assessed captopril's effects on MMP-2 expression in vitro and in vivo as well as its efficacy as an adjuvant in combination therapy regimens in vivo. METHODS: Following captopril treatment, MMP-2 protein expression and migratory capabilities of 9 L gliosarcoma cells were assessed in vitro via western blots and scratch wound assays, respectively. Rats were intracranially implanted with 9 L gliosarcoma tumors, and survival was assessed in the following groups: control; captopril (30 mg/kg/day); temozolomide (TMZ) (50 mg/kg/day), and captopril+TMZ. In vivo experiments were accompanied by immunohistochemistry for MMP-2 from brain tissue. RESULTS: In vitro, captopril decreased MMP-2 protein expression and reduced migratory capacity in 9 L gliosarcoma cells. In a gliosarcoma animal model, captopril decreased MMP-2 protein expression and extended survival as a TMZ adjuvant relative to untreated controls, captopril monotherapy, and TMZ monotherapy groups (27.5 versus 14 (p < 0.001), 16 (p < 0.001), and 23 (p = 0.018) days, respectively). CONCLUSIONS: Captopril decreases gliosarcoma cell migration, which may be mediated by reduction in MMP-2 protein expression. Captopril provided a survival advantage as a TMZ adjuvant in a rat intracranial gliosarcoma model. Captopril may represent a promising potential adjuvant to TMZ therapy in gliosarcoma as a modulator of the MMP-2 pathway.

Gliosarcoma vs. glioblastoma: a retrospective case series using molecular profiling.

BACKGROUND: Gliosarcoma (GS) refers to the presence of mesenchymal differentiation (as seen using light microscopy) in the setting of glioblastoma (GB, an astrocytoma, WHO Grade 4). Although the same approach to treatment is typically adopted for GS and GB, there remains some debate as to whether GS should be considered a discrete pathological entity. Differences between these tumors have not been clearly established at the molecular level. METHODS: Patients with GS (n=48) or GB (n=1229) underwent molecular profiling (MP) with a pan-cancer panel of tests as part of their clinical care. The methods employed included next-generation sequencing (NGS) of DNA and RNA, copy number variation (CNV) of DNA and immunohistochemistry (IHC). The MP comprised 1153 tests in total, although results for each test were not available for every tumor profiled. We analyzed this data retrospectively in order to determine if our results were in keeping with what is known about the pathogenesis of GS by contrast with GB. We also sought novel associations between the MP and GS vs. GB which might improve our understanding of pathogenesis of GS. RESULTS: Potentially meaningful associations (p<0.1, Fisher's exact test (FET)) were found for 14 of these tests in GS vs. GB. A novel finding was higher levels of proteins mediating immuno-evasion (PD-1, PD-L1) in GS. All of the differences we observed have been associated with epithelial-to-mesenchymal transition (EMT) in other tumor types. Many of the changes we saw in GS are novel in the setting of glial tumors, including copy number amplification in LYL1 and mutations in PTPN11. CONCLUSIONS: GS shows certain characteristics of EMT, by contrast with GB. Treatments targeting immuno-evasion may be of greater therapeutic value in GS relative to GB.

TNF-α mediated MEK-ERK signaling in invasion with putative network involving NF-κB and STAT-6: a new perspective in glioma.

Glioblastoma is the most common malignant primary brain tumor with poor prognosis. Invasion involves pro-inflammatory cytokines and major signaling hubs. Tumor necrosis factor-α (TNF-α) acts as a master switch in establishing an intricate link between inflammation and cancer. The present study attempted to explore the possible implication of MAPK extracellular signaling-regulated kinase kinase (MEK)-extracellular signaling-regulated kinase (ERK) signaling pathway and expression of nuclear factor-κB (NF-κB), signal transducers and activators of transcription-6 (STAT-6), ERK, and phosphorylated-ERK (p-ERK) signaling proteins in TNF-α microenvironment. U0126 and PD98059 were used to inhibit the MEK-ERK1/2 pathway. TNF-α stimulation enhanced invasion in U87MG, U251MG and patient-derived primary glioma cells, whereas cell viability was not altered. Matrix metalloproteinase-2 (MMP-2) activity was increased only in U251MG glioma cells. These data suggest that TNF-α microenvironment plays an important role in the invasion of U251MG, U87MG, and patient-derived primary glioma cells, without any cytotoxic effect. The MMP-2 activity is differentially regulated by TNF-α stimulation in these cells. TNF-α stimulation upregulated the protein expression of ERK-1, ERK-2 and also increased the level of p-ERK1/2. TNF-α stimulation further upregulated the expression of NF-κB1, STAT-6 in tandem with Ras-MEK signaling system in U87MG cells, which emphasized the possible involvement of these signaling hubs in the glioma microenvironment. MEK-ERK inhibitors significantly attenuated the invasion of U87MG cells mediated by the TNF-α stimulation, probably through their inhibitory impact on p-ERK1/2 and ERK-2. This study provides the possible rationale of invasion by glioma cells in a TNF-α-induced pro-inflammatory milieu, which involves direct role of MEK-ERK signaling, with possible implication of NF-κB and STAT-6.

Clinical and molecular characteristics of gliosarcoma and modern prognostic significance relative to conventional glioblastoma.

Gliosarcoma is a rare histopathologic variant of glioblastoma traditionally associated with a poor prognosis. While gliosarcoma may represent a distinct clinical entity given its unique histologic composition and molecular features, its relative prognostic significance remains uncertain. While treatment of gliosarcoma generally encompasses the same standardized approach used in glioblastoma, supporting evidence is limited given its rarity. Here, we characterized 32 cases of gliosarcoma and retrospectively evaluated survival relative to 451 glioblastoma patients diagnosed during the same era within the same institution. Overall, we identified 22 primary gliosarcomas, representing 4.7% of WHO Grade IV primary glioblastomas, and 10 secondary gliosarcomas. With median age of 62, patients were predominately Caucasian (87.5%) and male (65.6%). Tumors with available molecular profiling were primarily MGMT-unmethylated (87.5%), IDH-1-preserved (100%) and EGFR wild-type (100%). Interestingly, while no significant median survival difference between primary gliosarcoma and glioblastoma was observed across the entire cohort (11.0 vs. 14.8 months, p = 0.269), median survival was worse for gliosarcoma specifically among patients who received modern temozolomide-based (TMZ) chemoradiotherapy (11.0 vs. 17.3 months, p = 0.006). Matched-pair analysis also trended toward worse median survival among gliosarcomas (11.0 vs. 19.6 months, log-rank p = 0.177, Breslow p = 0.010). While adjuvant radiotherapy (HR 0.206, p = 0.035) and TMZ-based chemotherapy (HR 0.531, p = 0.000) appeared protective, gliosarcoma emerged as a significantly poor prognostic factor on multivariate analysis (HR 3.27, p = 0.012). Collectively, our results suggest that gliosarcoma may still portend worse prognosis even with modern trimodality therapy.

Expression of CD70 (CD27L) Is Associated With Epithelioid and Sarcomatous Features in IDH-Wild-Type Glioblastoma.

Glioblastoma is an aggressive, often recalcitrant disease. In the majority of cases, prognosis is dismal and current therapies only moderately prolong survival. Immunotherapy is increasingly being recognized as an effective treatment modality. CD70 is a transmembrane protein that shows restricted expression in tissue but has been described in various malignancies. Therapeutic targeting of CD70 has demonstrated antitumor efficacy and is in clinical trials. Here, we sought to characterize CD70 expression in a large cohort of gliomas (n = 205) using tissue microarrays. We identified a subset of tumors (n = 18, 8.8% of high-grade gliomas) exhibiting moderate-to-strong immunoreactivity that enriched for the IDH-wild-type glioblastoma variants gliosarcoma (n = 10) and the newly described epithelioid glioblastoma (n = 4). CD70 expression was associated with prolonged survival in gliosarcoma. Analysis of TCGA datasets showed significantly increased CD70 expression in mesenchymal tumors and prolonged survival in recurrent non-G-CIMP high-expressing tumors. In CD70+ gliomas, there was a significant increase in CD68/CD163/HLA-DR+ tumor-associated macrophages, but not CD27+ TIL. These results confirm prior in vitro studies and demonstrate expression in a clinical cohort. The absence of CD70 expression in the post-treatment setting may portend more clinically aggressive disease in gliosarcoma. However, larger-scale studies will be needed to characterize and validate this relationship.

Evaluation of permeability, doxorubicin delivery, and drug retention in a rat brain tumor model after ultrasound-induced blood-tumor barrier disruption.

Drug delivery in brain tumors is challenging because of the presence of blood-brain barrier (BBB) and the blood-tumor barrier (BTB). Focused ultrasound (FUS) combined with microbubbles can enhance the permeability of the BTB in brain tumors, as well as disrupting the BBB in the surrounding tissue. In this study, dynamic contrast-enhanced Magnetic Resonance Imaging (DCE-MRI) was used to characterize FUS-induced permeability changes in a rat glioma model and in the normal brain and to investigate the relationship between these changes and the resulting concentration of the chemotherapy agent doxorubicin (DOX). 9L gliosarcoma cells were implanted in both hemispheres in male rats. At day 10-12 after implantation, FUS-induced BTB disruption using 690kHz ultrasound and Definity microbubbles was performed in one of the tumors and in a normal brain region in each animal. After FUS, DOX was administered at a dose of 5.67mg/kg. The resulting DOX concentration was measured via fluorometry at 1 or 24h after FUS. The transfer coefficient Ktrans describing extravasation of the MRI contrast agent Gd-DTPA was significantly increased in both the sonicated tumors and in the normal brain tissue (P<0.001) between the two DCE-MRI acquisitions obtained before and after FUS, while no significant difference was found in the controls (non-sonicated tumor/normal brain tissue). DOX concentrations were also significantly larger than controls in both the sonicated tumors and in the normal tissue volumes at 1 and 24h after sonication. The DOX concentrations were significantly larger (P<0.01) in the control tumors harvested 1h after FUS than in those harvested at 24h, when the tumor concentrations were not significantly different than in the non-sonicated normal brain. In contrast, there was no significant difference in the DOX concentrations between the tumors harvested at 1 and 24h after FUS or in the concentrations measured in the brain at these time points. The transfer coefficient Ktrans for Gd-DTPA and the drug concentrations showed a good linear correlation (R2=0.56). Overall, these data suggest that FUS and microbubbles can not only increase DOX delivery across the BBB and BTB, but that it is retained in the tissue at significantly enhanced levels for at least 24h. Such enhanced retention may increase the potency of this chemotherapy agent and allow for reduced systemic doses. Furthermore, MRI-based estimates of Gd-DTPA transport across these barriers might be useful to estimate local DOX concentrations in the tumor and in the surrounding normal tissue.

Gliosarcomas lack BRAFV600E mutation, but a subset exhibit ß-catenin nuclear localization.

Gliosarcoma (GS) is a rare subtype of glioblastoma (GBM) characterized by both glial and mesenchymal components. Unlike GBM, there are no specific prognostic markers, and optimized treatments for patients with GS do not exist. Recent reports describe BRAFV600E mutation in malignant peripheral nerve sheath tumors, and aberrant Wnt signaling and CTNNB1 (ß-catenin gene) mutations have been described in GBM. We sought to determine whether GS tumors harbor BRAFV600E mutations or aberrant Wnt signaling, as indicated by nuclear localization of ß-catenin, by immunohistochemical detection. Forty-eight (48) cases of primary and secondary adult GS (including recurrent ones) were evaluated by immunohistochemical techniques for the presence of nuclear ß-catenin and the BRAFV600E mutation. A small subset (6/46, 13%) showed nuclear localization of ß-catenin. None of the cases harbored BRAFV600E mutations (0/48). These results are the first to describe the presence of Wnt signaling pathway abnormalities, manifested by nuclear ß-catenin, in a subset, as well as the lack of BRAFV600E mutation in GS. We propose a potential role for Wnt pathway alterations in the pathogenesis of a subset of GS.

Gliosarcoma with primitive neuroectodermal, osseous, cartilage and adipocyte differentiation: a case report.

We describe a rare case of gliosarcoma with primitive neuroectodermal, osseous, cartilage and adipocyte differentiation. A 57-year-old man experienced a month history of headache, nausea and vomiting. Worse yet, the headache has become more severe for the past 6 days. Magnetic resonance (MR) images disclosed a lesion with operative indications located in the right frontal lobe. Then the tumor was macroscopically totally removed. Histologically, the tumor showed two kinds of components. One kind of the tumor cells appeared typical astrocytic tumor cells with anaplastic appearance. The other kind of the tumor cells appeared sheets of small round hyperchromatic cells, which presented a kind of pancreatic neuroendocrine tumor (PNET)-like structure. These sheets of small round cells were surrounded by a large number of relative-sparse-spindle cells. Multiple separate distinct areas of adipose tissue, osteoid matrix laid down and cartilage tissue were also identified. Immunohistochemically, a portion of typical astrocytic tumor cells and some small round hyperchromatic cells showed GFAP positivity. Small round hyperchromatic cells were positive for S-100, Fli-1, Nestin, MAP-2 and Syn. A large amount of relative sparse spindle cells (sarcomatous areas) were positive for vimentin. In addition, reticulin staining demonstrated expression of reticular fibers in relative-sparse-spindle cells areas but not in the astrocytic tumor cells and small round hyperchromatic cells areas. Molecular cytogenetic analyses demonstrated PTEN allele loss and no evidence of amplification of EGFR in both the astrocytic tumor cells, PNET-like structure and sparse spindle cells areas. These data suggest that this tumor was a gliosarcoma with primitive neuroectodermal, osseous, cartilage and adipocyte differentiation. To our knowledge, this is a rare gliosarcoma , reporting our additional new case would add to the better understanding of this tumor.

Assessing Amide Proton Transfer (APT) MRI Contrast Origins in 9 L Gliosarcoma in the Rat Brain Using Proteomic Analysis.

PURPOSE: To investigate the biochemical origin of the amide photon transfer (APT)-weighted hyperintensity in brain tumors. PROCEDURES: Seven 9 L gliosarcoma-bearing rats were imaged at 4.7 T. Tumor and normal brain tissue samples of equal volumes were prepared with a coronal rat brain matrix and a tissue biopsy punch. The total tissue protein and the cytosolic subproteome were extracted from both samples. Protein samples were analyzed using two-dimensional gel electrophoresis, and the proteins with significant abundance changes were identified by mass spectrometry. RESULTS: There was a significant increase in the cytosolic protein concentration in the tumor, compared to normal brain regions, but the total protein concentrations were comparable. The protein profiles of the tumor and normal brain tissue differed significantly. Six cytosolic proteins, four endoplasmic reticulum proteins, and five secreted proteins were considerably upregulated in the tumor. CONCLUSIONS: Our experiments confirmed an increase in the cytosolic protein concentration in tumors and identified several key proteins that may cause APT-weighted hyperintensity.

A Phase III study of radiation therapy (RT) and O6-benzylguanine + BCNU versus RT and BCNU alone and methylation status in newly diagnosed glioblastoma and gliosarcoma: Southwest Oncology Group (SWOG) study S0001.

AIMS: To determine the efficacy of methylguanine methyltransferase (MGMT) depletion + BCNU [1,3-bis(2-chloroethyl)-1- nitrosourea: carmustine] therapy and the impact of methylation status in adults with glioblastoma multiforme (GBM) and gliosarcoma. METHODS: Methylation analysis was performed on GBM patients with adequate tissue samples. Patients with newly diagnosed GBM or gliosarcoma were eligible for this Phase III open-label clinical trial. At registration, patients were randomized to Arm 1, which consisted of therapy with O(6)-benzylguanine (O(6)-BG) + BCNU 40 mg/m(2) (reduced dose) + radiation therapy (RT) (O6BG + BCNU arm), or Arm 2, which consisted of therapy with BCNU 200 mg/m(2) + RT (BCNU arm). RESULTS: A total of 183 patients with newly diagnosed GBM or gliosarcoma from 42 U.S. institutions were enrolled in this study. Of these, 90 eligible patients received O(6)-BG + BCNU + RT and 89 received BCNU + RT. The trial was halted at the first interim analysis in accordance with the guidelines for stopping the study due to futility (<40 % improvement among patients on the O6BG + BCNU arm). Following adjustment for stratification factors, there was no significant difference in overall survival (OS) or progression-free survival (PFS) between the two groups (one sided p = 0.94 and p = 0.88, respectively). Median OS was 11 [95 % confidence interval (CI) 8-13] months for patients in the O6BG + BCNU arm and 10 (95 % CI 8-12) months for those in the BCNU arm. PFS was 4 months for patients in each arm. Adverse events were reported in both arms, with significantly more grade 4 and 5 events in the experimental arm. CONCLUSIONS: The addition of O(6)-BG to the standard regimen of radiation and BCNU for the treatment patients with newly diagnosed GBM and gliosarcoma did not provide added benefit and in fact caused additional toxicity.

Cell-density-dependent manifestation of partial characteristics for neuronal precursors in a newly established human gliosarcoma cell line.

Gliosarcoma cell line K308 was established from a primary tumor specimen removed from a 51-year-old male Han Chinese patient. Besides the typical characteristics of gliosarcoma cells, K308 cells express abundant glutaminase and can release large amount of glutamate. K308 exhibited cell-density-dependent expression of neuronal precursor markers, particularly nestin. At low density, the majority of K308 cells were nestin negative (approximately 70%) and nestin levels remained homogenous within each single-cell-derived colony when K308 proliferated. After reaching confluence, however, the majority of K308 cells turned nestin positive. These confluent K308 cells were also Sox2 positive and could form tumor spheres even in serum-containing media.

A bacterial gene, mms6, as a new reporter gene for magnetic resonance imaging of mammalian cells.

Magnetic resonance imaging (MRI) allows for noninvasive, deep tissue imaging with high spatial resolution, making it an attractive modality for in vivo cellular imaging. Since reporter genes can generate magnetic resonance (MR) contrast based on molecular activity, they offer a potentially powerful tool for cellular imaging. The mms6 gene was originally identified in magnetotactic bacteria (MTB), which is known to play a key role in magnetic crystal formation. The purpose of the present work was to investigate the possibility of using mms6 as an MR reporter gene. We established a transgenic mammalian cell line that stably expresses mms6. In vitro experiments show that mms6-expressing cells form clusters of nanoparticles within and outside membrane-enclosed structures and produce changes in MR contrast, most likely by increasing iron uptake of intracellular iron. Additionally, in vivo MRI experiments demonstrate that mms6-expressing tumors can be distinguished from parental tumors not expressing mms6, even in the absence of exogenous iron supplementation. Our results demonstrate that mms6 can function as an MR reporter gene with the potential to monitor gene expression and to visualize the proliferation, migration, and metastasis of tumor cells expressing it.

Highly stable polymer coated nano-clustered silver plates: a multimodal optical contrast agent for biomedical imaging.

Here, we present a new optical contrast agent based on silver nanoplate clusters embedded inside of a polymer nano matrix. Unlike nanosphere clusters, which have been well studied, nanoplate clusters have unique properties due to the different possible orientations of interaction between the individual plates, resulting in a significant broadening of the absorption spectra. These nanoclusters were immobilized inside of a polymer cladding so as to maintain their stability and optical properties under in vivo conditions. The polymer-coated silver nanoplate clusters show a lower toxicity compared to the uncoated nanoparticles. At high nanoparticle concentrations, cell death occurs mostly due to apoptosis. These nanoparticles were used for targeted fluorescence imaging in a rat glioma cell line by incorporating a fluorescent dye into the matrix, followed by conjugation of a tumor targeting an F3 peptide. We further used these nanoparticles as photoacoustic contrast agents in vivo to enhance the contrast of the vasculature structures in a rat ear model. We observed a contrast enhancement of over 90% following the nanoparticle injection. It is also shown that these NPs can serve as efficient contrast agents, with specific targeting abilities for broadband multimodal imaging that are usable for diagnostic applications and that extend into use as therapeutic agents as well.

[Gliosarcoma of cerebral hemispheres: a clinicopathologic study of 10 cases].

OBJECTIVE: To study the clinical and pathologic features of gliosarcoma of cerebral hemispheres. METHODS: The clinicopathologic features of 10 cases of gliosarcoma involving cerebral hemispheres were reviewed. Immunohistochemical study was carried out using EnVision method. RESULTS: The mean age of the patients was 54 years and the male-to-female ratio was 6 to 4. Clinical symptoms included headache (6/10), nausea/vomiting (5/10), and sensory or motor impairment (4/10). Nine of the cases were primary gliosarcoma, with maximum diameter ranging from 2.4 to 5.5 cm (mean = 4.2 cm). The remaining case represented secondary gliosarcoma involving skull base and extracranial tissues. Histologic examination showed a biphasic pattern in all cases. Regarding the glial component, there were 9 cases of pleomorphic glioblastoma and 1 case of giant cell glioblastoma. Reticulin stain was positive in all cases. Immunohistochemical study showed that the tumor cells variably expressed GFAP (10/10), p16 (4/10), EGFR (1/10), CD68 (1/10) and p53 (6/10). The Ki-67 index ranged from 15% to 70% (mean = 34%). Six patients had follow-up data available. One patient was disease-free for 45 months and 5 patients died of the disease at 3 to 17 months after the operation (mean duration of survival = 9 months). CONCLUSIONS: Gliosarcoma is a highly aggressive tumor, often locates in the deeper part cerebral hemispheres and has a relatively short duration of symptoms. It carries a poor prognosis. GFAP immunostain and reticulin stain are helpful in confirming the diagnosis. p53 and p16 are also expressed in some cases.

Increased expression of glutamate transporter GLT-1 in peritumoral tissue associated with prolonged survival and decreases in tumor growth in a rat model of experimental malignant glioma.

OBJECT: Gliomas are known to release excessive amounts of glutamate, inducing glutamate excitotoxic cell death in the peritumoral region and allowing the tumor to grow and to expand. Glutamate transporter upregulation has been shown to be neuroprotective by removing extracellular glutamate in a number of preclinical animal models of neurodegenerative diseases, including amyotrophic lateral sclerosis and Parkinson disease as well as psychiatric disorders such as depression. The authors therefore hypothesized that the protective mechanism of glutamate transporter upregulation would be useful for the treatment of gliomas as well. METHODS: In this study 9L gliosarcoma cells were treated with a glutamate transporter upregulating agent, thiamphenicol, an antibiotic approved in Europe, which has been shown previously to increase glutamate transporter expression and has recently been validated in a human Phase I biomarker trial for glutamate transporter upregulation. Cells were monitored in vitro for glutamate transporter levels and cell proliferation. In vivo, rats were injected intracranially with 9L cells and were treated with increasing doses of thiamphenicol. Animals were monitored for survival. In addition, postmortem brain tissue was analyzed for tumor size, glutamate transporter levels, and neuron count. RESULTS: Thiamphenicol showed little effects on proliferation of 9L gliosarcoma cells in vitro and did not change glutamate transporter levels in these cells. However, when delivered locally in an experimental glioma model in rats, thiamphenicol dose dependently (10-5000 µM) significantly increased survival up to 7 days and concomitantly decreased tumor size from 46.2 mm(2) to 10.2 mm(2) when compared with lesions in nontreated controls. Furthermore, immunohistochemical and biochemical analysis of peritumoral tissue confirmed an 84% increase in levels of glutamate transporter protein and a 72% increase in the number of neuronal cells in the tissue adjacent to the tumor. CONCLUSIONS: These results show that increasing glutamate transporter expression in peritumoral tissue is neuroprotective. It suggests that glutamate transporter upregulation for the treatment of gliomas should be further investigated and potentially be part of a combination therapy with standard chemotherapeutic agents.

Ridaifen-SB8, a novel tamoxifen derivative, induces apoptosis via reactive oxygen species-dependent signaling pathway.

Tamoxifen is an anticancer agent widely used for treatment of estrogen receptor (ERα)-positive breast cancer. We previously developed a novel synthesis of tamoxifen and its derivatives, named Ridaifens (RIDs). Some of them, including RID-SB8, exhibited a stronger anticancer activity than tamoxifen in ERα-positive MCF-7 cells while having lost the affinity for ERα, suggesting an ERα-independent anticancer mode of action. In this study, we investigated the underlying mechanism by which RID-SB8 exerts anticancer activity. As expected, anticancer activity of RID-SB8 was not influenced upon knockdown of ERα expression in MCF-7 cells. RID-SB8 exerted similar anticancer effects on thirteen ERα-negative cancer cell lines including human gliosarcoma SF539 cells. In SF539 cells, RID-SB8 triggered loss of mitochondrial membrane potential (ΔΨ(m)) and progression of apoptosis accompanied by activation of caspases and translocation of apoptosis-inducing factor (AIF) to the nucleus. Furthermore, it induced reactive oxygen species (ROS), and a ROS scavenger, N-acetylcysteine (NAC), canceled loss of ΔΨ(m) and progression of apoptosis triggered by RID-SB8. Using fifteen human cancer cell lines, we demonstrated a significant correlation between RID-SB8 concentration required for ROS production and that required for cytotoxic effect across these cell lines, but such correlation was not observed for tamoxifen. Finally, the selective induction of ROS and cytotoxic effect on cancer cells by RID-SB8 were confirmed. From these results, we concluded that RID-SB8 exerts an anticancer effect via a mode of action distinct from tamoxifen, and that RID-SB8 could become a promising anticancer lead compound which selectively induces ROS formation and apoptosis in cancer cells.

Gliosarcoma with ependymal and PNET-like differentiation.

A rare case of gliosarcoma which arose in the temporal lobe of a 39-yearold man was reported. The gliomatous area of the tumor showed ependymal differentiation, and also contained immature neuroectodermal tissue resembling a primitive neuroectodermal tumor (PNET) in addition to an ordinary glioblastomatous component. Tumor cells in the PNET-like component were immunoreactive for synaptophysin, CD99, neurogenin 3, and α-internexin, but not for glial fibrillary acidic protein (GFAP), Class III-ß tubulin, or Neu N. The mesenchymal area exhibited a compact fascicular proliferation of atypical spindle cells invested by fine reticulin fibrils. In addition, these cells were immunoreactive for Slug and Twist - transcription factors which are involved in the "epithelial-mesenchymal transition (EMT)" phenomenon. Gliosarcomas containing an ependymal or PNET-like component are rare, and to our knowledge, the present case is the first to be reported whose glial element exhibited differentiation toward these two components. The diverse differentiation in the glial element suggests that the tumor most likely originated from primitive neuroepithelial progenitor cells rather than from the neometaplasia of a glioblastoma. The immunoreactivity for transcription factors in the mesenchymal element indicated that EMT might be involved in the pathogenesis of this very rare type of gliosarcoma.

Synchrotron microbeam radiation therapy induces hypoxia in intracerebral gliosarcoma but not in the normal brain.

PURPOSE: Synchrotron microbeam radiation therapy (MRT) is an innovative irradiation modality based on spatial fractionation of a high-dose X-ray beam into lattices of microbeams. The increase in lifespan of brain tumor-bearing rats is associated with vascular damage but the physiological consequences of MRT on blood vessels have not been described. In this manuscript, we evaluate the oxygenation changes induced by MRT in an intracerebral 9L gliosarcoma model. METHODS: Tissue responses to MRT (two orthogonal arrays (2 × 400Gy)) were studied using magnetic resonance-based measurements of local blood oxygen saturation (MR_SO2) and quantitative immunohistology of RECA-1, Type-IV collagen and GLUT-1, marker of hypoxia. RESULTS: In tumors, MR_SO2 decreased by a factor of 2 in tumor between day 8 and day 45 after MRT. This correlated with tumor vascular remodeling, i.e. decrease in vessel density, increases in half-vessel distances (×5) and GLUT-1 immunoreactivity. Conversely, MRT did not change normal brain MR_SO2, although vessel inter-distances increased slightly. CONCLUSION: We provide new evidence for the differential effect of MRT on tumor vasculature, an effect that leads to tumor hypoxia. As hypothesized formerly, the vasculature of the normal brain exposed to MRT remains sufficiently perfused to prevent any hypoxia.