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Background Brain tumor surgery must balance the benefit of maximal resection against the risk of inflicting severe damage. The impact of increased resection is diagnosis-specific. However, the precise diagnosis is typically uncertain at surgery due to limitations of imaging and intraoperative histomorphological methods. Novel and accurate strategies for brain tumor classification are necessary to support personalized intraoperative neurosurgical treatment decisions. Here, we describe a fast and cost-efficient workflow for intraoperative classification of brain tumors based on DNA methylation profiles generated by low coverage nanopore sequencing and machine learning algorithms. Methods We evaluated 6 independent cohorts containing 105 patients, including 50 pediatric and 55 adult patients. Ultra-low coverage whole-genome sequencing was performed on nanopore flow cells. Data were analyzed using copy number variation and ad hoc random forest classifier for the genome-wide methylation-based classification of the tumor. Results Concordant classification was obtained between nanopore DNA methylation analysis and a full neuropathological evaluation in 93 of 105 (89%) cases. The analysis demonstrated correct diagnosis in 6/6 cases where frozen section evaluation was inconclusive. Results could be returned to the operating room at a median of 97 min (range 91-161 min). Precise classification of the tumor entity and subtype would have supported modification of the surgical strategy in 12 out of 20 patients evaluated intraoperatively. Conclusion Intraoperative nanopore sequencing combined with machine learning diagnostics was robust, sensitive, and rapid. This strategy allowed DNA methylation-based classification of the tumor to be returned to the surgeon within a timeframe that supports intraoperative decision making.

Purpose Constructed from a theoretical framework, the coordinated undermining of survival paths in glioblastoma (GBM) is a combination of nine drugs approved for non-oncological indications (CUSP9; aprepitant, auranofin, captopril, celecoxib, disulfiram, itraconazole, minocycline, quetiapine, and sertraline) combined with temozolomide (TMZ). The availability of these drugs outside of specialized treatment centers has led patients to embark on combination treatments without systematic follow-up. However, no experimental data on efficacy using the CUSP9 strategy in GBM have been reported. Methods Using patient-derived glioblastoma stem cell (GSC) cultures from 15 GBM patients, we described stem cell properties of individual cultures, determined the dose–response relationships of the drugs in the CUSP9, and assessed the efficacy the CUSP9 combination with TMZ in concentrations clinically achievable. The efficacy was evaluated by cell viability, cytotoxicity, and sphere-forming assays in both primary and recurrent GSC cultures. Results We found that CUSP9 with TMZ induced a combination effect compared to the drugs individually (p

Evidence suggests that the growth and therapeutic resistance of glioblastoma (GBM) may be enabled by a population of glioma stem cells (GSCs) that are regulated by typical stem cell pathways, including the WNT/&beta, catenin signaling pathway. We wanted to explore the effect of treating GSCs with a small-molecule inhibitor of tankyrase, G007-LK, which has been shown to be a potent modulator of the WNT/&beta, catenin and Hippo pathways in colon cancer. Four primary GSC cultures and two primary adult neural stem cell cultures were treated with G007-LK and subsequently evaluated through the measurement of growth characteristics, as well as the expression of WNT/&beta, catenin and Hippo signaling pathway-related proteins and genes. Treatment with G007-LK decreased in vitro proliferation and sphere formation in all four primary GSC cultures in a dose-dependent manner. G007-LK treatment altered the expression of key downstream WNT/&beta, catenin and Hippo signaling pathway-related proteins and genes. Finally, cotreatment with the established GBM chemotherapeutic compound temozolomide (TMZ) led to an additive reduction in sphere formation, suggesting that WNT/&beta, catenin signaling may contribute to TMZ resistance. These observations suggest that tankyrase inhibition may serve as a supplement to current GBM therapy, although more work is needed to determine the exact downstream mechanisms involved.

BACKGROUND: Tumor cell invasion is a hallmark of glioblastoma (GBM) and a major contributing factor for treatment failure, tumor recurrence, and the poor prognosis of GBM. Despite this, our understanding of the molecular machinery that drives invasion is limited. METHODS: Time-lapse imaging of patient-derived GBM cell invasion in a 3D collagen gel matrix, analysis of both the cellular invasive phenotype and single cell invasion pattern with microarray expression profiling. RESULTS: GBM invasion was maintained in a simplified 3D-milieue. Invasion was promoted by the presence of the tumorsphere graft. In the absence of this, the directed migration of cells subsided. The strength of the pro-invasive repulsive signaling was specific for a given patient-derived culture. In the highly invasive GBM cultures, the majority of cells had a neural progenitor-like phenotype, while the less invasive cultures had a higher diversity in cellular phenotypes. Microarray expression analysis of the non-invasive cells from the tumor core displayed a higher GFAP expression and a signature of genes containing VEGFA, hypoxia and chemo-repulsive signals. Cells of the invasive front expressed higher levels of CTGF, TNFRSF12A and genes involved in cell survival, migration and cell cycle pathways. A mesenchymal gene signature was associated with increased invasion. CONCLUSION: The GBM tumorsphere core promoted invasion, and the invasive front was dominated by a phenotypically defined cell population expressing genes regulating traits found in aggressive cancers. The detected cellular heterogeneity and transcriptional differences between the highly invasive and core cells identifies potential targets for manipulation of GBM invasion.

Background Despite the well described heterogeneity in glioblastoma (GBM), treatment is standardized, and clinical trials investigate treatment effects at population level. Genomics-driven oncology for stratified treatments allow clinical decision making in only a small minority of screened patients. Addressing tumor heterogeneity, we aimed to establish a clinical translational protocol in recurrent GBM (recGBM) utilizing autologous glioblastoma stem cell (GSC) cultures and automated high-throughput drug sensitivity and resistance testing (DSRT) for individualized treatment within the time available for clinical application. Results From ten patients undergoing surgery for recGBM, we established individual cell cultures and characterized the GSCs by functional assays. 7/10 GSC cultures could be serially expanded. The individual GSCs displayed intertumoral differences in their proliferative capacity, expression of stem cell markers and variation in their in vitro and in vivo morphology. We defined a time frame of 10 weeks from surgery to complete the entire pre-clinical work-up; establish individualized GSC cultures, evaluate drug sensitivity patterns of 525 anticancer drugs, and identify options for individualized treatment. Within the time frame for clinical translation 5/7 cultures reached sufficient cell yield for complete drug screening. The DSRT revealed significant intertumoral heterogeneity to anticancer drugs (p

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15-20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.

Background Clear identification of tumor subtype is the main predictor of patient outcome and ultimately what is considered an adequate level of surgical risk. At brain tumor resection, imaging modalities and intraoperative histology often give an ambigious diagnosis, complicating intraoperative surgical decision-making. Here, we report a nanopore DNA methylation analysis (NDMA) sequencing approach combined with machine learning for classification of tumor entities that could be used intraoperatively. Methods We analyzed 50 biopsies obtained from biobanked tissue (43, prospective) or sampled at surgery (7, intraoperative) from 20 female and 30 male patients with a median age of 8 years. DNA was extracted using spin columns, quantified on a Qubit fluorometer and assessed for purity using NanoDrop spectrophotometer. DNA was then barcoded with the Rapid Barcoding kit from Oxford Nanopore technologies and loaded onto a MinION flow cell. Sequencing was performed for 3 hours (intraoperative) and 24 hours (prospective). Raw reads were basecalled using the Guppy algorithm, then fed into a snakemake workflow (nanoDx pipeline). This generated a report showing the copy number profile, genome-wide methylation status and subclassification of the tumor according to the Heidelberg reference cohort. Results Twelve different tumor classes were discovered within our cohort spanning from WHO Grade I to Grade IV. The results generated by NDMA were concordant with standard neuropathological diagnosis in 43 out of 50 cases (86%). Of the discordant cases, six were due to the biological complexity of the tumor and one case was misclassified by the pipeline. NDMA enabled correct subclassification of 6/7 intraop cases within a mean of 129 minutes. Conclusion NDMA can accurately subclassify tumor entities intraoperatively and guide surgical procedures when preoperative imaging and frozen section evaluation are unclear.

INTRODUCTION Despite being able to characterize pediatric brain tumors such as medulloblastoma and high-grade gliomas using detailed molecular analysis tools, this knowledge hasn’t been translated to better treatment methods. In this project, we aim to create a biobank of pediatric brain tumors (PBTs), characterize samples using next generation molecular diagnostics and identify patient specific drug-treatment options using high-throughput drug screening (HTDS). METHODS To establish tumor spheres from biopsies, we mechanically dissociated the tissue and digested it in trypsin. The cells isolated were cultured in serum free DMEM medium. Immunocytochemistry analysis was done to compare the spheres and original tumor. After the second passage, DNA was extracted and subjected to low-pass whole genome nanopore sequencing. HTDS with a library of FDA/EMA-approved anticancer drugs and investigational compounds was also performed. RESULTS We’ve established tumor sphere cultures that grew to passage two and onwards from five juvenile pilocytic astrocytomas, two gangliogliomas and two midline gliomas. The spheres expressed markers of stem cells (Nestin), neurons (β3-tubulin) and glial (GFAP), similar to the original tumor. Copy number profiling and methylation-based classification of the spheres showed the same alterations and classification as the biopsy. HTDS revealed significant differences in drug sensitivity including patient-specific vulnerabilities to anticancer drugs. CONCLUSION We’ve created a protocol to generate tumor spheres from PBTs. We are also building a biobank comprising high and low grade PBTs. Our tumor spheres maintain the characteristics of the original tumor and can be used for further downstream analysis including drug screening.

Background A major barrier to effective treatment of glioblastoma (GBM) is the large intertumoral heterogeneity at the genetic and cellular level. In early phase clinical trials, patient heterogeneity in response to therapy is commonly observed; however, how tumor heterogeneity is reflected in individual drug sensitivities in the treatment-naïve glioblastoma stem cells (GSC) is unclear. Methods We cultured 12 patient-derived primary GBMs as tumorspheres and validated tumor stem cell properties by functional assays. Using automated high-throughput screening (HTS), we evaluated sensitivity to 461 anticancer drugs in a collection covering most FDA-approved anticancer drugs and investigational compounds with a broad range of molecular targets. Statistical analyses were performed using one-way ANOVA and Spearman correlation. Results Although tumor stem cell properties were confirmed in GSC cultures, their in vitro and in vivo morphology and behavior displayed considerable tumor-to-tumor variability. Drug screening revealed significant differences in the sensitivity to anticancer drugs (p

Exosomes are small extracellular vesicles around 30-100 nm in diameter that are secreted from cells and can be found in most body fluids. Exosomes can be a vital source of biomarkers as they contain various substances (e.g. lipids, RNAs, metabolites and proteins) that can reflect the cell of origin (e.g. cancer cells). For isolation of exosomes present in biological matrices, ultracentrifugation (UC)-based procedures are most common. Other approaches exist, including commercial kits developed for easy and low sample volume isolation. In this study, differential UC and an isolation kit from a major vendor (Total Exosome Isolation Reagent from Thermo Fisher Scientific) were compared. Exosomes were isolated from cell culture media of two different cell sources (patient derived cells from glioblastoma multiforme and the breast cancer cell line MDA-MB-231). For both isolation methods, transmission electron microscopy, dynamic light scattering and western blotting indicated the presence of exosomes. The kit- and UC isolates contained similar amounts of protein measured by the bicinchoninic acid (BCA) assay with absorbance at 562 nm. Using western blot, positive exosome markers were identified in all isolates, and additional exosome markers were identified using MS-based proteomics. For the glioblastoma exosome isolates, the number of proteins identified with liquid chromatography tandem MS (LC-MS/MS) was higher for the UC isolates than the kit isolates when injecting equal protein amounts, contrary to that for the breast cancer exosome isolates. However, negative exosome markers were also found in glioblastoma isolates using LC-MS/MS. Thus, we would not use the term “exosome isolation” as impurities may be present with both isolation methods. Notably, potential biomarkers for both diseases were identified in the isolates using LS-MS/MS. In our opinion, the two isolation methods had rather similar performance, although with some minor differences based on cell of origin.

Evidence indicates that the growth of glioblastoma (GBM), the most common and malignant primary brain cancer, is driven by glioma stem cells (GSCs) resistant to current treatment. As Wnt-signaling is pivotal in stem cell maintenance, we wanted to explore its role in GSCs with the objective of finding distinct signaling mechanisms that could serve as potential therapeutic targets. We compared gene expression in GSCs (n=9) and neural stem cells from the adult human brain (ahNSC; n=3) to identify dysregulated genes in the Wnt signaling pathway. This identified a six-gene Wnt signature present in all nine primary GSC cultures, and the combined expression of three of these genes (SFRP1, SFRP4 and FZD7) reduced median survival of glioma patients from 38 to 17 months. Treatment with recombinant SFRP1 protein in primary cell cultures downregulated nuclear β-catenin and decreased in vitro proliferation and sphere formation in a dose-dependent manner. Furthermore, expressional and functional analysis of SFRP1-treated GSCs revealed that SFRP1 halts cell cycling and induces apoptosis. These observations demonstrate that Wnt signaling is dysregulated in GSC, and that inhibition of the Wnt pathway could serve as a therapeutic strategy in the treatment of GBM.

Aim: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared. Materials & methods: ‘Case study’ exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed. Results: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC–MS. Western blotting and nanoLC–MS also revealed negative exosome markers regarding both isolation approaches. Conclusion: The two isolation methods had an overall similar performance, but we hesitate to use the term ‘exosome isolation’ as impurities may be present with both isolation methods. NanoLC–MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures., Lay abstract Exosomes are small vesicles that are released from biological cells. Exosomes are viewed as being tools for intracellular communication, and there is evidence that cancer cells can release exosomes to, for example, enable metastasis. Exosomes may be very useful for diagnostics, as the detection and characterization of exosomes may allow disease detection at early stages. However, exosomes must be isolated from blood or other biomaterials prior to analysis. This can be a challenging task, and we have here taken a critical look at two familiar isolation techniques, using samples from breast cancer cell lines and brain cancer cells (glioblastoma). We find that the two approaches have comparable performance, and contain protein biomarkers of their cells of origin. However, the isolated exosomes can contain contaminations, which may cloud the analysis in clinical settings. Hence, there is room for new approaches for the isolation of exosomes.

Background: Tissue microenvironment plays key roles in regulating the progression of aggressive tumors. Tumors are uncommon in the early embryo, suggesting that embryonic tissue microenvironments are nonpermissive for tumors. Yet, the effects of embryonic tissue microenvironments on tumor cells have not been extensively studied. We have, therefore, tested the behavior of human glioblastoma multiforme (GBM) cells transplanted into a central neural tissue microenvironment in the chicken embryo. Results: GBM cells were cultured as spheres to enrich for GBM stem cells (GSCs) and transduced with GFP for identification. Within the proliferative embryonic neural tissue, GSC-enriched GBM cells exhibited reduced proliferation and survival, altered gene expression, and formed no tumors, in marked contrast to their aggressive behavior in vitro and tumor formation in other tissue microenvironments including the chorioallantoic membrane of the chicken embryo and the brain of adult severe combined immunodeficiency (SCID) mice. Surviving cells in the spinal neural tube exhibited tumor-atypical expression profiles of neuron-, glia-, stem cell-, and tumor-related genes. Conclusions: Embryonic neural tissue provides a poor environment for GBM cell survival and tumor formation, and redirects differentiation toward a more benign phenotype. Understanding the anti-tumorigenic effects of this embryonic tissue microenvironment could provide opportunities to develop novel therapies for GBM treatment. Developmental Dynamics 242:1078-1093, 2013. © 2013 Wiley Periodicals, Inc.

Background The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting. Methods We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 107 cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456. Results Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test). Conclusion These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival. Electronic supplementary material The online version of this article (doi:10.1007/s00262-013-1453-3) contains supplementary material, which is available to authorized users.

Traditional in vitro culturing of tumor cells has been shown to induce changes so that cultures no longer represent the tumor of origin. Serum-free culturing conditions are used in a variety of cancers to propagate stem-like cells in vitro. Limited reports, however, exist on the effects of such propagation. We have compared cells from brain tumor biopsies cultivated under serum-free conditions at passages 2 and 10 to describe the effects of in vitro culturing. We were able to establish cell lines from 7 of 10 biopsies from patients with glioblastoma. The cell lines adapted to conditions and had 2.2 times increased population doubling rate at later passages. Karyotyping and comparative genomic hybridization analysis revealed that all examined cell lines had cytogenetic aberrations commonly found in glioblastomas, and there were only minor differences between tumor and early and late passages in the same culture. Whole-transcriptome analysis shows that tumors had interindividual differences. Changes in the overall expression patterns through passaging were modest, with a significant change in only 14 genes; the variation among cultures was, however, reduced through passages. The ability to differentiate differed among tumors but was maintained throughout passaging. The cells initiated tumors upon transplantation to immunodeficient mice with differing phenotypes, but a given cell culture maintained tumor phenotype after serial cultivation. The cultures established maintained individual characteristics specific to culture identity. Thus, each cell culture reflects an image of the tumor--or a personalized model--from which it was derived and remains representative after moderate expansion.

Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies. To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways. Nine genes: PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.

We recently reported that the endothelin (ET) receptor subtypes ET(A) and ET(B) are targeted to distinct intracellular destinations upon agonist stimulation (Bremnes, T., Paasche, J. D., Mehlum, A., Sandberg, C., Bremnes, B., and Attramadal, H. (2000) J. Biol. Chem. 275, 17596-17604). The ET(A) receptor was shown to follow the recycling route of transferrin, whereas ET(B) is targeted to lysosomes for degradation. In the present study we have investigated the mechanisms of ET receptor subtype-specific targeting to distinct intracellular trafficking pathways. Truncation mutants of the ET(A) and ET(B) receptors with deletions of the cytoplasmic carboxyl-terminal tail distal to the palmitoylation site were found to mediate inositol phosphate accumulation and to internalize upon agonist stimulation, although internalization occurred at a slower rate as compared with the wild-type receptors. However, the truncated ET(A) receptor was no longer able to undergo recycling. Rather, both truncation mutants were recognized by beta-arrestin for recruitment to endocytosis and were sorted to lysosomes by a dynamin-dependent internalization pathway. Furthermore, studies of chimeric ET(A) and ET(B) receptors where the cytoplasmic tail of ET(A) was swapped with the corresponding domain of ET(B), and vice versa, revealed that the cytoplasmic tail of ET(B) is required for efficient lysosomal sorting and that signals for targeting to recycling reside in the cytoplasmic tail of the ET(A) receptor.

The effects of endothelin (ET) are mediated via the G protein-coupled receptors ET(A) and ET(B). However, the mechanisms of ET receptor desensitization, internalization, and intracellular trafficking are poorly understood. The aim of the present study was to investigate the molecular mechanisms of ET receptor regulation and to characterize the intracellular pathways of ET-stimulated ET(A) and ET(B) receptors. By analysis of ET(A) and ET(B) receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed betaARK, beta-arrestin-1, beta-arrestin-2, or dynamin as well as dominant negative mutants of these regulators, we have demonstrated that both ET receptor subtypes follow an arrestin- and dynamin/clathrin-dependent mechanism of internalization. Fluorescence microscopy of Chinese hamster ovary and COS cells expressing green fluorescent protein (GFP)-tagged ET receptors revealed that the ET(A) and ET(B) subtypes were targeted to different intracellular routes after ET stimulation. While ET(A)-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ET(B)-GFP was targeted to lysosomes after ET-induced internalization. Both receptor subtypes colocalized with Rab5 in classical early endosomes, indicating that this compartment is a common early intermediate for the two ET receptors during intracellular transport. The distinct intracellular routes of ET-stimulated ET(A) and ET(B) receptors may explain the persistent signal response through the ET(A) receptor and the transient response through the ET(B) receptor. Furthermore, lysosomal targeting of the ET(B) receptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.

Sphere forming assays have been useful to enrich for stem like cells in a range of tumors. The robustness of this system contrasts the difficulties in defining a stem cell population based on cell surface markers. We have undertaken a study to describe the cellular and organizational composition of tumorspheres, directly comparing these to neurospheres derived from the adult human subventricular zone (SVZ). Primary cell cultures from brain tumors were found to contain variable fractions of cells positive for tumor stem cell markers (CD133 (2-93%)/SSEA1 (3-15%)/CXCR4 (1-72%)). All cultures produced tumors upon xenografting. Tumorspheres contained a heterogeneous population of cells, but were structurally organized with stem cell markers present at the core of spheres, with markers of more mature glial progenitors and astrocytes at more peripheral location. Ultrastructural studies showed that tumorspheres contained a higher fraction of electron dense cells in the core than the periphery (36% and 19%, respectively). Neurospheres also contained a heterogeneous cell population, but did not have an organization similar to tumorspheres. Although tumorspheres clearly display irregular and neoplastic cells, they establish an organized structure with an outward gradient of differentiation. We suggest that this organization is central in maintaining the tumor stem cell pool.

OBJECTIVE: To directly compare stem cells from the normal adult human brain (adult human neural stem cells [AHNSC]), Grade II astrocytomas (AC II), and glioblastoma multiforme (GBM), with respect to proliferative and tumor-forming capacity and differentiation potential. METHODS: Cells were isolated from tissue obtained during epilepsy surgery (AHNSCs) or tumor surgery (glioma stem cells [GSC]). They were cultured and investigated in vitro or after transplantation in immunodeficient mice. RESULTS: Under identical experimental conditions, the following were found: 1) GBM stem cells formed tumors after orthotopic transplantation; AHNSCs showed no sign of tumor formation; 2) GSCs showed a significantly higher growth rate and self-renewal capacity; 3) both the growth rate and telomerase expression were high in GSCs and correlated with malignancy grade (GBM higher than AC II); AHNSCs had low telomerase expression; 4) GSCs invaded normal neurospheres, not vice versa; 5) both AHNSCs and stem cells from AC II and GBM responded to differentiation cues with a dramatic decrease in the proliferation index (Ki-67); 6) GSCs differentiated faster than AHNSCs; 7) upon differentiation, AHNSCs produced normal glia and neurons; GSCs produced morphologically aberrant cells often expressing both glial and neuronal antigens; and 8) differentiation of AHNSCs resulted in 2 typical functional phenotypes: neurons (high electrical membrane resistance, ability to generate action potentials) and glial cells (low membrane resistance, no action potentials). In contrast, GSCs resulted in only 1 functional phenotype: cells with high electrical resistance and active membrane properties capable of generating action potentials. CONCLUSION: AHNSCs and stem cells from AC II and GBM differ with respect to proliferation, tumor-forming capacity, and rate and pattern of differentiation.

There is an increasing demand for rapid, sensitive and robust methods for toxicity testing of single chemicals, complex mixtures and environmental samples. The objective of this work was to validate and use a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes as a multi-endpoint in vitro bioassay for toxicity characterisation of river sediments from four areas of the Sava and Krupa Rivers (Slovenia). The endpoints were chosen to encompass acute toxicity (cytotoxicity) as well as sub-lethal biomarker and effect endpoints such as metabolic inhibition, DNA damage (Fast Micromethod), endocrine disruption (estrogenicity), and 7-ethoxyresorufin O-deethylase (EROD) activity. Results from these studies show that the primary hepatocyte culture was able to successfully detect effects of single model chemicals in all endpoints analysed. Furthermore, the bioassays were also able to discriminate between contaminated and less contaminated sediments for a number of endpoints such as cytotoxicity, metabolic inhibition and induction of EROD activity, although no increase in DNA damage and estrogenicity was observed above background at any site. The present study shows that primary fish hepatocytes may be used to determine multiple mechanisms of toxic action and that a holistic assessment of effects may improve our understanding of cellular toxicity of complex mixtures such as sediments.

Some environmental pollutants have the ability to alter the endocrine function in fish through interaction with the estrogen receptor (ER). Many of these chemicals are also able to interfere with the endocrine system through other mechanisms of action, however. The plasma sex steroid-binding protein (SBP), which is involved in regulating circulating levels of endogenous sex steroids, has recently been proposed to contribute to pollutant induced disruption of endocrine homeostasis. The objective of the present work was to determine whether industrial effluents contain chemicals that are able to modulate the endocrine system through interference with the function of the ER and SBP using in vitro biological assays (bioassays) from rainbow trout. The results show that solid phase extracts of process water (produced water) from an oil production facility in the North Sea and a land-based oil refinery contain chemicals that are able to induce estrogenic effects as well as displace natural sex steroid 17beta-estradiol from the SBP. The bioactive chemicals were found to be partly resistant to biological degradation, but the identity of the chemicals was not determined. The alkylphenol 4-tert-butylphenol, which is known to occur in effluents from various oil production facilities, was found to be estrogenic and displace 17beta-estradiol from the SBP and may thus contribute to the observed endocrine disrupting activity.

Adrenomedullin (AM) is a potent vasorelaxing peptide with natriuretic and diuretic actions. Recent data indicate that AM may function as an endogenous regulator of cardiac function. We investigated to what extent AM, the AM receptor subtypes, and AM receptor-associated proteins were regulated in cardiomyocytes and non-cardiomyocytes of rats with congestive heart failure (CHF), and whether such regulation was paralleled by corresponding alterations of functional responses to AM. Cardiomyocytes and non-cardiomyocytes were isolated from myocardial tissue of rats 7 days after induction of myocardial infarction or sham operation. AM immunoreactivity was found in cardiomyocytes, endothelial cells, and fibroblasts. Robust increase of AM mRNA levels was observed both in the cardiomyocytes and in the non-cardiomyocytes of CHF rats compared to that of sham-operated rats (2.7-fold and 3.7-fold, respectively, P

Background —Increased plasma adrenomedullin (ADM) levels have been reported in congestive heart failure (HF). The present study was designed to investigate myocardial regulation of the different components of the ADM signaling system (ADM, ADM receptor, and receptor-activity–modifying protein-2, RAMP-2) during ischemic HF in rats and to identify the cells in the myocardium displaying ADM-like immunoreactivity (ADM-ir). Furthermore, the effects of endothelin (ET) receptor antagonism on expression of the myocardial ADM system during HF were investigated. Methods and Results —Northern blot analysis revealed increased ADM mRNA expression in the nonischemic left ventricle, with maximal levels 28 days after induction of myocardial infarction (1.5-fold, P P P A /ET B receptor antagonist bosentan (100 mg · kg −1 · day −1 PO) for 15 days prevented the increase of RAMP-2 mRNA. Conclusions —The study demonstrates a concerted induction of several components of the myocardial ADM signaling system during postinfarction failure and that the vessels are the main source of myocardial ADM. Our observations indicate a role for ADM as an autocrine/paracrine factor during ventricular remodeling after myocardial infarction.

Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the most malignant human cancers (median survival 14 months from diagnosis despite combined surgery/irradiation/chemotherapy). Evidence from our and other groups indicates that glioblastoma stem-like cells (GSCs) contained within these tumors are critically important in GBM initiation, progression, and resistance to therapy. To identify new targets for glioblastoma treatment, we have systematically compared gene expression in nine primary GBM cell cultures to that in five primary neural stem cell cultures from the adult human brain (ahNSCs), using microarrays. Rigorous bioinformatic filtering identified 20 genes whose RNA expression levels were very high in primary GSC cultures but were undetectable in ahNSCs. The identified genes are involved in cell-cycle/division, epigenetic regulation, signaling and down-regulation of tumor-suppressors. Several of the candidate genes are implicated in cancer (breast, ovary and colon), while others have no known associations to cancer or have unknown functions. A total of nine new GBM primary cultures were further analyzed by real-time polymerase chain reaction (qPCR), Western blot and Immunohistochemistry on tissue sections and cell cultures. qPCR confirmed the increased expression of 17 of the candidate genes. These results were in good accordance with the information provided by public data bases (Rembrant, TCGA). Western blot performed on 17 candidates showed increased protein levels in 9 cases thus demarcating these as best candidates for molecular targeting. The rest of the proteins featured aberrant isoforms, probably due to alternate splicing, modifications, cleavage or degradation. Further bioinformatics analysis identified a subset of 5 candidates that are particularly highly up-regulated in GBM when compared to low-grade gliomas. To explore the functional importance of the potential target genes we established lentiviral-based shRNA delivery and started to test the potential of gene knock-downs (KDs) to inhibit growth of tumor cells. Our preliminary results look very promising. Three different lentiviral vectors, designed to silence a candidate gene encoding a tumor-suppressor binding protein kinase, were tested in three different primary GBM cultures and achieved a KD efficiency of 60–70%. We used proliferation essays to describe the growth speed of GBMs. To estimate relative numbers of sphere-forming cells (widely referred to as cancer stem cells) we performed sphere-forming assays (SFAs). The KD of the protein kinase gene resulted both in dramatic reduction of growth speed (50–70%) and in the number of sphere-forming cells (50–70%). In addition, the volume of the spheres was reduced to 50%. Cell cycle analysis revealed that there was doubling of the number of KD cells arrested in G2/M phase compared to non-silencing controls. There was also an increase (20–65%) in the proportion of apoptotic cells in 2 out of 3 GBM KD cultures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B202.

Glioblastoma Multiforme (GBM) is the most common primary brain malignancy and despite multimodal therapy including surgery, irradiation and chemotherapy median survival remains 12–15 months. Increasing body of evidence from our and other groups is confirming the existence of a stem-like cell population within these tumors believed to confer tumor growth, invasiveness, and therapy resistance. Adult human neural stem cells (aHNSCs) are suggested to be the cells of origin for the glioblastoma stem-like cells (GSCs). To identify new molecular targets we performed a micro-array analysis on the transcriptome of 33000 genes identifying differentially expressed genes in 5 aHNSC and 9 GSC cultures. Amongst the genes highly up-regulated in GBM, several were involved in cell division and the regulation of cell cycle. For further analysis, we chose the Chromatid Segreration Mediator (CSM), a centromere specific gene important for appropriate chromatid segregation during mitosis. This candidate gene was strongly expressed in GSCs but was almost undetectable in aHNSCs. Abundance of the CSM protein may lead to aneupleudy, which is a common feature of GBMs. The high levels of CSM mRNA were confirmed by analyzing 16 GSC cultures with micro-array and qPCR. CSM was also over-expressed on protein level, and was found to be highly immunopositive in glioblastoma biopsies compared to the normal human brain. In silico analysis, using Rembrandt, identifies CSM-gene over-expression as a negative prognostic indicator in glioma patients. In order to study the functional role of this gene in GBM, we used lentiviral-based knock-down (KD) technology to obtain specific down regulation of the gene. We transduced different shRNA constructs in 2 GBM cultures and achieved KD-efficieny of 55–65%. Growth of tumors was measured by proliferation assay and the relative number of sphere-forming cells (widely known as GSCs) was quantified with a sphere-formation assay. In our KD-cultures we observed a moderate reduction of proliferation (25%). Interestingly, the sphere forming capacity was dramatically reduced by 60–70%, indicating a significant decrease in number of GSCs. Cell-cycle analysis revealed that it was 30–40% less cells in G2/M-phase and 15–20% more cells in G0/G1-phase in the KD compared to non-silencing control. We find our results very promising and suggest that Chromatid Segregation Mediator gene might have potential as a novel therapeutic target for GBM management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B186.

Glioblastoma is the most common brain tumor. Median survival in unselected patients is

Background Glioblastomas are invasive therapy resistant brain tumors with extremely poor prognosis. The Glioma initiating cell (GIC) population contributes to therapeutic resistance and tumor recurrence. Targeting GIC-associated gene candidates could significantly impact GBM tumorigenicity. Here, we investigate a protein kinase, PBK/TOPK as a candidate for regulating growth, survival and in vivo tumorigenicity of GICs. Methods PBK is highly upregulated in GICs and GBM tissues as shown by RNA and protein analyses. We knocked down PBK using shRNA vectors and inhibited the function of PBK protein with a pharmacological PBK inhibitor, HITOPK-032. We assessed viability, tumorsphere formation and apoptosis in three patient derived GIC cultures. Results Gene knockdown of PBK led to decreased viability and sphere formation and in one culture an increase in apoptosis. Treatment of cells with inhibitor HITOPK-032 (5 μM and 10 μM) almost completely abolished growth and elicited a large increase in apoptosis in all three cultures. HI-TOPK-032 treatment (5 mg/kg and 10 mg/kg bodyweight) in vivo resulted in diminished growth of experimentally induced subcutaneous GBM tumors in mice. We also carried out multi-culture assays of cell survival to investigate the relative effects on GICs compared with the normal neural stem cells (NSCs) and their differentiated counterparts. Normal NSCs seemed to withstand treatment slightly better than the GICs. Conclusion Our study of identification and functional validation of PBK suggests that this candidate can be a promising molecular target for GBM treatment.