Human Glioblastoma Stem-like Cells Research Articles

Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

Glioblastoma (GB) is notoriously resistant to therapy. GB genesis and progression are driven by glioblastoma stem-like cells (GSCs). One goal for improving treatment efficacy and patient outcomes is targeting GSCs. Currently, there are no universal markers for GSCs. Glycoprotein A repetitions predominant (GARP), an anti-inflammatory protein expressed by activated regulatory T cells, was identified as a possible marker for GSCs. This study evaluated GARP for the detection of human GSCs utilizing a multidimensional experimental design that replicated several features of GB: (1) intratumoral heterogeneity, (2) cellular hierarchy (GSCs with varied degrees of self-renewal and differentiation), and (3) longitudinal GSC evolution during GB recurrence (GSCs from patient-matched newly diagnosed and recurrent GB). Our results indicate that GARP is expressed by GSCs across various cellular states and disease stages. GSCs with an increased GARP expression had reduced self-renewal but no alterations in proliferative capacity or differentiation commitment. Rather, GARP correlated inversely with the expression of GFAP and PDGFR-α, markers of astrocyte or oligodendrocyte differentiation. GARP had an abnormal nuclear localization (GARPNU+) in GSCs and was negatively associated with patient survival. The uniformity of GARP/GARPNU+ expression across different types of GSCs suggests a potential use of GARP as a marker to identify GSCs.

Dauricine attenuates Oct4/sonic hedgehog co-activated stemness and induces reactive oxygen species-mediated mitochondrial apoptosis via AKT/β-catenin signaling in human neuroblastoma and glioblastoma stem-like cells.

Neuroblastoma and glioblastoma are primary malignant tumors of the nervous system, with frequent relapse and limited clinical therapeutic drugs. The failure of their treatment is due to the tumor cells exhibiting cancer stem-like cells (CSLCs) properties. Octamer binding transcription factor 4 (Oct4) is involved in mediating CSLCs, our previous work found that Oct4-driven reprogramming of astrocytes into induced neural stem cells was potentiated with continuous sonic hedgehog (Shh) stimulation. In this study, we aimed to study the importance of Oct4 and Shh combination in the stemness properties induction of neuroblastoma and glioblastoma cells, and evaluate the anti-stemness effect of dauricine (DAU), a natural product of bis-benzylisoquinoline alkaloid. The effect of Oct4 and Shh co-activation on cancer stemness was evaluated by tumor spheres formation model and flow cytometry analysis. Then the effects of DAU on SH-SY5Y and T98-G cells were assessed by the MTT, colony formation, and tumor spheres formation model. DAU acts on Oct4 were verified using the Western blotting, MTT, and so on. Mechanistic studies were explored by siRNA transfection assay, Western blotting, and flow cytometry analysis. We identified that Shh effectively improved Oct4-mediated generation of stemness in SH-SY5Y and T98-G cells, and Oct4 and Shh co-activation promoted cell growth, the resistance of apoptosis. In addition, DAU, a natural product, was found to be able to attenuate Oct4/Shh co-activated stemness and induce cell cycle arrest and apoptosis via blocking AKT/β-catenin signaling in neuroblastoma and glioblastoma, which contributed to the neuroblastoma and glioblastoma cells growth inhibition by DAU. In summary, our results indicated that the treatment of DAU may be served as a potential therapeutic method in neuroblastoma and glioblastoma.

EPCO-19. ADAPTIVE RESPONSES TO GENOME-WIDE DNA DAMAGE RESULT IN TOPOLOGIC GENOME REORGANIZATION IN GLIOBLASTOMA

Abstract In glioblastoma, treatment with radiation and chemotherapy leads to DNA-damage and most DNA breaks are faithfully repaired, but the impact on the epigenome is largely unknown. Using newly developed tools to enable these studies, we hypothesize that genome-wide DNA damage leads to local alterations in DNA-methylation, genome organization, and results in persistent gene-expression alterations near sites of repaired damage. We use patient-derived human glioblastoma stem-like cells (GSCs) as a model. DNA breaks are induced using (i) irradiation or (ii) a novel “multi-cut” CRISPR-Cas9 DNA break system followed by multi-omic profiling. With radiation, we find significant and wide-spread alterations in DNA-methylation after treating multiple glioblastoma cultures. However, it is challenging to study local alterations around sites of radiation induced damage because breaks are introduced at different sites in each cell, resulting in stochastic DNA methylation alterations. To circumvent this issue, we developed a multi-cut CRISPR-Cas9 DNA break system that targets 142 or 483 pre-defined loci. Induction of pre-mapped genome-wide cuts reproduces a similar level of toxicity as standard doses of radiation. To assess repair efficiency and confirm induction of breaks, we performed targeted sequencing of the 142 or 483 sites to allow for high coverage sequencing. To understand how DNA damage may lead to regional epigenetic and 3D chromatin organization changes, we performed HiC, Methylation-seq, ChIP-seq of the chromatin organizing factor CTCF and enhancer marker H3K27ac, as well as RNA-seq, before and after cut induction. Our findings show significant mega-base scale alterations in chromatin contacts centered around cut sites, enrichment of DNA methylation alterations at regulatory elements and altered gene-expression. The findings here provide a mechanistic view of the interplay between genome-wide DNA damage, DNA methylation and genome re-organization, and have wide ranging implications for the effect of DNA damage on the epigenome in glioblastoma.

DNA Damage Drives DNA Methylation and 3D Chromatin Organization Alterations in Glioblastoma

<h3>Purpose/Objective(s)</h3> The objective of this study is to determine how the epigenome is locally altered after DNA damage and investigate the impact of these changes on 3D chromatin organization, gene regulation and treatment resistance. In glioblastoma, treatment with radiation and chemotherapy leads to DNA-damage and most DNA breaks are faithfully repaired, but the impact on the epigenome is largely unknown. Since epigenetic alterations such as DNA-methylation can impact the binding of factors involved in chromatin folding, and transcription factors that bind regulatory elements, the downstream impact on gene regulation could lead to the emergence of treatment resistance. Using newly developed tools to enable these studies, we hypothesize that genome-wide DNA damage leads to local alterations in DNA-methylation, genome organization, and results in persistent gene-expression alterations near sites of repaired damage. <h3>Materials/Methods</h3> We use patient-derived human glioblastoma stem-like cells (GSCs) as a model. DNA breaks are induced using (i) irradiation or (ii) a novel "multi-cut" CRISPR-Cas9 DNA break system. Genomic profiling methods include HiC (genome-wide chromosome conformation capture), DNA-methylation profiling (bisulfite sequencing), targeting DNA-seq and RNA-seq. <h3>Results</h3> With radiation, we find significant and wide-spread alterations in DNA-methylation after treating multiple glioblastoma cultures. It is difficult to study local alterations around sites of radiation induced damage because breaks are introduced at different sites in each cell, resulting in stochastic DNA methylation alterations. To circumvent this issue, we developed a multi-cut CRISPR-Cas9 DNA break system that targets 142 or 483 pre-defined loci. Induction of pre-mapped genome-wide cuts reproduces a similar level of toxicity as standard doses of radiation. To assess repair efficiency (i.e., deletions, translocations), we employed a custom panel of DNA probes that flank the 142 or 483 sites to allow for high coverage sequencing at each site following cut induction. To understand how DNA damage may lead to local epigenetic alterations and 3D chromatin organization changes, we performed HiC (genome-wide chromosome conformation capture), before and after cut induction, and subjected cultures to further downstream analysis. Our findings show significant mega-base scale alterations in chromatin contacts centered around cut sites, enrichment of DNA methylation alterations and altered gene-expression. <h3>Conclusion</h3> The multi-cut CRISPR-Cas9 DNA break system, which we develop and characterize here, can be rapidly employed in cell lines or animal models to study pre-mapped genome-wide DNA damage, and has widespread applicability in the radiobiology and DNA damage and repair fields. The findings here provide a mechanistic view of the interplay between genome-wide DNA damage, DNA methylation and genome re-organization, and have wide-ranging implications for the effect of DNA damage on the epigenome.

49P Antitumor effects of cationic dendritic molecules and their complexes with microRNA in glioblastoma stem-like cells

An important way for anti-tumor drug development is using of molecular targets involved in the regulatory pathways during tumorogenesis. As it was found microRNAs (miRs) are active players in this processes. Manipulations with miR-34a and onco-miR-21 demonstrated their effectiveness as antitumor intervention in glioblastoma (GBM), so stable and efficient nanocarriers should be found. One of the promising approach is using of dendrimers – highly symmetrical hyperbranched polymers. The aim of our study was evaluation of effects of treatment by complexes of dendrimers with miRs on human glioblastoma stem-like cells (GSCs).

CBIO-14. SINGLE CELL RNA SEQUENCING ANALYSIS OF HUMAN GLIOBLASTOMA STEM-LIKE CELL CULTURES AND XENOGRAFT TUMORS

Abstract Single cell RNA-seq (scRNA-seq) studies for glioma have yielded critical insight into intratumoral heterogeneity and developmental gene expression patterns for primary gliomas. One key conclusion from these studies is that each tumor represents a complex, yet maligned, neuro-developmental ecosystem, harboring diverse cell types, which presumably contribute to tumor growth and homeostasis in specific ways (e.g., vascular mimicry, immune evasion, recreating NSC niches, neural injury responses, etc.). Here, to better understand experimental models of human glioblastoma (GB), we performed single cell RNA-seq analysis of human GB stem-like cells (GSCs) of distinct tumor subtypes (mesenchymal and proneural) during their in vitro culture in serum-free conditions and also during tumor formation in immunocompromised mice. This analysis revealed surprising differences between in vitro and in vivo grown GSCs. Among our results, we find that in vivo mesenchymal GSCs are capable of transitioning to proneural-like states, while proneural GSCs are capable of transitioning to mesenchymal states. We characterize cycling cells based on expression of and G2/M and S phase makers, estimate RNA velocity, and examine different developmental trajectories arising in vitro and in vivo. We also compare and discuss different analysis pipelines for scRNA-seq data.

P10.02 Combined methods of a micropump system and a chronic cranial window allows tumor observation with multi photon laser scanning microscopy under continuous treatment

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

The Radiosensitizing Effect of AZD0530 in Glioblastoma and Glioblastoma Stem-Like Cells.

AZD0530, a potent small-molecule inhibitor of the Src kinase family, is an anticancer drug used in the treatment of various cancers. In the case of glioblastoma (GBM), where resistance to radiotherapy frequently occurs, Src kinase is known as one of the molecules responsible for imparting radioresistance to GBM. Thus, we evaluated the effect of AZD0530 on the radiosensitivity of human GBM cells and human glioblastoma stem-like cells (GSCs). We show that Src activity of GBM and GSC is increased by radiation and inhibited by AZD0530, and using clonogenic assays, AZD0530 enhances the radiosensitivity of GBM and GSCs. Also, AZD0530 induced a prolongation of radiation-induced γH2AX without specific cell cycle and mitotic index changes, suggesting that AZD0530-induced radiosensitization in GBM cells and GSCs results from the inhibition of DNA repair. In addition, AZD0530 was shown to inhibit the radiation-induced EGFR/PI3K/AKT pathway, which is known to promote and regulate radioresistance and survival of GBM cells by radiation. Finally, mice bearing orthotopic xenografts initiated from GBM cells were then used to evaluate the in vivo response to AZD0530 and radiation. The combination of AZD0530 and radiation showed the longest median survival compared with any single modality. Thus, these results show that AZD0530 enhances the radiosensitivity of GBM cells and GSCs and suggest the possibility of AZD0530 as a clinical radiosensitizer for treatment of GBM.

Rapid Processing and Drug Evaluation in Glioblastoma Patient-Derived Organoid Models with 4D Bioprinted Arrays

SummaryGlioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.

Curcumin Loaded in Niosomal Nanoparticles Improved the Anti-tumor Effects of Free Curcumin on Glioblastoma Stem-like Cells: an In Vitro Study

Using a novel curcumin-loaded niosome nanoparticle (CM-NP), the present study was designed to evaluate the effect of curcumin on human glioblastoma stem-like cells (GSCs). CM-NP has a diameter of ~ 60 nm and a zeta potential of ~ − 35 mV with a constant physicochemical stability. The cytotoxic effects of free curcumin (CM) and CM-NP were investigated on GSCs obtained during the removal of a brain tumor. Both CM and CM-NP caused a dose-dependent decrease in cell proliferation and viability of GSCs. The IC50 values of CM and CM-NP on GSCs were 50 and 137 μg/ml after 24 h, respectively. CM-NP exerted significantly higher effects on GSC viability, apoptosis, cell cycle arrest, and the expression of Bax, a pro-apoptotic marker, compared with CM. In addition, the migration of GSCs was significantly impaired following the administration of CM-NP compared with CM. Furthermore, CM-NP significantly increased the values of reactive oxygen species and decreased the mRNA expressions of NF-κB and IL-6 of GSCs compared with CM. Our data also revealed that CM-NP could significantly reduce the invasiveness of GSCs compared with CM, possibly via MCP-1-mediated pathways. In addition, CM-NP exhibited a significantly greater inhibitory effect on colony formation of GSCs compared with CM. These data indicate that CM-NP exhibited stronger anti-tumor effects on GSCs than CM. Although further in vivo investigations are warranted, our results suggest that CM-NP could be an ideal carrier to deliver curcumin for potential therapeutic approaches into glioblastoma.

RND1 regulates migration of human glioblastoma stem-like cells according to their anatomical localization and defines a prognostic signature in glioblastoma.

Despite post-operative radio-chemotherapy, glioblastoma systematically locally recurs. Tumors contacting the periventricular zone (PVZ) show earlier and more distant relapses than tumors not contacting the PVZ. Since glioblastoma stem-like cells (GSCs) have been proposed to play a major role in glioblastoma recurrence, we decided to test whether GSC migration properties could be different according to their anatomical location (PVZ+/PVZ–). For that purpose, we established paired cultures of GSCs from the cortical area (CT) and the PVZ of glioblastoma patient tumors. We demonstrated that PVZ GSCs possess higher migration and invasion capacities than CT GSCs. We highlighted specific transcriptomic profiles in PVZ versus CT populations and identified a down-regulation of the RhoGTPase, RND1 in PVZ GSCs compared to CT GSCs. Overexpression of RND1, dramatically inhibited PVZ GSC migration and conversely, downregulation of RND1 increased CT GSC migration. Additionally, transcriptomic analyses also revealed a down-regulation of RND1 in glioblastoma compared to normal brain. Using the glioblastoma TCGA database, low levels of RND1 were also shown to correlate with a decreased overall survival of patients. Finally, based on signaling pathways activated in patients with low levels of RND1, we identified an RND1low signature of six genes (MET, LAMC1, ITGA5, COL5A1, COL3A1, COL1A2) that is an independent prognostic factor in glioblastoma. These findings contribute to explain the shorter time to progression of patients with PVZ involvement and, point out genes that establish the RND1low signature as key targets genes to impede tumor relapse after treatment.

Abstract 4171: Preferential radioresistance of glioblastoma stem-like tumor cells that migrate to the olfactory bulb

Abstract Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (GSCs) simulate many of the characteristics of GBMs in situ including extensive invasiveness and migration. Using a GSC orthotopic brain tumor model, we tested the hypothesis that location within the brain microenvironment influences the radiosensitivity of GBM cells. At 35 days after implantation of CD133+ NSC11 GSCs into the right striatum of nude mice, tumor cells are detectable throughout the striatum, the corpus callosum and the olfactory bulb. To evaluate radioresponse of tumor cells in each brain region, mice were injected with the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine), which is incorporated into DNA during S-phase and allows for the identification of proliferating cells. Specifically, NSC11 brain tumor xenografts received a single dose of 10Gy, which results in an approximate 13-day survival advantage, followed by 3 daily intraperitoneal (IP) injections of CldU initiated every 4 days following irradiation. Subsequent immunohistochemical analyses of CldU incorporation along with the GSC marker SOX2 was then used to determine the percentage of proliferating tumor cells in the corpus callosum, striatum, and olfactory bulb. When CldU injections were initiated 4 days post-10Gy, 4% of tumor cells in the corpus callosum, 0.75% in the striatum, and 35% in the olfactory bulb were CldU positive. These values can be compared to 55%, 53%, and 56% CldU positive tumor cells in the corpus callosum, striatum, and olfactory bulb, respectively, of the sham irradiated tumors. These data indicate that by 4 days after irradiation significantly more tumor cells in the olfactory bulb were proliferating than in the corpus callosum or striatum. CldU injections initiated 12 days post-10Gy resulted in 7% of tumor cells in the corpus callosum, 4% in the striatum, and 60% in the olfactory bulb being CldU positive. Finally, when CldU delivery was begun 20 days post-10Gy approximately 17% of tumor cells in the corpus callosum, 15% in the striatum, and 85% in the olfactory bulb were CldU positive. These data suggest that after irradiation, GBM tumor cells located in the olfactory bulb recover and begin to proliferate sooner than those in the striatum or corpus callosum. As a measure of radiation-induced DNA double strand breaks, γH2AX foci were quantified in the tumor cells located in the corpus callosum, striatum, and olfactory bulb on day 35 post-implantation of NSC11 cells. At 0.5h after irradiation, no difference was detected in the mean number of γH2AX foci per tumor cell in the corpus callosum, striatum, or olfactory bulb; no consistent differences were detected out to 6h post-irradiation. While the mechanism remains to be defined, analysis of tumor cell proliferation indicates a preferential radioresistance of GBM cells located in the olfactory bulb. Citation Format: Cindy R. Timme, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. Preferential radioresistance of glioblastoma stem-like tumor cells that migrate to the olfactory bulb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4171.

Correction: Wnt5a Drives an Invasive Phenotype in Human Glioblastoma Stem-like Cells.

Correction: Wnt5a Drives an Invasive Phenotype in Human Glioblastoma Stem-like Cells.

Abstract 5851: Detection of proliferating tumor cells in orthotopic xenografts initiated from glioblastoma stem-like cells and the influence of radiation

Abstract Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (GSCs) simulate many of the characteristics of GBMs in situ including extensive invasiveness, phenotypic heterogeneity and radioresistance. As an approach to investigating the relationship between these aspects of GBM biology and tumor cell proliferation, we used the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine), which is incorporated into DNA during S-phase, to identify proliferating cells in orthotopic brain tumor xenografts. Specifically, CD133+ NSC11 GSCs were implanted into the right striatum of nude mice; at various times after tumor cell implantation CldU was delivered in a series of daily intraperitoneal (IP) injections. Subsequent immunohistochemical analyses of CldU incorporation along with the GSC marker SOX2 was then used to determine the percentage of proliferating tumor cells in the mouse brain. NSC11 tumors are first detectable using bioluminescent imaging (BLI) at day 21 post-implantation. After 4 daily injections of CldU beginning on Day 21, approximately 60% and 30% of tumor cells were CldU positive in the white matter (corpus callosum) and grey matter (striatum), respectively. However, when CldU injections were initiated on Day 28 post-implantation, approximately 80% of cells were positive in both tissue locations after 4 doses, which corresponds to a rapid tumor growth phase according to bioluminescent imaging. Tumor cells were also detected in the left hemisphere and had lower percentages of CldU positive cells as compared to the right hemisphere (site of implantation), which illustrates the extensive invasive capacity of this GSC line and is consistent with the “grow or go” concept whereby GBM cells switch between proliferative and migratory states. NSC11 cells also displayed the hallmark GBM cell migration along white matter tracts. To determine the pattern of proliferating tumor cell after irradiation, NSC11 brain tumor xenografts received three daily doses of 12Gy, which results in an approximate 10-day survival advantage, followed by a series of 4 daily IP injections of CldU. After the 4 doses of CldU, approximately 2% of tumors cells were CldU positive in the white matter of the 3x12Gy-treated tumors and no proliferating cells were detected in the grey matter. These values can be compared to 50% and 40% CldU positive cells in the white and grey matter, respectively, of the sham irradiated tumors. When the CldU injections were initiated 4 days post-3x12Gy, discrete pockets of CldU positive tumor cells began appearing in both white and grey matter from 7-8 days after the completion of radiation treatment. These pockets of proliferating cells may reflect radioresistant subpopulations and provide a framework to further characterize radioresistance based on phenotype and/or microenvironmental niche. Citation Format: Cindy R. Timme, Marzia Shah, Kevin Camphausen, Philip J. Tofilon. Detection of proliferating tumor cells in orthotopic xenografts initiated from glioblastoma stem-like cells and the influence of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5851. doi:10.1158/1538-7445.AM2017-5851

PNIPAAm-co-Jeffamine® (PNJ) scaffolds as invitro models for niche enrichment of glioblastoma stem-like cells.

Glioblastoma (GBM) is the most common adult primary brain tumor, and the 5-year survival rate is less than 5%. GBM malignancy is driven in part by a population of GBM stem-like cells (GSCs) that exhibit indefinite self-renewal capacity, multipotent differentiation, expression of neural stem cell markers, and resistance to conventional treatments. GSCs are enriched in specialized niche microenvironments that regulate stem phenotypes and support GSC radioresistance. Therefore, identifying GSC-niche interactions that regulate stem phenotypes may present a unique target for disrupting the maintenance and persistence of this treatment resistant population. In this work, we engineered 3D scaffolds from temperature responsive poly(N-isopropylacrylamide-co-Jeffamine M-1000® acrylamide), or PNJ copolymers, as a platform for enriching stem-specific phenotypes in two molecularly distinct human patient-derived GSC cell lines. Notably, we observed that, compared to conventional neurosphere cultures, PNJ cultured GSCs maintained multipotency and exhibited enhanced self-renewal capacity. Concurrent increases in expression of proteins known to regulate self-renewal, invasion, and stem maintenance in GSCs (NESTIN, EGFR, CD44) suggest that PNJ scaffolds effectively enrich the GSC population. We further observed that PNJ cultured GSCs exhibited increased resistance to radiation treatment compared to GSCs cultured in standard neurosphere conditions. GSC radioresistance is supported invivo by niche microenvironments, and this remains a significant barrier to effectively treating these highly tumorigenic cells. Taken in sum, these data indicate that the microenvironment created by synthetic PNJ scaffolds models niche enrichment of GSCs in patient-derived GBM cell lines, and presents tissue engineering opportunities for studying clinically important behaviors such as radioresistance invitro.

Bisacodyl and its cytotoxic activity on human glioblastoma stem-like cells. Implication of inositol 1,4,5-triphosphate receptor dependent calcium signaling

Glioblastoma is the most common malignant brain tumor. The heterogeneity at the cellular level, metabolic specificities and plasticity of the cancer cells are a challenge for glioblastoma treatment. Identification of cancer cells endowed with stem properties and able to propagate the tumor in animal xenografts has opened a new paradigm in cancer therapy. Thus, to increase efficacy and avoid tumor recurrence, therapies need to target not only the differentiated cells of the tumor mass, but also the cancer stem-like cells. These therapies need to be effective on cells present in the hypoxic, slightly acidic microenvironment found within tumors. Such a microenvironment is known to favor more aggressive undifferentiated phenotypes and a slow-growing “quiescent state” that preserves the cells from chemotherapeutic agents, which mostly target proliferating cells. Based on these considerations, we performed a differential screening of the Prestwick Chemical Library of approved drugs on both proliferating and quiescent glioblastoma stem-like cells and identified bisacodyl as a cytotoxic agent with selectivity for quiescent glioblastoma stem-like cells. In the present study we further characterize bisacodyl activity and show its efficacy in vitro on clonal macro-tumorospheres, as well as in vivo in glioblastoma mouse models. Our work further suggests that bisacodyl acts through inhibition of Ca2+ release from the InsP3 receptors.

A driver role for GABA metabolism in controlling stem and proliferative cell state through GHB production in glioma

Cell populations with differing proliferative, stem-like and tumorigenic states co-exist in most tumors and especially malignant gliomas. Whether metabolic variations can drive this heterogeneity by controlling dynamic changes in cell states is unknown. Metabolite profiling of human adult glioblastoma stem-like cells upon loss of their tumorigenicity revealed a switch in the catabolism of the GABA neurotransmitter toward enhanced production and secretion of its by-product GHB (4-hydroxybutyrate). This switch was driven by succinic semialdehyde dehydrogenase (SSADH) downregulation. Enhancing GHB levels via SSADH downregulation or GHB supplementation triggered cell conversion into a less aggressive phenotypic state. GHB affected adult glioblastoma cells with varying molecular profiles, along with cells from pediatric pontine gliomas. In all cell types, GHB acted by inhibiting α-ketoglutarate-dependent Ten–eleven Translocations (TET) activity, resulting in decreased levels of the 5-hydroxymethylcytosine epigenetic mark. In patients, low SSADH expression was correlated with high GHB/α-ketoglutarate ratios, and distinguished weakly proliferative/differentiated glioblastoma territories from proliferative/non-differentiated territories. Our findings support an active participation of metabolic variations in the genesis of tumor heterogeneity.

Use of an anti-viral drug, Ribavirin, as an anti-glioblastoma therapeutic.

The median survival for glioblastoma patients is ~15 months despite aggressive surgery and radio-chemotherapy approaches. Thus, developing new therapeutics is necessary to improve the treatment of these invasive brain tumors, which are known to show high levels of the eukaryotic initiation factor, eIF4E, a potent oncogene. Ribavirin, the only clinically approved drug known to target eIF4E, is an anti-viral molecule currently used in hepatitis C treatment. Here, we report the effect of ribavirin on proliferation, cell cycle, cell death and migration of several human and murine glioma cell lines, as well as human glioblastoma stem-like cells, in vitro. In addition, we tested ribavirin efficacy in vivo, alone and in combination with temozolomide and radiation. Our work showed that ribavirin inhibits glioma cell growth and migration, and increases cell cycle arrest and cell death, potentially through modulation of the eIF4E, EZH2 and ERK pathways. We also demonstrate that ribavirin treatment in combination with temozolomide or irradiation increases cell death in glioma cells. Finally and most importantly, ribavirin treatment in vivo significantly enhances chemo-radiotherapy efficacy and improves survival of rats and mice orthotopically implanted with gliosarcoma tumors or glioma stem-like cells, respectively. On the basis of these results, we propose that ribavirin represents a new therapeutic option for glioblastoma patients as an enhancer of the cytotoxic effects of temozolomide and radiotherapy.

OS1.6 Tumor Microtubes contribute to resistance against surgical lesions, and chemotherapy in malignant glioma

AbstractBackground:The current standard for the treatment of astrocytomas, including glioblastomas, consists of tumor resection, followed by radio- and chemotherapy. As we could recently demonstrate, a dense multicellular tumor network formed by intercellular tumor microtubes (TMs) greatly contributes to radioresistance. The neuronal growth-associated protein 43 (GAP-43) is involved in this mechanism. Here we asked whether similar resistance mechanisms can be found with respect to surgical lesions, and chemotherapy.Methods:Cranial windows were implanted into 8–10 weeks old male NMRI nude mice. Human glioblastoma stem-like cells (GBMSCs; stably transduced with GFP, RFP, YFP, shRNA for GAP-43, control vector, and/or H2B-GFP to label tumor cell nuclei) were stereotactically injected into the mouse’s brain. In established tumors, 100mg/kg temozolomide (TMZ) was applied to mice p.o. for three consecutive days (day 0,1,2). For surgical lesion experiments, a regional injury was set to the brain by a 26 gauge Hamilton syringe.Results:Similar to radiotherapy, GBMSCs that were not part of the TM-connected tumor cell network died in relevant numbers 7 days after chemotherapy, while TM-connected tumor cells survived. The number of TM-connected tumor cells even increased twofold after chemotherapy, which speaks for an adapative resistance mechanism. It is currently under investigation whether knock down of GAP-43 increases overall efficacy of TMZ therapy. After surgical resection, the lesioned area was rapidly re-colonized with TM-extending tumor cells, with a striking increase in tumor cells at the site of lesion that by far extended the tumor cell densitiy in unlesioned tumor areas. Qantification revealed that the tumor cell nuclear density increased to numbers five-fold above values before surfical intervention in this area, evident as early as five days (488.3%; P=0.033; 95% CI, 50.3–726.4), and extending to 14 days (523.9%; P=0.03; 95% CI, 66.7–781.1) after surgical procedure. This apparent,,repair response” gradually diminished with increasing distance to the surgical lesion site. Finally, while dexamethasone did not relevantly change these reponses, GAP-43 knock down reduced them significantly.Conclusion:TMs convey primary and adaptive resistance to TMZ chemotherapy, in accordance to what we have shown before for radiotherapy. They are also involved in a strong repair response at the very site of a surgical lesion. Reducing the ability of gliomas to repair themselves, and to resist genotoxic stress by interfering with TM formation and function appears as a promising avenue to improve treatment efficacies in these callenging diseases.

Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells.

MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs.