Search

Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., (©2021 American Association for Cancer Research.)

Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.

Purpose: Glioblastoma (GBM) is characterized by extensive clonal diversity suggesting the presence of tumor cells with varying degrees of treatment sensitivity. Radiotherapy is an integral part of glioblastoma treatment. Whether GBMs are comprised of spatially distinct cellular populations with uniform or varying degrees of radiosensitivity has not been established., Methods: Spatially distinct regions of three GBMs (J3, J7 and J14) were resected and unique cell lines were derived from each region. DNA from cell lines, corresponding tumor fragments, and patient blood was extracted for whole exome sequencing. Variants, clonal composition, and functional implications were compared and analyzed with superFreq and IPA. Limiting dilution assays were performed on cell lines to measure intrinsic radiosensitivity., Results: Based on WES, cell lines generated from different regions of the same tumor were more closely correlated with their tumor of origin than the other GBMs. Variant and clonal composition comparisons showed that cell lines from distinct tumors displayed increasing levels of ITH with J3 and J14 having the lowest and highest, respectively. The radiosensitivities of the cell lines generated from the J3 tumor were similar as were those generated from the J7 tumor. However, the radiosensitivities of the 2 cell lines generated from the J14 tumor (J14T3 and J14T6) were significantly different with J14T6 being more sensitive than J14T3., Conclusion: Data suggest a tumor dependent ITH in radiosensitivity. The existence of ITH in radiosensitivity may impact not only the initial therapeutic response but also the effectiveness of retreatment protocols.

Purpose: The various microenvironments that exist within the brain combined with the invasive nature of glioblastoma (GBM) creates the potential for a topographic influence on tumor cell radiosensitivity. The aim of this study was to determine whether specific brain microenvironments differentially influence tumor cell radioresponse., Methods and Materials: GBM stem-like cells were implanted into the right striatum of nude mice. To measure radiosensitivity, proliferation status of individual tumor cells was determined according to the incorporation of 5-chloro-2'-deoxyuridine delivered at 4, 12, and 20 days after brain irradiation. As an additional measure of radiosensitivity, the percentage of human cells in the right hemisphere and the olfactory bulb were defined using digital droplet polymerase chain reaction. Targeted gene expression profiling was accomplished using NanoString analysis., Results: Tumor cells were detected throughout the striatum, corpus callosum, and olfactory bulb. After an initial loss of proliferating tumor cells in the corpus callosum and striatum after irradiation, there was only a minor recovery by 20 days. In contrast, the proliferation of tumor cells located in the olfactory bulb began to recover at 4 days and returned to unirradiated levels by day 12 postirradiation. The percentage of human cells in the right hemisphere and the olfactory bulb after irradiation also suggested that the tumor cells in the olfactory bulb were relatively radioresistant. Gene expression profiling identified consistent differences between tumor cells residing in the olfactory bulb and those in the right hemisphere., Conclusions: These results suggest that the olfactory bulb provides a radioresistant niche for GBM cells., (Published by Elsevier Inc.)

A consequence of the intratumor heterogeneity (ITH) of glioblastoma (GBM) is the susceptibility to treatment-driven evolution. To determine the potential of radiotherapy to influence GBM evolution, we used orthotopic xenografts initiated from CD133 + GBM stem-like cells (GSC). Toward this end, orthotopic xenografts grown in nude mice were exposed to a fractionated radiation protocol, which resulted in a significant increase in animal survival. Brain tumors from control and irradiated mice were then collected at morbidity and compared in terms of growth pattern, clonal diversity, and genomic architecture. In mice that received fractionated radiation, tumors were less invasive, with more clearly demarcated borders and tumor core hypercellularity as compared with controls, suggesting a fundamental change in tumor biology. Viral integration site analysis indicated a reduction in clonal diversity in the irradiated tumors, implying a decrease in ITH. Changes in clonal diversity were not detected after irradiation of GSCs in vitro , suggesting that the radiation-induced reduction in ITH was dependent on the brain microenvironment. Whole-exome sequencing revealed differences in mutation patterns between control and irradiated tumors, which included modifications in the presence and clonality of driver mutations associated with GBM. Moreover, changes in the distribution of mutations as a function of subpopulation size between control and irradiated tumors were consistent with subclone expansion and contraction, that is, subpopulation evolution. Taken together, these results indicate that radiation drives the evolution of the GSC-initiated orthotopic xenografts and suggest that radiation-driven evolution may have therapeutic implications for recurrent GBM. SIGNIFICANCE: Radiation drives the evolution of glioblastoma orthotopic xenografts; when translated to the clinic, this may have therapeutic implications for recurrent tumors., (©2019 American Association for Cancer Research.)

Analysis of the radiation-induced translatome of glioblastoma stem-like cells (GSC) identified an interacting network in which XPO1 serves as a major hub protein. To determine whether this nuclear export protein provides a target for radiosensitization, we defined the effects of clinically relevant XPO1 inhibitor selinexor on the radiosensitivity of glioblastoma cells. As determined by clonogenic survival analysis, selinexor enhanced the radiosensitivity of GSCs but not normal fibroblast cell lines. On the basis of γH2AX foci and neutral comet analyses, selinexor inhibited the repair of radiation-induced DNA double-strand breaks in GSCs, suggesting that the selinexor-induced radiosensitization is mediated by an inhibition of DNA repair. Consistent with a role for XPO1 in the nuclear to cytoplasm export of rRNA, selinexor reduced 5S and 18S rRNA nuclear export in GSCs, which was accompanied by a decrease in gene translation efficiency, as determined from polysome profiles, as well as in protein synthesis. In contrast, rRNA nuclear export and protein synthesis were not reduced in normal cells treated with selinexor. Orthotopic xenografts initiated from a GSC line were then used to define the in vivo response to selinexor and radiation. Treatment of mice bearing orthotopic xenografts with selinexor decreased tumor translational efficiency as determined from polysome profiles. Although selinexor treatment alone had no effect on the survival of mice with brain tumors, it significantly enhanced the radiation-induced prolongation of survival. These results indicate that selinexor enhances the radiosensitivity of glioblastoma cells and suggest that this effect involves the global inhibition of gene translation. Mol Cancer Ther; 17(8); 1717-26. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Radiotherapy is a primary treatment modality for glioblastomas (GBM). Because DNA-PKcs is a critical factor in the repair of radiation-induced double strand breaks (DSB), this study evaluated the potential of VX-984, a new DNA-PKcs inhibitor, to enhance the radiosensitivity of GBM cells. Treatment of the established GBM cell line U251 and the GBM stem-like cell (GSC) line NSC11 with VX-984 under in vitro conditions resulted in a concentration-dependent inhibition of radiation-induced DNA-PKcs phosphorylation. In a similar concentration-dependent manner, VX-984 treatment enhanced the radiosensitivity of each GBM cell line as defined by clonogenic analysis. As determined by γH2AX expression and neutral comet analyses, VX-984 inhibited the repair of radiation-induced DNA double-strand break in U251 and NSC11 GBM cells, suggesting that the VX-984-induced radiosensitization is mediated by an inhibition of DNA repair. Extending these results to an in vivo model, treatment of mice with VX-984 inhibited radiation-induced DNA-PKcs phosphorylation in orthotopic brain tumor xenografts, indicating that this compound crosses the blood-brain tumor barrier at sufficient concentrations. For mice bearing U251 or NSC11 brain tumors, VX-984 treatment alone had no significant effect on overall survival; radiation alone increased survival. The survival of mice receiving the combination protocol was significantly increased as compared with control and as compared with radiation alone. These results indicate that VX-984 enhances the radiosensitivity of brain tumor xenografts and suggest that it may be of benefit in the therapeutic management of GBM. Mol Cancer Ther; 17(6); 1207-16. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo. Moreover, we observed high expression of Forkhead box protein M1 (FOXM1) in irradiated GBM cells both in vitro and in vivo. Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells, intrigued us to validate it as a radiosensitizing target. Here we show that FOXM1 inhibition radiosensitizes GBM cells by abrogating genes associated with cell cycle progression and DNA repair, suggesting its role in cellular response to radiation. Further, we demonstrate that radiation induced stimulation of FOXM1 expression is dependent on STAT3 activation. Co-immunoprecipitation and co-localization assays revealed physical interaction of FOXM1 with phosphorylated STAT3 under radiation treatment. In conclusion, we hypothesize that FOXM1 regulates radioresistance via STAT3 in GBM cells. We also, show GBM patients with high FOXM1 expression have poor prognosis. Collectively our observations might open novel opportunities for targeting FOXM1 for effective GBM therapy.

Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Although cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F-cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line-specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for glioblastoma radiosensitization. Cancer Res; 76(10); 3078-87. ©2016 AACR., (©2016 American Association for Cancer Research.)

Toward developing a model system for investigating the role of the microenvironment in the radioresistance of glioblastoma (GBM), human glioblastoma stem-like cells (GSCs) were grown in coculture with human astrocytes. Using a trans-well assay, survival analyses showed that astrocytes significantly decreased the radiosensitivity of GSCs compared to standard culture conditions. In addition, when irradiated in coculture, the initial level of radiation-induced γH2AX foci in GSCs was reduced and foci dispersal was enhanced suggesting that the presence of astrocytes influenced the induction and repair of DNA double-strand breaks. These data indicate that astrocytes can decrease the radiosensitivity of GSCs in vitro via a paracrine-based mechanism and further support a role for the microenvironment as a determinant of GBM radioresponse. Chemokine profiling of coculture media identified a number of bioactive molecules not present under standard culture conditions. The gene expression profiles of GSCs grown in coculture were significantly different as compared to GSCs grown alone. These analyses were consistent with an astrocyte-mediated modification in GSC phenotype and, moreover, suggested a number of potential targets for GSC radiosensitization that were unique to coculture conditions. Along these lines, STAT3 was activated in GSCs grown with astrocytes; the JAK/STAT3 inhibitor WP1066 enhanced the radiosensitivity of GSCs under coculture conditions and when grown as orthotopic xenografts. Further, this coculture system may also provide an approach for identifying additional targets for GBM radiosensitization., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Purpose: Valproic acid (VPA) is an antiepileptic agent with histone deacetylase inhibitor (HDACi) activity shown to sensitize glioblastoma (GBM) cells to radiation in preclinical models. We evaluated the addition of VPA to standard radiation therapy (RT) plus temozolomide (TMZ) in patients with newly diagnosed GBM., Methods and Materials: Thirty-seven patients with newly diagnosed GBM were enrolled between July 2006 and April 2013. Patients received VPA, 25 mg/kg orally, divided into 2 daily doses concurrent with RT and TMZ. The first dose of VPA was given 1 week before the first day of RT at 10 to 15 mg/kg/day and subsequently increased up to 25 mg/kg/day over the week prior to radiation. VPA- and TMZ-related acute toxicities were evaluated using Common Toxicity Criteria version 3.0 (National Cancer Institute Cancer Therapy Evaluation Program) and Cancer Radiation Morbidity Scoring Scheme for toxicity and adverse event reporting (Radiation Therapy Oncology Group/European Organization for Research and Treatment)., Results: A total of 81% of patients took VPA according to protocol. Median overall survival (OS) was 29.6 months (range: 21-63.8 months), and median progression-free survival (PFS) was 10.5 months (range: 6.8-51.2 months). OS at 6, 12, and 24 months was 97%, 86%, and 56%, respectively. PFS at 6, 12, and 24 months was 70%, 43%, and 38% respectively. The most common grade 3/4 toxicities of VPA in conjunction with RT/TMZ therapy were blood and bone marrow toxicity (32%), neurological toxicity (11%), and metabolic and laboratory toxicity (8%). Younger age and class V recursive partitioning analysis (RPA) results were significant for both OS and PFS. VPA levels were not correlated with grade 3 or 4 toxicity levels., Conclusions: Addition of VPA to concurrent RT/TMZ in patients with newly diagnosed GBM was well tolerated. Additionally, VPA may result in improved outcomes compared to historical data and merits further study., (Published by Elsevier Inc.)

Background: The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs)., Methods: mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation., Results: Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments., Conclusions: These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in the United States of America (USA) with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signalling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumour growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM., (Published by Elsevier Ltd.)

Glioblastomas (GBMs) are characterized as highly invasive; the contribution of GBM stem-like cells (GSCs) to the invasive phenotype, however, has not been completely defined. Towards this end, we have defined the invasion potential of CD133+ GSCs and their differentiated CD133- counterparts grown under standard in vitro conditions and in co-culture with astrocytes. Using a trans-well assay, astrocytes or astrocyte conditioned media in the bottom chamber significantly increased the invasion of GSCs yet had no effect on CD133- cells. In addition, a monolayer invasion assay showed that the GSCs invaded farther into an astrocyte monolayer than their differentiated progeny. Gene expression profiles were generated from two GSC lines grown in trans-well culture with astrocytes in the bottom chamber or directly in contact with astrocyte monolayers. In each co-culture model, genes whose expression was commonly increased in both GSC lines involved cell movement and included a number of genes that have been previously associated with tumor cell invasion. Similar gene expression modifications were not detected in CD133- cells co-cultured under the same conditions with astrocytes. Finally, evaluation of the secretome of astrocytes grown in monolayer identified a number of chemokines and cytokines associated with tumor cell invasion. These data suggest that astrocytes enhance the invasion of CD133+ GSCs and provide additional support for a critical role of brain microenvironment in the regulation of GBM biology.

Brain tumor xenografts initiated from glioblastoma (GBM) CD133(+) tumor stem-like cells (TSCs) are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133(+) cells and their differentiated (CD133(-)) progeny. Under in vitro conditions, no difference was detected between CD133(+) and CD133(-) cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133(+) cells compared to their CD133(-) counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133(+) cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133(+) cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumour. Patients afflicted with this disease unfortunately have a very poor prognosis, and fewer than 5% of patients survive for 5 years from the time of diagnosis. Therefore, improved therapies to treat this disease are sorely needed. One such class of drugs that have generated great enthusiasm for the treatment of numerous malignancies, including GBM, is histone deacetylase (HDAC) inhibitors. Pre-clinical data have demonstrated the efficacy of various HDAC inhibitors as anticancer agents, with the greatest effects shown when HDAC inhibitors are used in combination with other therapies. As a result of encouraging pre-clinical data, numerous HDAC inhibitors are under investigation in clinical trials, either as monotherapies or in conjunction with other treatments such as chemotherapy, biologic therapy or radiation therapy. In fact, two actively studied HDAC inhibitors, vorinostat and depsipeptide, were recently approved for the treatment of refractory cutaneous T cell lymphoma. In this review, we first present a patient with GBM, and then discuss the pathogenesis, epidemiology and current treatment options of GBM. Finally, we examine the translation of pre-clinical studies that have demonstrated HDAC inhibitors as potent radiosensitizers in in vitro and in vivo models, to a phase II clinical trial combining the HDAC inhibitor, valproic acid, along with temozolomide and radiation therapy for the treatment of GBM., (Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd No claim to US government works.)

Purpose: Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (TSC) simulate the biological characteristics of GBMs in situ. Therefore, to determine whether the brain microenvironment affects the intrinsic radiosensitivity of GBM cells, we compared the radioresponse of GBM TSCs grown in vitro and as brain tumor xenografts., Experimental Design: As indicators of DNA double-strand breaks (DSB), γH2AX, and 53BP1 foci were defined after irradiation of 2 GBM TSC lines grown in vitro and as orthotopic xenografts in nude mice. Microarray analysis was conducted to compare gene expression patterns under each growth condition., Results: Dispersal of radiation-induced γH2AX and 53BP1 foci was faster in the tumor cells grown as orthotopic xenografts compared with cells irradiated in vitro. In addition, cells irradiated in vivo were approximately 3-fold less susceptible to foci induction as compared with cells grown in vitro. Microarray analysis revealed a significant number of genes whose expression was commonly affected in the 2 GBM models by orthotopic growth conditions. Consistent with the decrease in sensitivity to foci induction, genes related to reactive oxygen species (ROS) metabolism were expressed at higher levels in the brain tumor xenografts., Conclusion: γH2AX and 53BP1 foci analyses indicate that GBM cells irradiated within orthotopic xenografts have a greater capacity to repair DSBs and are less susceptible to their induction than tumor cells irradiated under in vitro growth conditions. Because DSB induction and repair are critical determinants of radiosensitivity, these results imply that the brain microenvironment contributes to GBM radioresistance., (©2010 AACR.)

Purpose: CD133+ glioblastoma tumor stem-like cells (TSC) have been defined as radioresistant. However, although previously classified relative to CD133- cells, the radiosensitivity of CD133+ TSCs with respect to the standard glioblastoma model, established glioma cell lines, has not been determined. Therefore, to better understand the radioresponse of this cancer stem cell, we have used established cell lines as a framework for defining their in vitro radioresponse., Experimental Design: The intrinsic radiosensitivity of CD133+ TSC cultures and established glioma cell lines was determined by clonogenic assay. The TSCs and established cell lines were also compared in terms of DNA double-strand break (DSB) repair capacity and cell cycle checkpoint activation., Results: Based on clonogenic analysis, each of the six TSC cultures evaluated was more sensitive to radiation than the established glioma cell lines. Consistent with increased radiosensitivity, the DSB repair capacity as defined by neutral comet assay and gammaH2AX and Rad51 foci was significantly reduced in TSCs compared with the cell lines. Although G2 checkpoint activation was intact, in contrast to the cell lines, DNA synthesis was not inhibited in TSCs after irradiation, indicating the absence of the intra-S-phase checkpoint., Conclusions: These data indicate that the mechanisms through which CD133+ TSCs respond to radiation are significantly different from those of the traditional glioblastoma in vitro model, established glioma cell lines. If TSCs play a critical role in glioblastoma treatment response, then such differences are likely to be of consequence in the development and testing of radiosensitizing agents.

In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell-like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133(+) cells sorted from three GB neurosphere cultures at 7% O(2) reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133(+) cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell-like phenotype of CD133(+) GB cells.

Purpose: Poly (ADP-ribose) polymerase (PARP) inhibitors are undergoing clinical evaluation for cancer therapy. Because PARP inhibition has been shown to enhance tumor cell sensitivity to radiation, we investigated the in vitro and in vivo effects of the novel PARP inhibitor E7016., Experimental Design: The effect of E7016 on the in vitro radiosensitivity of tumor cell lines was evaluated using clonogenic survival. DNA damage and repair were measured using gammaH2AX foci and neutral comet assay. Mitotic catastrophe was determined by immunostaining. Tumor growth delay was evaluated in mice for the effect of E7016 on in vivo (U251) tumor radiosensitivity., Results: Cell lines exposed to E7016 preirradiation yielded an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 from 1.4 to 1.7. To assess DNA double-strand breaks repair, gammaH2AX measured at 24 hours postirradiation had significantly more foci per cell in the E7016/irradiation group versus irradiation alone. Neutral comet assay further suggested unrepaired double-strand breaks with significantly greater DNA damage at 6 hours postirradiation in the combination group versus irradiation alone. Mitotic catastrophe staining revealed a significantly greater number of cells staining positive at 24 hours postirradiation in the combination group. In vivo, mice treated with E7016/irradiation/temozolomide had an additional growth delay of six days compared with the combination of temozolomide and irradiation., Conclusions: These results indicate that E7016 can enhance tumor cell radiosensitivity in vitro and in vivo through the inhibition of DNA repair. Moreover, enhanced growth delay with the addition of E7016 to temozolomide and radiotherapy in a glioma mouse model suggests a potential role for this drug in the treatment of glioblastoma multiforme.

Background: Nestin is an intermediate filament protein that has been implicated in early stages of neuronal lineage commitment. Based on the heterogeneous expression of nestin in GBM and its potential to serve as a marker for a dedifferentiated, and perhaps more aggressive phenotype, the Radiation Therapy Oncology Group (RTOG) sought to determine the prognostic value of nestin expression in newly diagnosed GBM patients treated on prior prospective RTOG clinical trials., Methods: Tissue microarrays were prepared from 156 patients enrolled in these trials. These specimens were stained using a mouse monoclonal antibody specific for nestin and expression was measured by computerized quantitative image analysis using the Ariol SL-50 system. The parameters measured included both staining intensity and the relative area of expression within a specimen. This resulted into 3 categories: low, intermediate, and high nestin expression, which was then correlated with clinical outcome., Results: A total of 153 of the 156 samples were evaluable for this study. There were no statistically significant differences between pretreatment patient characteristics and nestin expression. There was no statistically significant difference in either overall survival or progression-free survival (PFS) demonstrated, although a trend in decreased PFS was observed with high nestin expression (p = 0.06)., Conclusion: Although the correlation of nestin expression and histologic grade in glioma is of considerable interest, the presented data does not support its prognostic value in newly diagnosed GBM. Further studies evaluating nestin expression may be more informative when studied in lower grade glioma, in the context of markers more specific to tumor stem cells, and using more recent specimens from patients treated with temozolomide in conjunction with radiation.

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the growth and progression of a variety of human cancers. Although COX-2 overexpression has been observed in human gliomas, the prognostic or clinical relevance of this overexpression has not been investigated to date. In addition, no study has analyzed the relationship between COX-2 expression and other molecular alterations in gliomas. Consequently, we examined COX-2 expression by immunohistochemistry in tumor specimens from 66 patients with low- and high-grade astrocytomas and correlated the percentage of COX-2 expression with patient survival. We also analyzed the relative importance of COX-2 expression in comparison with other clinicopathological features (age and tumor grade) and other molecular alterations commonly found in gliomas (high MIB-1 level, p53 alteration, loss of retinoblastoma (Rb) protein or p16, and high bcl-2 level). Kaplan-Meier analyses demonstrated that high COX-2 expression (>50% of cells stained positive) correlated with poor survival for the study group as a whole (P < 0.0001) and for those with glioblastoma multiforme in particular (P < 0.03). Cox regression analyses demonstrated that COX-2 expression was the strongest predictor of outcome, independent of all other variables. In addition, high COX-2 expression correlated with increasing histological grade but did not correlate with positive p53 immunostaining, bcl-2 expression, loss of p16 or retinoblastoma protein expression, or high MIB-1 expression. These findings indicate that high COX-2 expression in tumor cells is associated with clinically more aggressive gliomas and is a strong predictor of poor survival.