Primary Glioblastoma Cells Research Articles

AEG-1 promotes mesenchymal transition through the activation of Rho GTPases in human glioblastoma cells.

Despite growing evidence indicating that astrocyte elevated gene-1(AEG-1) plays pivotal roles in tumor progression in various types of human cancers including brain tumors; to date, its role in the regulation of mesenchymal transition is not clear in glioblastoma. In the present study, we investigated the contribution of AEG-1 to stress fiber formation and then the acquisition of mesenchymal characteristics of glioblastoma cells. Gain- and loss-of-function studies in normal immortalized primary human fetal astrocytes(IM-PHFAs) and glioblastoma cells revealed that overexpression of AEG-1 increased expression of mesenchymal markers including N-cadherin and two mesenchymal transition‑inducing transcription factors ZEB1 and Slug but decreased epithelial markers E-cadherin and ZO-1. In addition, knockdown of AEG-1 suppressed invasive ability and migration of glioblastoma cells. Overexpression of AEG-1 also induced stress fiber formation and activated the Rho GTPase signaling pathways in glioblastoma cells. Consistently, treatment with an RhoA inhibitor decreased AEG-1-mediated stress fiber formation in glioblastoma cells. Collectively, our findings suggest that AEG-1 promotes mesenchymal transition in glioblastoma through the regulation of the Rho signaling pathway, resulting in tumor invasion, a primary characteristic of malignant brain tumors.

Multi-omics analysis of primary glioblastoma cell lines shows recapitulation of pivotal molecular features of parental tumors.

Glioblastoma (GBM) is the deadliest primary brain cancer in adults. Emerging innovative therapies hold promise for personalized cancer treatment. Improving therapeutic options depends on research relying on relevant preclinical models. In this line we have established in the setting of the GlioTex project (GBM and Experimental Therapeutics) a GBM patient-derived cell line (GBM-PDCL) library. A multi-omic approach was used to determine the molecular landscape of PDCL and the extent to which they represent GBM tumors. Single nucleotide polymorphism array, expression arrays, exome sequencing, and RNA sequencing were used to measure and compare the molecular landscapes of 20 samples representing 10 human GBM tumors and paired GBM-PDCLs. Copy number variations were similar for a median of 85% of the genome and for 59% of the major focal events. Somatic point mutations were similar in a median of 41%. Mutations in GBM driver and "druggable" genes were maintained in 67% of events. Mutations that were not conserved in the PDCL were mainly low allelic fraction and/or non-driver mutations. Based on RNA expression profiling, PDCLs cluster closely to their parental tumor with overexpression of pathways associated with cancer progression in PDCL. Overall, PDCLs recapitulate pivotal molecular alterations of paired-parental tumors supporting their use as a preclinical model of GBM. However, some driver aberrations are lost or gained in the passage from tumor to PDCL. Our results support using PDCL as a relevant preclinical model of GBM. Further investigations of changes between PDCLs and their parental tumor may provide insights into GBM biology.

Janus face-like effects of Aurora B inhibition: antitumoral mode of action versus induction of aneuploid progeny.

The mitotic Aurora B kinase is overexpressed in tumors and various inhibitors for Aurora B are currently under clinical assessments. However, when considering Aurora B kinase inhibitors as anticancer drugs, their mode of action and the role of p53 status as a possible predictive factor for response still needs to be investigated. In this study, we analyzed the effects of selective Aurora B inhibition using AZD1152-HQPA/Barasertib (AZD1152) on HCT116 cells, U87-MG, corresponding isogenic p53-deficient cells and a primary glioblastoma cell line. AZD1152 treatment caused polyploidy and non-apoptotic cell death in all cell lines irrespective of p53 status and was accompanied by poly-merotelic kinetochore-microtubule attachments and DNA damage. In p53 wild-type cells a DNA damage response induced an inefficient pseudo-G1 cell cycle arrest, which was not able to halt ongoing endoreplication of cells. Of note, release of tumor cells from AZD1152 resulted in recovery of aneuploid progenies bearing numerical and structural chromosomal aberrations. Yet, AZD1152 treatment enhanced death receptor TRAIL-R2 levels in all tumor cell lines investigated. A concomitant increase of the activating natural killer (NK) cell ligand MIC A/B in p53-deficient cells and an induction of FAS/CD95 in cells containing p53 rendered AZD1152-treated cells more susceptible for NK-cell-mediated lysis. Our study mechanistically explains a p53-independent mode of action of a chemical Aurora B inhibitor and suggests a potential triggering of antitumoral immune responses, following polyploidization of tumor cells, which might constrain recovery of aneuploid tumor cells.

Topoisomerase IIβ mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs.

BackgroundGlioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation.MethodsIn this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage.ResultsWe found several differentially expressed genes and we identified topoisomerase IIβ (Top2β) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2β was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2β expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2β increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2β depletion in NCH421k cells.ConclusionWe suggest that Top2β may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0339-9) contains supplementary material, which is available to authorized users.

Abstract LB-308: Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation

Abstract IDH1 R132H mutations are considered to play a key role in the development of low grade gliomas and therefrom derived secondary glioblastomas (GBM). Wild type IDH1 converts isocitrate to α-ketoglutarate (a-KG) while reducing NADP+. IDH1R132H has a neomorphic enzymatic function using a-KG to generate high amounts of the oncometabolite D-2-hydroxyglutarate (D-2-HG). While the effects of D-2-HG have been subject to intense research, D-2-HG independent effects of IDH1R132H on energy homeostasis and redox status are not well studied. Here we demonstrate that IDH1R132H transduction but not D-2-HG alone leads to significantly decreased Krebs cycle metabolite concentrations and proliferation in U87 and the primary GBM cell line HT7606 as well as in immortalized astrocytes SVGp12. Furthermore, IDH1R132H mutation, but not D-2-HG treatment, resulted in a significant drop in NADPH levels in tumor cells (U87 and HT7606), whereas immortalized astrocytes retained normal NADPH levels. Since NAPDH levels can be restored via the reaction of the NAD-kinase, we analyzed NAD levels and enzymes involved in NAD-synthesis in our cell lines. Indeed we found a significant drop of NAD levels and in the activity of the NAD-dependent enzyme sirtuin in IDH1R132H mutant U87 and HT7606 but not in NADPH stable SVGp12-IDH1R132H. Interestingly, there were marked differences in expression of NAD-synthesis enzymes between the different cell-lines. In particular, NAMPT-levels were much higher in U87 and HT7606 then in astrocytes (SVGp12) and significantly decreased in U87-IDH1R132H and HT7606-IDH1R132H. Importantly, we also found decreased levels of NAMPT in primary tumor tissues and patient derived glioma cell lines with IDH1 R132H compared to wild type gliomas. Altogether our results for the first time show that the IDH1 mutation directly affects energy homeostasis and redox status in a cell-type dependent manner. We hypothesize that this leads to a drop in NADPH and NAD-levels during malignant progression, resulting in a disadvantage for proliferating tumor cells. This is in line with the favorable prognosis and good response to chemo- and radiation therapy clinically observed in IDH-mutated gliomas. Our findings suggest that the impaired metabolism in IDH1-mutant tumors might be a promising target for future therapies. Citation Format: Evelin Schrock, Julia Biedermann, Khalil Abou-El-Ardat, Matthias Lehmann, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Matthias Meinhardt, William P. J. Leenders, Christel Herold-Mende, Graeme Eisenhofer, Simone P. Niclou, Leoni Kunz-Schughart, Achim Temme, Barbara Klink. Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-308.

Methionine and Kynurenine Activate Oncogenic Kinases in Glioblastoma, and Methionine Deprivation Compromises Proliferation.

We employed a metabolomics-based approach with the goal to better understand the molecular signatures of glioblastoma cells and tissues, with an aim toward identifying potential targetable biomarkers for developing more effective and novel therapies. We used liquid chromatography coupled with mass spectrometry (LC-MS/Q-TOF and LC-MS/QQQ) for the discovery and validation of metabolites from primary and established glioblastoma cells, glioblastoma tissues, and normal human astrocytes. We identified tryptophan, methionine, kynurenine, and 5-methylthioadenosine as differentially regulated metabolites (DRM) in glioblastoma cells compared with normal human astrocytes (NHAs). Unlike NHAs, glioblastoma cells depend on dietary methionine for proliferation, colony formation, survival, and to maintain a deregulated methylome (SAM:SAH ratio). In methylthioadenosine phosphorylase (MTAP)-deficient glioblastoma cells, expression of MTAP transgene did not alter methionine dependency, but compromised tumor growth in vivo We discovered that a lack of the kynurenine-metabolizing enzymes kynurenine monooxygenase and/or kynureninase promotes the accumulation of kynurenine, which triggers immune evasion in glioblastoma cells. In silico analysis of the identified DRMs mapped the activation of key oncogenic kinases that promotes tumorigenesis in glioblastoma. We validated this result by demonstrating that the exogenous addition of DRMs to glioblastoma cells in vitro results in oncogene activation as well as the simultaneous downregulation of Ser/Thr phosphatase PP2A. We have connected a four-metabolite signature, implicated in the methionine and kynurenine pathways, to the promotion and maintenance of glioblastoma. Together, our data suggest that these metabolites and their respective metabolic pathways serve as potential therapeutic targets for glioblastoma. Clin Cancer Res; 22(14); 3513-23. ©2016 AACR.

386 - Induction of senescence in primary glioblastoma cells by serum and TGFβ

386 - Induction of senescence in primary glioblastoma cells by serum and TGFβ

P.106 Thrombospondin 1 mediates transforming growth factor beta induced premature senescence in primary glioblastoma cells

Background: Glioblastoma is the most common primary malignant brain tumor. Primary Glioblastoma (PriGO) cells are key drivers of glioblastoma. Senescence is the irreversible growth arrest of cells with continued metabolic activity. Recently, I discovered PriGO cells undergo premature senescence in response to Fetal Bovine Serum (FBS). Determining the underlying molecular mechanisms may allow development of novel therapeutic strategies to decrease the malignant potential of glioblastoma. Methods: Global gene expression changes in PriGO cells treated with serum were analyzed and compared to untreated cells. Senescence was determined by the Senescence-Associated-Beta-Galactosidase (SA-B-Gal) assay. Results: PriGO cells treated with serum demonstrated increased expression of genes in the Transforming Growth Factor Beta (TGF-B) pathway, such as Thrombospondin 1 (TSP1), compared to untreated cells. TGF-B treatment of PriGO cells significantly increased senescence compared to untreated cells. Treatment of PriGO cells with serum and the TGF-B inhibitor SB431542 led to a decrease in senescence compared to serum only treated cells. Treatment of PriGO cells with serum and the TSP1 inhibitor LSKL led to a reduction in senescence compared to serum only treated cells. Conclusions: Our data identifies TGF-B as an important component of serum responsible for inducing senescence in PriGO cells. Furthermore, TGF-B induced senescence in PriGO cells is in part mediated by TSP1.

Connection between Proliferation Rate and Temozolomide Sensitivity of Primary Glioblastoma Cell Culture and Expression of YB-1 and LRP/MVP.

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

198. CD4+ IL13Rα2-Specific CAR T Cells Exhibit Potent Effector Function Against Glioblastoma

Adoptive T cell transfer, including chimeric antigen receptor (CAR) T cell therapy, has been successfully applied to the treatment of multiple types of cancer, but the variation in response rates among patients remains an important issue. Recent studies have demonstrated that CAR T cells vary in function and persistence, depending on the specific T cell subpopulation that is engineered and the phenotype of the final T cell product. Specifically, a starting population of central memory T cells (Tcm) has been shown to improve persistence and efficacy of CAR T cell therapy. Tcm consist of both CD4+ and CD8+ pools, but little is known about the contribution of each subset for CAR T cell function. Notably, although CD8+ T cells have long been considered the primary cytotoxic effector population, CD4+ T cells have also been shown to mediate anti-tumor response against some solid tumors which is CD8-independent. It is thus critical to identify the optimal composition of CD4+ and CD8+ CAR T cells for mediating the maximal anti-tumor effect. Here, using both in vitro and in vivo glioblastoma (GBM) models, we examined the anti-tumor activity of IL13Rα2-specific CAR T cells engineered from purified CD4+ or CD8+ Tcm pools. Surprisingly, we discovered a superior antitumor response from CD4+ IL13Rα2-CAR T cells as compared to CD8+ CAR T cells. After co-culture with primary patient-derived GBM cells in vitro, CD4+ CAR T cells displayed more potent tumor killing as compared to CD8+ CAR T cells, especially when effector cell numbers were more limited. Consistently, CD4+ CAR T cells maintained better effector function in vitro when re-challenged with tumor. These CD4+ CAR T cells produced higher levels of cytokine as compared to CD8+ T cells, a difference that was further maintained after tumor re-challenge. We also assessed whether the CD4 versus CD8 composition of the IL13Rα2-CAR T cell product would impact therapeutic efficacy in vivo using primary patient-derived GBM lines orthotopically implanted in NSG mice. When injected intracranial into GBM-bearing mice, the CD4+ CAR T cells resulted in durable antitumor efficacy, mediating tumor-free survival for over 200 days in 7of 7 experimental animals, which was unaffected by mixing with 10% of CD8+ CAR T cells. While CD8+ CAR T cells, as well as 50%-50% or 10%-90% CD4-CD8 mixed populations, also mediated significant antitumor activity, the majority of mice recurred and showed a less durable therapeutic response. These results demonstrate that CD4+ CAR T cells can have potent effector function and suggest that, for at least IL13Rα2-CARs, CD4+ T cells mediated superior anti-tumor immune responses as compared to CD8+ T cells. These studies provide important insight to further elucidate the optimal composition of the CAR T cell product for the treatment of cancer.

654. Oncolytic Vesicular Stomatitis Virus Retargeted to the Tumor Stem Cell Marker CD133

Therapy resistance and tumor recurrence are often linked to cancer stem cells (CSCs) which represent a minor proportion of highly refractory cancer-propagating cells within a tumor. A putative marker of CSCs is CD133 (prominin-1) which has been suggested to define the tumorigenic population in glioma and hepatocellular cancer (HCC). Moreover, poor prognosis correlates with CD133 expression in cancer biopsies. CD133 is therefore a prime target for novel anti-tumoral strategies. We have previously developed a strategy by which the envelope protein complex of oncolytic measles virus (MV) can be engineered to use CD133 as receptor for cell entry. Notably, MV-CD133 was more effective in killing tumors than non-targeted MV when treating tumors with a high percentage of CD133-positive cells (Bach et al., 2013; Can Res 73, 865). We have meanwhile extended the receptor usage of MV-CD133 to CD46 to effectively infect both CD133-negative and -positive tumor cells. Moreover, to take advantage of the faster replication kinetics and superior cytotoxic activity of the vesicular stomatitis virus (VSV), we used the MV envelope glycoproteins to develop hybrid viruses. The resulting oncolytic virus VSV-CD133 is deleted for its glycoprotein gene G and instead equipped with the mutated MV hemagglutinin (H) and fusion (F) glycoproteins displaying a single-chain antibody (scFv) specific for CD133. The data show that VSV-CD133 infects CD133-positive tumor cells as selectively as MV-CD133, while MV-CD133/CD46 in addition infected CD133−/CD46+ tumor cells. We further studied the cytotoxic activity of VSV-CD133 compared to its MV counterpart. Even though both were effective at killing tumor cells, VSV-CD133 induced cell death more rapidly and at lower MOI than MV-CD133. In a mouse tumor model with subcutaneously transplanted HuH7 cells VSV-CD133 reduced the tumor burden more efficiently than MV-CD133 or MV-CD133/CD46. Most importantly, this increase in efficacy did not come at the expense of safety, as CD133+ human hematopoietic stem cells (HSCs) were neither susceptible to infection nor impaired in growth and differentiation into hematopoietic lineages. Ongoing studies will determine whether the superior oncolytic effects of VSV-CD133 translate into prolonged survival in a mouse model with orthotopically transplanted human primary glioblastoma cells.

Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo.

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

The transcriptional modulator HMGA2 promotes stemness and tumorigenicity in glioblastoma

Glioblastoma (GBM) contains a population of stem-like cells that promote tumor invasion and resistance to therapy. Identifying and targeting stem cell factors in GBM may lead to the development of more effective therapies. High Mobility Group AT-hook 2 (HMGA2) is a transcriptional modulator that mediates motility and self-renewal in normal and cancer stem cells. We identified increased expression of HMGA2 in the majority of primary human GBM tumors and cell lines compared to normal brain. Additionally, HMGA2 expression was increased in CD133+ GBM neurosphere cells compared to CD133− cells. Targeting HMGA2 with lentiviral short hairpin RNA (shRNA) led to decreased GBM stemness, invasion, and tumorigenicity. Ectopic expression of HMGA2 in GBM cell lines promoted stemness, invasion, and tumorigenicity. Our data suggests that targeting HMGA2 in GBM may be therapeutically beneficial.

Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements.

In near infrared fluorescence-guided surgical oncology, it is challenging to distinguish healthy from cancerous tissue. One promising research avenue consists in the analysis of the exogenous fluorophores’ lifetime, which are however in the (sub-)nanosecond range. We have integrated a single-photon pixel array, based on standard CMOS SPADs (single-photon avalanche diodes), in a compact, time-gated measurement system, named FluoCam. In vivo measurements were carried out with indocyanine green (ICG)-modified derivatives targeting the αvβ3 integrin, initially on a genetically engineered mouse model of melanoma injected with ICG conjugated with tetrameric cyclic pentapeptide (ICG−E[c(RGD f K)4]), then on mice carrying tumour xenografts of U87-MG (a human primary glioblastoma cell line) injected with monomeric ICG−c(RGD f K). Measurements on tumor, muscle and tail locations allowed us to demonstrate the feasibility of in vivo lifetime measurements with the FluoCam, to determine the characteristic lifetimes (around 500 ps) and subtle lifetime differences between bound and unbound ICG-modified fluorophores (10% level), as well as to estimate the available photon fluxes under realistic conditions.

CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells.

Glioblastoma (GBM) is one of the most aggressive types of cancer with limited therapeutic options and unfavorable prognosis. Stemness and non-classical epithelial-to-mesenchymal transition (ncEMT) features underlie the switch from normal to neoplastic states as well as resistance of tumor clones to current therapies. Therefore, identification of ligand/receptor systems maintaining this privileged state is needed to devise efficient cancer therapies. In this study, we show that the expression of CD95 associates with stemness and EMT features in GBM tumors and cells and serves as a prognostic biomarker. CD95 expression increases in tumors and with tumor relapse as compared with non-tumor tissue. Recruitment of the activating PI3K subunit, p85, to CD95 death domain is required for maintenance of EMT-related transcripts. A combination of the current GBM therapy, temozolomide, with a CD95 inhibitor dramatically abrogates tumor sphere formation. This study molecularly dissects the role of CD95 in GBM cells and contributes the rational for CD95 inhibition as a GBM therapy.

Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry

Non-viral, biomaterial-mediated gene delivery has the potential to treat many diseases, but is limited by low efficacy. Elucidating the bottlenecks of plasmid mass transfer can enable an improved understanding of biomaterial structure–function relationships, leading to next-generation rationally designed non-viral gene delivery vectors. As proof of principle, we transfected human primary glioblastoma cells using a poly(beta-amino ester) complexed with eGFP plasmid DNA. The polyplexes transfected 70.6±0.6% of the cells with 101±3% viability. The amount of DNA within the cytoplasm, nuclear envelope, and nuclei was assessed at multiple time points using fluorescent dye conjugated plasmid up to 24h post-transfection using a quantitative multi-well plate-based flow cytometry assay. Conversion to plasmid counts and degradation kinetics were accounted for via quantitative PCR (plasmid degradation rate constants were determined to be 0.62h−1 and 0.084h−1 for fast and slow phases respectively). Quantitative cellular uptake, nuclear association, and nuclear uptake rate constants were determined by using a four-compartment first order mass-action model. The rate limiting step for these poly(beta-amino ester)/DNA polyplex nanoparticles was determined to be cellular uptake (7.5×10−4h−1) and only 0.1% of the added dose was taken up by the human brain cancer cells, whereas 12% of internalized DNA successfully entered the nucleus (the rate of nuclear internalization of nuclear associated plasmid was 1.1h−1). We describe an efficient new method for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles using flow cytometry to improve understanding and design of polymeric gene delivery nanoparticles. Statement of SignificanceIn this work, a quantitative high throughput flow cytometry-based assay and computational modeling approach was developed for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles. This method is significant as it can be used to elucidate structure–function relationships of gene delivery nanoparticles and improve their efficiency. This method was applied to a particular type of biodegradable polymer, a poly(beta-amino ester), that transfected human brain cancer cells with high efficacy and without cytotoxicity. A four-compartment first order mass-action kinetics model was found to model the experimental transport data well without requiring external fitting parameters. Quantitative rate constants were identified for the intracellular transport, including DNA degradation rate from polyplexes, cellular uptake rate, and nuclear uptake rate, with cellular uptake identified as the rate-limiting step.

Differential response of patient-derived primary glioblastoma cells to environmental stiffness.

The ability of cancer cells to sense external mechanical forces has emerged as a significant factor in the promotion of cancer invasion. Currently there are conflicting reports in the literature with regard to whether glioblastoma (GBM) brain cancer cell migration and invasion is rigidity-sensitive. In order to address this question we have compared the rigidity-response of primary patient-derived GBM lines. Cells were plated on polyacrylamide gels of defined rigidity that reflect the diversity of the brain tissue mechanical environment, and cell morphology and migration were analysed by time-lapse microscopy. Invasiveness was assessed in multicellular spheroids embedded in 3D matrigel cultures. Our data reveal a range of rigidity-dependent responses between the patient-derived cell lines, from reduced migration on the most compliant tissue stiffness to those that are insensitive to substrate rigidity and are equally migratory irrespective of the underlying substrate stiffness. Notably, the rigidity-insensitive GBM cells show the greatest invasive capacity in soft 3D matrigel cultures. Collectively our data confirm both rigidity-dependent and independent behaviour in primary GBM patient-derived cells.

Staphylococcus aureus Enterotoxin B Down-Regulates the Expression of Transforming Growth Factor-Beta (TGF-β) Signaling Transducers in Human Glioblastoma

BackgroundIt has been revealed that Staphylococcus aureus enterotoxin B (SEB) may feature anti-cancer and anti-metastatic advantages due to its ability to modify cell immunity processes and signaling pathways. Glioblastoma is one of the most aggressive human cancers; it has a high mortality nature, which makes it an attractive area for the development of novel therapies.ObjectivesWe examined whether the SEB could exert its growth inhibitory effects on glioblastoma cells partially through the manipulation of a key tumor growth factor termed transforming growth factor-beta (TGF-β).Materials and MethodsA human primary glioblastoma cell line, U87, was treated with different concentrations of SEB. The cell quantity was measured by the MTT assay at different exposure times. For molecular assessments, total ribonucleic acid (RNA) was extracted from either non-treated or SEB-treated cells. Subsequently, the gene expression of TGF-β transducers, smad2/3, at the messenger RNA (mRNA) level, was analyzed via a quantitative real-time polymerase chain reaction (qPCR) using the SYBR Green method. Significant differences between cell viability and gene expression levels were determined (Prism 5.0 software) using one-way analyses of variance (ANOVA) test.ResultsWe reported that SEB could effectively down-regulate smad2/3 expression in glioblastoma cells at concentrations as quantity as 1 μg/mL and 2 μg/mL (P < 0.05 and P < 0.01, respectively). The SEB concentrations effective at regulating smad2/3 expression were correlated with those used to inhibit the proliferation of glioblastoma cells. Our results also showed that SEB was able to decrease smad2/3 expression at the mRNA level in a concentration- and time-dependent manner.ConclusionsWe suggested that SEB could represent an agent that can significantly decrease smad2/3 expression in glioblastoma cells, leading to moderate TGF-β growth signaling and the reduction of tumor cell proliferation.

Cell cycle progression in glioblastoma cells is unaffected by pathophysiological levels of hypoxia.

Hypoxia is associated with the increased malignancy of a broad range of solid tumours. While very severe hypoxia has been widely shown to induce cell cycle arrest, the impact of pathophysiological hypoxia on tumour cell proliferation is poorly understood. The aim of this study was to investigate the effect of different oxygen levels on glioblastoma (GBM) cell proliferation and survival. GBM is an extremely aggressive brain tumour with a heterogeneous oxygenation pattern. The effects of a range of oxygen tensions on GBM cell lines and primary cells were assessed using flow cytometry. Results indicate that cell cycle distribution and viability are unaffected by long term exposure (24–96 h) to pathophysiological levels of oxygen (1–8% O2). Both transient cell cycle arrest and small amounts of cell death could only be detected when cells were exposed to severe hypoxia (0.1% O2). No significant changes in p21 protein expression levels were detected. These findings reinforce the importance of using physiologically relevant oxygen tensions when investigating tumour hypoxia, and help to explain how solid tumours can be both hypoxic and highly proliferative, as is the case with GBM.