Patient-derived Glioma Cell Lines Research Articles

HGG-32. UNCOVERING THERAPEUTIC VULNERABILITIES IN MISMATCH REPAIR-DEFICIENT GLIOMAS

INTRODUCTIONWe have observed that approximately 26% of recurrent gliomas acquire hypermutation following treatment with temozolomide (TMZ). Intriguingly, 91% of these tumors harbor mutations in mismatch repair (MMR) genes. Strategies to target MMR-deficient gliomas thus stand to impact a large number of patients.METHODSWe ablated the MMR genes MSH2, MSH6, MLH1, and PMS2 using an all-in-one sgRNA-CRISPR/Cas9 expression vector to generate panels of isogenic MMR knockouts in patient-derived glioma cell lines. We have characterized the phenotype of these MMR-deficient glioma cells, and leveraged high-throughput drug screens to identify therapeutic vulnerabilities induced by loss of MMR.RESULTSWe demonstrate that sgRNA-CRISPR/Cas9 targeting of either MSH2 or MLH1 – the two obligatory components of the MutSα and MutLα complexes, respectively – also results in loss of protein expression of their respective binding partner MSH6 or PMS2. Moreover, we show that loss of each MMR component confers resistance to TMZ while maintaining sensitivity to the alkylating nitrosourea CCNU. Furthermore, we show that long-term TMZ treatment of MSH2 and MSH6 knockouts in an MGMT-methylated line induces hypermutation with enrichment of C > T mutations but not in MMR wild-type controls. Lastly, loss of MSH2 or MLH1 confers differential dependencies to small molecule inhibitors.CONCLUSIONSCRISPR/Cas9 knockout of individual MMR pathway members allows us to systematically study the response of MMR-deficient cells to alkylating agents in an isogenic context. MMR deficiencies in glioma confer dependencies to small molecule treatment, which may inform future therapies for MMR-deficient tumors.

DDIS-37. SMALL MOLECULE AUTOPHAGY ACTIVATORS IN GROWTH INHIBITION OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is one of the most lethal human tumors, and despite considerable advances in multimodality treatments consisting of surgical removal, radiation and chemotherapy, overall survival is 14 to 16 months after diagnosis. Therefore, there is a need to target non-apoptotic death pathways and inhibit pro-survival signaling mechanisms that contribute to both tumorigenesis and treatment resistance. Depending on tumor type and environment, autophagy modulation plays an important role in tumor cell survival and inhibition pathways. Increasing evidence supports that chemical modulation of autophagy inhibition and activation holds a therapeutic potential and these studies have led to the initiation of multiple clinical trials combining chemotherapeutic agents and autophagy inhibitors and activators for various cancer types. However, recent studies have also demonstrated a therapeutic potential for enhancers of autophagy in GBM. This clearly demonstrates the need for further investigation into the autophagy induced mechanism of GBM, and the development of better treatments. We have identified a new chemical entity VMY-BC-1 that showed promising effects in both serum and patient-derived glioma cell lines. Drug target engagement studies identified an autophagy induction protein, UNC-51 like kinase 1 (ULK1), as the main target of VMY-BC-1, in which VMY-BC-1 covalently bound to the protein’s active pocket and increased ULK1 phosphorylation.ULK1 is a component of the ULK1 complex (ULK1-ATG13-FIP200) that is essential for inducing autophagy, a lysosome-dependent mechanism of intracellular degradation for maintaining cellular homeostasis. Additionally, our molecule cross talks to the apoptotic and DNA damage pathways, which could potentially overcome treatment resistance.VMY-BC-1 is orally bioavailable, brain penetrant, and inhibits cell invasion and tumor growth in GBM flank models, making this small molecule an ideal candidate for further development. We hypothesize that activating ULK1-modulated autophagy will be a therapeutic strategy for GBM growth inhibition

IMMU-34. ATRX MUTATIONS PREDICT RESPONSE TO INNATE BASED THERAPY IN GLIOMA

Abstract BACKGROUND Innate based immunotherapies are becoming increasingly important for treating brain tumor patients. Gliomas carry recurrent mutations in regulatory genes that control innate immune signaling responses. About 71% of adult WHO grade II and III gliomas and 57% of secondary glioblastomas also carry a loss-of-function mutation in the ATRX gene. ATRX is a SWI-SNF chromatin remodeling protein that has major roles in processes such as cell cycle regulation and maintenance of genomic stability. Recent studies have implicated ATRX in dysfunctional innate immune signaling in cancer cells. However, the role of ATRX in mediating innate immune responses has not been investigated in gliomas. METHODS AND RESULTS Human and mouse glioma cell lines from a variety of genetic contexts have been examined including models which carry IDH/ATRX mutations, IDH 1p-/19q- and ATRX -/- status. Additionally, using Crispr-Cas9 technology and cloning cell lines with ATRX deletions, we have derived a series of immune competent and nude mice models. Treating these cell lines with double-stranded RNA based innate stimuli led to an enhanced early induction in phospho-interferon regulatory factor 3 (IRF3) and late induction in phospho-STAT1 in the ATRX knockout (KO) cell lines. A differential increase in interferon-stimulated gene 15 (ISG15) release was also noted in the ATRX KO cell lines, further suggesting that ATRX deletion may enable a potent activation of type I interferon production. A combination of patient-derived glioma cell lines in xenograft models and syngeneic murine glioma models derived from ATRX KO cell lines and controls confirm a survival advantage in both immuno-competent mice and xenografts. Our models are under evaluation with PVSRIPO and other innate based RNA therapies. CONCLUSION Our data suggests that ATRX mutations may confer sensitivity to RNA-based innate immune signaling agonists in gliomas. This potential vulnerability can be targeted in future therapies.

P11.46 Discovery of a new HDAC6 inhibitor for the treatment of glioblastoma

Abstract BACKGROUND Glioblastoma’s origin and development is not only associated to genetic alterations, but also to epigenetic changes. Indeed, an altered expression or activity of epigenetic enzymes such as histone deacetylases (HDAC) has been associated to cancer stem cell activity, which has been widely described as a major feature for therapy resistance and tumor recurrence. In particular, inhibition of HDAC6 is an increasingly attractive pharmacological strategy, due to its association with low toxicity. Thus, the aim of the present study was to determine the impact of a new HDAC6-selective-inhibitor in glioblastoma and glioma stem cells. MATERIAL AND METHODS To test the effect of QTX compound in glioblastoma and glioma stem cell lines, cell viability after 72h of treatment was studied by MTT assay. After evaluation of IC50, QTX in vitro activity was analyzed, focusing on proliferation, apoptosis and stemness of U87-MG cell line and confirmed in a patient-derived glioma stem cell line. In vivo antitumor effect was evaluated using U87-MG cells xenografted in immunocompromised mice; after tumor formation, 5 mice were randomly selected as control group and another 5 for QTX treatment (intraperitoneal administration of 50 mg/kg; 5 days of dosing / 2 days off for 2 weeks). Mice weight was measured daily and tumor volume every two days. RESULTS We demonstrated that QTX reduces viability of all tested glioblastoma cells, even more greatly than normal astrocytes. Indeed, QTX diminishes proliferation and induces apoptosis in both conventional and patient-derived glioma cell lines. In particular, this effect was accompanied by a reduction of self-renewal properties of glioma stem cells. Interestingly, QTX in vitro activity was more effective comparing to the pan-inhibitor SAHA or the HDAC6-selective inhibitor Tubastatin A. Furthermore, QTX delayed tumor initiation and progression in vivo, without presenting significant side effects. CONCLUSION QTX compound presents a promising anti-tumor effect both in vitro and in vivo in glioblastoma, at least in part, inhibiting glioma stem cell activity.

Abstract 248: Dynamic multi-OMICS analysis of glioblastoma cells reveals context of vulnerability to neddylation inhibition by pevonedistat

Abstract As the most lethal primary brain tumor, glioblastoma multiforme (GBM) calls for novel therapeutic development. Global over activation of neddylation (a post-translational modification) has recently been found in GBM patients and has correlated with shorter patient survival. Significant accumulation of neddylation in recurrent GBM tissues indicates its importance in tumorigenesis and tumor progression. Analogous to the ubiquitination pathway, neddylation is essential to many protein regulation and biological processes. Although most well-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), non-cullin NEDD8 substrates have been investigated in recent years. Neddylation and subsequent degradation of PARC, p53, MDM2 and EGFR exemplify the broader functional role of neddylation. The neddylation inhibitor MLN4924 targets NEDD8 Activating Enzyme (NAE), an upstream activator of neddylation, and, as a result, induces cell cycle arrest, apoptosis and senescence in cancer cells. In this work, we investigated the context of vulnerability to Pevonedistat (MLN4924) in GBM by comparing the dynamic response of sensitive and non-sensitive cells using transcriptomics and proteomics profiling, using long-established and patient derived glioma cell lines. Efficacy of MLN4924 in glioma cell models was evaluated by measuring cell viability (CellTiterGlo®), colony formation efficiency, and cell cycle progression (flow cytometry with propidium iodide staining). GB1 (IC50= 0.28 μM), LN18 (IC50 = 0.19 μM), and GBM43 (IC50= 0.45 μM) were established as sensitive and M059K (IC50= 5.5 μM), SNU1105 (IC50 = 20.9 μM), and GBM39 (IC50= 10.3 μM) as non-sensitive cell lines based on IC50 values. Western blot analysis of known cell cycle regulatory pathways and DNA damage response pathway did not show significant dynamic differences between sensitive and non-sensitive glioma cell models. To discover genomic and/or proteomic markers of differential response we collected RNA and protein for LN18 (sensitive) and SNU1105 (Non-sensitive) cells after 0, 2, 8 and 24 h treatment with MLN4924 at 100 nM and 500 nM concentration for transcriptomics and proteomics analysis. RNA sequencing was utilized for dynamic transcriptomic analysis. Cell lysates were processed using bottom-up proteomics workflow and the data was acquired on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. Proteins were identified by querying spectral data against canonical and RNA-Seq predicted proteins and differential analysis was carried out to identify candidate determinats of vulnerability. An understanding of determinants of vulnerability to MLN4924 will expand knowledge of neddylation’s role in cancer and may point to signatures of GBM patients most likely to respond to this targeted intervention. Citation Format: Shayesteh R. Ferdosi, Brett Taylor, Nanyun Tang, Rita Bybee, Sen Peng, Victoria David-Dirgo, Krystine Garcia-Mansfield, Ritin Sharma, Patrick Pirrotte, Michael Berens, Harshil Dhruv. Dynamic multi-OMICS analysis of glioblastoma cells reveals context of vulnerability to neddylation inhibition by pevonedistat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 248.

Abstract 5443: Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas

Abstract Gliomas with mutations in isocitrate dehydrogenase 1 (IDH1mut) have global genomic hypermethylation, are less aggressive than IDH1 wild-type (IDH1wt) gliomas, and generally grow poorly in vitro and in vivo. Yet little data exist that connect specific hypermethylation events to this unique phenotype. We previously reported that the gene encoding Tissue Factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas relative to IDH1wt gliomas. TF was originally described in blood coagulation, but also can enhance tumor malignancy via protease-activated receptor 2 (PAR2) signaling, though its role in gliomas is not as well understood. In this study, we further explored the significance of TF on malignancy in IDH1wt and IDH1mut gliomas, and showed that receptor tyrosine kinases (RTKs) and β-catenin are downstream effectors of TF-PAR2 signaling. Studies were performed using 6 patient derived glioma cell lines, 3 IDH1wt (GBM6, GBM12, GBM43) and 3 IDH1mut (TB09, BT142, GBM164). DNA methylation was assessed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using conditioned medium. Markers of malignancy included BrdU incorporation for cell proliferation, transwell invasion, and soft agar colony formation. Nude mice (N≥6/group) were transplanted with glioma cells and monitored for growth using bioluminescence imaging. The TF gene, F3, was significantly hypermethylated in IDH1mut cells compared to IDH1wt cells, with corresponding lower TF protein in IDH1mut cells. Treatment of IDH1mut glioma cells with a demethylating agent, decitabine, increased F3 transcription 5-fold and PCA 19-fold in IDH1mut cells. TF increased the in vivo "take rate" of IDH1mut GBM164 from 0% to 100% (P=0.0001, Fisher's exact test). Conversely, TF knockdown in IDH1wt cells greatly reduced cell proliferation, invasion, colony formation, and extended median survival of engrafted mice by 86% (P=0.001). In both IDH1wt and IDH1mut cells, TF-PAR2 activated β-catenin, ERK1/2, and Akt, and was associated with multiple pathways that increase high-grade behavior. Disrupting TF-PAR2 signaling had the greatest impact on tumor growth in IDH1wt gliomas driven by epidermal growth factor receptor (EGFR), a major receptor tyrosine kinase (RTK) involved in glioma malignancy. TF-PAR2 transactivated EGFR through a Src-dependent intracellular pathway, even when extracellular stimulation of EGFR was blocked, and further enhanced phosphorylation of the constitutively activated EGFRvIII. In IDH1mut cells, TF-PAR2 signaled through another RTK, PDGFR. In contrast, TF-PAR2 stimulated invasion in all glioma cell lines through an RTK-independent, β-catenin-dependent mechanism. These results demonstrate the importance of TF-PAR2 in gliomas, and show that its suppression is a critical component of the IDH1mut phenotype. Citation Format: Dusten J. Unruh, Snezana Mirkov, Seamus Caragher, Jann Sarkaria, Atique Ahmed, C. David James, Craig Horbinski. Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas [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 5443.

TMOD-14. ESTABLISHMENT AND MOLECULAR CHARACTERIZATION OF PATIENT DERIVED CELL LINES FROM LOW GRADE GLIOMA

Low Grade Gliomas (LGGs), although they are rare in incidence, usually affect younger adults (20-40 years of age) with patient survival varying between 5-15 years. The in vitro culturing of LGG and subsequent mutational profiling would be an invaluable tool for: (1) understanding mechanisms underpinning progression; (2) identifying novel diagnostic, prognostic and predictive biomarkers; and (3) identifying new treatment strategies using drug screening. Our aim is to develop and characterize patient derived LGG in vitro models through phenotypic, genetic and epigenetic analyses. Fresh LGG specimens were collected from the Prince of Wales Private Hospital, Sydney. All tumor samples were dissociated and cultured in serum free media supplemented with EGF and FGF. The minimum time to expand the cells from one passage to the next was at least 3 weeks. Successful cell lines were harvested at different passages. Molecular characterization of the tissue and cell lines were conducted using Sanger sequencing, Western blot, immunocytochemistry, short tandem repeat analyses and methylation arrays. 15 IDH1 mutant LGG cell lines were developed from 34 freshly collected specimens. IDH1 mutation exclusively reverted back to wild type in all cell lines within several passages. Further molecular characterization of LGG associated genes (for example p53, ATRX, TERT) and epigenetic studies through methylation arrays of paired tumor tissue samples and cell lines enabled us to better determine the selective pressures associated with in vitro culturing of patient derived glioma cell lines. We have established and characterized LGG cell lines derived from patient tumor material and provided a unique resource to better understand LGG biology and improve targeted treatment strategies whilst also detailing the extent to which in vitro models of patient derived glioma cell lines mimic their tumor of origin.

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.

Combined treatment of Nimotuzumab and rapamycin is effective against temozolomide-resistant human gliomas regardless of the EGFR mutation status.

BackgroundThe treatment of glioblastoma multiforme (GBM) is an unmet clinical need. The 5-year survival rate of patients with GBM is less than 3%. Temozolomide (TMZ) remains the standard first-line treatment regimen for gliomas despite the fact that more than 90% of recurrent gliomas do not respond to TMZ after repeated exposure. We have also independently shown that many of the Asian-derived glioma cell lines and primary cells derived from Singaporean high-grade glioma patients are indeed resistant to TMZ. This issue highlights the need to develop new effective anti-cancer treatment strategies. In a recent study, wild-type epidermal growth factor receptor (wtEGFR) has been shown to phosphorylate a truncated EGFR (known as EGFRvIII), leading to the phosphorylation of STAT proteins and progression in gliomagenesis. Despite the fact that combination of EGFR targeting drugs and rapamycin has been used before, the effect of mono-treatment of Nimotuzumab, rapamycin and combination therapy in human glioma expressing different types of EGFR is not well-studied. Herein, we evaluated the efficacy of dual blockage using monoclonal antibody against EGFR (Nimotuzumab) and an mTOR inhibitor (rapamycin) in Caucasian patient-derived human glioma cell lines, Asian patient-derived human glioma cell lines, primary glioma cells derived from the Mayo GBM xenografts, and primary short-term glioma culture derived from high-grade glioma patients.MethodsThe combination effect of Nimotuzumab and rapamycin was examined in a series of primary human glioma cell lines and glioma cell lines. The cell viability was compared to TMZ treatment alone. Endogenous expressions of EGFR in various GBM cells were determined by western blotting.ResultsThe results showed that combination of Nimotuzumab with rapamycin significantly enhanced the therapeutic efficacy of human glioma cells compared to single treatment. More importantly, many of the Asian patient-derived glioma cell lines and primary cells derived from Singaporean high-grade gliomas, which showed resistance to TMZ, were susceptible to the combined treatments.ConclusionsIn conclusion, our results strongly suggest that combination usage of Nimotuzumab and rapamycin exert higher cytotoxic activities than TMZ. Our data suggest that this combination may provide an alternative treatment for TMZ-resistant gliomas regardless of the EGFR status.

Abstract 447: The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection

Abstract Introduction: In GBM IDH mutations are associated with relatively prolonged survival compared to other subtypes. The mutations in both IDH1 result in the abnormal production of 2-hydroxyglutarate (2-HG). Although the subject of intense study, the function of 2-HG in GBM is not entirely understood. The investigation of IDH mutant tumors has been hampered by the lack of appropriate models. We have now utilized the neurosphere culture method to generate cells from 7 different tumors bearing IDH1 mutations. This study tests the validity of these lines. We utilize both these primary cultures and mouse neurospheres to test the hypotheses that mutant IDH1 is pro-proliferative and influences cell fate. Methods: We successfully propagated and obtained microarray expression data on 7 neurosphere lines bearing IDH1 mutations and 61 IDH1 wildtype cell lines for comparison. We additionally performed methylation sequencing on 3 IDH1 mutant and 3 IDH1 wildtype lines to identify sets of genes that are consistently methylated and down-regulated in these IDH1mutant cell lines. We treated 6 IDH1 mutant lines with a small molecule IDH1 mutant inhibitor that prevents 2-HG formation to to determine effects on proliferation and whether down-regulated genes were rescued by the inhibition of 2-HG formation. Finally we treated fetal murine neural progenitors with exogenous 2-HG to determine the effects on differentiation status as well as on gene expression. Results: Gene expression studies of the 7 IDH1 mutant neurosphere lines revealed 103 genes that were 4-fold down-regulated in IDH1mutant tumors. Of those 103 genes, 32 were also found to be methylated and down-regulated in IDH1mutant brain tumor samples found in public databases. Methylation sequencing revealed that 31 of these 32 genes were heavily methylated in our three IDH1mutant cell lines, while only 12 of these genes were methylated in the IDH1WT cell lines. Treatment of IDH1mutant neurosphere cultures with a small molecule inhibitor resulted in diminished growth capacity. Microarray analysis demonstrated that only one of the downregulated genes was was consistently rescued, Inhibitor of DNA binding 2 (ID2). Application of exogenous 2-HG to murine neurosphere cultures promototed the appearance of oligodendrocytes and neurons, and also induced downregulation of ID2 and upregulation of Olig 2. Discussion: Our data suggest that there is likely a conserved set of “key gateway genes” that are frequently down-regulated in these IDH1mutant tumors and that patient-derived glioma cell lines maintain these epigenetic changes. Inhibition of 2-HG formation may be an effective therapy evidenced by the fact that tumor cells grow slower without 2-HG production. However inhibition of 2-HG production did not appear to reverse the majority of epigenetic changes in gene expression. The Id2 gene is consistently rescued by 2-HG inhibition and may be an important target for future tumor therapy. Citation Format: Matthew C. Garrett, Jack Mottahedeh, Jantzen Sperry, Ascia Eskin, Giovanni Coppola, Ya-shin Shih, Albert Lai, Arthur Chou, Linda Liau, Rob Prins, Timothy Cloughesy, Hong Wu, Stanley Nelson, Harley Kornblum. The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 447. doi:10.1158/1538-7445.AM2014-447

Solid Lipid Nanoparticles for Potential Doxorubicin Delivery in Glioblastoma Treatment: Preliminary In Vitro Studies

The major obstacle to glioblastoma pharmacological therapy is the overcoming of the blood-brain barrier (BBB). In literature, several strategies have been proposed to overcome the BBB: in this experimental work, solid lipid nanoparticles (SLN), prepared according to fatty acid coacervation technique, are proposed as the vehicle for doxorubicin (Dox), to enhance its permeation through an artificial model of BBB. The in vitro cytotoxicity of Dox-loaded SLN has been measured on three different commercial and patient-derived glioma cell lines. Dox was entrapped within SLN thanks to hydrophobic ion pairing with negatively charged surfactants, used as counterions. Results indicate that Dox entrapped in SLN maintains its cytotoxic activity toward glioma cell lines; moreover, its permeation through hCMEC/D3 cell monolayer, assumed as a model of the BBB, was increased when the drug was entrapped in SLN. In conclusion, SLN proved to be a promising vehicle for the delivery of Dox to the brain in glioblastoma treatment.

Abstract B60: Interleukin-1 expression and inflammasome activation in human malignant glioma

Abstract Introduction: Interleukin 1 (IL-1) is a major proinflammatory cytokine produced by myeloid cells and is implicated in neurodegeneration. In human brain, IL-1 is expressed by microglia and contributes to neuroinflammation and neuronal toxicity. IL-1 is also the major activator of human astrocyte and glioma cells. In this study, we investigated the mechanism of IL-1 α and IL-1β expression and activation in human glioma cells. Methods: Human glioblastoma cell lines (U87, U251, and SN19) as well as patient-derived glioma cell lines were stimulated with cytokines (IL-1/IFNγ) or TLR ligands (LPS or poly IC) and the expression of IL-1α and IL-1β was studied by real-time PCR, western blot analysis, and ELISA. The glioma secretome was investigated using quantitative mass spectrometry. Results: We found that while primary human astrocytes show IL-1 translational block (no protein made despite high level of mRNA), but GBM cells responded strongly to IL-1 itself to produce large amounts of IL-1α and IL-1β mRNA and proteins. Furthermore, GBM cells processed pro-IL-1β (31kDa) to mature IL-1β (17kDa) by activating the NALP3 inflammasome. Both ATP and nigericin induced IL-1β processing by upregulating NALP3. Proteomics analysis of IL-1 stimulated GBM cell secretome revealed several highly upregulated proteins (IL-8, MCP-1, tenascin-C, pentraxin 3, galectin 1, and MMP2), all of which are implicated in glioma progression. Furthermore, many extracellular matrix proteins (ECM) and ECM-related proteins were downregulated by IL-1 in the glioma secretome. Discussion: We propose that the ability of malignant glioma cells to produce IL-1 protein can set off chronic smoldering inflammation and induce epithelial-mesenchymal transition (EMT) resulting in increased migratory capacity, a unique gene signature, and increased immune signaling (NF-κB, pStat3). There is potential for IL-1 to be a biomarker/surrogate marker for a subset of GBM and that IL-1 blockade could benefit GBM patients. Citation Format: Leonid Tarassishin, Jihyeon Lim, Sunhee C. Lee. Interleukin-1 expression and inflammasome activation in human malignant glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B60.