Primary Human Glioblastoma Cells Research Articles

Hypofractionated Radiation Therapy Suppresses Radioresistance in U87 Human Glioma Cells by Inhibiting Yap1 and Hsp90 Proteins.

Radiotherapy plays a vital role in the management of high-grade gliomas. However, the radio resistance of glioma cells limits the effect of radiation and drives recurrence inside the irradiated tumor volume leading to poor outcomes for patients. High-grade glioma cell radioresistance significantly contributes to radiotherapy failure, highlighting the importance of identifying predictive biomarkers for radioresistance. An increasing body of evidence complies with the Yes Associated Protein 1 (Yap-1) and heat shock protein 90 (Hsp90) as biomarkers for radioresistance in glioma cells. A number of studies suggest the potential of radioresistance-associated factors as biomarkers and/ or novel therapeutic targets in glioma cells. Thus, it is essential for glioblastoma patients to identify robust druggable targets involved in radioresistance, optimizing irradiation protocol, and understanding their underlying molecular mechanisms. Therefore, in the present study, we hypothesized that hypofractionated Gamma Knife radiation therapy (HF-GKRT) could target Yap-1 and Hsp90 and downregulate the mechanism of radioresistance in high-grade glioma cells. For this purpose, expression levels of radioresistance markers Yap-1 and Hsp90 were evaluated after treatment with HF-GKRT, and this was compared with single fraction Gamma Knife radiation therapy (SF-GKRT) in U87MG primary human glioblastoma cell line model. This would help design a novel radiation therapy regimen for glioblastoma patients by reducing the risk of radioresistance.

Abstract 5564: The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling

Abstract Glioblastoma (GBM), the most aggressive brain cancer which is usually fatal within a year after diagnosis, poses a significant therapeutic challenge with conventional treatments providing limited success. Despite advancements in immune checkpoint blockade, PD-L1 inhibitors exhibit suboptimal efficacy in recurrent GBMs with recent clinical trials demonstrating only 8% objective responses in patients. Utilizing the R2 Genomic Analysis Visualization Platform, we identified a positive 8-18% correlation between Pyk2 and PD-L1 gene expression in human GBM specimens. We hypothesize that Pyk2 signaling is involved in the regulation of PD-L1 expression in GBM cells. In this study, primary human glioblastoma cell lines and GL261 mouse glioma cells were assessed employing CRISPR/Cas9 Pyk2 knockout (Pyk2KO) combined with pharmacological Pyk2 inhibition. Flow cytometric analysis revealed a 35% decrease in PD-L1 expression in Pyk2KO GL261 cells compared to wild-type cells. SiRNA knockdown against Pyk2 in primary human GBM cells demonstrated a similar response, with a 50% decrease in PD-L1 expression compared to MOCK control cells. Additionally, the application of the Pyk2/FAK inhibitor defactinib at a concentration of 0.2 μM reduced PD-L1 expression by 32% in both GL261 and primary human GBM cells. These findings highlight Pyk2 as a potential therapeutic target for modulating the immunosuppressive microenvironment in GBM and enhancing the effectiveness of immune checkpoint blockade therapies. This study was supported by: NIH Grant 1SC1GM122691 and PRSTRT2022. Citation Format: Tyrel Porter, Lilia Kucheryavykh. The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5564.

ABCC4 suppresses glioblastoma progression and recurrence by restraining cGMP-PKG signalling.

Cyclic nucleotides are critical mediators of cellular signalling in glioblastoma. However, the clinical relevance and mechanisms of regulating cyclic nucleotides in glioblastoma progression and recurrence have yet to be thoroughly explored. In silico, mRNA, and protein level analyses identified the primary regulator of cyclic nucleotides in recurrent human glioblastoma. Lentiviral and pharmacological manipulations examined the functional impact of cyclic nucleotide signalling in human glioma cell lines and primary glioblastoma cells. An orthotopic xenograft mice model coupled with aspirin hydrogels verified the in vivo outcome of targeting cyclic nucleotide signalling. Elevated intracellular levels of cGMP, instead of cAMP, due to a lower substrate efflux from ATP-binding cassette sub-family C member 4 (ABCC4) is engaged in the recurrence of glioblastoma. ABCC4 gene expression is negatively associated with recurrence and overall survival outcomes in glioblastoma specimens. ABCC4 loss-of-function activates cGMP-PKG signalling, promoting malignancy in glioblastoma cells and xenografts. Hydrogels loaded with aspirin, inhibiting glioblastoma progression partly by upregulating ABCC4 expressions, augment the efficacy of standard-of-care therapies in orthotopic glioblastoma xenografts. ABCC4, repressing the cGMP-PKG signalling pathway, is a tumour suppressor in glioblastoma progression and recurrence. Aspirin hydrogels impede glioblastoma progression through ABCC4 restoration and constitute a viable translational approach.

Formulation of multicomponent inclusion complex of cyclodextrin-amino acid with Chrysin: Physicochemical characterization, cell viability and apoptosis assessment in human primary glioblastoma cell line

Chrysin (CR) is a water-insoluble drug reported for different therapeutic effects. The microwave irradiation method was used in this study to create a multicomponent inclusion complex (CR-MC) containing CR (drug) and carrier hydroxyl propyl beta cyclodextrin (HP β CD) and L-arginine (LA). The prepared inclusion complex (CR-MC) was evaluated for dissolution study and results were compared with chrysin physical mixture (CR-PM). Further, the samples were assessed for infra-red (IR), nuclear magnetic resonance (NMR), differential scanning calorimeter (DSC), scanning electron microscope (SEM) and molecular docking. Finally, the cell viability, reactive oxygen species and flow cytometer studies were also assessed to check the potential of the prepared inclusion complex on the human primary glioblastoma cell line (U87-MG cell). The phase solubility findings revealed a stability constant (773 mol L−1) as well as a complexation efficiency of 0.027. The dissolution study displayed a significant increase in CR release from CR-MC (99.03 ± 0.39%) > CR-PM (70.58 ± 1.16%) > pure CR (35.29 ± 1.55%). NMR and IR spectral data revealed no interaction between CR and carriers. SEM and DSC study results revealed the conversion into amorphous form. The molecular docking results illustrated a high docking score, which supports the findings of complex formation. The cell viability, reactive oxygen species, and flow cytometry studies results showed enhanced activity from CR-MC against the tested human primary glioblastoma cell line. From the results it has been observed that chrysin solubility significantly increased after complexation and there in vitro activity also enhanced against cancer cell line.

Antiproliferative effect of boldine on neural progenitor cells and on glioblastoma cells.

The subventricular zone (SVZ) is a brain region that contains neural stem cells and progenitor cells (NSCs/NPCs) from which new neurons and glial cells are formed during adulthood in mammals. Recent data indicate that SVZ NSCs are the cell type that acquires the initial tumorigenic mutation in glioblastoma (GBM), the most aggressive form of malignant glioma. NSCs/NPCs of the SVZ present hemichannel activity whose function has not yet been fully elucidated. In this work, we aimed to analyze whether hemichannel-mediated communication affects proliferation of SVZ NPCs and GBM cells. For that purpose, we used boldine, an alkaloid derived from the boldo tree (Peumus boldus), that inhibits connexin and pannexin hemichannels, but without affecting gap junctional communication. Boldine treatment (50 μM) of rat SVZ NPCs grown as neurospheres effectively inhibited dye uptake through hemichannels and induced a significant reduction in neurosphere diameter and in bromodeoxyuridine (BrdU) incorporation. However, the differentiation pattern was not modified by the treatment. Experiments with specific blockers for hemichannels formed by connexin subunits (D4) or pannexin 1 (probenecid) revealed that probenecid, but not D4, produced a decrease in BrdU incorporation similar to that obtained with boldine. These results suggest that inhibition of pannexin 1 hemichannels could be partially responsible for the antiproliferative effect of boldine on SVZ NPCs. Analysis of the effect of boldine (25-600 μM) on different types of primary human GBM cells (GBM59, GBM96, and U87-MG) showed a concentration-dependent decrease in GBM cell growth. Boldine treatment also induced a significant inhibition of hemichannel activity in GBM cells. Altogether, we provide evidence of an antimitotic action of boldine in SVZ NPCs and in GBM cells which may be due, at least in part, to its hemichannel blocking function. These results could be of relevance for future possible strategies in GBM aimed to suppress the proliferation of mutated NSCs or glioma stem cells that might remain in the brain after tumor resection.

Aloe-Emodin Overcomes Anti-Cancer Drug Resistance to Temozolomide and Prevents Colony Formation and Migration in Primary Human Glioblastoma Cell Lines NULU and ZAR.

Glioblastoma, the most dangerous and aggressive type of CNS tumor, appears resistant to many chemotherapy drugs. In the patient-derived glioma cell lines NULU and ZAR, which exhibit drug-resistant phenotypes, we investigated the effect of combined AE (Aloe-emodin) and TMZ (temozolomide) and found a significant additive inhibitory effect on cell growth and a promising cytotoxic effect on both cell lines compared to treatment with single agents. We also examined the effect of combined AE and TMZ treatment on the drug-resistance protein MGMT. The results suggest that using AE combined with traditional drugs restores drug resistance in both primary resistant cell lines (NULU and ZAR). Furthermore, migration assays and scratch tests showed that the combined use of AE and TMZ can slow down the colony formation and migration of glioblastoma cells. These convincing results suggest that AE could be a natural adjuvant agent to potentiate the effects of traditional drugs (TMZ) and overcome drug resistance in glioblastoma cells.

Perivascular invasion of primary human glioblastoma cells in organotypic human brain slices: human cells migrating in human brain.

Glioblastoma (GBM) is an aggressive primary brain cancer. Lack of effective therapy is related to its highly invasive nature. GBM invasion has been studied with reductionist systems that do not fully recapitulate the cytoarchitecture of the brain. We describe a human-derived brain organotypic model to study the migratory properties of GBMIDH-wild type ex vivo. Non-tumor brain samples were obtained from patients undergoing surgery (n = 7). Organotypic brain slices were prepared, and green fluorescent protein (GFP)-labeled primary human GBM IDH-wild type cells (GBM276, GBM612, GBM965) were placed on the organotypic slice. Migration was evaluated via microscopy and immunohistochemistry. After placement, cells migrated towards blood vessels; initially migrating with limited directionality, sending processes in different directions, and increasing their speed upon contact with the vessel. Once merged, migration speed decreased and continued to decrease with time (p < 0.001). After perivascular localization, migration is limited along the blood vessels in both directions. The percentage of cells that contact blood vessels and then continue to migrate along the vessel was 92.5% (-3.9/ + 2.9)% while the percentage of cells that migrate along the blood vessel and leave was 7.5% (-2.9/ + 3.9) (95% CI, Clopper-Pearson (exact); n = 256 cells from six organotypic cultures); these percentages are significantly different from the random (50%) null hypothesis (z = 13.6; p < 10-7). Further, cells increase their speed in response to a decrease in oxygen tension from atmospheric normoxia (20% O2) to anoxia (1% O2) (p = 0.033). Human organotypic models can accurately study cell migrationex vivo. GBM IDH-wild type cells migrate toward the perivascular space in blood vessels and their migratory parameters change once they contact vascular structures and under hypoxic conditions. This model allows the evaluation of GBM invasion, considering the human brain microenvironment when cells are removed from their native niche after surgery.

Mapping the functional expression of auxiliary subunits of KCa1.1 in glioblastoma

Glioblastoma (GBM) is the most aggressive glial tumor, where ion channels, including KCa1.1, are candidates for new therapeutic options. Since the auxiliary subunits linked to KCa1.1 in GBM are largely unknown we used electrophysiology combined with pharmacology and gene silencing to address the functional expression of KCa1.1/β subunits complexes in both primary tumor cells and in the glioblastoma cell line U-87 MG. The pattern of the sensitivity (activation/inhibition) of the whole-cell currents to paxilline, lithocholic acid, arachidonic acid, and iberiotoxin; the presence of inactivation of the whole-cell current along with the loss of the outward rectification upon exposure to the reducing agent DTT collectively argue that KCa1.1/β3 complex is expressed in U-87 MG. Similar results were found using human primary glioblastoma cells isolated from patient samples. Silencing the β3 subunit expression inhibited carbachol-induced Ca2+ transients in U-87 MG thereby indicating the role of the KCa1.1/β3 in the Ca2+ signaling of glioblastoma cells. Functional expression of the KCa1.1/β3 complex, on the other hand, lacks cell cycle dependence. We suggest that the KCa1.1/β3 complex may have diagnostic and therapeutic potential in glioblastoma in the future.

ANGI-07. THE DUAL ROLE CD97 PLAYS IN GLIOBLASTOMA INVASION AND PROLIFERATION

Abstract INTRODUCTION Increased levels of adhesion G Protein-Coupled Receptor(GPCR) CD97 have been detected in multiple metastatic cancers. Glioblastoma(GBM), also overexpress this receptor, and its levels positively correlate with more aggressive GBM invasion. Here, using primary human GBM cells we investigated the effects of CD97 knockdown and overexpression in-vitro and in-vivo. METHODS CD97 consists of two distinct domains: the large extracellular fragment interacting with ECM and other cell surface receptors and the GPCR core that activates heterotrimeric G Proteins G12/13. To investigate the mechanism of action of CD97, we generated a CD97 knockdown (shCD97) and overexpression (CD97-WT) constructs. Additionally, we generated two functional mutants: a constitutively active mutant lacking the N-Terminus (dNTF) and dominant-negative mutant (H436A) that cannot activate G-Proteins downstream. Glioma Stem Cells(GSC) were transduced by control and CD97 lentiviral constructs and implanted into mouse brains. Once tumors formed the brains were harvested, sliced, and immunostained to evaluate invasion and proliferation. Comparing mutants to control, dunnett’s multiple comparisons test was used for statistical analysis. RESULTS Similar to CD97-WT, we found that both dNTF and H436A, increase tumor volume(p&amp;lt; 0.05). In contrast, the shCD97 mutant decreased tumor size(p&amp;lt; 0.05). Interestingly, only dNTF significantly increased tumor cell proliferation(p&amp;lt; 0.005), yet CD97-WT and H436A affected proliferation of non-tumor cells in the tumor bulk, indicating that this process requires the N-Terminus Fragment. In addition, over-expression of CD97-WT dramatically increases tumor invasion along myelinated fibers of corpus collosum, and tumor spread in the brain tissue(p&amp;lt; 0.05). Yet, the H436A mutant produced large, compact, and non-invasive tumors with sharp borders(p&amp;lt; 0.05) indicating that cell migration requires activation of G12/13. Surprisingly, immunostaining revealed that CD97 knockdown cells displayed invasive phenotype. CONCLUSIONS Our results demonstrate the dual role CD97 plays in GBM invasion and proliferation, alluding to the molecular mechanisms that can potentially be targeted in GBM treatment.

Establishment and characteristics of GWH04, a new primary human glioblastoma cell line.

Glioblastoma multiforme (GBM) is a common and fatal disease of the central nervous system. GBM cell lines are fundamental tools used in GBM research. The establishment of novel continuous GBM cell lines with clear genetic backgrounds could facilitate the exploration of molecular mechanisms and the screening and evaluation of antitumor drugs in GBM studies. In the present study, a novel primary glioblastoma cell line was established, named GWH04, from a patient with GBM, and its STR genotype and various tumor parameters were examined. The STR information of GWH04 was identical to that of the original primary tumor tissue. Compared with existing cell lines, GWH04 had a similar in vitro proliferation rate as the U87 cell line, but a faster rate than the GL15 cell line, and substantial soft agar clone-formation capacity and subcutaneous and intracranial tumorigenic capacity. For drug sensitivity test, half maximal inhibitory concentration assays for multiple drugs were performed in these three cell lines, and GWH04 cells were found to be resistant to temozolomide. Aneuploid karyotype with a median of 84 chromosomes was possessed by GWH04, as well as chromosomal structural abnormalities, such as broken chromosomes, double centromere chromosomes, homogeneous staining regions, and double microbodies. Gene sequencing further revealed the mutational status of genes TP53, PTEN, PDGFRA, ERBB2, BRCA1, NF1, and MLH1 and the promoter region of telomerase reverse transcriptase (C228T) in this cell line. Altogether, these results indicated that GWH04 will be a useful tool for human GBM studies both in vitro and in vivo.

The Anti-Glioma Effect of Juglone Derivatives through ROS Generation.

Juglone has been extensively reported as a natural antitumor pigment. However, it is easy to be oxidized due to active hydroxy in the quinone. Here, we designed some new juglone derivatives, as the hydroxy was replaced by methyl (D1), allyl (D2), butyl (D3), and benzyl (D4) groups. Nuclear magnetic resonance spectra and mass spectrometry were applied to confirm the derivatives and oxidative products of juglone. U87 and U251 cell lines were used for tests in vitro, and primary human glioblastoma cells were applied for in vivo experiments. The CCK8 and EdU assay demonstrated the anti-tumor effect of the four derivatives, and IC50 for U87 was 3.99, 3.28, 7.60, and 11.84 μM, respectively. In U251, IC50 was 7.00, 5.43, 8.64, and 18.05 μM, respectively. D2 and D3 were further selected, and flow cytometry showed that apoptosis rates were increased after D2 or D3 treatment via ROS generation. Potential targets were predicted by network pharmacology analysis, most of which were associated with apoptosis, cell cycle, and metabolism pathway. CDC25B and DUSP1 were two of the most likely candidates for targets. The orthotopic glioblastoma model was established to evaluate the anti-glioma effect and side-effect of juglone derivatives, and the in vivo experiments confirmed the anti-glioma effects of juglone derivatives. In conclusion, new derivatives of juglone were created via chemical group substitution and could inhibit glioma cell viability and proliferation and induce apoptosis rate via ROS generation.

Cucurbitacin B and Evodiamine imparts anti‐neoplastic effects against Glioblastoma Multiforme via PI3‐K/AKT signaling pathway

Glioblastoma Multiforme (GBM) is a highly malignant and aggressive brain tumor. Patients’ survival is around 6 to 12 months even with chemotherapy and surgery. Temozolomide is a first line chemotherapeutic treatment prescribed along with surgery but due to the high resistance to the drug and a low patient survival rate, new drugs and drug combinations must continue to be investigated and developed. Evodiamine, a marketed supplement, and Cucurbitacin B, a compound obtained from natural plants, have been identified as promising candidates to treat various neoplasms but has not been well studied in GBM, and especially in combination. The present study investigated the anti‐proliferating effects of Evodiamine (Evo) and Cucurbitacin‐B (CuB) individually and in combination in U‐87MG cell line (Human Primary Glioblastoma cell line) and elucidated probable mechanisms of action via PI3K/AKT pathway in GBM. Results demonstrated that both CuB and Evo individually decreased the U‐87MG cell viability in a dose dependent manner. The effect was pronounced with drug combination. CuB and Evo imparted their effect via the PI3K/P‐AKT signaling pathway. In addition, Cells treated with CuB and Evo showed a decrease in Bcl2, an anti‐apoptotic factor, and an increase in Bax, a pro‐apoptotic factor, expression; a hallmark of induction of apoptosis. The increased expression of Cleaved Caspase‐3 and p‐c Jun have also been observed. Overall, Cucurbitacin and evodiamine individually and especially in combination proved to be potential anti‐neoplastic agents in U‐87MG cells and may be considered as potential adjuvant therapies for GBM.

Ageritin-The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy.

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.

Intratumoral administration of the antisecretory peptide AF16 cures murine gliomas and modulates macrophage functions

Glioblastoma has remained the deadliest primary brain tumor while its current therapy offers only modest survival prolongation. Immunotherapy has failed to record notable benefits in routine glioblastoma treatment. Conventionally, immunotherapy relies on T cells as tumor-killing agents; however, T cells are outnumbered by macrophages in glioblastoma microenvironment. In this study, we explore the effect of AF16, a peptide from the endogenous antisecretory factor protein, on the survival of glioma-bearing mice, the tumor size, and characteristics of the tumor microenvironment with specific focus on macrophages. We elucidate the effect of AF16 on the inflammation-related secretome of human and murine macrophages, as well as human glioblastoma cells. In our results, AF16 alone and in combination with temozolomide leads to cure in immunocompetent mice with orthotopic GL261 gliomas, as well as prolonged survival in immunocompromised mice. We recorded decreased tumor size and changes in infiltration of macrophages and T cells in the murine glioma microenvironment. Human and murine macrophages increased expression of proinflammatory markers in response to AF16 treatment and the same effect was seen in human primary glioblastoma cells. In summary, we present AF16 as an immunomodulatory factor stimulating pro-inflammatory macrophages with a potential to be implemented in glioblastoma treatment protocols.

Bee Venom Effect on Glioblastoma Cells Viability and Gelatinase Secretion.

BackgroundThe involvement of MMP-2 and MMP-9 in the pathogenesis of various kinds of cancers including glioblastoma is well documented. The evaluation of the anticancer potential of honey bee (Apis mellifera) venom (BV) consisting of the inhibition of MMP-2 and MMP-9 secretion in a glioblastoma cell culture model was the aim of the study.Methods8-MG-BA and GAMG human primary glioblastoma cell lines vs. HT-22 mouse hippocampal neuronal cells were applied for the study. The BV dose (0.5, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, and 5.0 μg/ml) and time-dependent (24, 48, 72 h) cytotoxicity was evaluated with the tetrazolium-based colorimetric assay (MTT test). MMP-2 and MMP-9 activities in the cell culture medium under different BV concentrations were determined by gelatin zymography.ResultsA dose and time-dependent BV effect on cytotoxicity of both glioblastoma cell lines and hippocampus line was observed. The weakest, but statistically important effect was exerted by BV on HT-22 cells. The greatest cytotoxic effect of BV was observed on the 8-MG-BA line, where a statistically significant reduction in viability was observed at the lowest BV dose and the shortest incubation time. The reduction of both gelatinases secretion was observed at 8-MG-BA and GAMG lines without significant effect of HT-22 cell line.ConclusionIn vitro studies indicate that BV has both cytotoxic and inhibitory effects on the secretion of MMP-2 and MMP-9 in selected lines of glioma, suggesting anticancer properties of BV.

Gene-Directed Enzyme/Prodrug Therapy of Rat Brain Tumor Mediated by Human Mesenchymal Stem Cell Suicide Gene Extracellular Vesicles In Vitro and In Vivo.

Simple SummaryExtracellular vesicles— exosomes—secreted by human mesenchymal stem/stromal cells are able to cross the blood–brain barrier and internalize glioblastoma cells. We prepared exosomes possessing a gene message, the product of which is able to convert nontoxic 5-fluorocytosine to cytotoxic drug 5-fluorouracil. Such therapeutic exosomes administered intranasally, intraperitoneally, or subcutaneously to rats bearing intracerebral glioblastoma cells inhibited their growth. The treatment cured a significant number of animals.MSC-driven, gene-directed enzyme prodrug therapy (GDEPT) mediated by extracellular vesicles (EV) represents a new paradigm—cell-free GDEPT tumor therapy. In this study, we tested the efficacy of yeast cytosine deaminase::uracilphosphoribosyl transferase (yCD::UPRT-MSC)-exosomes, in the form of conditioned medium (CM) to inhibit the growth of C6 glioblastoma cells both in vitro and in vivo. MSCs isolated from human adipose tissue, umbilical cord, or dental pulp engineered to express the yCD::UPRT gene secreted yCD::UPRT-MSC-exosomes that in the presence of the prodrug 5-fluorocytosine (5-FC), inhibited the growth of rat C6 glioblastoma cells and human primary glioblastoma cells in vitro in a dose-dependent manner. CM from these cells injected repeatedly either intraperitoneally (i.p.) or subcutaneously (s.c.), applied intranasally (i.n.), or infused continuously by an ALZET osmotic pump, inhibited the growth of cerebral C6 glioblastomas in rats. A significant number of rats were cured when CM containing yCD::UPRT-MSC-exosomes conjugated with 5-FC was repeatedly injected i.p. or applied i.n. Cured rats were subsequently resistant to challenges with higher doses of C6 cells. Our data have shown that cell-free GDEPT tumor therapy mediated by the yCD::UPRT-MSC suicide gene EVs for high-grade glioblastomas represents a safer and more practical approach that is worthy of further investigation.

Chemical synthesis of 4'-thio and 4'-sulfinyl pyrimidine nucleoside analogues.

Analogues of the canonical nucleosides required for nucleic acid synthesis have a longstanding presence and proven capability within antiviral and anticancer research. 4'-Thionucleosides, that incorporate bioisosteric replacement of furanose oxygen with sulfur, represent an important chemotype within this field. Established herein is synthetic capability towards a common 4-thioribose building block that enables access to thio-ribo and thio-arabino pyrimidine nucleosides, alongside their 4'-sulfinyl derivatives. In addition, this building block methodology is templated to deliver 4'-thio and 4'-sulfinyl analogues of the established anticancer drug gemcitabine. Cytotoxic capability of these new analogues is evaluated against human pancreatic cancer and human primary glioblastoma cell lines, with observed activities ranging from low μM to >200 μM; explanation for this reduced activity, compared to established nucleoside analogues, is yet unclear. Access to these chemotypes, with thiohemiaminal linkages, will enable a wider exploration of purine and triphosphate analogues and the application of such materials for potential resistance towards relevant hydrolytic enzymes within nucleic acid biochemistries.

Brusatol Inhibits Proliferation and Invasion of Glioblastoma by Down-Regulating the Expression of ECM1.

Brusatol (Bru), a Chinese herbal extract, has a variety of anti-tumor effects. However, little is known regarding its role and underlying mechanism in glioblastoma cells. Here, we found that Bru could inhibit the proliferation of glioblastoma cells in vivo and in vitro. Besides, it also had an inhibitory effect on human primary glioblastoma cells. RNA-seq analysis indicated that Bru possibly achieved these effects through inhibiting the expression of extracellular matrix protein 1 (ECM1). Down-regulating the expression of ECM1 via transfecting siRNA could weaken the proliferation and invasion of glioblastoma cells and promote the inhibitory effect of Bru treatment. Lentivirus-mediated overexpression of ECM1 could effectively reverse this weakening effect. Our findings indicated that Bru could inhibit the proliferation and invasion of glioblastoma cells by suppressing the expression of ECM1, and Bru might be a novel effective anticancer drug for glioblastoma cells.

α‐Tocopherol‐Conjugated, Open Chain Sugar‐Mimicking Cationic Lipids: Design, Synthesis and In–Vitro Gene Transfection Properties

AbstractSugar based cationic lipids are promising carriers of nucleic acids into various cells. Particularly, abundant hydroxyl functional groups in the sugar‐like moieties are believed to enhance target oriented uptake as well as biocompatibility. Towards this direction, we have synthesized three new cationic lipid analogues with or without sugar‐like multi‐hydroxyl functional head groups. Liposomal formulations of these lipids were developed and thoroughly characterized by means of DNA binding ability, size and zeta potential. Optimal lipid‐DNA complexes were determined by their transfection efficiency in HEK‐293 (Human embryonic kidney) cells. 1,4 gluconic γ‐lactone derived lipid (TS1) and ascorbic acid derived lipid (TS2), have exhibited 25‐fold and 12‐fold increase in transfected HEPG2 (human liver cancer) cells, respectively, when compared with a control lipid without sugar‐like moiety (TA). However, this property was reversed in the case of U87 (human primary glioblastoma) cell line. The results point out that the transfection efficiencies of sugar alcohol‐based cationic lipids are cell‐dependant. Cytotoxicity of the lipid complexes revealed that the sugar‐mimicking cationic lipids are non‐toxic as ≈80 % viable cells were observed in the treated cell lines. In conclusion, the current paper adds knowledge on the sugar based cationic lipids and reiterates that these lipids could yield potential applications towards liver targeted gene delivery.

EXTH-33. RETROVIRAL REPLICATING VECTORS PSEUDOTYPED WITH GIBBON APE LEUKEMIA VIRUS ENVELOPE FOR PRODRUG ACTIVATOR GENE THERAPY IN PRECLINICAL GLIOMA MODELS

Abstract Amphotropic retroviral replicating vector (RRV) Toca 511, expressing the yeast cytosine deaminase (CD) prodrug activator gene, showed promising evidence of therapeutic benefit and increased survival in early-phase trials for recurrent high-grade glioma. While a multi-center Phase 3 trial did not meet its overall endpoints, highly statistically significant survival was observed within predetermined patient subgroups compared to matched randomized control patients, and clinical investigation is on-going. Hence it is worthwhile to consider strategies aimed at enhancing therapeutic efficacy, such as delivering combinations of multiple transgenes. However, RRVs encoated with the same envelope compete for the same cancer cell surface receptors. We have now developed novel RRV encoated (‘pseudotyped’) with a heterologous envelope derived from Gibbon ape leukemia virus (GALV), which utilizes a different cell surface receptor from the native amphotropic retrovirus envelope for cellular entry. RRV(GALV) vectors expressing either GFP or HSV thymidine kinase (TK) were constructed, and efficient replication and transgene expression was observed in &amp;gt; 90% of both established and primary human glioblastoma cells within 14 days after initial infection at 0.01 (1%) multiplicity of infection (MOI). Genomic stability of RRV(GALV) vectors was also confirmed over prolonged propagation. Established and primary human glioblastoma cells infected with RRV(GALV)-TK vector showed ≥ 50%-90% reduction in cell viability after exposure to Ganciclovir prodrug in the range of 1µM-100µM for 5 days, as compared to uninfected control cells or cells infected with RRV(GALV)-GFP control vector. Furthermore, dual infection with RRV(GALV)-TK and amphotropic RRV-CD (Toca 511) resulted in synergistic cytotoxicity upon simultaneous exposure to their respective prodrugs. Further data will be presented from on-going studies evaluating these vectors in intracerebral glioblastoma models. These results indicate that GALV envelope-pseudotyped RRV can efficiently deliver prodrug activator gene therapy in experimental glioma models, and open the door to combinatorial gene therapy regimens with this vector platform.