Anti-glioma Therapies Research Articles

Inhibitors of CK2 Kinase for the Treatment of Gliomas and Other Brain Tumors

Protein kinase CK2 has become a target of experimental antiglioma therapies as laboratory data are almost uniformly favorable and the number of synthetized CK2 inhibitors is rapidly growing. The evidence of their use for other brain tumors is on the increase as well. Great expectations are entrusted in naturally occurring compounds capable of inhibiting CK2 kinase. These compounds are extracted and purified by means of biochemistry methods and are amenable for innovative drug delivery systems as well. CK2 kinase inhibitors have been proven suitable for combined therapies with other investigational antiglioma agents and treatment modalities. However a greater share of efforts should be undertaken towards inhibiting functions relatively specific for glial tumors including infiltrative growth and invasiveness or maintenance of glioma initiating cells. Many of these function appear to converge on mTOR and JAK/STAT pathways which are being meticulously studied in this respect. Protein kinase CK2 holds therapeutic promise especially when combined with other agents aimed at molecular signatures of gliomas.

MODL-14. IMAGING, HISTOLOGICAL AND MOLECULAR CHARACTERIZATION AND COMPARISON OF POST-ABLATION RECURRENT TUMOR WITH THE PRIMARY TUMOR IN A PRECLINICAL GLIOBLASTOMA MODEL

Abstract Recurrent glioblastoma (rGBM) is highly aggressive and invasive. A reliable preclinical model that recapitulates these features is not presently available. The objective was to generate a preclinical rGBM model, characterize and compare its imaging, histological and molecular signatures in comparison to the primary tumor. Immune-suppressed, RNU/RNU female rats were implanted with U251N tumor cells in one brain hemisphere (n=33). Tumor progression in all rats was followed by longitudinal dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI). In 24 rats the tumor was ablated under diffusion-weighted imaging (DWI)-guided laser interstitial thermal therapy (LITT) at post-implantation 2-weeks. Cohorts from twenty ablated rats were euthanized at post-LITT 24 h, 2- and 4-weeks and, along with 5 unablated controls, used for hematoxylin and eosin (H&E) and Ki67 staining. Tissues from 4 other unablated and 4 recurrent tumors at post-LITT 2-weeks were used for RNAseq. All the rats survived the LITT procedure. Unablated controls showed increased tumor burden by post-implantation 2 weeks and were euthanized. In the LITT group, MRI showed little tumor tissue at 24 h, evidence of recurrence at 2 weeks and significant tumor tissue at 4 weeks and matched with histological evidence for tumor recurrence. Compared to the primary tumor, H&E staining showed increased vascular hyperplasia, mitotic bodies and hypoxic regions with pseudopalisading necrosis in the recurrent tumor. Increased KI67 staining in recurrent tumors suggested higher rates of proliferation and evidence of infiltration into host tissue. Pathway analyses demonstrated differentially expressed genes in the canonical pathways of hypoxia-inducible factor-alpha (HIF-1α), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and OX40 signaling. The genes for the following functions were significantly affected: cell cycle, cellular movement and cell morphology. Reliable preclinical rGBM models are few. These data suggest that this model replicates the features of human rGBM and can be useful in testing putative anti-glioma therapies.

Human neural stem cells repress glioma cell progression in a paracrine manner by downregulating the Wnt/β-catenin signalling pathway.

Neural stem cells (NSCs) play crucial roles in neurological disorders and tissue injury repair through exerting paracrine effects. However, the effects of NSC-derived factors on glioma progression remain unclear. This study aimed to evaluate the effects of human NSC-conditioned medium (NSC-CM) on the behaviour of glioma cells using an in vitro co-culture system. Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays revealed that NSC-CM inhibited glioma cell proliferation and growth in a foetal bovine serum (FBS)-independent manner. In addition, our wound healing assay demonstrated that NSC-CM repressed glioma cell migration, while results from transwell and 3D spheroid invasion assays indicated that NSC-CM also reduced the invasion capacity of glioma cells. Flow cytometry showed that NSC-CM prevented cell cycle progression from the G1 to S phase and promoted apoptosis. Western blotting was used to show that the expression of Wnt/β-catenin pathway-related proteins, including β-catenin, c-Myc, cyclin D1, CD44, and Met, was remarkably decreased in NSC-CM-treated glioma cells. Furthermore, the addition of a Wnt/β-catenin pathway activator, CHIR99021, significantly induced the expression of β-catenin and Met and increased the proliferative and invasive capabilities of control medium-treated glioma cells but not those of NSC-CM-treated glioma cells. The use of enzyme-linked immunosorbent assays (ELISA) revealed the secretion of some anti-tumour factors in human and rat NSCs, including interferon-α and dickkopf-1. Our data suggest that NSC-CM partially inhibits glioma cell progression by downregulating Wnt/β-catenin signalling. This study may serve as a basis for developing future anti-glioma therapies based on NSC derivatives.

Medicinal Plants for Glioblastoma Treatment.

Glioblastoma, an aggressive brain cancer, demonstrates the least life expectancy among all brain cancers. Because of the regulation of diverse signaling pathways in cancers, the chemotherapeutic approaches used to suppress their multiplication and spread are restricted. Sensitivity towards chemotherapeutic agents has been developed because of the pathological and drug-evading abilities of these diverse mechanisms. As a result, the identification and exploration of strategies or treatments, which can overcome such refractory obstacles to improve glioblastoma response to treatment as well as recovery, is essential. Medicinal herbs contain a wide variety of bioactive compounds, which could trigger aggressive brain cancers, regulate their anti-cancer mechanisms and immune responses to assist in cancer elimination, and cause cell death. Numerous tumor-causing proteins, which facilitate invasion as well as metastasis of cancer, tolerance of chemotherapies, and angiogenesis, are also inhibited by these phytochemicals. Such herbs remain valuable for glioblastoma prevention and its incidence by effectively being used as anti-glioma therapies. This review thus presents the latest findings on medicinal plants using which the extracts or bioactive components are being used against glioblastoma, their mechanism of functioning, pharmacological description, and recent clinical studies conducted on them.

DDRE-22. DISRUPTING REDOX BALANCE VIA THE TETRAHYDROBIOPTERIN PATHWAY IN GLIOBLASTOMA

Abstract Aberrant redox statuses are observed in glioblastoma (GBM), and we previously identified GTP cyclohydrolase I (GCH1) to be a redox regulator upregulated in brain tumor initiating cells (BTICs). GCH1 is a rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a cofactor that produces catecholamine precursors and nitric oxide (NO) and, once used, becomes 7,8-dihydrobiopterin (BH2). Regeneration of BH2 into BH4 by dihydrofolate reductase (DHFR) helps to maintain proper BH4/BH2 ratios for redox balance. Although the BH4 pathway has traditionally been studied in the vasculature system for its regulation of NO, our previous work and that of others suggests the GCH1/BH4 pathway plays a critical redox role including in neoplastic cells. In silico analysis of primary and recurrent gliomas indicate high expression of BH4 related enzymes that correlated with worse patient survival in both primary and recurrent gliomas. The observed elevation of the BH4 pathway not only emphasizes its importance, but a therapeutic opportunity for improving survival in glioma patients. By repurposing FDA approved drugs known to cross the blood brain barrier and previously suggested as anti-glioma therapies, combining inhibitors for the de novo synthesis (sulfasalazine) and regeneration (pyrimethamine) of BH4 could prove to be an effective strategy for targeting the GCH1/BH4 through redox disruption. Preliminary data BTICs isolated from patient derived xenografts (PDXs) indicated reduced viability when treated with sulfasalazine (SASP) and pyrimethamine (PYR). Furthermore, we observed lower/depleted levels of BH4 relative to BH2 when BTICs were treated with SASP and PYR. Lastly, there is an increase in mitochondrial ROS upon SASP and PYR treatment, suggesting dysregulated redox states. Importantly, temozolomide resistant GBM cells remained sensitive to SASP and PYR. Taken together, our preliminary data suggests the plausibility of targeting the GCH1/BH4 pathway with SASP and PYR to disrupt redox balance in glioma through the depletion of BH4.

Targeting the Regulatory Subunit R2Alpha of Protein Kinase A in Human Glioblastoma through shRNA-Expressing Lentiviral Vectors.

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.

A Key Pathway to Cancer Resilience: The Role of Autophagy in Glioblastomas.

There are no effective strategies for the successful treatment of glioblastomas (GBM). Current therapeutic modalities effectively target bulk tumor cells but leave behind marginal GBM cells that escape from the surgical margins and radiotherapy field, exhibiting high migratory phenotype and resistance to all available anti-glioma therapies. Drug resistance is mostly driven by tumor cell plasticity: a concept associated with reactivating transcriptional programs in response to adverse and dynamic conditions from the tumor microenvironment. Autophagy, or “self-eating”, pathway is an emerging target for cancer therapy and has been regarded as one of the key drivers of cell plasticity in response to energy demanding stress conditions. Many studies shed light on the importance of autophagy as an adaptive mechanism, protecting GBM cells from unfavorable conditions, while others recognize that autophagy can kill those cells by triggering a non-apoptotic cell death program, called ‘autophagy cell death’ (ACD). In this review, we carefully analyzed literature data and conclude that there is no clear evidence indicating the presence of ACD under pathophysiological settings in GBM disease. It seems to be exclusively induced by excessive (supra-physiological) stress signals, mostly from in vitro cell culture studies. Instead, pre-clinical and clinical data indicate that autophagy is an emblematic example of the ‘dark-side’ of a rescue pathway that contributes profoundly to a pro-tumoral adaptive response. From a standpoint of treating the real human disease, only combinatorial therapy targeting autophagy with cytotoxic drugs in the adjuvant setting for GBM patients, associated with the development of less toxic and more specific autophagy inhibitors, may inhibit adaptive response and enhance the sensibility of glioma cells to conventional therapies.

Abstract B95: PD-L1-mediated nanotherapeutic targeting of glioma-infiltrating myeloid cells synergizes with radiotherapy for glioblastoma

Abstract Massive infiltration of tumor-associated myeloid cells (TAMCs), reaching 30-50% of the tumor mass, is a distinctive characteristic of glioblastoma (GBM). These cells are a critical player in generating highly immunosuppressive microenvironment in GBM. Thus, therapeutic targeting of TAMCs might dampen the immunosuppression and improve the outcome of antiglioma therapies. In this work, we developed a novel nano-immunotherapy platform to actively and specifically target glioma-infiltrating TAMCs in vivo. Based on our finding that glioma-infiltrating TAMCs exhibit the highest expression of PD-L1 among all the immune cell infiltrates, we designed a lipid nanoparticle system (LNP) with surface-engineered anti-PD-L1 therapeutic antibody. Decoration of anti-PD-L1 antibody rendered our LNP highly capable of specifically targeting TAMCs and, interestingly, routing PD-L1 to lysosomal degradation. Co-encapsulation of dinaciclib, a cyclin-dependent kinase inhibitor, into LNP caused a dramatic elimination of TAMCs and inhibition of immunosuppression in glioma. Notably, the targeting efficiency of TAMC-targeted LNP was further increased when combined with radiation therapy (RT), a standard care of GBM. The superior therapeutic efficacy in mice treated by RT+LNP combination over the RT or LNP monotherapy was demonstrated in two syngeneic murine glioma models, GL261 and CT2A. We also demonstrated that our LNP successfully targeted TAMCs harvested from human resection specimens, which further validated the translational potential and clinical relevance of our nano-immunotherapy approach. In conclusion, our work established a new nanotechnology-based immunotherapeutic approach with great potential to improve the clinical outcome in patients with GBM. Citation Format: Peng Zhang, Jason Miska, Catalina Lee-Chang, Aida Rashidi, Wojciech K. Panek, Shejuan An, Markella Zannikou, Aurora Lopez-Rosas, Yu Han, Ting Xiao, Irina V. Balyasnikova, Maciej S. Lesniak. PD-L1-mediated nanotherapeutic targeting of glioma-infiltrating myeloid cells synergizes with radiotherapy for glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B95.

Common Molecular Alterations in Canine Oligodendroglioma and Human Malignant Gliomas and Potential Novel Therapeutic Targets.

Spontaneous canine (Canis lupus) oligodendroglioma (ODG) holds tremendous potential as an immunocompetent large animal model of human malignant gliomas (MG). However, the feasibility of utilizing this model in pre-clinical studies depends on a thorough understanding of the similarities and differences of the molecular pathways associated with gliomas between the two species. We have previously shown that canine ODG has an immune landscape and expression pattern of commonly described oncogenes similar to that of human MG. In the current study, we performed a comprehensive analysis of canine ODG RNAseq data from 4 dogs with ODG and 2 normal controls to identify highly dysregulated genes in canine tumors. We then evaluated the expression of these genes in human MG using Xena Browser, a publicly available database. STRING-database inquiry was used in order to determine the suggested protein associations of these differentially expressed genes as well as the dysregulated pathways commonly enriched by the protein products of these genes in both canine ODG and human MG. Our results revealed that 3,712 (23%) of the 15,895 differentially expressed genes demonstrated significant up- or downregulation (log2-fold change > 2.0). Of the 3,712 altered genes, ~50% were upregulated (n = 1858) and ~50% were downregulated (n = 1854). Most of these genes were also found to have altered expression in human MG. Protein association and pathway analysis revealed common pathways enriched by members of the up- and downregulated gene categories in both species. In summary, we demonstrate that a similar pattern of gene dysregulation characterizes both human MG and canine ODG and provide additional support for the use of the canine model in order to therapeutically target these common genes. The results of such therapeutic targeting in the canine model can serve to more accurately predict the efficacy of anti-glioma therapies in human patients.

Immunologic and gene expression profiles of spontaneous canine oligodendrogliomas.

Malignant glioma (MG), the most common primary brain tumor in adults, is extremely aggressive and uniformly fatal. Several treatment strategies have shown significant preclinical promise in murine models of glioma; however, none have produced meaningful clinical responses in human patients. We hypothesize that introduction of an additional preclinical animal model better approximating the complexity of human MG, particularly in interactions with host immune responses, will bridge the existing gap between these two stages of testing. Here, we characterize the immunologic landscape and gene expression profiles of spontaneous canine glioma and evaluate its potential for serving as such a translational model. RNA in situ hybridization, flowcytometry, and RNA sequencing were used to evaluate immune cell presence and gene expression in healthy and glioma-bearing canines. Similar to human MGs, canine gliomas demonstrated increased intratumoral immune cell infiltration (CD4+, CD8+ and CD4+Foxp3+ T cells). The peripheral blood of glioma-bearing dogs also contained a relatively greater proportion of CD4+Foxp3+ regulatory T cells and plasmacytoid dendritic cells. Tumors were strongly positive for PD-L1 expression and glioma-bearing animals also possessed a greater proportion of immune cells expressing the immune checkpoint receptors CTLA-4 and PD-1. Analysis of differentially expressed genes in our canine populations revealed several genetic changes paralleling those known to occur in human disease. Naturally occurring canine glioma has many characteristics closely resembling human disease, particularly with respect to genetic dysregulation and host immune responses to tumors, supporting its use as a translational model in the preclinical testing of prospective anti-glioma therapies proven successful in murine studies.

Electrophysiology of glioma: a Rho GTPase-activating protein reduces tumor growth and spares neuron structure and function.

Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies.

CK2 inhibition induced PDK4-AMPK axis regulates metabolic adaptation and survival responses in glioma

Understanding mechanisms that link aberrant metabolic adaptation and pro-survival responses in glioma cells is crucial towards the development of new anti-glioma therapies. As we have previously reported that CK2 is associated with glioma cell survival, we evaluated its involvement in the regulation of glucose metabolism. Inhibition of CK2 increased the expression of metabolic regulators, PDK4 and AMPK along with the key cellular energy sensor CREB. This increase was concomitant with altered metabolic profile as characterized by decreased glucose uptake in a PDK4 and AMPK dependent manner. Increased PDK4 expression was CREB dependent, as exogenous inhibition of CREB functions abrogated CK2 inhibitor mediated increase in PDK4 expression. Interestingly, PDK4 regulated AMPK phosphorylation which in turn affected cell viability in CK2 inhibitor treated glioma cells. CK2 inhibitor 4,5,6,7-Tetrabromobenzotriazole (TBB) significantly retarded the growth of glioma xenografts in athymic nude mouse model. Coherent with the in vitro findings, elevated senescence, pAMPK and PDK4 levels were also observed in TBB-treated xenograft tissue. Taken together, CK2 inhibition in glioma cells drives the PDK4-AMPK axis to affect metabolic profile that has a strong bearing on their survival.

Abstract A08: Telomerase inhibition brings metabolic compromises in glioblastoma multiforme (GBM)

Abstract Introduction: A defining characteristic of cancer cells is their ability to circumvent multiple regulatory mechanisms that normally restrict cell proliferation. Understanding the links between aberrant metabolic adaptation and prosurvival responses in glioma cells is crucial towards the development of new anti-glioma therapies. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase holoenzyme. The telomere maintenance or telomerase activity is mainly driven by hTERT and hTERC. A large number of reports suggest an ever increasing role of telomere maintenance in cancer progression. We sought to evaluate its involvement in the regulation of metabolic status in these cells. Methods: Human glioma cell lines A172 and U87MG Cells were grown in DMEM supplemented with 10% FBS. On attaining semi confluence, cells were switched to SFM (serum free media) for 4 hrs and further treated with Telomerase inhibitor for 24hrs. Glioma cell lines were transiently transfected with lipofectamine 2000 and lipofectamine RNAi max reagents. Cells were harvested 48hrs post transfection. Heterotropic glioma xenografts were generated upon injecting U87MG cells subcutaneously in flank region of nude mice. After 20 days of injection, prominent tumor volume was visible. Thereafter, mice were grouped and administered either with PBS or Telomerase inhibitor for 20 days on every alternate day. Mice were sacrificed and tissues were processed for subsequent experiments. Histochemically identified TERT mutant and wild type human glioma samples were also used to unravel the ongoing signaling cascades. Results: Inhibition of telomerase resulted in metabolic compromises. TERT was found to regulate the expression of an important transcription factor responsibe for maintenance of metabolic and antioxidant proteins. The expression of this transcription factor was concomitant with altered metabolic profile as characterized by decreased pentose phosphate pathway. TERT regulated expression of PPP genes seemed to have its effect on prosurvival and antioxidant molecules. Conclusions: hTERT plays a key role in survival responses by modulating the metabolic status of glioma cells both in vitro and in vivo. By switching the signals under the control of a metabolic sensor (transcription factor) that modifies energy status to govern anti proliferative environment, hTERT regulates both survival responses and energy status. Citation Format: Fahim Ahmad, Ellora Sen. Telomerase inhibition brings metabolic compromises in glioblastoma multiforme (GBM). [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A08.

DDEL-19PENETRATION OF HOMING PEPTIDE-FUNCTIONALIZED NANOPARTICLES TO GLIOMA SPHEROIDS IN VITRO

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain malignancy. Even when subjected to multimodal treatment using surgery, chemo- and radiotherapy, the majority of GBM patients die within a year after diagnosis. Affinity targeting of anti-glioma drugs to malignant lesions, in particular to disseminated cells hiding behind the blood-brain-barrier, may dramatically improve efficacy of antiglioma therapies. Upon intravenous administration, tumor penetrating peptides (TPP-s) home to malignant lesions and extravasate into tumor parenchyma. Interestingly, these peptides retain their activity to penetrate excised tumor explants in short-term ex vivo assays. Here, we report development of an in vitro 3D penetration assay of homing peptide-functionalized silver nanoparticles (AgNP) in U87 glioma spheroids. U87 spheroids were incubated with fluorescent silver nanoparticles functionalized with prototypic tumor penetrating peptides, iRGD (CRGDKGPDC) and RPARPAR, or control peptides. Subsequently, spheroids were incubated with a non-toxic etching solution that selectively removes the extracellular AgNP with no effect on internalized AgNP. Fluorescence imaging and Matlab -based quantitative analysis of spheroid cryosections and of live spheroids was used to assess distribution of AgNP with and without etching treatment. Compared to control nanoparticles, iRGD and RPARPAR functionalized AgNP showed dramatically increased binding and penetration of the particles in the U87 spheroids. The binding was inhibited by peptide receptor-blocking anti NRP-1 and anti-av-integrin antibodies (but not by control antibodies), suggesting specific effect of AgNP surface peptides. Establishment and validation of 3D in vitro tumor spheroid penetration assay may provide a standardized system for evaluation tumor homing and penetration ability of homing peptides and to allow medium- throughput mechanistic and experimental therapy studies.

The role of integrins in glioma biology and anti-glioma therapies.

Integrins are a group of molecules expressed by various cells including glioma cells and endothelial cells within the tumor. There are 18 known alpha and beta integrin subunits which form a heterodimer. Integrins regulate different cellular processes such as proliferation, adhesion, motility and survival as shown in numerous preclinical models. Furthermore, integrins control the activity of the transforming growth factor (TGF)-beta pathway and are involved in the process of angiogenesis which is indispensable for continued tumor growth. Because of the high expression levels of some integrins on glioma cells and their numerous functions, inhibition of integrin signaling has been considered a promising strategy for the treatment of glioma patients. Besides blocking antibodies which are currently under clinical investigation in other cancer entities, the integrin inhibitor cilengitide has been tested within several trials in glioblastoma patients over the last years. Cilengitide is a cyclic RDG peptide which targets integrins alphvbeta3 and alphavbeta5. Based on the results of smaller, initial trials suggesting an activity of cilengitide against glioblastoma, 2 larger trials were subsequently performed. However, both trials, which combined temozolomide-based chemoradiation with cilengitide failed to demonstrate an improved outcome with the addition of cilengitide. Ongoing translational analyses suggest that integrin levels in the tumor tissue are neither prognostic nor predict response to cilengitide. While the clinical development of cilengitide has been stopped, integrin inhibition with more effective agents may still be a promising approach in clinical neurooncology.

Recent advances in the role of toll-like receptors and TLR agonists in immunotherapy for human glioma.

Gliomas are extremely aggressive brain tumors with a very poor prognosis. One of the more promising strategies for the treatment of human gliomas is targeted immunotherapy where antigens that are unique to the tumors are exploited to generate vaccines. The approach, however, is complicated by the fact that human gliomas escape immune surveillance by creating an immune suppressed microenvironment. In order to oppose the glioma imposed immune suppression, molecules and pathways involved in immune cell maturation, expansion, and migration are under intensive clinical investigation as adjuvant therapy. Toll-like receptors (TLRs) mediate many of these functions in immune cell types, and TLR agonists, thus, are currently primary candidate molecules to be used as important adjuvants in a variety of cancers. In animal models for glioma, TLR agonists have exhibited antitumor properties by facilitating antigen presentation and stimulating innate and adaptive immunity. In clinical trials, several TLR agonists have achieved survival benefit, and many more trials are recruiting or ongoing. However, a second complicating factor is that TLRs are also expressed on cancer cells where they can participate instead in a variety of tumor promoting activities including cell growth, proliferation, invasion, migration, and even stem cell maintenance. TLR agonists can, therefore, possibly play dual roles in tumor biology. Here, how TLRs and TLR agonists function in glioma biology and in anti-glioma therapies is summarized in an effort to provide a current picture of the sophisticated relationship of glioma with the immune system and the implications for immunotherapy.

SC-17 * BMP4-SECRETING hAdMSCs ENGINEERED WITH NANOPARTICLES: A NON-VIRAL MSC-BASED THERAPY FOR GLIOBLASTOMA

Mesenchymal stem cells (MSCs) have attracted significant attention as effective delivery vehicles for targeting brain disorders. We have shown that MSCs derived from human adipose tissue (hAMSCs) and genetically modified with viruses display high anti-glioma tropism and can deliver bone morphogenetic protein 4 (BMP4), a therapeutic agent that reduces the clonogenic ability of glioma stem cells. Virus-safety concerns have led to the development of biodegradable nanoparticles that can transfect human cells. We identify nanoparticles for engineering hAMCSs to secrete BMP4 both in vitro and in vivo to treat brain cancer. hAMSCs were transfected using poly (beta-amino ester-PBAE)-based nanoparticles. Transfection efficacy and toxicity were evaluated to determine the optimal formulation to transfect hAMSCs with a BMP4-expressing plasmid. In vitro functional assays evaluated the effect of the transfection on hAMSC phenotype. In vivo imaging and histological analyses tracked engineered hAMSCs (NP-AMSCs) in the brain after local and systemic administration to rodents with orthotopic human tumors. Optimized nanoparticles transfected hAMSCs with 75 ± 2% efficacy and 71 ± 7% viability which was significantly superior (p < 0.0001) to LipofectamineTM 2000(16 ± 3% transfection, 54 ± 5% viability) the leading commercial product. PCR and Western blot analysis confirmed BMP4 production from NP-hAMSCs (52.5 ng protein/106 NP-hAMSCs). NP-hAMSCs were significantly better in migration and invasion (p = 0.005) than hAMSCs transduced with lentivirus. Flow cytometry showed that NP-hAMSCs retain multipotency. Intranasal, intravenous, and local delivery of luciferase- and GFP-expressing NP-hAMSCs in a rodent model of human glioma showed transgene expression 5 days after injection and NP-hAMSC migration towards the brain and within the tumor. We demonstrate that PBAE-nanoparticle formulations effectively transfect hAMSCs to produce BMP4. NP-AMSCs migrate towards the brain and penetrate the tumor in a model of human glioma after local and systemic administration. NP-engineered BMP4-secreting hAMSCs can be a safe and effective alternative to viral transduction for stem cell-based anti-glioma gene therapies.

Blocking immunosuppressive checkpoints for glioma therapy: the more the Merrier!

Immunosuppressive checkpoints mediated by IDO, CTLA4, and PD1/PDL1 play a critical role in glioma progression and the efficacy of immunotherapies. Combined blockade of these immunosuppressive checkpoints in a glioma model elicited long-term survival. This combined blockade adds to the armamentarium of anti-glioma therapies, which could be implemented in clinical trials. Clin Cancer Res; 20(20); 5147-9. ©2014 AACR.

P2X7 receptors are a potential novel target for anti-glioma therapies

Human gliomas pose significant morbidity and mortality to those afflicted by them, and currently there are no curative treatment modalities available for these highly invasive tumours. With the approval from the human ethics committee, patients diagnosed with brain tumour (glioma) were recruited for this study. At the time of surgical resection, freshly resected tumour as well as ‘peri-tumour’ tissue were taken directly from theatre to the laboratory and were successfully cultured. Confocal fluorescence microscopy techniques and immunohistochemistry were used for characterization of human glioma cultures. Dye uptake experiments and confocal microscopy were utilized for P2X7 receptor (P2X7R) pore activity. We reveal human glioma cultures to contain microglia in close association with glioma (tumour) cells. Both glioma cells and microglia were found to express the purinergic, ATP sensing, P2X7R. P2X7R protein expression was increased in microglia derived from tumour when compared to ‘peri-tumour’ tissue. The pore capacity of P2X7R in tumour-associated microglia was functional, as evidenced by dye uptake experiments. Importantly, inhibition of P2X7R with the synthetic antagonist, brilliant blue G (BBG) resulted in a significant decrease in the number of glioma cells in culture. P2X7R was found to be over-expressed in grade IV human gliomas and its pore capacity was functional. Antagonism of P2X7R with BBG resulted in a decrease in tumour cell number. This identifies P2X7R as a promising therapeutic target to combat human glioma proliferation.

Effect of silencing thymosin beta 4 gene expression on stemness and invasiveness in glioblastoma.

2081 Background: Thymosin β4 (TB4) is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. TB4 gene silencing promotes differentiation of neural progenitor cells whereas TB4 overexpression initiates cortical folding of developing brain hemispheres. However, a role of TB4 in malignant gliomas has not yet been investigated. Methods: We first analyzed TB4 expression on tissue microarrays and performed REMBRANDT and TCGA database interrogations. We analyzed TB4 expression in a panel of 8 long-term glioma cell lines and 7 glioma-initiating cell lines. Using lentiviral transduction, we modulated TB4 expression in LNT-229, U87MG and the glioma-initiating cell line GS-2. We studied clonogenic survival, migration, invasion, self-renewal, differentiation capacity of TB4-depleted or TB4-overexpressing glioma cells in vitro and tumorigenicity upon orthotopic implantation in vivo. Finally, we performed an Affymetrix gene chip analysis to unravel the molecular network of TB4 signaling effects. Results: TB4 expression increased with the grade of malignancy in gliomas and correlated with patient survival. In vitro, TB4 gene silencing by lentiviral transduction decreased migration, invasion, growth and self-renewal, and promoted differentiation and the susceptibility to undergo apoptotic cell death upon nutrient depletion in LNT-229, U87MG and the glioma stem-cell line GS2, respectively. In vivo, survival of nude mice bearing tumors derived from TB4-depleted glioma cells was improved and the tumorigenicity of the GS2 glioma stem-cell line was decreased. The gene expression pattern was shifted from the mesenchymal towards the pro-neural gene signature upon TB4 gene silencing. The clustering of differentially regulated genes involved TGF-β and p53 signaling networks. Conclusions: TB4 may be a key regulator of malignancy in glioblastoma and therefore a novel candidate molecular target for anti-glioma therapies.