Presence Of Glioma Stem Cells Research Articles

Targeted therapies for Glioblastoma multiforme (GBM): State-of-the-art and future prospects.

Glioblastoma multiforme (GBM) remains one of the most aggressive and lethal forms of brain cancer, characterized by rapid growth and resistance to conventional therapies. The present review explores the latest advancements in targeted therapies for GBM, emphasizing the critical role of the blood-brain barrier (BBB), blood-brain-tumor barrier, tumor microenvironment, and genetic mutations in influencing treatment outcomes. The impact of the key hallmarks of GBM, for example, chemoresistance, hypoxia, and the presence of glioma stem cells on the disease progression and multidrug resistance are discussed in detail. The major focus is on the innovative strategies aimed at overcoming these challenges, such as the use of monoclonal antibodies, small-molecule inhibitors, and novel drug delivery systems designed to enhance drug penetration across the BBB. Additionally, the potential of immunotherapy, specifically immune checkpoint inhibitors and vaccine-based approaches, to improve patient prognosis was explored. Recent clinical trials and preclinical studies are reviewed to provide a comprehensive overview of the current landscape and future prospects in GBM treatment. The integration of advanced computational models and personalized medicine approaches is also considered, aiming to tailor therapies to individual patient profiles for better efficacy. Overall, while significant progress has been made in understanding and targeting the complex biology of GBM, continued research and clinical innovation are imperative to develop more effective and sustainable therapeutic options for patients battling this formidable disease.

Harnessing the role of aberrant cell signaling pathways in glioblastoma multiforme: a prospect towards the targeted therapy.

Glioblastoma Multiforme (GBM), designated as grade IV by the World Health Organization, is the most aggressive and challenging brain tumor within the central nervous system. Around 80% of GBM patients have a poor prognosis, with a median survival of 12-15months. Approximately 90% of GBM cases originate from normal glial cells via oncogenic processes, while the remainder arise from low-grade tumors. GBM is notorious for its heterogeneity, high recurrence rates, invasiveness, and aggressive behavior. Its malignancy is driven by increased invasive migration, proliferation, angiogenesis, and reduced apoptosis. Throughout various stages of central nervous system (CNS) development, pivotal signaling pathways, including Wnt/β-catenin, Sonic hedgehog signaling (Shh), PI3K/AKT/mTOR, Ras/Raf/MAPK/ERK, STAT3, NF-КB, TGF-β, and Notch signaling, orchestrate the growth, proliferation, differentiation, and migration of neural progenitor cells in the brain. Numerous upstream and downstream regulators within these signaling pathways have been identified as significant contributors to the development of human malignancies. Disruptions or aberrant activations in these pathways are linked to gliomagenesis, enhancing the invasiveness, progression, and aggressiveness of GBM, along with epithelial to mesenchymal transition (EMT) and the presence of glioma stem cells (GSCs). Traditional GBM treatment involves surgery, radiotherapy, and chemotherapy with Temozolomide (TMZ). However, most patients experience tumor recurrence, leading to low survival rates. This review provides an overview of the major cell signaling pathways involved in gliomagenesis. Furthermore, we explore the signaling pathways leading to therapy resistance and target key molecules within these signaling pathways, paving the way for the development of novel therapeutic approaches.

Elevated α-1,2-mannosidase MAN1C1 in glioma stem cells and its implications for immunological changes and prognosis in glioma patients

Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumor, and the presence of glioma stem cells (GSCs) has been linked to its resistance to treatments and recurrence. Additionally, aberrant glycosylation has been implicated in the aggressiveness of cancers. However, the influence and underlying mechanism of N-glycosylation on the GSC phenotype and GBM malignancy remain elusive. Here, we performed an in-silico analysis approach on publicly available datasets to examine the function of N-glycosylation-related genes in GSCs and gliomas, accompanied by a qRT-PCR validation experiment. We found that high α-1,2-mannosidase MAN1C1 is associated with immunological functions and worse survival of glioma patients. Differential gene expression analysis and qRT-PCR validation revealed that MAN1C1 is highly expressed in GSCs. Furthermore, higher MAN1C1 expression predicts worse outcomes in glioma patients. Also, MAN1C1 expression is increased in the perinecrotic region of GBM and is associated with immunological and inflammatory functions, a hallmark of the GBM mesenchymal subtype. Further analysis confirmed that MAN1C1 expression is closely associated with infiltrating immune cells and disrupted immune response in the GBM microenvironment. These suggest that MAN1C1 is a potential biomarker for gliomas and may be important as an immunotherapeutic target for GBM.

Gambogic acid impairs the maintenance and therapeutic resistance of glioma stem cells by targeting B-cell-specific Moloney leukemia virus insert site 1

BackgroundGlioblastoma (GBM) is the most common and lethal primary brain tumor with low effectiveness of available treatments. The tumor heterogeneity and therapeutic resistance are largely due to the presence of glioma stem cells (GSCs). Therefore, eliminating GSCs can overcome the progression, relapse, and resistance of GBM. Previous studies have shown that gambogic acid (GA), a natural active ingredient, has anti-glioma properties. Nonetheless, it is still unclear whether it has an inhibitory effect on GSCs and what its target might be. This study aimed to investigate the anti-tumor effects of GA on GSCs. In addition, this study found the target of GA in GSCs and elucidated the potential specific mechanisms by conducting both in vitro and in vivo experiments.B-cell-specific Moloney leukemia virus insert site 1 (BMI1) is a key stem cell factor of the polycomb group (PcG) family with important effects on the development, recurrence, and chemoresistance of several cancers. In both normal and cancer stem cells, BMI1 maintains stem cell self-renewal by regulating the cell cycle, cellular immortalization, and senescence. Its high expression in a variety of cancers correlates with poor clinical prognosis and chemoresistance. These mechanisms of BMI1 make it a potential therapeutic target for cancer therapy, and future studies may further reveal the specific roles of BMI1 mechanism and provide a basis for the development of new cancer therapeutic strategies. PurposeThis study investigated the in vitro and in vivo effects of GA in inducing apoptosis in GSCs and inhibiting GSCs self-renewal, as well as its underlying mechanisms. MethodsThis study synthesized biotinylated gambogic acid for the first time and angled for the target of gambogic acid using LC-MS/MS analysis, which has not been reported previously. Human-derived glioma stem cells GSC123 and GSC111 were used for in vitro studies, analyzing functions and mechanisms via microscale thermophoresis (MST), Annexin V/PI staining, Western blotting, immunofluorescence, and co-immunoprecipitation. The orthotopic glioma mouse model was used to assess the anti-tumor effects of GA in vivo. ResultsThis study demonstrated that GA is a specific inhibitor of BMI1, a key regulator controlling stem cell growth and self-renewal. GA binds to BMI1′s RING domain, accelerating K51-dependent degradation and suppressing H2A ubiquitination. Importantly, GA induces apoptosis, and inhibits GSC self-renewal, but minimally impacts neural progenitor cells (NPCs). GA can also be combined effectively with temozolomide and radiotherapy to increase their sensitivities in resistant cells. Furthermore, exogenous induction of BMI1 expression significantly hinders the disruption of GSCs by GA. In vivo, GA inhibits tumorigenicity, enhances the effect of temozolomide, and reduces BMI1 expression. ConclusionThese findings suggest that GA is a potential candidate for targeting GSCs and therefore be used to treat GBM.

Lactoferrin/CD133 antibody conjugated nanostructured lipid carriers for dual targeting of blood–brain-barrier and glioblastoma stem cells

The main reasons for the difficulty in curing and high recurrence rate of glioblastoma multiforme (GBM) include: 1. The difficulty of chemotherapy drugs in penetrating the blood-brain barrier (BBB) to target tumor cells; 2. The presence of glioma stem cells (GSCs) leading to chemotherapy resistance. Therefore, breaking through the limitations of the BBB and overcoming the drug resistance caused by GSCs are the main strategies to address this problem. This study presents our results on the development of lactoferrin (Lf)/CD133 antibody conjugated nanostructured lipid carriers (Lf/CD133-NLCS) for simultaneously targeting BBB and GSCs. Temozolomide (TMZ) loaded Lf/CD133-NLCS (Lf/CD133-NLCS-TMZ) exhibited high-efficiencyin vitroanti-tumor effects toward malignant glioma cells (U87-MG) and GSCs, while demonstrating no significant toxicity to normal cells at concentrations lower than 200 μg ml-1. The results of thein vitrotargeting GBM study revealed a notably higher cellular uptake of Lf/CD133-NLCS-TMZ in U87-MG cells and GSCs in comparison to Lf/CD133 unconjugated counterpart (NLCS-TMZ). In addition, increased BBB permeability were confirmed for Lf/CD133-NLCS-TMZ compared to NLCS-TMZ bothin vitroandin vivo. Taking together, Lf/CD133-NLCS-TMZ show great potential for dual targeting of BBB and GSCs, as well as GBM therapy based on this strategy.

Research progress on function and mechanism of long non-coding RNA in glioma.

This review aimed to comprehensively summarize the role of long non-coding RNA (lncRNA) in gliomas, the most common malignant tumors in the central nervous system, and explore their potential clinical applications. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic search using the PubMed database was conducted forty studies met the inclusion and exclusion criteria and were analyzed for type of intervention, the study's design, participants' demographics, and outcomes, including attrition. Gliomas, originating within the central nervous system, account for 40-45% of intracranial tumors. Despite advances in neurosurgical techniques, precise radiotherapy, and chemotherapy, the prognosis for glioma patients remains suboptimal. The review highlights the crucial regulatory role of lncRNA in gliomas. Differential expression of various lncRNAs, such as INHEG, SATB2-AS1, PSMB8-AS1, LINC01018, and SPRY4-IT1, has been observed in gliomas, suggesting their involvement in promoting or inhibiting tumorigenesis. Additionally, lncRNAs play roles in glioma characteristics such as proliferation, invasion, migration, angiogenesis, and the presence of glioma stem cells. The potential clinical applications of lncRNA in gliomas involve their association with tumor grading, diameter, metastasis, and family history. This review emphasizes the importance of understanding the molecular mechanisms involving lncRNA in gliomas. The identification of specific lncRNAs associated with gliomas provides potential molecular markers for diagnosis, differentiation, treatment, and prognosis evaluation. Further research is needed to uncover additional key lncRNAs and their underlying mechanisms, ultimately contributing to the improvement of glioma diagnosis and treatment.

STEM-21. DETERMINATION OF RACIAL DISPARITIES IN GLIOMA THROUGH RAP1B/NOTCH -INDUCED TUMORIGENESIS AND STEMNESS

Abstract BACKGROUND The incidence of GBM and survival rates after diagnosis vary significantly by race, with Caucasian Americans (CAs) having higher incidence and lower survival rates than African Americans (AAs). Accumulating evidence suggests that the failure of current therapies is partly attributed to the presence of glioma stem cells (GSC), leading to tumor progression and recurrence. Our recent work demonstrates that Ras-associated protein 1 (Rap1), a Ras-like small GTP-binding protein superfamily member, has roles in glioma cell proliferation and invasion. METHODS 1) The TCGA database was analyzed to investigate the predictive role of Rap1b in glioma patients. 2) The immunohistochemical staining for Rap1b1 was performed using the rabbit polyclonal anti-Rap1b in glioma tissue microarrays (TMA) from 76 glioma patients with 10 normal individuals. The staining intensity of cells in TMA was evaluated in three different bright fields (≥100 cells/field). The semi-quantitative HSCORE was calculated for Rap1b. 3) The mechanism of the oncogenic role of Rap1b in glioma was examined using in vitro cell culture. RESULTS 1. TCGA data showed that Rap1b mRNA is highly expressed in GBM patients compared to normal controls, indicating that Rap1b is a potential oncotarget in GBM. Expression of Rap1b is associated with worse survival in glioma patients, especially in CAs, but not in AAs. 2. The TMA data showed that elevated Rap1b protein expression correlates with grades in glioma patients. 3. In vitro studies showed that GSCs exhibit high levels of Rap1b, silencing Rap1b decreases glioma cell growth and invasion through G1/S arrest and the induction of apoptosis, as well as reduces Notch1 signaling in glioma cells; while potentiates GBM cell response to TMZ. CONCLUSION This study provided new knowledge of Rap1b’s potential as a therapeutic drug target for GBM therapy and revealed biological diversity across races and unaddressed health disparities.

Targeting EZH2 regulates the biological characteristics of glioma stem cells via the Notch1 pathway.

Glioma is the most common malignant brain tumor, and its behavior is closely related to the presence of glioma stem cells (GSCs). We found that the enhancer of zeste homolog 2 (EZH2) is highly expressed in glioma and that its expression is correlated with the prognosis of glioblastoma multiforme (GBM) in two databases: The Cancer Genome Atlas and the Chinese Glioma Genome Atlas. Additionally, EZH2 is known to regulate the stemness-associated gene expression, proliferation, and invasion ability of GSCs, which may be achieved through the activation of the STAT3 and Notch1 pathways. Furthermore, we demonstrated the effect of the EZH2-specific inhibitor GSK126 on GSCs; these results not only corroborate our hypothesis, but also provide a potential novel treatment approach for glioma.

Interrogating drug action and immune response in organotypic glioma microenvironment.

2068 Background: The glioma tumor microenvironment is characteristic of a high level of heterogeneity and a profoundly immune suppressive phenotype as driven by the presence of glioma stem cells and abundant innate immune cells of the myeloid lineages, rendering glioblastomas (GBM) recalcitrant to conventional treatments and immunotherapies. To address the full complexity of the brain tumor with its diverse components, we have developed an organotypic slice culture system to overcome several limitations of the other preclinical models and allow interrogation of drug action and immunomodulatory agents in human GBM ex vivo. Methods: We performed extensive characterization of the organotypic glioma slice system and optimization of the ex vivo tissue culture conditions, and determined the longitudinal dynamics of slice cell viability, relative tumor versus immune cellularity, and responses to a selection of therapeutic agents and cytokines by multiplexed flow cytometry and single cell transcriptome (scRNA seq) profiling. Results: Single cell transcriptomic analysis showed that both tumor and immune cell heterogeneity were preserved ex vivo for the two-week observation time, which exhibited tumor-type specific gene expression profiles suggesting cooperative interactions among cell populations. Consistent with the findings in fresh tumor specimens and an immune suppressive microenvironment, we found a scarcity of T lymphocytes in IDH mutant gliomas, whereas abundant T cells were found in recurrent GBM slices that persisted ex vivo. Additional analyses revealed that T cell infiltration in GBM was associated with the mesenchymal-like molecular phenotype, which exhibited remarkable intratumor heterogeneity highlighting the presence of stem-like cells and a unique cell population expressing aberrant cancer genes and antigen presenting molecules. We further found that T cells in organotypic GBM slices acquired a potent effector phenotype in response to cytokine-induced reprogramming of microglia and tumor-associated macrophages. In addition, scRNA seq also revealed a population of cancer-associated fibroblast-like cells that are present in all tumors sequenced, which may function to mediate the interactions between immune and tumor cells as our data suggested. Conclusions: We demonstrated that the organotypic slice culture system we developed is compatible with genomic, pharmacological, and immunotherapeutic manipulations and provided data showing that organotypic slice culture preserves glioma microenvironment ex vivo allowing acquisition of crucial information of response of heterogeneous patient tumor tissue to the agent tested. This method represents a unique mechanism by which therapy development can be facilitated in an ex vivo setting.

Glioblastoma Metabolism: Insights and Therapeutic Strategies.

Tumor metabolism is emerging as a potential target for cancer therapies. This new approach holds particular promise for the treatment of glioblastoma, a highly lethal brain tumor that is resistant to conventional treatments, for which improving therapeutic strategies is a major challenge. The presence of glioma stem cells is a critical factor in therapy resistance, thus making it essential to eliminate these cells for the long-term survival of cancer patients. Recent advancements in our understanding of cancer metabolism have shown that glioblastoma metabolism is highly heterogeneous, and that cancer stem cells exhibit specific metabolic traits that support their unique functionality. The objective of this review is to examine the metabolic changes in glioblastoma and investigate the role of specific metabolic processes in tumorigenesis, as well as associated therapeutic approaches, with a particular focus on glioma stem cell populations.

Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells.

Glioblastoma (GBM) is both the most common and aggressive type of primary brain tumor, associated with high mortality rates and resistance to conventional therapy. Despite recent advancements in knowledge and molecular profiling, recurrence of GBM is nearly inevitable. This recurrence has been attributed to the presence of glioma stem cells (GSCs), a small fraction of cells resistant to standard-of-care treatments and capable of self-renewal and tumor initiation. Therefore, targeting these cancer stem cells will allow for the development of more effective therapeutic strategies against GBM. We have previously identified several 7-amino acid length peptides which specifically target GSCs through in vitro and in vivo phage display biopanning. We have combined two of these peptides to create a dual peptide construct (EV), and demonstrated its ability to bind GSCs in vitro and target intracranial GBM in mouse models. A peptide pull-down performed with peptide EV followed by mass spectrometry determined N-cadherin as the binding partner of the peptide, which was validated by enzyme-linked immunosorbent assay and surface plasmon resonance. To develop cytotoxic cellular products aimed at specifically targeting GSCs, chimeric antigen receptors (CARs) were engineered containing the peptide EV in place of the single-chain variable fragment (scFv) as the antigen-binding domain. EV CAR-transduced T cells demonstrated specific reactivity towards GSCs by production of interferon-gamma when exposed to GSCs, in addition to the induction of GSC-specific apoptosis as illustrated by Annexin-V staining. These results exemplify the use of phage display biopanning for the isolation of GSC-targeting peptides, and their potential application in the development of novel cytotoxic therapies for GBM.

TRPML2 Mucolipin Channels Drive the Response of Glioma Stem Cells to Temozolomide and Affect the Overall Survival in Glioblastoma Patients

The survival of patients with glioblastoma (GBM) is poor. The main cause is the presence of glioma stem cells (GSCs), exceptionally resistant to temozolomide (TMZ) treatment. This last may be related to the heterogeneous expression of ion channels, among them TRPML2. Its mRNA expression was evaluated in two different neural stem cell (NS/PC) lines and sixteen GBM stem-like cells by qRT-PCR. The response to TMZ was evaluated in undifferentiated or differentiated GSCs, and in TRPML2-induced or silenced GSCs. The relationship between TRPML2 expression and responsiveness to TMZ treatment was evaluated by MTT assay showing that increased TRPML2 mRNA levels are associated with resistance to TMZ. This research was deepened by qRT-PCR and western blot analysis. PI3K/AKT and JAK/STAT pathways as well as ABC and SLC drug transporters were involved. Finally, the relationship between TRPML2 expression and overall survival (OS) and progression-free survival (PFS) in patient-derived GSCs was evaluated by Kaplan-Meier analysis. The expression of TRPML2 mRNA correlates with worse OS and PFS in GBM patients. Thus, the expression of TRPML2 in GSCs influences the responsiveness to TMZ in vitro and affects OS and PFS in GBM patients.

ARPC1B promotes mesenchymal phenotype maintenance and radiotherapy resistance by blocking TRIM21-mediated degradation of IFI16 and HuR in glioma stem cells

BackgroundIntratumoral heterogeneity is the primary challenge in the treatment of glioblastoma (GBM). The presence of glioma stem cells (GSCs) and their conversion between different molecular phenotypes contribute to the complexity of heterogeneity, culminating in preferential resistance to radiotherapy. ARP2/3 (actin-related protein-2/3) complexes (ARPs) are associated with cancer migration, invasion and differentiation, while the implications of ARPs in the phenotype and resistance to radiotherapy of GSCs remain unclear.MethodsWe screened the expression of ARPs in TCGA-GBM and CGGA-GBM databases. Tumor sphere formation assays and limiting dilution assays were applied to assess the implications of ARPC1B in tumorigenesis. Apoptosis, comet, γ-H2AX immunofluorescence (IF), and cell cycle distribution assays were used to evaluate the effect of ARPC1B on radiotherapy resistance. Immunoprecipitation (IP) and mass spectrometry analysis were used to detect ARPC1B-interacting proteins. Immune blot assays were performed to evaluate protein ubiquitination, and deletion mutant constructs were designed to determine the binding sites of protein interactions. The Spearman correlation algorithm was performed to screen for drugs that indicated cell sensitivity by the expression of ARPC1B. An intracranial xenograft GSC mouse model was used to investigate the role of ARPC1B in vivo.ResultsWe concluded that ARPC1B was significantly upregulated in MES-GBM/GSCs and was correlated with a poor prognosis. Both in vitro and in vivo assays indicated that knockdown of ARPC1B in MES-GSCs reduced tumorigenicity and resistance to IR treatment, whereas overexpression of ARPC1B in PN-GSCs exhibited the opposite effects. Mechanistically, ARPC1B interacted with IFI16 and HuR to maintain protein stability. In detail, the Pyrin of IFI16 and RRM2 of HuR were implicated in binding to ARPC1B, which counteracted TRIM21-mediated degradation of ubiquitination to IFI16 and HuR. Additionally, the function of ARPC1B was dependent on IFI16-induced activation of NF-κB pathway and HuR-induced activation of STAT3 pathway. Finally, we screened AZD6738, an ataxia telangiectasia mutated and rad3-related (ATR) inhibitor, based on the expression of ARPC1B. In addition to ARPC1B expression reflecting cellular sensitivity to AZD6738, the combination of AZD6738 and radiotherapy exhibited potent antitumor effects both in vitro and in vivo.ConclusionARPC1B promoted MES phenotype maintenance and radiotherapy resistance by inhibiting TRIM21-mediated degradation of IFI16 and HuR, thereby activating the NF-κB and STAT3 signaling pathways, respectively. AZD6738, identified based on ARPC1B expression, exhibited excellent anti-GSC activity in combination with radiotherapy.

TMET-05. MAGMAS FACILITATES METABOLIC CHANGES INDUCED BY STRESSORS IN GLIOBLASTOMA

Abstract The dynamic nature of tumor microenvironments contributes to tumor heterogeneity generating subpopulations of cells that are resistant to treatment in glioblastoma (GBM). The high recurrent rate of GBM tumors in patients can partially be explained by the presence of glioma stem cells (GSCs), which are thought to give rise to resistant clones against chemotherapy. As solid tumors expand, cancer cells can disrupt the tumor microenviroment by disrupting the blood brain barrier. Pericytes and astrocytes detach from the vascular endothelial cells, forming leaky vessels, which leads to thrombosis and eventually necrosis. Necrosis is a hallmark signature of GBM, as oxygen and nutrient supply runs low which can be observed through contrast imaging. Cancer cells go through a phenotypic change by upregulating stemness genes and glycolytic metabolism. Cells migrate away from hypoxic and nutrient deprived regions forming pseudopalisading cells which are an indication of cells becoming more invasive and malignant. Mitochondrial protein trafficking is a tightly regulated mechanism which selectively allows specific peptides carrying a mitochondrial targeting sequence (MTS) to be transported through the TOM40 and TIM23 complexes. Magmas, a TIM23 subunit, negatively regulates DNAJC19 by inhibiting its stimulatory activity on Hsp70 in the mitochondrial matrix. The regulation of the ATPase activity on Hsp70 is critical for processing pre-cursor proteins through the TIM23 complex into the mitochondrial matrix. Our laboratory has uncovered a novel role of Magmas activity in GBM cells under serum starved conditions in vitro. Magmas is downregulated in serum starved cells which allows for an increase of mitochondrial matrix proteins, which include key subunits important for forming electron transport chain complexes. This influx of ETC proteins can explain how cells are able to reduce aerobic glycolysis and increase oxidative phosphorylation (OXPHOS), a mechanism that can be exploited for potential therapeutic treatment in patients with GBM.

Dual-drug loaded nanomedicine hydrogel as a therapeutic platform to target both residual glioblastoma and glioma stem cells

Glioblastoma (GBM) recurrences are inevitable, and mainly originate from residual tumor cells and the presence of glioma stem cells (GSC) around the resection cavity borders. We previously showed that the local treatment of GBM with nanomedicine-based Lauroyl-gemcitabine lipid nanocapsules (GemC12-LNC) hydrogel delayed tumor onset in various preclinical models and can be used as a scaffold to deliver multiple drugs. However, it does not inhibit tumor relapse in the long-term. In this work, we aim at encapsulating an anti-GSC molecule in the GemC12-LNC hydrogel to eliminate both GBM cells and GSC. We performed a screening on GBM cell lines (GL261 and U-87 MG) and patient-derived GSC (GBM9) to select the anti-GSC molecule that could act synergically with GemC12. Based on our results, salinomycin (Sal) and curcumin (Cur) were selected for further development. Both GemC12-Sal-LNC and GemC12-Cur LNC showed similar size (55 nm), zeta potential (- 2 mV) and viscoelastic properties compared to the GemC12-LNC hydrogel. Encapsulation efficiency was above 95 %. Moreover, the GemC12-Sal-LNC hydrogel was stable for at least 6 months and released both drugs over 30 days in vitro. Both hydrogels inhibited the growth of GL261 and U-87 MG spheroids. Flow cytometry analysis showed that Sal reduced the GSC population in GL261 and U-87 MG cells. Our results show that the co-encapsulation of Sal in the GemC12-LNC hydrogel can reduce both GBM cells and GSC, and therefore might be promising to avoid the onset of GBM recurrences.

Small Molecules and Immunotherapy Agents for Enhancing Radiotherapy in Glioblastoma.

Glioblastoma (GBM) is an aggressive primary brain tumor that is associated with a poor prognosis and quality of life. The standard of care has changed minimally over the past two decades and currently consists of surgery followed by radiotherapy (RT), concomitant and adjuvant temozolomide, and tumor treating fields (TTF). Factors such as tumor hypoxia and the presence of glioma stem cells contribute to the radioresistant nature of GBM. In this review, we discuss the current treatment modalities, mechanisms of radioresistance, and studies that have evaluated promising radiosensitizers. Specifically, we highlight small molecules and immunotherapy agents that have been studied in conjunction with RT in clinical trials. Recent preclinical studies involving GBM radiosensitizers are also discussed.

Targeting Histone Deacetylase‐7 as a Novel Therapeutic Strategy for Glioblastoma Multiforme

BackgroundGlioblastoma multiforme (GBM) is the most aggressive primary brain tumor. GBM has an ominous prognosis with a survival rate of 14‐15 months after diagnosis and despite worldwide initiatives to optimize therapeutic approaches, GBM is still among the most challenging diseases to treat and the fastest to relapse in clinical oncology. The inevitable treatment resistance and relapse are mainly attributed to the presence of glioma stem cells (GSCs), which have the ability to self‐renew, indefinitely proliferate and to differentiate into different cell types. We hypothesize that HDAC7, as an epigenetic regulator, is playing a crucial mechanistic role in GBM and constitutes a novel druggable target for this universally fatal disease. By uncovering the HDAC7 interactome in the GSC nucleus, we provide mechanistic insights into the role of HDAC7 in regulating cancer stemness and possible unprecedented targeting strategies.MethodologyWe quantified the HDAC7 expression in tissue samples from 80 GBM patients admitted at Rhode Island Hospital using Custom Nanostring Panels and correlated the HDAC7 expression to overall patient survival. Also, we compared HDAC7 mRNA expression in GBM to the non‐tumor samples from TCGA data using GlioVis webtool. Next, we performed siRNA knock‐down (KD) of HDAC7 in GSCs followed by RNA‐seq and transcriptomic, gene enrichment and stemness enrichment analysis. We used mass spectrometry (Mod Spec) in si‐HDAC7 KD versus Control to quantify the potential enzymatic de‐acetylation properties of HDAC7 on the global post‐translational modifications of histones. Finally, to unravel the protein partners for HDAC7 in the GSC nucleus, we performed Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME).ResultsSurvival analysis of GBM patients showed significant decrease in survival for patients with high HDAC7 mRNA expression. In addition, GlioVis webtool revealed that HDAC7 expression is significantly higher in GBM tumors relative to non‐tumor samples. Differential gene expression analysis of RNASeq performed on HDAC7 siRNA KD GSCs versus control, revealed 4963 differentially expressed genes with the Gene Ontology enrichment showing significant enrichment for cell cycle and cell division suppression in HDAC7 KD GSCs. Additionally, 653 genes that were downregulated in HDAC7 KD GSCS were found to play significant role in stemness processes of embryonic stem cells and embryonal carcinoma stem cells. Mass spectrometry on GSCs following HDAC7 siRNA KD, revealed non‐significant de‐acetylation changes on global histone marks suggesting that HDAC7 has minimal or no histone deacetylation activity. Finally, RIME revealed novel protein partners in the proximity of 2.5 A° to HDAC7 in the GSCs nucleus. The newly discovered protein complex of HDAC7 suggests that HDAC7 regulates heterochromatin formation and spreading, which mechanistically control transcriptional programs of GSCs.ConclusionOur data suggest that HDAC7 plays a pivotal role in GBM. It regulates expression of transcripts that define cancer stemness, while inhibition of HDAC7 results in global increase in heterochromatin and transcriptional repression in GSCs. This study supports HDAC7 as a novel druggable target for GBM in a preclinical setting.

IMMU-42. OPTIMIZING IMMUNOTHERAPY FOR GLIOMA USING CYTOKINE-ACTIVATED NATURAL KILLER CELLS

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy associated with a 12-15 month survival after surgery and radio-chemotherapy. Utilizing adoptive cellular immunotherapy using natural killer (NK) cells has developed over the past two decades for a variety of hematologic malignancies. This approach in solid malignancies is limited by questions of cell dose versus tumor burden, insufficient tumor infiltration, and a tumor microenvironment that suppresses NK cell function. METHODS We isolated NK cells from healthy volunteers and activated them using IL-2, -15, -12, -18, then perform cytotoxic assays in the presence of glioma stem cells. We also tested the efficacy of the NK cells with intracranial delivery in a pre-clinical murine model of glioma. We tested various concentrations of IL-2 and IL-15 on the cytokine culture platform. RESULTS In this study, we demonstrate human NK cells, activated using a cytokine cocktail of interleukins-2, -15, -12, and -18, exert strong cytotoxic events against glioma cell lines. To further examine the efficacy of activated NK cells in vitro, we utilized intracranially xenografted glioma lines and demonstrated a survival benefit with tumor bed injections of these cytokine-activated NK cells (p=0.0089). We were able to confirm that NK cells cultured with low doses (200u IL2; 50ng/ml IL15) of both cytokines are just as effective as higher doses. This is important, as in vivoexhaustion of NK cells stimulated with high doses of either cytokine has been well validated. We also found that low-dose irradiation (4Gy) of glioma cells prior to co-culture with cytokine-activated NK cells promoted increased targeted glioma cell killing within 4 hours(32% cell killing). CONCLUSIONS These findings suggest that in a clinical study, injection of cytokine-activated NK cells into the glioblastoma tumor bed could be used as adjuvant treatment following either stereotactic radiation or surgical resection.

STEM-10. UPSTREAM REGULATORS OF THE GLIOMA STEM CELL PHENOTYPE

Abstract Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Recurrence of the disease is attributed in part to the presence of Glioma Stem Cells (GSC), which are resistant to chemo- and radiotherapy and can initiate tumor formation. Molecularly, GSCs resemble the mesenchymal subtype that is associated with worse prognosis. GSCs share many characteristics with Neural Stem Cells (NSCs) including proliferative potential, migratory capacity, telomerase activity, diverse progeny and similar gene signature, however differ fundamentally from NSCs in their tumor forming ability. RNA-Seq analysis of GSCs and NSCs illustrates significant enrichment in GSCs of transcription factors (TFs) known to be dysregulated in cancer, chief among them being SIX1, a developmental TF with documented roles in progression of multiple cancers. Overexpression of SIX1 in A172 GBM cells enhances proliferation, promotes resistance to radiotherapy and alters expression of a core set of 4 developmental TFs (POU3F2, SALL2, OLIG2 and SOX2) capable to reprogram differentiated GBM cells into GSCs (Suva et al., 2014). Analysis of SIX1 in surgical samples from glioma patients illustrates that high expression of SIX1 correlates with tumor grade, finding corroborated in the TCGA and CGGA data sets. Surprisingly, primary NSCs, after extended time in culture, increase their proliferation rate, acquire a mesenchymal transcriptional signature akin to GSCs, including high expression of SIX1, and form deadly tumors when implanted into the brains of mice. Comparing the epigenetic landscape of transformed and normal NSCs we identify a significant enrichment of accessible chromatin at promoter and enhancer loci in transformed NSCs, including at regulatory regions of SIX1 and of genes that define mesenchymal GBM. These data suggest that SIX1 may represent an upstream regulator of the GSC phenotype and may drive malignant transformation of NSCs. Genetic and epigenetic loss of function analyses are ongoing to test this hypothesis.

Anticarin β Inhibits Human Glioma Progression by Suppressing Cancer Stemness via STAT3.

Glioma is the most common form of malignant brain cancer. It is very difficult to cure malignant glioma because of the presence of glioma stem cells, which are a barrier to cure, have high tumorigenesis, associated with drug resistance, and responsible for relapse by regulating stemness genes. In this study, our results demonstrated that anticarin β, a natural compound from Antiaris toxicaria, can effectively and selectively suppress proliferation and cause apoptosis in glioma cells, which has an IC50 that is 100 times lower than that in mouse normal neural stem cells. Importantly, cell sphere formation assay and real time-quantitative analysis reveal that anticarin β inhibits cancer stemness by modulating related stemness gene expression. Additionally, anticarin β induces DNA damage to regulate the oncogene expression of signal transducer and activator of transcription 3 (STAT3), Akt, mitogen-activated protein kinases (MAPKs), and eventually leading to apoptosis. Furthermore, anticarin β effectively inhibits glioma growth and prolongs the lifts pan of tumor-bearing mice without systemic toxicity in the orthotopic xenograft mice model. These results suggest that anticarin β is a promising candidate inhibitor for malignant glioma.