Glioma Stem Cell Models Research Articles

Single-Cell Heterogeneity of EGFR Pathway Is Linked to Unique Signatures of Drug Response and Malignancy in Patient Derived Glioblastoma Stem Cells.

In glioblastoma, the therapeutically intractable and resistant phenotypes can be derived from glioma stem cells, which often have different underlying mechanisms from non-stem glioma cells. Aberrant signaling across the EGFR-PTEN-AKT-mTOR pathways have been shown as common drivers of glioblastoma. Revealing the inter and intra-cellular heterogeneity within glioma stem cell populations in relations to signaling patterns through these pathways may be key to precision diagnostic and therapeutic targeting of these cells. Single cell parallel proteomic heterogeneity profiling of the EGFR-PTEN-AKT-mTOR pathways was conducted in a panel of fifteen glioma stem cell models derived from patient glioblastoma biopsies. The analysis included 59,464 data points from 14,866 cells and identified forty-nine molecularly distinct signaling phenotypes. High content bioinformatics resolved two unique patient clusters diverging on EGFR expression and AKT/TORC1 activation. Phenotypic validation indicated drug responsive phenotypes to EGFR blocking in the high EGFR expressing cluster with lower tumor initiating potential in comparison to the AKT/TORC1 activated cluster. High EGFR expression trended with improved patient prognosis while AKT/TORC1 activated samples trended with poorer patient outcomes. Genetic heterogeneity was observed in both clusters with proneural, classical and mesenchymal subtypes observed. Quantitative single cell heterogeneity profiling reveals divergent EGFR-PTEN-AKT-mTOR pathways of patient derived glioma stem cells, which would inform future research and personalized therapeutic strategies.

DDDR-32. EXPLORING ANDROGENIC MODULATION IN GLIOBLASTOMA: POTENCY AND SELECTIVITY OF NOVEL NORETHINDRONE DERIVATIVES

Abstract BACKGROUND Glioblastoma (GBM) represents the most frequently diagnosed malignant CNS tumor in adults and remains incurable. Despite the identification of several oncogenic drivers contributing to tumor formation, targeted therapy with brain-penetrant inhibitors has afforded little progress in survival outcomes. Interestingly, gender has been shown to increase the incidence of GBM, with men having roughly a 50-60% increase in incidence compared to women in the US. This statistic led to research on the involvement of and subsequent inhibition of androgen synthesis and signaling in cell culture models and tumor samples. METHODS To further investigate the potential involvement of steroidal hormones in GBM proliferation, our lab synthesized a series of norethindrone (NE) derivatives with varied molecular weights, steric bulk, and lipophilicity to modulate the androgenic activity of NE. The small library was tested in several GBM cell lines, including U87, A172, and T98G cells, using cell viability assays. RESULTS One derivative, a lipophilically modified version of NE synthesized via a one-pot click reaction, was shown to have improved potency compared with parental NE (>5-fold). The lipophilic NE derivative had IC50 value of 22 uM, 15 uM, and 35 uM in the U87, A172, and T98G cells, respectively. This molecule was tested in immortalized normal human astrocyte (NHA) cells and found to have an IC50 value of 34 uM suggesting some selectivity. DISCUSSION Given this derivative’s activity in GBM models and modest selectivity, we plan to screen it in additional GBM cell lines (LN-18 and U251) and eventually in glioma stem cell models. Future research will focus on identifying key targets involved in the mechanism of action, including the impact of lipophilic addition to the steroid nucleus and its effect on androgenic activity and downstream signaling. We will investigate these effects using RPPA and co-treatments with androgen receptor antagonists and 5-alpha reductase inhibitors.

Glioma Stem Cells as Promoter of Glioma Progression: A Systematic Review of Molecular Pathways and Targeted Therapies.

Gliomas' aggressive nature and resistance to therapy make them a major problem in oncology. Gliomas continue to have dismal prognoses despite significant advancements in medical science, and traditional treatments like surgery, radiation (RT), and chemotherapy (CT) frequently prove to be ineffective. After glioma stem cells (GSCs) were discovered, the traditional view of gliomas as homogeneous masses changed. GSCs are essential for tumor growth, treatment resistance, and recurrence. These cells' distinct capacities for differentiation and self-renewal are changing our knowledge of the biology of gliomas. This systematic literature review aims to uncover the molecular mechanisms driving glioma progression associated with GSCs. The systematic review adhered to PRISMA guidelines, with a thorough literature search conducted on PubMed, Ovid MED-LINE, and Ovid EMBASE. The first literature search was performed on 1 March 2024, and the search was updated on 15 May 2024. Employing MeSH terms and Boolean operators, the search focused on molecular mechanisms associated with GCSs-mediated glioma progression. Inclusion criteria encompassed English language studies, preclinical studies, and clinical trials. A number of 957 papers were initially identified, of which 65 studies spanning from 2005 to 2024 were finally included in the review. The main GSC model distribution is arranged in decreasing order of frequency: U87: 20 studies (32.0%); U251: 13 studies (20.0%); A172: 4 studies (6.2%); and T98G: 2 studies (3.17%). From most to least frequent, the distribution of the primary GSC pathway is as follows: Notch: 8 studies (12.3%); STAT3: 6 studies (9.2%); Wnt/β-catenin: 6 studies (9.2%); HIF: 5 studies (7.7%); and PI3K/AKT: 4 studies (6.2%). The distribution of molecular effects, from most to least common, is as follows: inhibition of differentiation: 22 studies (33.8%); increased proliferation: 18 studies (27.7%); enhanced invasive ability: 15 studies (23.1%); increased self-renewal: 5 studies (7.7%); and inhibition of apoptosis: 3 studies (4.6%). This work highlights GSC heterogeneity and the dynamic interplay within the glioblastoma microenvironment, underscoring the need for a tailored approach. A few key pathways influencing GSC behavior are JAK/STAT3, PI3K/AKT, Wnt/β-catenin, and Notch. Therapy may target these pathways. This research urges more study to fill in knowledge gaps in the biology of GSCs and translate findings into useful treatment approaches that could improve GBM patient outcomes.

MODL-08. ESTABLISHING IN VITRO MODELS TO RECAPITULATE PROTEIN-LEVEL PROGNOSTIC CELL PHENOTYPES FROM PRIMARY HUMAN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a highly aggressive primary central nervous system tumor for which median survival continues to be low, despite multimodal treatment and surgical resection. Heterogeneity at many levels (genomic, cytoarchitectural, and metabolic) likely contributes to these poor outcomes, and single-cell approaches can help to identify prognostically meaningful cells and features within heterogeneous tumors at each of these levels. Recent studies using mass cytometry (CyTOF) and the accompanying analysis pipeline Risk Assessment Population Identification (RAPID) measured per-cell protein levels in > 2 million cells from 28 IDH1/2-wildtype primary glioblastoma tumors and identified 2 phenotypes within these tumors that are present in different abundances across patients and have contrasting prognostic value. Increased abundance of Glioblastoma Negative Prognostic (GNP) cells within these tumors is predictive of shorter overall survival (OS HR = 1.07 [95% CI 1.02–1.12], p = 0.003), while the converse is true for Glioblastoma Positive Prognostic (GPP) cells (OS HR = 0.93 [0.87–1.0], p = 0.05). To effectively study these phenotypes, reproducible in vitro models are needed that recapitulate the total protein expression and phosphorylation events that distinguish GNP and GPP cells. Using available RNA transcript data, a candidate subset of glioma stem cell models were identified and are now being profiled using standardized mass-tagged antibody panels. These culture models will be quantitatively compared against established GNP and GPP populations in patient tissue samples using the Marker Enrichment Modeling statistic as well as other machine learning tools. Development of these models will be critical to future experiments aimed at identifying possible mechanistic therapies that effectively eliminate each of these identified populations, as well as aligning these protein-level phenotypes to other genomic and functional data in glioblastoma samples and models.

D-Mannose Slows Glioma Growth by Modulating Myeloperoxidase Activity.

Simple SummaryInflammation and oxidative stress are important host defense responses. However, while the host response can be cytotoxic and kill tumor cells, tumor cells can also alter and exploit the host immune environment to further their survival. Thus, the host response can impact both tumor suppression and progression. Modulating the tumor–host response interaction to favor tumor suppression would be highly desirable. D-mannose has been found to have anti-inflammatory properties and can block signaling related to myeloperoxidase (MPO), a highly oxidizing pro-inflammatory enzyme secreted in host defense. However, the effect of D-mannose on host immune response in the glioma microenvironment has not been explored. We found that D-mannose slowed glioma growth by increasing MPO activity and oxidative stress in the glioma microenvironment. Our findings revealed that D-mannose may be able to shift the host immune response toward tumor suppression and could be a potential new therapeutic direction for these difficult-to-treat tumors. Host immune response in the tumor microenvironment plays key roles in tumorigenesis. We hypothesized that D-mannose, a simple sugar with anti-inflammatory properties, could decrease oxidative stress and slow glioma progression. Using a glioma stem cell model in immunocompetent mice, we induced gliomas in the brain and tracked MPO activity in vivo with and without D-mannose treatment. As expected, we found that D-mannose treatment decreased the number of MPO+ cells and slowed glioma progression compared to PBS-treated control animals with gliomas. Unexpectedly, instead of decreasing MPO activity, D-mannose increased MPO activity in vivo, revealing that D-mannose boosted the MPO activity per MPO+ cell. On the other hand, D-glucose had no effect on MPO activity. To better understand this effect, we examined the effect of D-mannose on bone marrow-derived myeloid cells. We found that D-mannose modulated MPO activity via two mechanisms: directly via N-glycosylation of MPO, which boosted the MPO activity of each molecule, and indirectly by increasing H2O2 production, the main substrate for MPO. This increased host immune response acted to reduce tumor size, suggesting that increasing MPO activity such as through D-mannose administration may be a potential new therapeutic direction for glioma treatment.

TAMI-24. INHIBITION OF GBM INVASION BY THE Α-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID (AMPA) GLUTAMATE RECEPTOR ANTAGONIST PERAMPANEL

Abstract BACKGROUND Extensive tumor cell invasion within the brain represents a major problem for effective treatment of glioblastomas (GBMs). The invasive processes can be divided into three types: Collective cell invasion, perivascular infiltration, and single-cell invasion into the brain parenchyma. GBM cells can form synapses with neural cells pointing at an extensive communication network between brain and GBM cells which can be mediated via the metabolites Glutamine and Glutamate both needed for GBM cell proliferation. In this context, it has been shown in preclinical models that Perampanel, an antiepileptic agent, functioning as non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonist, has an inhibitory effect on GBM growth. To delineate how Perampanel affects GBM invasion, we utilised a highly characterized 3D GBM-brain organoid invasion model where single-cell invasion was studied in real-time following Perampanel treatment. METHODS A brain coculture model, consisting of rat brain organoids expressing various markers of the human adult brain, where confronted with GFP-labelled tumor cells. By using time-lapse confocal microscopy, we quantified single-cell invasion patterns and speed of invasion using two glioma stem cell models (BG5 and BG7). RESULTS Perampanel treatment significantly reduces tumor cell invasion into the brain organoids with the strongest effect seen in the most invasive GBM (BG5). The single-tumor cell invasion ratio was reduced by 72 % compared to the control (p= 0.0033). In contrast, collective cell invasion was reduced by 19 % (p= 0.028). Statistical analysis was performed using an unpaired sample t-test. CONCLUSION The AMPA glutamate receptor antagonist Perampanel significantly inhibits GBM invasion, suggesting an important role of the glutamate-glutamine cycle between the GBM cells and neurons in the invasion process. Moreover, this communication and exchange of metabolites seem to be more prominent where single GBM cells invade into the brain parenchyma compared to areas where collective invasion take place.

OS06.6A Inhibition of GBM invasion by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonist Perampanel

Abstract BACKGROUND Extensive tumor cell invasion within the brain represents a major problem for effective treatment of glioblastomas (GBMs). The invasive processes can be divided into three types: Collective cell invasion, perivascular infiltration and single-cell invasion into the brain parenchyma. It has recently been shown that GBM cells have the ability to form synapses with neural cells pointing at an extensive communication network between brain cells GBM cells. This communication network can be mediated via the metabolites glutamine and glutamate both needed for GBM cell proliferation. In this context, it has been shown in preclinical models that Perampanel, an antiepileptic agent, functioning as non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonist, has an inhibitory effect on GBM growth. In order to delineate how Perampanel affects GBM invasion, we here utilised a highly characterized 3D GBM-brain organoid invasion model where single-cell invasion was studied in real-time following Perampanel treatment. MATERIAL AND METHODS A brain coculture model, consisting of rat brain organoids expressing various markers of the human adult brain, where confronted with GFP-labelled tumor cells. By using time-lapse confocal microscopy, we quantified single-cell invasion patterns and speed of invasion using two glioma stem cell models (GSCs; BG5 and BG7). RESULTS Perampanel treatment significantly reduces tumor cell invasion into the brain organoids with the strongest effect seen in the most invasive GBM (BG5). Here, the single-tumor cell invasion ratio was reduced by 72 % compared to the control group (p=0.0033). In contrast, collective cell invasion was reduced by 19 % (p=0.028). Statistical analysis was performed using an unpaired sample t-test. CONCLUSION The AMPA glutamate receptor antagonist Perampanel significantly inhibits GBM invasion, suggesting an important role of the glutamate-glutamine cycle between the GBM cells and neurons in the invasion process. Moreover, this communication and exchange of metabolites seems to be more prominent where single GBM cells invade into the brain parenchyma compared to areas where collective invasion take place.

RGFP966, a histone deacetylase 3 inhibitor, promotes glioma stem cell differentiation by blocking TGF-β signaling via SMAD7

Glioma stem cells (GSC) play a major role in drug resistance and tumor recurrence. Using a genetic screen with a set of shRNAs that can target chromatin regulators in a GSC model, we have HDAC3 as a major negative regulator of GSC differentiation. Inhibition of HDAC3 using a pharmacological inhibitor or a siRNA led to the induction of GSC differentiation into astrocytes. Consequently, HDAC3-inhibition also caused a strong reduction of tumor-promoting and self-renewal capabilities of GSCs. These phenotypes were highly associated with an increased acetylation of SMAD7, which protected its ubiquitination. SMAD7 inhibits a TGF-β signaling axis that is required for maintaining stemness. These results demonstrate that HDAC3 appears to be a proper target in anti-glioma therapy.

Indoleamine 2,3-dioxygenase 1 is highly expressed in glioma stem cells

Recent research has revealed that glioblastoma (GBM) avoids the immune system via strong expression of indoleamine 2,3-dioxygenase 1 (IDO1). IDO1, an enzyme involved in tryptophan metabolism, is now proposed as a new target in GBM treatment, since several reports have demonstrated that IDO1 expression is related to GBM malignancy. On the other hand, it is well known that glioma stem cells (GSCs) are strongly related to the malignancy of GBM. However, there is as yet no report evaluating the relationship between GSCs and IDO1. We therefore examined the expression levels of IDO1 in GSCs in order to identify a new therapeutic target for GBM based on the immune systems of GSCs. In the present study, we employed human GBM cell lines (U-138MG, U-251MG) and patient-derived GSC model cell lines (0125-GSC, 0222-GSC). GSC model cell lines Rev-U-138MG and Rev-U-251MG were established by culturing U-138MG and U-251MG in serum-free media, while differentiated GBM model cell lines 0125-DGC and 0222-DGC were established by culturing 0125-GSC and 0222-GSC in serum-containing media. The expression levels of stem cell markers (Nanog, Nestin, Oct4 and Sox2) and IDO1 protein and mRNA were determined. Rev-U-138MG and Rev-U-251MG formed spheres and their expression levels of stem cell markers were increased as compared to U-138MG and U-251MG. On the other hand, 0125-DGC and 0222-DGC suffered breakdown of sphere formation, despite the original 0125-GSC and 0222-GSC forming spheres, and their expression levels of the markers were decreased. IDO1 expressions were strongly recognized in Rev-U-138MG, Rev-U-251MG, 0125-GSC and 0222-GSC as compared to U-138MG, U-251MG, 0125-DGC and 0222-DGC. These findings demonstrate that GSCs exhibit treatment resistance with immunosuppression via high expression levels of IDO1, and could represent a novel target for GBM treatment.

Abstract 1917: High-throughput evaluation of treatment response in patient-derived glioma stem cell models

Abstract Although significant progresses in molecular oncology are being made using conventional cell lines, most therapies still fail in phase III clinical trials. Patient-derived models are being used more frequently as they are more faithfully representing the genomic features of primary tumors. However, one by one test of each model from large biobanks is extremely economy and time consuming. In this study, each of a panel of patient-derived glioblastoma stem cell (GSC) models was uniquely tagged by a lentiviral Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding system. Barcoded GSCs were then pooled evenly and following by radiation treatment (RT) in vitro. Amplicon sequencing was employed to count the barcodes distribution, which represent the relative cell number. The results showed that this approach faithfully identified the RT resistant GSCs from a mixing pool when comparing to the results from canonical clonogenic assay. In addition, a fluorescence marker will be switched by delivery of corresponding barcodes targeting CRISPR so that we can re-isolate interested cell models from the treated pool for investigating the treatment sensitivity and resistance mechanism. This study will provide a robust approach for therapeutic discovery take advantage of patient-derived models from large biobanks. Citation Format: Ze-yan Zhang, Yingwen Ding, Ravesanker Ezhilarasan, Jie Yang, Lihong Long, Lawrence Bronk, Qianghu Wang, Erik P. Sulman. High-throughput evaluation of treatment response in patient-derived glioma stem cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1917.

Convection-enhanced delivery of sulfasalazine prolongs survival in a glioma stem cell brain tumor model.

Expression of CD44 in glioma cells was previously correlated with tumor grade and is considered a stem cell marker. CD44 stabilizes the cystine-glutamate transporter (xCT) and inhibits apoptosis in cancer stem cells (CSCs). Recently it was found that Sulfasalazine (SSZ), an anti-inflammatory drug, acts as an inhibitor of xCT and therefore has potential as a targeted therapy for CSCs. In this study, we tested an efficacy of SSZ against glioma stem cell model developed in rats. As poor penetration of blood-brain barrier resulted in insufficient efficacy of systemic SSZ treatment, SSZ was delivered locally with convection-enhanced delivery (CED). In vitro, expression of CD44 in glioma cells and efficacy of SSZ against glioma cells and glioma stem cells were confirmed. SSZ demonstrated anti-proliferative activity in a dose dependent manner against these cells. This activity was partially reversible with the addition of antioxidant, N-acetyl-L-cysteine, to the medium. In vivo, CED successfully delivered SSZ into the rat brain parenchyma. When delivered at 5mM concentration, which was the highest possible concentration when SSZ was dissolved in water, CED of SSZ resulted in almost no tissue damage. Against highly malignant bRiTs-G3 brain tumor xenografted rat model; the glioma stem cell model, CED of SSZ at 5mM concentration induced apoptosis and prolonged survival. Consequently, CED of SSZ induced glioma stem cell death without evidence of tissue damage to normal brain parenchyma. This strategy may be a promising targeted treatment against glioma stem cells.

3D bioprinted glioma stem cells for brain tumor model and applications of drug susceptibility

Glioma is still difficult to treat because of its high malignancy, high recurrence rate, and high resistance to anticancer drugs. An alternative method for research of gliomagenesis and drug resistance is to use in vitro tumor model that closely mimics the in vivo tumor microenvironment. In this study, we established a 3D bioprinted glioma stem cell model, using modified porous gelatin/alginate/fibrinogen hydrogel that mimics the extracellular matrix. Glioma stem cells achieved a survival rate of 86.92%, and proliferated with high cellular activity immediately following bioprinting. During the in vitro culture period, the printed glioma stem cells not only maintained their inherent characteristics of cancer stem cells (Nestin), but also showed differentiation potential (glial fibrillary acidic protein and β-tubulin III). In order to verify the vascularization potential of glioma stem cells, tumor angiogenesis biomarker, vascular endothelial growth factor was detected by immunohistochemistry, and its expression increased from week one to three during the culture period. Drug-sensitivity results showed that 3D printed tumor model was more resistant to temozolomide than 2D monolayer model at TMZ concentrations of 400–1600 μg ml−1. In summary, 3D bioprinted glioma model provides a novel alternative tool for studying gliomagenesis, glioma stem cell biology, drug resistance, and anticancer drug susceptibility in vitro.

Microscopic DTI accurately identifies early glioma cell migration: correlation with multimodal imaging in a new glioma stem cell model.

Monitoring glioma cell infiltration in the brain is critical for diagnosis and therapy. Using a new glioma Glio6 mouse model derived from human stem cells we show how diffusion tensor imaging (DTI) may predict glioma cell migration/invasion. In vivo multiparametric MRI was performed at one, two and three months of Glio6 glioma growth (Glio6 (n=6), sham (n=3)). This longitudinal study reveals the existence of a time window to study glioma cell/migration/invasion selectively. Indeed, at two months only Glio6 cell invasion was detected, while tumor mass formation, edema, blood-brain barrier leakage and tumor angiogenesis were detected later, at three months. To robustly confirm the potential of DTI for detecting glioma cell migration/invasion, a microscopic 3D-DTI (80μm isotropic spatial resolution) technique was developed and applied to fixed mouse brains (Glio6 (n=6), sham (n=3)). DTI changes were predominant in the corpus callosum (CC), a known path of cell migration. Fractional anisotropy (FA) and perpendicular diffusivity (D⊥ ) changes derived from ex vivo microscopic 3D-DTI were significant at two months of tumor growth. In the caudate putamen an FA increase of +38% (p<0.001) was observed, while in the CC a-28% decrease in FA (p<0.005) and a+95% increase in D⊥ (p<0.005) were observed. In the CC, DTI changes and fluorescent Glio6 cell density obtained by two-photon microscopy in the same brains were correlated (p<0.001, r=0.69), validating FA and D⊥ as early quantitative biomarkers to detect glioma cell migration/invasion. The origin of DTI changes was assessed by electron microscopy of the same tract, showing axon bundle disorganization. During the first two months, Glio6 cells display a migratory phenotype without being associated with the constitution of a brain tumor mass. This offers a unique opportunity to apply microscopic 3D-DTI and to validate DTI parameters FA and D⊥ as biomarkers for glioma cell invasion.

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models.

Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4(+) and CD8(+) T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas.

Senescence from glioma stem cell differentiation promotes tumor growth

Glioblastoma (GBM) is a lethal brain tumor composed of heterogeneous cellular populations including glioma stem cells (GSCs) and differentiated non-stem glioma cells (NSGCs). While GSCs are involved in tumor initiation and propagation, NSGCs' role remains elusive. Here, we demonstrate that NSGCs undergo senescence and secrete pro-angiogenic proteins, boosting the GSC-derived tumor formation in vivo. We used a GSC model that maintains stemness in neurospheres, but loses the stemness and differentiates into NSGCs upon serum stimulation. These NSGCs downregulated telomerase, shortened telomeres, and eventually became senescent. The senescent NSGCs released pro-angiogenic proteins, including vascular endothelial growth factors and senescence-associated interleukins, such as IL-6 and IL-8. Conditioned medium from senescent NSGCs promoted proliferation of brain microvascular endothelial cells, and mixed implantation of GSCs and senescent NSGCs into mice enhanced the tumorigenic potential of GSCs. The senescent NSGCs seem to be clinically relevant, because both clinical samples and xenografts of GBM contained tumor cells that expressed the senescence markers. Our data suggest that senescent NSGCs promote malignant progression of GBM in part via paracrine effects of the secreted proteins.

Abstract 1385: TGF beta regulates tumor resistance to antiangiogenic therapy through POSTN in glioma stem cell models

Abstract Periostin (osteoblast-specific factor 2, POSTN) is a 90-kDa ECM (extracellular matrix) protein containing an amino-terminal EMI, tandem repeat of four fascilin-domains and carboxyl-terminal domain including a heparin-binding site. Stromal POSTN plays a key role in regulating cancer stem cell (CSC) maintenance and expansion during metastatic colonization. Recently, it has been reported that POSTN functions as a progression associated and prognostic biomarker in glioma via inducing an invasive and proliferative phenotype. POSTN mRNA expression was significantly higher in grade IV gliomas than in grade II and grade III tumors. POSTN interacts with several integrin receptors such as αvβ1 and αvβ3 to regulate cellular response including cell proliferation, EMT (epithelial-mesenchymal transition) and cell migration. We demonstrated in previous studies that bevacizumab (Bevacizumab, Roche/Genentech) increased glioblastoma invasion in vivo. In the present study, we investigated the role of POSTN and its receptor in tumor invasion and resistance to antiangiogenic therapy. POSTN expression after treatment with avastin in vivo was increased as measured by Western blot and immunofluorescence. Stable knockdown of POSTN expression using specific shRNA abrogated expression of EMT (CAMK2N1, COL1A2, KRT14, COL3A1 and MMP-9) and angiogenesis-related (ANGPTL4, VEGFA, CXCL5, HPSE and EFNA3) genes compared to controls. VEGF expression was decreased in POSTN shRNA, and POSTN shRNA infected cells decreased invasion in both GSC11 and GSC272 glioma stem cell lines. TGF beta1 increased secretion of POSTN and phosphorylation of smad3 through a decrease in POSTN binding to smad3. Moreover, recombinant POSTN increased glioma cell invasion in an intergrin β1 receptor-dependent fashion. In animal experiments, median survival of animals implanted with GSC272-control cells was 63 days, while GSC272-bevacizumab was 84.5 days and knock down of POSTN was 84 days (p&amp;lt;0.05). Treatment with bevacizumab increased survival of POSTN shPOSTN tumors to 114.5 days compare to control (p&amp;lt;0.01). Tumor volume was decreased in the bevacizumab treatment (1.35 mm3) and POSTN shRNA (0.05 mm3) groups compared to controls (7.04 mm3). However, no tumor was observed in mice treated with both POSTN shRNA and bevacizumab. Bevacizumab increased TGF beta expression in GSC272 tumor tissue whereas tumors from POSTN shRNA mice treated with bevacizumab did not show evidence of TGF beta expression. Collectively, our data suggests that TGF beta increases POSTN secretion which appears to play an important role in glioma invasion and resistance to antiangiogenic therapy. Citation Format: Soon Young Park, Yuji Piao, Ningyi Tiao, Verlene Henry, Jianwen Dong, John Frederick de Groot. TGF beta regulates tumor resistance to antiangiogenic therapy through POSTN in glioma stem cell models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2015-1385

Abstract LB-314: CSF-1R inhibitor JNJ-28312141 reduces antiangiogenic therapy-induced myeloid cell infiltration and mesenchymal shift

Abstract Glioblastoma is the most common and malignant primary brain tumor in adults. Despite maximal tumor resection, radiation and chemotherapy, the tumor's high degree of infiltration leads to 100% recurrence and an average life expectancy after diagnosis of about 12 to 14 months. Vascular endothelial growth factor (VEGF) and other pro-angiogenic factors have been identified as critical mediators of angiogenesis in glioblastoma. Inhibition of angiogenesis results in a high radiographic response rate but ultimately all patients’ progress on this therapy. An increase in monocyte/macrophage recruitment has been associated with resistance to antiangiogenic therapy in preclinical mouse xenograft models and in patients. We hypothesized that combining antiangiogenic therapy with therapies inhibiting macrophage infiltration may delay resistance and prolong animal survival. In our glioma stem cell model, we evaluated the impact of bevacizumab alone and in combination with JNJ-28312141 (a novel colony-stimulating factor-1 receptor/FMS-related receptor tyrosine kinase-3 inhibitor) on the recruitment of myeloid cells, microvascular density, and animal survival. Four days after orthotopic implantation of 1× 105 NSC11 glioma stem cells, mice were randomized to treatment with bevacizumab, JNJ-28312141, or the combination of bevacizumab plus JNJ-28312141 and followed for survival (n=8-10 per group). JNJ-28312141 did not prolong survival compared to control. However, JNJ-28312141 combined with bevacizumab significantly reduced tumor vascularity (microvascular density measured using Factor VIII) and prolonged survival compared with bevacizumab alone (p=0.04). We compared the F4/80+ myeloid cell population in xenograft tumors using immunohistochemistry and immunofluorescence. The number of F4/80+ myeloid cells was significantly less in mice treated with JNJ-28312141 and bevacizumab compared with mice treated with bevacizumab alone (p&amp;lt;0.01). To evaluate the impact of treatment on the expression of mesenchymal markers, we quantified YKL-40 protein expression using immunofluorescence. Bevacizumab increased the expression of YKL-40 compared to controls and this increase in expression was significant blocked in the mice treated with the combination of bevacizumab and JNJ -28312141. These results suggest that resistance to antiangiogenic therapy can be partly overcome in glioblastoma by combining anti-VEGF therapy with an inhibitor of macrophages. In our orthotopic glioma model, CSF-1R inhibition is associated with a decrease in the recruitment and infiltration of F4/80+ cells into tumor and a decrease in mesenchymal marker expression. Our studies provide a new strategy to prolong the efficacy of antiangiogenic therapy and accelerate the integration of combination therapies into clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-314. doi:1538-7445.AM2012-LB-314

Low-Dose Radiation Enhances Survivin-Mediated Virotherapy against Malignant Glioma Stem Cells

To improve the efficacy and selectivity of virotherapy for malignant glioma, we designed a strategy to amplify adenoviral replication in conjunction with radiotherapy using a radioinducible promoter. First, we compared the radiation-inducible activity of FLT-1, vascular endothelial growth factor, DR5, Cox2, and survivin. We then examined the capacity of the optimal promoter to modulate transgene expression followed by E1A activity in vitro and in vivo in a glioma stem cell model. In the presence of radiation, survivin mRNA activity increased 10-fold. Luciferase transgene expression was dose dependent and optimal at 2 Gy. A novel oncolytic adenovirus, CRAd-Survivin-pk7, showed significant toxicity and replication against a panel of passaged and primary CD133(+) glioma stem cells. On delivery of radiation, the toxicity associated with CRAd-Survivin-pk7 increased by 20% to 50% (P < 0.05). At the same time, the level of E1A activity increased 3- to 10-fold. In vivo, treatment of U373MG CD133(+) stem cells with CRAd-Survivin-pk7 and radiation significantly inhibited tumor growth (P < 0.05). At the same time, the level of E1A activity was 100-fold increased versus CRAd-Survivin-pk7 alone. Selected genes linked to radioinducible promoters whose expression can be regulated by ionizing radiation may improve the therapeutic ratio of virotherapy. In this study, we have identified a new radioinducible promoter, survivin, which greatly enhances the activity of an oncolytic adenovirus in the presence of low-dose radiotherapy.