GBM Models Research Articles

Targeted Brain Tumor Radiotherapy Using an Auger Emitter.

Glioblastoma multiforme is a highly aggressive form of brain cancer whose location, tendency to infiltrate healthy surrounding tissue, and heterogeneity significantly limit survival, with scant progress having been made in recent decades. 123I-MAPi (Iodine-123 Meitner-Auger PARP1 inhibitor) is a precise therapeutic tool composed of a PARP1 inhibitor radiolabeled with an Auger- and gamma-emitting iodine isotope. Here, the PARP inhibitor, which binds to the DNA repair enzyme PARP1, specifically targets cancer cells, sparing healthy tissue, and carries a radioactive payload within reach of the cancer cells' DNA. The high relative biological efficacy of Auger electrons within their short range of action is leveraged to inflict DNA damage and cell death with high precision. The gamma ray emission of 123I-MAPi allows for the imaging of tumor progression and therapy response, and for patient dosimetry calculation. Here we demonstrated the efficacy and specificity of this small-molecule radiotheranostic in a complex preclinical model. In vitro and in vivo studies demonstrate high tumor uptake and a prolonged survival in mice treated with 123I-MAPi when compared with vehicle controls. Different methods of drug delivery were investigated to develop this technology for clinical applications, including convection enhanced delivery and intrathecal injection. Taken together, these results represent the first full characterization of an Auger-emitting PARP inhibitor which demonstrate a survival benefit in mouse models of GBM and confirm the high potential of 123I-MAPi for clinical translation.

Predicting postoperative delirium after microvascular decompression surgery with machine learning

ObjectiveThe aim of this study was to predict early delirium after microvascular decompression using machine learning. DesignRetrospective cohort study. SettingSecond Hospital of Lanzhou University. PatientsThis study involved 912 patients with primary cranial nerve disease who had undergone microvascular decompression surgery between July 2007 and June 2018. InterventionsNone. MeasurementsWe collected data on preoperative, intraoperative, and postoperative variables. Statistical analysis was conducted in R, and the model was constructed with python. The machine learning model was run using the following models: decision tree, logistic regression, random forest, gbm, and GBDT models. Results912 patients were enrolled in this study, 221 of which (24.2%) had postoperative delirium. The machine learning Gbm algorithm finds that the first five factors accounting for the weight of postoperative delirium are CBZ use duration, hgb, serum CBZ level measured 24 h before surgery, preoperative CBZ dose, and BUN. Through machine learning five algorithms to build prediction models, we found the following values for the training group: Logistic algorithm (AUC value = 0.925, accuracy = 0.900); Forest algorithm (AUC value = 0.994, accuracy = 0.948); GradientBoosting algorithm (AUC value = 0.994, accuracy = 0.970) and DecisionTree algorithm (aucvalue = 0.902, accuracy = 0.861); Gbm algorithm (AUC value = 0.979, accuracy = 0.944). The test group had the following values: Logistic algorithm (aucvalue = 0.920, accuracy = 0.901); DecisionTree algorithm (aucvalue = 0.888, accuracy = 0.883); Forest algorithm (aucvalue = 0.963, accuracy = 0.909); GradientBoostingc algorithm (aucvalue = 0.962, accuracy = 0.923); Gbm algorithm (AUC value = 0.956, accuracy = 0.920). ConclusionMachine learning algorithms predict the occurrence of delirium after microvascular decompression with an accuracy rate of 96.7%. And the major risk factors for the development of post-cardiac delirium are carbamazepine, hgb, and BUN.

Phase I trial of drug resistant immunotherapy: A first-in-class combination of MGMT-modified γδ t cells and temozolomide chemotherapy in newly diagnosed glioblastoma multiforme.

TPS3150 Background: Temozolomide (TMZ) transiently upregulates GBM-specific stress-induced NKG2D ligands that are targeted by innate immune effector cells. Leveraging this effect is problematic, however, due to the lymphodepleting effects of TMZ.Genetic modification of ex vivo expanded and activated with an MGMT-expressing lentivector allows simultaneous chemotherapy and γδ T cell therapy that targets the tumor when NKG2DL are maximally expressed. We have termed this Drug Resistant Immunotherapy (DRI). Patient-derived xenograft mouse models of both primary and recurrent GBM treated with DRI have shown a significant survival advantage that were otherwise impervious to either cell therapy or TMZ. These preclinical findings and associated safety data provide the rationale to initiate a Phase I trial of DRI in primary GBM. Methods: This first in human study will evaluate the safety and optimal dosing frequency of the DRI with TMZ (NCT04165941).Eligibility criteria include the following: GBM eligible for resection, ≥18y, adequate organ and marrow function, and KPS≥70. Six to 12patients with newly diagnosed GBM are being enrolled in a 3 + 3 design into 1 of 2 fixed dose levels (DL) of DRI. Following tumor resection and immediately prior to induction chemo/radiotherapy, an apheresis product is collected and γδ T cells expanded in Zoledronic Acid (Novartis) and rhIL-12 (Miltenyi) and transduced with a P140K-MGMT lentivector (Miltenyi Lentigen, Gaithersburg, MD), harvested, and cryopreserved. At initiation of maintenance phase TMZ therapy, patients receive 150mg/m2 intravenous TMZ concurrently with intracranial injection of 1 x 107 γδ T cells (DL1) delivered through a Rickham reservoir previously inserted into the tumor cavity at resection. The patient then receives 4 daily doses of oral TMZ followed by 24d rest. Treatment cycles escalate from 1 to 3 (DL2) DRI doses following a safety observation period and absence of dose limiting toxicity. Maintenance TMZ treatment will continue for 6 cycles. Safety evaluations consist of routine laboratory analyses, clinical measurements (physical exams, vital signs), neurological function and evidence DRI γδ T cell related toxicity. Peripheral blood will be obtained for comprehensive immuno-phenotyping and T cell function analysis. Clinical benefit of DRI will be characterized by evaluating responses (CR, PR, SD and PD) and determining progression-free, median, and overall survival. As of February 2020, enrollment into DL 1 is ongoing. Clinical trial information: NCT04165941 .

Combination immunotherapy of αPD-1 and extended half-life IL-2 clears established GL261 gliomas in an MHC class I independent fashion

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12 months post diagnosis despite aggressive treatment. Immunotherapy approaches to treat GBM are being developed, but the unique immune environment of the brain and the immunosuppressive features of GBM have complicated early efforts. Even αPD-1 checkpoint blockade, highly useful in the context of melanoma, has proved ineffective in the treatment of GBM. In order to promote the efficacy of αPD-1 against the GL261 murine model of GBM, we supplemented this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated dangerously high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. While neither αPD-1 nor MSA-IL-2 are effective monotherapies against the GL261 model tumor, we have found this combination immunotherapy to provide durable tumor clearance and to create immunologic memory capable of resisting GL261 rechallenge. Most strikingly, this combination therapy is independent of the CD8 T cell function, clearing established GL261 tumors in mice incapable of MHC class I restricted antigen presentation. Independence of MHC class I restricted antigen presentation makes this combination immunotherapy using αPD-1 checkpoint blockade both highly novel and translatable to clinical use.

Intracranial tumors lead to sequestration of T cells in the bone marrow

Abstract Glioblastoma (GBM) is the most common primary, malignant brain tumor and is universally fatal. Immunotherapeutic intervention has shown some success in immunogenic solid tumors, but little efficacy in GBM. Tumor-imposed T cell deficits remain a major obstacle. Recently, we demonstrated that GBM patients exhibit local and systemic T cell dysfunction, severe lymphopenia and contracted lymphoid organs. These observations are recapitulated in multiple murine models of GBM. Although T cell numbers are decreased in blood, lymph nodes, and spleen, we find large numbers of T cells sequestered in the bone marrow of both patients and mice with GBM. T cell sequestration occurs not only in the setting of GBM, but with nearly any cancer placed intracranially. Intriguingly, none of the tumors tested, including GBM, induce sequestration of T cells in the bone marrow when implanted subcutaneously. These data suggest that the capacity to sequester T cells resides within the brain and is usurped by tumors of the intracranial compartment. Such capacities may well represent a novel mechanism of immune privilege. Sequestration of T cells follows loss of sphingosine-1 phosphate-receptor 1 (S1P1) from the T cell surface. Understanding the drivers of S1P1 loss will provide crucial insight for how brain tumors facilitate systemic immune alterations and permit the development of more effective immune-based strategies for brain tumor patients.

Antigen-presenting cells lead to severe T cell exhaustion in glioblastoma

Abstract Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Median survival remains a bleak 15–17 months from time of diagnosis, calling for new therapeutic strategies. Current immunotherapeutic efficacy continues to be hindered by the robust immunosuppression of the GBM tumor microenvironment (TME). T cells, critical for tumor clearance, are particularly affected and many take on an exhausted phenotype within the tumor. We have previously observed significantly worse exhaustion in models of glioma relative to other solid tumor models, suggesting a TME-specific phenomenon. In an orthotopic murine model of GBM, T cell exhaustion was identified using surface expression of inhibitory ligands, PD1, TIM3, and LAG3. Functional status was examined using intracellular flow cytometry for IFNy, TNFa, and IL-2. We consistently observed T cell exhaustion among antigen-experienced tumor-specific CD8+ T cells, suggesting that tumor-imposed exhaustion is dependent upon T cells encountering their antigen. It remains undetermined, however, which cell within the GBM microenvironment provokes a uniquely severe T cell exhaustion phenotype. Using CRISPR-Cas9 gene editing, we modified tumor cells to lack antigen-presentation capacities and revealed persistence of T cell exhaustion. In contrast, when the same capacity was removed from infiltrating, hematopoietic cells, T cells in the GBM microenvironment were less exhausted and retained their function. This suggests antigen-presenting cells (APC) are critical for T cell exhaustion within GBM. Our findings uncover an important role for APCs in eliciting T cell exhaustion, which will provide novel interactions for targeted immunotherapies.

Therapeutic targeting of CD38 enhances temozolomide activity in glioblastoma cells and modulates their tumor microenvironment by eliciting an enhanced CD8+ T cell response

Abstract Glioblastoma (GBM) is an aggressive primary malignant brain tumor with poor prognosis. Surgical resection followed by chemoradiotherapy (temozolomide [TMZ] + radiation) results in a median survival of only 12-15 months. Recently, immunotherapy with targeted antibodies, therapeutic vaccination and adoptive cell therapy offers a promising option for treating GBM. Herein we observed that CD38, a catalytic receptor and regulator of NAD homeostasis, is expressed on GBM cells and their microenvironment in primary patient-derived GBM tissues. Blacher et al have shown that blocking CD38 enzymatic functions results in reduced GBM cell growth. This evidence prompted us to investigate the antitumor and immune engaging effects of a clinical grade anti CD38 therapeutic monoclonal antibody (Daratumumab/DARA) in preclinical models of GBM. We demonstrated that DARA significantly enhanced the direct apoptotic effect of TMZ and induced ADCC/CDC of GBM cells, irrespective of the presence of microglia. In an in vitro 3D simulated GBM + T cell coculture system, DARA induced activation (CD69/GITR upregulation/decreased PD1) and enhanced proliferation of CD8+ T cells/cTL activity against GBM cells. DARA also resulted in increased IFNγ and decreased IL-10; suggesting an improved antitumor cytokine milieu and ensued in downregulated inflammatory cytokine associated JAK2/STAT3 signaling pathway. Finally, we tested TMZ+DARA in a human GBM xenograft in vivo model and noted significantly prolonged survival of mice [TMZ+DARA vs. vehicle: 98.13±1.88 vs. 63.63±3.77 days, p<0.001]. Altogether, these observations led us to investigate the clinical activity of DARA+ TMZ in GBM patients in a phase I/II clinical trial.

Imaging CXCR4 Expression with Iodinated and Brominated Cyclam Derivatives

PurposeCXCR4 is one of several “chemokine” receptors expressed on malignant tumors (including GBM and PCNSL) and hematopoietic stem cells. Although 68Ga-pentixafor and 68Ga-NOTA-NFB have been shown to effectively image CXCR4 expression in myeloma and other systemic malignancies, imaging CXCR4 expression in brain tumors has been more limited due to the blood-brain barrier (BBB) and a considerable fraction of CXCR4 staining is intracellular.MethodsWe synthesized 6 iodinated and brominated cyclam derivatives with high affinity (low nM range) for CXCR4, since structure-based estimates of lipophilicity suggested rapid transfer across the BBB and tumor cell membranes.ResultsWe tested 3 iodinated and 3 brominated cyclam derivatives in several CXCR4(+) and CXCR4(−) cell lines, with and without cold ligand blocking. To validate these novel radiolabeled cyclam derivatives for diagnostic CXCR4 imaging efficacy in brain tumors, we established appropriated murine models of intracranial GBM and PCNSL. Based on initial studies, 131I-HZ262 and 76Br-HZ270-1 were shown to be the most avidly accumulated radioligands. 76Br-HZ270-1 was selected for further study in the U87-CXCR4 and PCNSL #15 intracranial tumor models, because of its high uptake (9.5 ± 1.3 %ID/g, SD) and low non-specific uptake (1.6 ± 0.7 %ID/g, SD) in the s.c. U87-CXCR4 tumor models. However, imaging CXCR4 expression in intracranial U87-CXCR4 and PCNSL #15 tumors with 76Br-HZ270-1 was unsuccessful, following either i.v. or spinal-CSF injection.ConclusionsImaging CXCR4 expression with halogenated cyclam derivatives was successful in s.c. located tumors, but not in CNS located tumors. This was largely due to the following: (i) the hydrophilicity of the radiolabeled analogues—as reflected in the “measured” radiotracer distribution (LogD) in octanol/PBS—which stands in contrast to the structure-based estimate of LogP, which was the rationale for initiating the study and (ii) the presence of a modest BTB in intracranial U87-CXCR4 gliomas and an intact BBB/BTB in the intracranial PCNSL animal model.

Localized Metabolomic Gradients in Patient-Derived Xenograft Models of Glioblastoma.

Glioblastoma (GBM) is increasingly recognized as a disease involving dysfunctional cellular metabolism. GBMs are known to be complex heterogeneous systems containing multiple distinct cell populations and are supported by an aberrant network of blood vessels. A better understanding of GBM metabolism, its variation with respect to the tumor microenvironment, and resulting regional changes in chemical composition is required. This may shed light on the observed heterogeneous drug distribution, which cannot be fully described by limited or uneven disruption of the blood-brain barrier. In this work, we used mass spectrometry imaging (MSI) to map metabolites and lipids in patient-derived xenograft models of GBM. A data analysis workflow revealed that distinctive spectral signatures were detected from different regions of the intracranial tumor model. A series of long-chain acylcarnitines were identified and detected with increased intensity at the tumor edge. A 3D MSI dataset demonstrated that these molecules were observed throughout the entire tumor/normal interface and were not confined to a single plane. mRNA sequencing demonstrated that hallmark genes related to fatty acid metabolism were highly expressed in samples with higher acylcarnitine content. These data suggest that cells in the core and the edge of the tumor undergo different fatty acid metabolism, resulting in different chemical environments within the tumor. This may influence drug distribution through changes in tissue drug affinity or transport and constitute an important consideration for therapeutic strategies in the treatment of GBM. SIGNIFICANCE: GBM tumors exhibit a metabolic gradient that should be taken into consideration when designing therapeutic strategies for treatment.See related commentary by Tan and Weljie, p. 1231.

Metabolite Imaging at the Margin: Visualizing Metabolic Tumor Gradients Using Mass Spectrometry.

Glioblastoma multiforme (GBM) tumors are highly metabolic and vascularized, yet little has been reported regarding the spatial localization of metabolic activity within these tumors. A mass spectrometry imaging (MSI) study by Randall and colleagues in this issue provides provocative observations of metabolic gradients in xenograft GBM models. The intensity of acylcarnitines is dramatically increased at tumor margins, which interface with normal tissue, but not in tumor margins at the edge of the brain. A secondary examination of drug metabolites suggests that the observed metabolic gradients are pharmacologically relevant. These findings underscore previously undescribed spatial metabolic heterogeneity in GBM biology and opportunities for MSI investigations.See related article by Randall et al., p. 1258.

Abstract B5: Reversing epigenetic gene silencing to overcome immune evasion in CNS malignancies

Abstract Glioblastoma (GBM) is a lethal brain malignancy, and standard of care only offers a modest survival benefit. Emerging evidence of immune surveillance in the brain has prompted investigation of immunotherapy as a potential treatment strategy. However, preliminary studies have demonstrated impaired immune cell trafficking to the tumor, making GBMs immunologically “cold.” One mechanism of immune evasion by the tumor is to epigenetically silence the expression of chemoattractant cytokines. Previous studies in ovarian and prostate cancer have successfully established the ability of GSK126, a small-molecule inhibitor of histone methyl transferase EZH2, to promote tumor T-cell infiltration and subsequent antitumor response. Mechanistically, GSK126 was shown to promote expression of interferon gamma (IFN gamma)-induced chemokines CXCL9 and CXCL10. In the present study we evaluated the therapeutic efficacy of GSK126 in preclinical models of GBM. Our studies determined that treatment of murine and human glioblastoma cell lines with GSK126 increased IFN gamma-mediated gene expression of CXCL9 and CXCL10. Transwell migration assays performed using human T cells demonstrated increased T-cell migration when exposed to conditioned medium from tumor cells treated with GSK126 and IFN gamma. In vivo, efficacy of GSK126 was tested in combination with anti-PD-1 antibody in C57Bl/6 immunocompetent mice subcutaneously implanted with the syngeneic glioma cell line CT2A. Mice treated with the combination of GSK126 and anti-PD-1 antibody showed a significant decrease in tumor volume and improved overall survival. Tumors treated with combination therapy showed increased infiltration of CD8+ IFN gamma expressing T cells and increased CXCR3+ migratory T cells in the tumor-draining lymph node. Interestingly, treatment of mice with intracranial CT2A tumors with GSK126 alone resulted in small tumors with better prognosis. Treatment-related survival benefit was lost in immune-compromised mice, suggesting that efficacy depended upon drug-related increased lymphocyte trafficking and subsequent T cell-mediated antitumor response. Analysis of post-treatment tumor by LC-MS confirmed that GSK126 crosses the blood-brain barrier. Taken together, our data suggest a potential synergistic therapeutic role for GSK126 in glioblastoma by enhancing current immunotherapy regimens with reversing the epigenetic changes that enable immune cell evasion with resultant increases in immune cell trafficking to the tumor. Citation Format: Nivedita M. Ratnam, Heather M. Sonnemann, Mark R. Gilbert, Amber J. Giles. Reversing epigenetic gene silencing to overcome immune evasion in CNS malignancies [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 B5.

Radiation Responses of 2D and 3D Glioblastoma Cells: A Novel, 3D-specific Radioprotective Role of VEGF/Akt Signaling through Functional Activation of NHEJ.

Glioblastoma is resistant to conventional treatments and has dismal prognosis. Despite promising in vitro data, molecular targeted agents have failed to improve outcomes in patients, indicating that conventional two-dimensional (2D) in vitro models of GBM do not recapitulate the clinical scenario. Responses of primary glioblastoma stem-like cells (GSC) to radiation in combination with EGFR, VEGF, and Akt inhibition were investigated in conventional 2D cultures and a three-dimensional (3D) in vitro model of GBM that recapitulates key GBM clinical features. VEGF deprivation had no effect on radiation responses of 2D GSCs, but enhanced radiosensitivity of GSC cultures in 3D. The opposite effects were observed for EGFR inhibition. Detailed analysis of VEGF and EGF signaling demonstrated a radioprotective role of Akt that correlates with VEGF in 3D and with EGFR in 2D. In all cases, positive correlations were observed between increased radiosensitivity, markers of unrepaired DNA damage and persistent phospho-DNA-PK nuclear foci. Conversely, increased numbers of Rad51 foci were observed in radioresistant populations, indicating a novel role for VEGF/Akt signaling in influencing radiosensitivity by regulating the balance between nonhomologous end-joining and homologous recombination-mediated DNA repair. Differential activation of tyrosine kinase receptors in 2D and 3D models of GBM explains the well documented discrepancy between preclinical and clinical effects of EGFR inhibitors. Data obtained from our 3D model identify novel determinants and mechanisms of DNA repair and radiosensitivity in GBM, and confirm Akt as a promising therapeutic target in this cancer of unmet need.

Preclinical evaluation of binimetinib (MEK162) delivered via polymeric nanocarriers in combination with radiation and temozolomide in glioma

Background and purposeGlioblastoma multiforme (GBM) is the most aggressive subtype of malignant gliomas, with an average survival rate of 15 months after diagnosis. More than 90% of all GBMs have activating mutations in the MAPK/ERK pathway. Recently, we showed the allosteric MEK1/2 inhibitor binimetinib (MEK162) to inhibit cell proliferation and to enhance the effect of radiation in preclinical human GBM models. Because the free drug cannot pass the blood–brain barrier (BBB), we investigated the use of nanocarriers for transport of the drug through the BBB and its efficacy when combined with radiotherapy and temozolomide (TMZ) in glioma spheroids.MethodsIn vitro studies were performed using multicellular U87 human GBM spheroids. Polymeric nanocarriers (polymersomes) were loaded with MEK162. The interaction between nanocarrier delivered MEK162, irradiation and TMZ was studied on the kinetics of spheroid growth and on protein expression in the MAPK/ERK pathway. BBB passaging was evaluated in a transwell system with human cerebral microvascular endothelial (hCMEC/D3) cells.ResultsMEK162 loaded polymersomes inhibited spheroid growth. A synergistic effect was found in combination with fractionated irradiation and an additive effect with TMZ on spheroid volume reduction. Fluorescent labeled polymersomes were taken up by human cerebral microvascular endothelial cells and passed the BBB in vitro.ConclusionMEK162 loaded polymersomes are taken up by multicellular spheroids. The nanocarrier delivered drug reduced spheroid growth and inhibited its molecular target. MEK162 delivered via polymersomes showed interaction with irradiation and TMZ. The polymersomes crossed the in vitro BBB model and therewith offer exciting challenges ahead for delivery of therapeutics agents to brain tumours.

TMOD-06. MODELLING ADULT AND PAEDIATRIC GLIOBLASTOMA MULTIFORME (GBM) USING A GENE SCREEN-BASED APPROACH IN MICE AND HUMAN IPSC-DERIVED CEREBRAL ORGANOIDS

Abstract Glioblastoma multiforme (GBM) represent the most devastating form of high-grade glioma (HGG) affecting adults and children. Despite a multi-therapeutic approach consisting in surgery, radio- and chemo-therapy the prognosis remains poor. Several models such as xenograft, animal models and recently organoids, have been developed in order to investigate the physiopathology of GBM. Although several mouse models (where either gain- or loss-of function of genes/pathway found altered in patients induce tumour formation) have been generated many aspects of how the tumour is formed, evolves, infiltrates and recurs after treatments remain unclear. Another challenge is the creation of proper GBM models showing intratumor heterogeneity found in the patient tumour but missing in the animal model generated so far. To overcome the lack of that human tumour characteristic, we decided to generate new model of GBM using a gene screen-based approach in mice. Firstly, we analysed the genes found amplified or mutated in GBM patients; secondly, we over-expressed the candidate genes (combination of multiple genes found to be mutated or amplified in specific GBM patients) in the subventricular zone (SVZ) of P2 mouse brain. Thirdly, we analysed the formation of tumour after 2 months. We over-expressed roughly 50 combinations in newborn mice and found that only 3 successfully led to the formation of lesions positive for proliferation and brain tumour marker (i.e. GFAP). We are currently characterising the tumours by DNA methylation analysis and RNA-seq (for further confirmation of the cancer subtype and intratumor heterogeneity) or investigating the cell of origin of the tumour. Moreover, we are testing the tumorigenicity of specific in GBM-amplified/mutated gene combinations in hiPSC-derived cerebal organoids. Our data suggest that a gene screen-based approach can be used for quickly and easily assaying the tumorigenicity of genes found amplified/mutated in GBM patients as well as the biology behind such complex process.

TMOD-08. SERUM ESTABLISHED CANCER STEM CELL CULTURES REPRESENT UNIQUE MODELS OF GLIOBLASTOMA INTER-TUMOR HETEROGENEITY

Abstract Glioblastoma (GBM) is the most frequent primary malignant brain tumor. The invasive and heterogeneous nature of GBM stem cells (GSCs) provide the basis for extreme therapy resistance of GBM and the majority of patients survive less than one year from diagnosis. Relevant models of GBM are important for basic research and drug screening and development, and the current consensus in the field is that GSCs are most faithfully maintained under serum-free neural stem cell (NSC) conditions. We have used serum-free NSC media to explant 230 patient-derived GBM samples, and have in parallel also explanted the same samples in regular serum-containing media. We found, accordingly, that serum-free media was most effective in generating sustainable cultures (137/230, 60%), called serum-free (SF) cultures. Interestingly, we also found that that there was a subgroup of GBM specimens that could only be established in serum-containing media (31/230, 13%), called exclusive serum-containing (eSC) cultures. We characterized a number of the eSC cultures and found that they expressed typical GSC markers such as MSI-1, BMI-1, nestin, CD44 and SOX2. They also displayed all functional characteristics of GSCs, i.e. extended proliferation, sustained self-renewal and orthotopic tumor initiation. Molecular analyses showed that the eSC cultures were most closely related to but separated from mesenchymal subtype GBM. In summary we have established GSC cultures that would have evaded modeling under serum-free conditions, representing unique cell models of GBM inter-tumor heterogeneity. Key words: Glioblastoma, serum cultures, stem-like cells, GBM heterogeneity

IMMU-15. A UNIQUE SELF-STIMULATIVE PROPERTY OF CD70 CAR ADVANCES T CELL FUNCTION AND ANTI-TUMOR RESPONSE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is a refractory brain tumor that desperately needs new therapeutic interventions. Our group identified CD70 as a novel target of CAR-T therapy for this malignancy. We demonstrate that CD70 is overexpressed by low-/high-grade gliomas and associated with poor survival for patients; CD70 promotes CD8 specific cell death and tumor-associated macrophage infiltration in gliomas. The CD70 CAR (using CD27, a natural costimulatory receptor of T cells as an antigen-binding region) T cells can efficiently eradicate CD70 positive tumors in syngeneic and xenograft mouse models. OBJECTIVE To evaluate the properties of CD70 CAR-transduced T cells in GBM treatment. METHODS CD70 CAR or IL13Rα2 CAR was linked with fluorescent reporter gene EGFP, and cloned into a retroviral vector (pMSGV8). In vitro T cell culture and flow cytometry were used to evaluate the self-enrichment property and susceptibility to TCR stimulation of the CAR T cells. KI67, Bcl-2, CD70 gene expression was tested by qPCR to measure the proliferation/apoptosis properties of the CAR T cells. Cytokine profile was analyzed by ELISA. The anti-tumor response was evaluated using Xenograft mouse models. RESULTS Compared with IL13Rα2 CAR T cells, the frequency of CD70 CAR T cells was significantly increased 3 weeks post transduction, and approximately 100 to 150-fold CD70 CAR T cell expansion without additional stimuli was achieved in vitro. The expanded CD70 CAR T cells were mostly (up to 85%) CD8+ T cells three weeks post CAR transduction. Enhanced proliferative capacity and production of IL-2, IFN-γ, and TNF-α of the CD70 CAR-transduced T cells upon anti-CD3/CD28 stimulation were also revealed. Results from animal models show that the CD70 CAR T cells present superior in vivo persistence and antitumor efficacy. CONCLUSION We show the auto-stimulative property, as well as superior T cell function and antitumor efficacy of CD70 CAR T cells in GBM models.

ATIM-22. PHASE I TRIAL OF DRUG RESISTANT IMMUNOTHERAPY: A FIRST-IN-CLASS COMBINATION OF MGMT-MODIFIEDγδ T CELLS AND TEMOZOLOMIDE CHEMOTHERAPY IN NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME

Abstract INTRODUCTION Alkylating agents such as Temozolomide (TMZ) significantly upregulate stress-induced NKG2D ligands targeted by innate immune effector cells. Genetic modification of γδT cells with an MGMT-expressing lentivector abrogates TMZ-induced lymphodepletion allowing simultaneous chemotherapy and γδT cell therapy in a process we have termed Drug Resistant Immunotherapy (DRI) resulting in a significant improvement in long-term survival in patient-derived xenograft (PDXT) models of primary and recurrent GBM. We have now initiated a Phase I clinical trial of DRI in primary GBM (BB-IND-17928) to determine the safety of this approach. METHODS Effector γδT cells are manufactured from a fractional apheresis product obtained immediately prior to post-resection chemo/radiotherapy. Cultures are propagated in a Miltenyi Prodigy® bioreactor using media containing Zoledronic Acid and rhIL-12, transduced with a P140K-MGMT lentivector, maintained for up to 14 days, harvested, and cryopreserved. At the initiation of maintenance TMZ therapy, patients receive 150mg/m2 intravenous TMZ and 1 x 107 γδT cells delivered through a Rickham reservoir inserted into the tumor cavity at primary resection. Treatment frequency escalates from one to three 28-day cycles in a standard 3 x 3 design. RESULTS Closed system manufacturing in the Prodigy bioreactor produced 4.5 – 11.9 x 108 γδT cells (44–103 fold expansion) from the apheresis starting material (0.6–1.9% γδT cells) with vector copy numbers of 0.9 - 2.1 copies/cell and in vitro cytotoxicity against K562 44.3% - 77.9% at E:T 40:1. Products were successfully cryopreserved and retained in vivo potency against GBM PDXT in mice over single-agent TMZ (p= 0.0286). All cell products met sterility and identity criteria. CONCLUSIONS These findings show that genetically modified γδT cells can be manufactured to clinical scale under GMP compliance in an automated bioreactor and cryopreserved to produce multiple doses of functionally-competent γδT cells. Patient accrual will initiate in 2019.

TMIC-58. THE CELLULAR AND MOLECULAR BASIS FOR MESENCHYMAL TRANSFORMATION IN GLIOMAS

Abstract Mesenchymal gliomas are the most aggressive tumors that carry the worst prognosis. The origins of mesenchymal cells within brain tumors, remains poorly understood. They could originate either from invading mesenchymal cells, from perivascular smooth muscle actin+ cells, or from a mesenchymal transformation of tumor cells. Identifying the origin and function of mesenchymal cells within gliomas is essential as these cells contribute to increased glioma aggressiveness and tumor progression. In this study we used human biopsies and implantable and genetically engineered mouse models (GEMM) of GBM to study tumor mesenchymal transformation. GBM implantable models were used to analyze the molecular landscape by laser microdissection followed by RNA-Seq and bioinformatics analysis. Time lapse confocal imagining was implemented to analyze GBM cells dynamics. Our results indicate the existence of a complex intratumoral and peritumoral dynamic organization of glioma cells (i.e., Oncostreams). Multicellular structures of elongated cells compatible with mesenchymal differentiation. These structures play important roles in intratumoral movements, peritumoral invasion of normal brain, and overall glioma progression. We also show that oncostreams are molecularly distinct and display increased expression of mesenchymal genes such as Col1a1. Knocking down of Col1a1 in a GEMM of aggressive gliomas reduced tumor progression and significantly increased animal survival. Histological examination confirmed absence of Col1a1, and absence of morphologically identifiable oncostreams. Our results show that tumor cells, especially within oncostreams, display a fibroblastic-like morphology and express proteins typical of mesenchymal cells. The knockout of Col1a1 from tumoral cells eliminated oncostreams from tumors and delayed tumor progression. These data suggest that tumor cells expressing mesenchymal genes regulate the organization of mesenchymal multicellular structures, and determine glioma progression. We propose that inhibiting mesenchymal transformation of glioma cells will assist in the treatment of glioblastoma.

SCIDOT-24. INCREASING INDUCED NEURAL STEM CELL PERSISTENCE IN THE TUMOR RESECTION CAVITY

Abstract Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults. Afflicted patients have a median survival rate of only 15 months, and patient survival statistics have remained stagnant for over three decades. Current standard of care includes maximal safe tumor resection, chemotherapy, and radiation. Nevertheless, GBM is aggressive, making recurrence and deeper tumor infiltration inevitable. Neural stem cells transdifferentiated from a skin biopsy, i.e. induced neural stem cells (iNSCs), have the innate ability to home to tumors, and, when engineered with cytotoxic proteins, can actively kill cancer cells. However, direct injection of these cells into the hostile immune environment of the tumor resection cavity results in an accelerated clearance rate and therefore a shortened therapeutic window. To combat this clearance issue, we investigated the FDA-approved hemostatic matrix, Floseal, as a cell delivery platform to increase iNSC persistence. In vitro, SEM imaging showed homogeneous iNSC distribution throughout Floseal. Using our surgical resection model of GBM in mice, we delivered iNSCs into the post-surgical cavity in Floseal and by direct injection to model current clinical delivery strategies. Serial kinetic imaging showed that human iNSC persistence delivered into the cavity in Floseal persisted over 90 days, while cells directly injected into the brain parenchyma persist less than 20 days. When we investigated the impact on tumor kill, we found the increase in persistence increased survival of GBM-bearing mice more than 30 days compared to control cells. Light-sheet microscopy showed wild-type neural stem cells migrate into invasive GBM-8 tumors in the contralateral hemisphere, and we are now using this approach to validate the homing of iNSCs delivered via Floseal. Administration of iNSCs encapsulated in the biocompatible Floseal matrix offers a promising, clinically-translatable therapeutic strategy for GBM.

CBMT-45. SEX-SPECIFIC METABOLIC ADAPTIONS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. GBM occurs more commonly in males, but female patients survive significantly longer. Understanding the molecular mechanisms that underlie those sex differences could support novel treatment strategies. In this regard, we found that male and female GBM patient samples differ in their metabolite abundance and that male patients exhibit a significantly higher abundance of TCA cycle metabolites. We confirmed those findings in a murine model of GBM, which has previously yielded important insights into sexual dimorphism in GBM. Strikingly, sex differences in TCA cycle flux were entirely driven by glutamine flux, not glucose flux, suggesting a sex-specific role for glutamine in GBM. Metabolic manipulation through glutamine deprivation resulted in a greater growth inhibition in male GBM cells. Glutamine itself can be utilized for anabolic reactions or it can be converted to glutamate by glutaminase. Only male GBM cells were sensitive to pharmacological glutaminase inhibition with BPTES or CB-839, suggesting that male GBM cells are glutamate dependent while female GBM cells are not. Concordantly, we found significantly higher glutaminase levels in male GBM cells. Furthermore, we found that numerous metabolites (including NADH, ATP, and glutathione) involved in cellular processes downstream of glutamate were more abundant in male GBM cells. In contrast, female GBM cells were resistant to low glutamine conditions and glutaminase inhibitors unless glutamine-synthase activity was disrupted, suggesting that glutamine synthesis might play a more prominent role in female GBM. Together, these data indicate that male and female GBM differ in their metabolic adaptions. Male GBM utilize glutamate to fuel the TCA cycle and mitochondrial activity while female GBM synthesize and utilize glutamine itself. This sexual dimorphism in metabolic reprogramming reveals novel sex specific metabolic targets for GBM and underlines the importance of considering sex in metabolic targeting approaches.