Preclinical Glioma Models Research Articles

Adaptation of laser interstitial thermal therapy for tumor ablation under MRI monitoring in a rat orthotopic model of glioblastoma.

Laser interstitial thermal therapy (LITT) under magnetic resonance imaging (MRI) monitoring is being increasingly used in cytoreductive surgery of recurrent brain tumors and tumors located in eloquent brain areas. The objective of this study was to adapt this technique to an animal glioma model. A rat model of U251 glioblastoma (GBM) was employed. Tumor location and extent were determined by MRI and dynamic contrast-enhanced (DCE) MRI. A day after assessing tumor appearance, tumors were ablated during diffusion-weighted imaging (DWI)-MRI using a Visualase LITT system (n = 5). Brain images were obtained immediately after ablation and again at 24h post-ablation to confirm the efficacy of tumor cytoablation. Untreated tumors served as controls (n = 3). Rats were injected with fluorescent isothiocyanate (FITC) dextran and Evans blue that circulated for 10min after post-LITT MRI. The brains were then removed for fluorescence microscopy and histopathology evaluations using hematoxylin and eosin (H&E) and major histocompatibility complex (MHC) staining. All rats showed a space-occupying tumor with T2 and T1 contrast-enhancement at pre-LITT imaging. The rats that underwent the LITT procedure showed a well-demarcated ablation zone with near-complete ablation of tumor tissue and with peri-ablation contrast enhancement at 24h post-ablation. Tumor cytoreduction by ablation as seen on MRI was confirmed by H&E and MHC staining. Data showed that tumor cytoablation using MRI-monitored LITT was possible in preclinical glioma models. Real-time MRI monitoring facilitated visualizing and controlling the area of ablation as it is otherwise performed in clinical applications.

SYST-03. A PHASE I/II STUDY TO EVALUATE SAFETY AND PRELIMINARY ACTIVITY OF THE TUMOR-TARGETING ANTIBODY-CYTOKINE FUSION PROTEIN L19TNF IN PATIENTS WITH GLIOBLASTOMA AT FIRST RELAPSE

Abstract Treatment options for recurrent glioblastoma are limited and there is a need for novel effective therapies. We previously demonstrated encouraging anti-tumor activity with the targeted delivery of tumor necrosis factor a (TNF) in preclinical orthotopic glioma models. TNF- a is a potent pro-inflammatory cytokine which may trigger strong anti-tumor immunity. However, its systemic administration at therapeutically active doses is hampered by toxic side effects. L19TNF is a fully human antibody-cytokine fusion protein, comprising TNF- a fused to the antibody L19 that binds a tumor-specific epitope of the extracellular matrix protein fibronectin. This allows a targeted delivery of therapeutically relevant doses of TNF to the tumor site upon intravenous administration while sparing healthy tissues. In this phase I/II open label, non-randomized, monocentric study, we investigated the safety and preliminary activity of L19TNF for patients with isocitrate dehydrogenase (IDH) wildtype World Health Organization (WHO) grade III / IV glioma at first relapse. Twenty patients were enrolled from 2019-2020 and treated with intravenous infusions of L19TNF. In the phase I part of the study, 6 patients were assigned to two different dose levels of L19TNF and a dose of 13 µg/kg was established as the recommended dose. In the phase II part 14 patients were treated at the recommended dose. No dose-limiting toxicities were observed and survival follow-up is ongoing. In almost all patients, we observed treatment-associated emerging tumor necrosis. For patients that had re-surgery at progression on or after treatment with L19TNF, we demonstrated increased numbers of tumor-infiltrating lymphocytes compared to the tumor tissue obtained at primary diagnosis. Translational studies to better understand the effects of L19TNF on a molecular and immunophenotypic level are ongoing. ClinicalTrials.gov Identifier: NCT 03779230

Agonistic CD40 therapy induces tertiary lymphoid structures but impairs responses to checkpoint blockade in glioma

Gliomas are brain tumors characterized by an immunosuppressive microenvironment. Immunostimulatory agonistic CD40 antibodies (αCD40) are in clinical development for solid tumors, but are yet to be evaluated for glioma. Here, we demonstrate that systemic delivery of αCD40 in preclinical glioma models induces the formation of tertiary lymphoid structures (TLS) in proximity of meningeal tissue. In treatment-naïve glioma patients, the presence of TLS correlates with increased T cell infiltration. However, systemic delivery of αCD40 induces hypofunctional T cells and impairs the response to immune checkpoint inhibitors in pre-clinical glioma models. This is associated with a systemic induction of suppressive CD11b+ B cells post-αCD40 treatment, which accumulate in the tumor microenvironment. Our work unveils the pleiotropic effects of αCD40 therapy in glioma and reveals that immunotherapies can modulate TLS formation in the brain, opening up for future opportunities to regulate the immune response.

Abstract 1489: Deposition of genetically engineered T cell attracting antigen presenting cells in the glioma microenvironment with low intensity pulsed ultrasound-based blood-brain barrier opening triggers therapeutic responses in preclinical glioma models

Abstract Introduction: In contrast to brain metastases, gliomas have few antigen-presenting cells (APCs) in the tumor microenvironment, which may limit T cell effector responses. We hypothesized that: (1) an APC could be modified to express a T cell attracting chemokine that could enhance localized immune activation, and (2) that ultrasound-based blood-brain barrier (BBB) opening would enhance delivery of these APCs at the site of tumor antigens. Methods: Murine F4/80 and CD11c expressing APCs were isolated from the bone marrow of C57BL/6 mice and modified via lentiviral transduction to encode the cDNA of immune cell chemokines CXCL9 or CXCL10 and methionine-deficient green fluorescent protein (co-expression) for cell tagging. C57BL/6 mice were implanted with GL261 cells and treated with 1 x 106 CXCL9 or CXCL10 over-expressing APCs intracranially (i.c.), or intravenously (i.v.) with or without ultrasound-based BBB opening. To determine how the CXCL10 expressing APCs were altering the tumor microenvironment, immune cells were isolated from the glioma implanted hemisphere of all treatment groups and analyzed by multicolor flow cytometry. Results: Transduced APCs produced 3,392 pg/ml and 2,987 pg/ml of CXCL9 or CXCL10, respectively. Mice tolerated the treatments well without any adverse events, behavioral changes, or neurological toxicity. Survival analysis of mice treated with CXCL9 expressing APCs using i.c., or i.v. with and without BBB opening ultrasound showed no significant survival benefit compared with PBS-treated control mice. Mice treated i.c. with CXCL10-expressing APCs had significantly improved survival relative to the PBS control group (p<0.05), demonstrating that this cellular therapy required access to the tumor microenvironment in order to be effective. Notably, the most therapeutically efficacious administration route was when the CXCL10-expressing APCs were administered i.v. before ultrasound-based BBB opening even relative to the i.c. administered group (p<0.05). Intravenous administration of CXCL10 APCs combined with ultrasound-based BBB opening also significantly increased the percentage of T cells in the glioma microenvironment relative to all other treatment groups. Conclusions: The delivery of CXCL10-secreting APCs to the glioma microenvironment with ultrasound-based BBB opening was superior to delivery with direct i.c. injection. BBB disruption may have triggered more diffuse dispersal of the APCs throughout the tumor microenvironment or positioned these cells in closer proximity to the T cells emigrating from the vascular space into the localized glioma microenvironment. Citation Format: Aria Sabbagh, Kevin Beccaria, Xiaoyang Ling, Anantha Marisetty, Martina Ott, Ling-Yuan Kong, Dexing Fang, Jun Wei, Carole Desseaux, Guillaume Bouchoux, Michael Canney, Alexandre Carpentier, Amy B. Heimberger. Deposition of genetically engineered T cell attracting antigen presenting cells in the glioma microenvironment with low intensity pulsed ultrasound-based blood-brain barrier opening triggers therapeutic responses in preclinical glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1489.

Abstract 1553: Intratumoral delivery of STING agonist results in radiographic response in spontaneous canine high-grade glioma

Abstract Introduction: STING (stimulator of interferon genes) agonists can increase T cell infiltration into immunologically “cold” tumors such as gliomas through proinflammatory activation of suppressive tumor stroma, re-education of tumor-supportive M2 macrophages toward a proinflammatory M1 phenotype and can reverse the suppressive phenotype of myeloid-derived suppressor cells. Preclinical murine glioma models have not correlated with immunotherapy responses in human glioma subjects in part because of poor correlative immunobiology, but recent “omic” profiling has demonstrated a marked association of spontaneously arising canine gliomas with those in human subjects. Methods: We have created a novel STING agonist IACS-8779 that has shown robust activation of the STING pathway with equivalent or superior systemic anti-tumor responses relative to clinical benchmark compounds in murine models. A dose escalation clinical trial was conducted in client-owned canines (n=4) with newly-diagnosed presumed high-grade glioma identified radiographically on standard MRI sequences obtained on a 3T magnet. Each dog received 2 injections intratumorally at an interval of 4-6 weeks. The dose of IACS-8779 was sequentially increased over the course of the 6 injections from 5ug to 20ug, whereas the injection volume was fixed at 50uL. Injections were targeted to the tumors by image guidance, and injections were performed at a rate of 2uL/minute. Neurological status was monitored during the inter-injection interval. MRI was repeated at intervals of 4-6 weeks and radiographic responses were volumetrically analyzed. Results: All dogs tolerated the injections well with clinical symptoms remaining stable between the first and second injection. The survival times of the first 2 dogs were 2.5 and 9 months from the date of onset of signs, whereas the remaining dogs are still alive. The first dog, treated twice at a dose of 5ug, had progressive disease on serial MRI; whereas the second (5 and 10ug) and third (15 and 20ug) dogs had stable disease. The fourth dog treated at 20ug showed a volumetric change of 0.785cm3 pre-treatment to 0.194cm3 (>75%) after a single dose 5 weeks after the first injection. Conclusion: In support of previously published data showing effectiveness of IACS-8779 for treatment of orthotopic murine tumors (Ager CR, Bioorg Med Chem Lett. 2019), these data indicate that IACS-8779 is well-tolerated for repeated intratumoral injections up to 20ug in 50uL in a large animals (canines) with spontaneous glioma. Further, we have shown that a single injection of 20ug IACS-8779 can produce a radiographic response in a spontaneously arising high-grade glioma in the dog. Citation Format: C. Elizabeth Boudreau, Cynthia Kassab, Martina Ott, Chase M. DeRay, Jonathan Levine, Michael Curran, Amy B. Heimberger. Intratumoral delivery of STING agonist results in radiographic response in spontaneous canine high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1553.

Argyrin F Treatment‐Induced Vulnerabilities Lead to a Novel Combination Therapy in Experimental Glioma

AbstractGlioblastoma are incurable primary tumors of the central nervous system that frequently harbor molecular alterations in the retinoblastoma pathway with subsequent cell cycle abnormalities. It is aimed to investigate the anti‐glioma activity of the novel cell cycle‐stabilizing compound Argyrin F and its potential treatment‐induced vulnerabilities to exploit possibilities for novel rational combination therapies. Human and murine glioma cells are used, cytotoxicity and clonogenic survival assays, cell cycle analyses, and immunoblots are performed. Residual freshly resected tissue is used for a preclinical glioma model ex vivo, i.e., patient‐derived microtumors (PDMs) for co‐culture experiments with autologous tumor‐infiltrating lymphocytes (TILs). Mass spectrometry‐based immunopeptidomics are performed and the orthotopic syngeneic SMA560/VM/Dk glioma mouse model is used. Argyrin F‐induced cell cycle arrest and reduced clonogenic survival is observed. Argyrin F‐treated experimental glioma in vivo displays 4.6‐fold more glioma‐infiltrating CD8+ T cells. A distinctive treatment‐induced immunopeptidome is discovered. The combination of Argyrin F plus PD‐1 antibody increases cellular toxicity in PDM/TILs co‐cultures ex vivo and prolonged overall survival compared with monotherapies in vivo. Thus, Argyrin F leads to anti‐glioma effects and increases the immunogenicity, paving the way for a novel combination therapy of Argyrin F plus PD‐1 blockade.

DNA Repair Mechanisms and Therapeutic Targets in Glioma.

This review discusses current and investigative strategies for targeting DNA repair in the management of glioma. Recent strategies in glioma treatment rely on the production of overwhelming DNA damage and inhibition of repair mechanisms, resulting in lethal cytotoxicity. Many strategies are effective in preclinical glioma models while clinical feasibility remains under investigation. The presence of glioma biomarkers, including IDH mutation and/or MGMT promoter methylation, may confer particular susceptibility to DNA damage and inhibition of repair. These biomarkers have been adopted as eligibility criteria in the design of multiple ongoing clinical trials. Targeting DNA repair mechanisms with novel agents or therapeutic combinations is a promising approach to the treatment of glioma. Further investigations are underway to optimize this approach in the clinical setting.

HGG-40. FOCUSED ULTRASOUND ENHANCES ETOPOSIDE DELIVERY IN A MURINE PONTINE GLIOMA MODEL

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain cancer with limited treatment options and poor survival. The delivery of systemic therapies in this disease is severely limited by the blood-brain barrier (BBB). Focused ultrasound combined with intravenous microbubbles (FUS+MB) can effectively open the BBB permitting the entry of drugs across the cerebrovasculature. Etoposide is a chemotherapy frequently used in pediatric oncology with well-established anti-tumor effects but limited efficacy when administered systemically in DIPG. Given that FUS+MB in DIPG is not well studied, our goal was to determine the feasibility of ultrasound-mediated BBB opening and etoposide delivery in a preclinical murine pontine glioma model. MethodsA syngeneic, orthotopic model was established by stereotactic injection of PDGF-B+PTEN-/-p53-/- murine glioma cells into the pons of B6 albino mice. Mice were randomly divided into control (n=6) or FUS+MB groups (n=6). A single-element, spherical-segment FUS transducer (center frequency=1.5MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to sonicate the tumor and its margins on post-injection day 14. Immediately after treatment, 5 mg/kg of intraperitoneal etoposide was administered to all 12 mice. All animals underwent cardiac puncture and blood sampling, followed by transcardiac perfusion and brain harvesting. Liquid chromatography-mass spectometry was performed on both serum and tumor tissue to measure etoposide levels. ResultsContrast-enhanced MRI demonstrated successful BBB opening in all FUS+MB mice. Compared to control (mean=20.98ng/g), etoposide concentration in the sonicated tumor tissue (mean=164.77ng/g) was nearly eight times greater. Lastly, the mean brain tumor–to-serum ratio was more than fivefold higher in the treated mice (1.50%) compared with the control mice (0.28%) (P<0.005). ConclusionsFUS+MB is hereby shown successful in BBB opening and enhanced delivery of etoposide in a preclinical pontine glioma model.

EPCT-14. GD2 CAR T-CELLS MEDIATE CLINICAL ACTIVITY AND MANAGEABLE TOXICITY IN CHILDREN AND YOUNG ADULTS WITH H3K27M-MUTATED DIPG AND SPINAL CORD DMG

BackgroundWe previously discovered high expression of the disialoganglioside GD2 on H3K27M+ gliomas and demonstrated preclinical efficacy of intravenous (IV) GD2-targeted chimeric antigen receptor (CAR) T-cells in preclinical models of H3K27M-mutated diffuse intrinsic pontine glioma (DIPG) and diffuse midline gliomas (DMGs). We are now conducting a Phase I clinical trial (NCT04196413) of autologous GD2-targeting CAR T-cells for H3K27M+ DIPG and spinal cord DMG. Here we present the results of subjects treated at dose level 1 (DL1; 1 million GD2-CAR T-cells/kg IV). MethodsFour patients (3 DIPG, 1 spinal DMG; ages 4–25; 1M/3F) were enrolled at DL1. Three subjects with H3K27M+ DIPG received 1e6 GD2-CAR T-cells/kg IV on study. One patient with spinal DMG enrolled but became ineligible after manufacturing and was treated on an eIND at DL1. An Ommaya reservoir was placed in all subjects for therapeutic monitoring of intracranial pressure. Subjects underwent lymphodepletion with fludarabine/cyclophosphamide and remained inpatient for at least two weeks post-infusion. ResultsAll subjects developed cytokine release syndrome (Grade 1–3) manifested by fever, tachycardia and hypotension. Other toxicities included ICANS (Grade 1–2) and neurological symptoms/signs mediated by intratumoral inflammation which we have termed Tumor Inflammation-Associated Neurotoxicity (TIAN). No evidence of on-target, off-tumor toxicity was observed in any patients. No dose-limiting toxicities occurred. CAR T cells trafficked to the CNS and were detected in CSF and blood. 3/4 patients exhibited marked improvement or resolution of neurological deficits and radiographic improvement. The patient treated on an eIND exhibited >90% reduction in spinal DMG volume but progressed by month 3. Re-treatment of this subject via intracerebroventricular administration resulted in a second reduction in spinal DMG volume by ~80%. ConclusionsGD2-CAR T-cells at DL1 demonstrate a tolerable safety profile in patients with H3K27M+ DIPG/DMG with clear signs of T-cell expansion and activity including clinical responses.

Opening of the Blood-Brain Barrier Using Low-Intensity Pulsed Ultrasound Enhances Responses to Immunotherapy in Preclinical Glioma Models.

The blood-brain barrier (BBB) inhibits adequate dosing/penetration of therapeutic agents to malignancies in the brain. Low-intensity pulsed ultrasound (LIPU) is a safe therapeutic method of temporary BBB disruption (BBBD) to enhance chemotherapeutic delivery to the tumor and surrounding brain parenchyma for treatment of glioblastoma. We investigated if LIPU could enhance therapeutic efficacy of anti-PD-1 in C57BL/6 mice bearing intracranial GL261 gliomas, epidermal growth factor receptor variant III (EGFRvIII) chimeric antigen receptor (CAR) T cells in NSG mice with EGFRvIII-U87 gliomas, and a genetically engineered antigen-presenting cell (APC)-based therapy producing the T-cell attracting chemokine CXCL10 in the GL261-bearing mice. Mice treated with anti-PD-1 and LIPU-induced BBBD had a median survival duration of 58 days compared with 39 days for mice treated with anti-PD-1, and long-term survivors all remained alive after contralateral hemisphere rechallenge. CAR T-cell administration with LIPU-induced BBBD resulted in significant increases in CAR T-cell delivery to the CNS after 24 (P < 0.005) and 72 (P < 0.001) hours and increased median survival by greater than 129%, in comparison with CAR T cells alone. Local deposition of CXCL10-secreting APCs in the glioma microenvironment with LIPU enhanced T-cell glioma infiltration during the therapeutic window (P = 0.004) and markedly enhanced survival (P < 0.05). LIPU increases immune therapeutic delivery to the tumor microenvironment with an associated increase in survival and is an emerging technique for enhancing novel therapies in the brain.

Focused ultrasound mediated blood\u2013brain barrier opening is safe and feasible in a murine pontine glioma model

Drug delivery in diffuse intrinsic pontine glioma is significantly limited by the blood-brain barrier (BBB). Focused ultrasound (FUS), when combined with the administration of microbubbles can effectively open the BBB permitting the entry of drugs across the cerebrovasculature into the brainstem. Given that the utility of FUS in brainstem malignancies remains unknown, the purpose of our study was to determine the safety and feasibility of this technique in a murine pontine glioma model. A syngeneic orthotopic model was developed by stereotactic injection of PDGF-B+PTEN−/−p53−/− murine glioma cells into the pons of B6 mice. A single-element, spherical-segment 1.5 MHz ultrasound transducer driven by a function generator through a power amplifier was used with concurrent intravenous microbubble injection for tumor sonication. Mice were randomly assigned to control, FUS and double-FUS groups. Pulse and respiratory rates were continuously monitored during treatment. BBB opening was confirmed with gadolinium-enhanced MRI and Evans blue. Kondziela inverted screen testing and sequential weight lifting measured motor function before and after sonication. A subset of animals were treated with etoposide following ultrasound. Mice were either sacrificed for tissue analysis or serially monitored for survival with daily weights. FUS successfully caused BBB opening while preserving normal cardiorespiratory and motor function. Furthermore, the degree of intra-tumoral hemorrhage and inflammation on H&E in control and treated mice was similar. There was also no difference in weight loss and survival between the groups (p > 0.05). Lastly, FUS increased intra-tumoral etoposide concentration by more than fivefold. FUS is a safe and feasible technique for repeated BBB opening and etoposide delivery in a preclinical pontine glioma model.

Cannabidiol inhibits human glioma by induction of lethal mitophagy through activating TRPV4

ABSTRACT Glioma is the most common primary malignant brain tumor with poor survival and limited therapeutic options. The non-psychoactive phytocannabinoid cannabidiol (CBD) has been shown to be effective against glioma; however, the molecular target and mechanism of action of CBD in glioma are poorly understood. Here we investigated the molecular mechanisms underlying the antitumor effect of CBD in preclinical models of human glioma. Our results showed that CBD induced autophagic rather than apoptotic cell death in glioma cells. We also showed that CBD induced mitochondrial dysfunction and lethal mitophagy arrest, leading to autophagic cell death. Mechanistically, calcium flux induced by CBD through TRPV4 (transient receptor potential cation channel subfamily V member 4) activation played a key role in mitophagy initiation. We further confirmed TRPV4 levels correlated with both tumor grade and poor survival in glioma patients. Transcriptome analysis and other results demonstrated that ER stress and the ATF4-DDIT3-TRIB3-AKT-MTOR axis downstream of TRPV4 were involved in CBD-induced mitophagy in glioma cells. Lastly, CBD and temozolomide combination therapy in patient-derived neurosphere cultures and mouse orthotopic models showed significant synergistic effect in both controlling tumor size and improving survival. Altogether, these findings showed for the first time that the antitumor effect of CBD in glioma is caused by lethal mitophagy and identified TRPV4 as a molecular target and potential biomarker of CBD in glioma. Given the low toxicity and high tolerability of CBD, we therefore propose CBD should be tested clinically for glioma, both alone and in combination with temozolomide. Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; Baf-A1: bafilomycin A1; CANX: calnexin; CASP3: caspase 3; CAT: catalase; CBD: cannabidiol; CQ: chloroquine; DDIT3: DNA damage inducible transcript 3; ER: endoplasmic reticulum; GBM: glioblastoma multiforme; GFP: green fluorescent protein; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; SLC8A1: solute carrier family 8 member A1; SQSTM1: sequestosome 1; TCGA: The cancer genome atlas; TEM: transmission electron microscopy; TMZ: temozolomide; TRIB3: tribbles pseudokinase 3; TRPC: transient receptor potential cation channel subfamily C; TRPV4: transient receptor potential cation channel subfamily V member 4

Targeting Isocitrate Dehydrogenase Mutations in Cancer: Emerging Evidence and Diverging Strategies

Isocitrate dehydrogenase (IDH) active-site mutations cause a neomorphic enzyme activity that results in the formation of supraphysiologic concentrations of D-2-hydroxyglutarate (D-2HG). D-2HG is thought to be an oncometabolite that drives the formation of cancers in a variety of tissue types by altering the epigenetic state of progenitor cells by inhibiting enzymes involved in histone and DNA demethylation. This model has led to the development of pharmacologic inhibitors of mutant IDH activity for anticancer therapy, which are now being tested in several clinical trials. Emerging evidence in preclinical glioma models suggests that the epigenetic changes induced by D-2HG may persist even after mutant IDH activity is inhibited and D-2HG has returned to basal levels. Therefore, these results have raised questions as to whether the exploitation of downstream synthetic lethal vulnerabilities, rather than direct inhibition of mutant IDH1, will prove to be a superior therapeutic strategy. In this review, we summarize the preclinical evidence in gliomas and other models on the induction and persistence of D-2HG-induced hypermethylation of DNA and histones, and we examine emerging lines of evidence related to altered DNA repair mechanisms in mutant IDH tumors and their potential for therapeutic exploitation.

Neurooncology: 2021 update.

This article briefly presents 10 topics that were selected by the author as 'top 10 discoveries' published in 2020 in the broader field of neurooncological pathology including neurosciences as well as clinical neurooncology of interest for neurooncological pathology. The selected topics concern new information on the molecular characteristics of gliomas (infratentorial IDH-mutant diffuse astrocytomas, pediatric low-grade gliomas, infant-type high-grade gliomas, hypermutation in gliomas), the immunological aspects of the brain tumor microenvironment (TME), the impact of the TME on preclinical glioma models, and the importance of lymphatic drainage on brain tumor surveillance. Furthermore, important papers were published on two 'new' genetic syndromes predisposing to medulloblastoma, on liquid biopsy-based diagnosis of central nervous system (CNS) tumors, and on the 'microbiome' in glioblastomas (and other cancers). In the last part of this review, a dozen of papers are given as examples of papers that did not make it to the top 10 list of the author, underscoring the subjective component in the selection process. Acknowledging that 2020 will be remembered as the year in which the world changed because of the COVID-19 pandemic, some of the consequences of this pandemic for neurooncological pathology are briefly discussed as well. Hopefully, this review forms an incentive to appreciate the wealth of information provided by the papers that were used as building blocks for the present manuscript.

A novel integrated system using patient-derived glioma cerebral organoids and xenografts for disease modeling and drug screening.

A physiologically relevant glioma tumor model is important to the study of disease progression and screening drug candidates. However, current preclinical glioma models lack the brain microenvironment, and the established tumor cell lines do not represent glioma biology and cannot be used to evaluate the therapeutic effect. Here, we reported a real-time integrated system by generating 3D ex vivo cerebral organoids and in vivo xenograft tumors based on glioma patient-derived tissues and cells. Our system faithfully recapitulated the histological features, response to chemotherapy drugs, and clinical progression of their corresponding parental tumors. Additionally, our model successfully identified a case from a grade II astrocytoma patient with typical grade IV GBM features in both organoids and xenograft models, which mimicked the disease progression of this patient. Further genomic and transcriptomic characterization was associated with individual clinical features. We have demonstrated the "GBM-&Normal-like" signature to predict prognosis. In conclusion, we developed an integrated system of parallel models from patient-derived glioma cerebral organoids and xenografts for understanding the glioma biology and prediction of response to chemotherapy drugs, which might lead to a new strategy for personalized treatment for this deadly disease.

MODL-09. FEASIBILITY OF ACUTE SLICE CULTURE-SINGLE CELL SEQUENCING DRUG SCREENING AS A TOOL TO SELECT THERAPY FOR CHILDREN WITH RELAPSED BRAIN TUMORS

Children with relapsed brain tumors are less responsive to treatment. These children often receive therapies without having any robust predictive method of potential benefit. Acute slice culturing(ASC) is a methodology permitting freshly operated tumor to undergo a culturing process preserving the tumor’s micro-environment. With the current study, we investigated the feasibility of obtaining therapeutically meaningful data in a timely manner (3–5 days), performing direct drug testing and single cell sequencing using ASC. Previously, we have combined ex vivo slices of intact, patient-derived Glioblastoma tissue with single-cell RNA-seq for small-scale drug screening and assessment of patient and cell type-specific drug responses. We generated slices from preclinical mouse glioma models and surgical specimens from adult Glioblastoma patients, as well as from children with relapsed Ependymomas, Medulloblastomas, and Gliomas. We demonstrated that these acute slices preserved both the tumor heterogeneity and tumor microenvironment observed in single-cell RNA-seq of cells directly isolated from tumor tissue. Testing drug responses, we then treated tissue slices from the Glioblastoma mouse models and different patients with multiple drugs and combinations. This technique allowed us to identify drug-induced transcriptional responses in specific subpopulations of tumor cells, patient-specific drug sensitivities, and drug effects conserved in both mouse and human tumors. Preliminary data suggests that we can apply this procedure within 5–7 days and provide real-time drug screening/single cell sequencing ASC results to Recurrent/ Progressive pediatric Low-Grade Gliomas, High Grade Gliomas, Ependymomas and Medulloblastomas.

DDEL-13. FOCUSED ULTRASOUND MEDIATED BLOOD BRAIN BARRIER DISRUPTION IN A MURINE MODEL OF PONTINE GLIOMA: A SAFETY AND FEASIBILITY STUDY

BACKGROUNDDrug delivery remains a major obstacle in DIPG, as the blood brain barrier (BBB) limits the penetration of systemic therapies to the brainstem. Focused ultrasound (FUS) is an exciting new technology that, when combined with microbubbles, can open the BBB permitting the entry of drugs across the cerebrovasculature. Given that the utility of FUS in brainstem tumors remains unknown, the purpose of our study was to determine the safety and feasibility of this technique in a murine pontine glioma model.METHODSA syngeneic orthotopic model was established by stereotactic injection of PDGF-B+PTEN-/-p53-/- murine glioma cells (10,000/1ul) into the pons of B6 albino mice. A single-element, spherical-segment FUS transducer (center frequency=1.5MHz) driven by a function generator through a power amplifier (acoustic pressure=0.7MPa) was used with concurrent intravenous microbubble injection (FUS+MB) to sonicate the tumor on post-injection day 14. BBB opening was confirmed with gadolinium-enhanced MRI and Evans blue. Kondziela inverted screen (KIS) testing was completed to measure motor function. Mice were either immediately sacrificed for histopathological assessment or serially monitored for survival.RESULTSIn mice treated with FUS (n=11), there was no measured deficit in KIS testing. Additionally, the degree of intra-tumoral hemorrhage and inflammation on H&E in control (n=5) and treated mice (n=5) was similar. Lastly, there was no difference in survival between the groups (control, n=6, median=26 days; FUS, n=6, median=25 days, p>0.05).CONCLUSIONFUS+MB is a safe and feasible technique to open the BBB in a preclinical pontine glioma model.

EXTH-51. ANTI-INVASIVE EFFICACY AND SURVIVAL BENEFIT OF THE YAP-TEAD INHIBITOR VERTEPORFIN IN PRECLINICAL GLIOBLASTOMA MODELS

Abstract Glioblastoma (GBM) remains an incurable disease, in large part due to its malignant infiltrative spread, and current clinical therapy fails to target the invasive nature of tumor cells in disease progression and recurrence. Here, we use the YAP-TEAD inhibitor Verteporfin to target a convergence point for regulating tumor invasion/metastasis and establish the robust anti-invasive therapeutic efficacy of this FDA-approved drug and its survival benefit across several preclinical glioma models. Using patient-derived GBM cells and orthotopic xenograft models (PDX), we show that Verteporfin treatment disrupts YAP/TAZ-TEAD activity and processes related to cell adhesion, migration and epithelial-mesenchymal transition. In-vitro, Verteporfin impairs tumor migration, invasion and motility dynamics. In-vivo, intraperitoneal administration of Verteporfin in mice with orthotopic PDX tumors shows consistent drug accumulation within the brain and decreased infiltrative tumor burden, across three independent experiments. Interestingly, PDX tumors with impaired invasion after Verteporfin treatment downregulate CDH2 and ITGB1 adhesion protein levels within the tumor microenvironment. Finally, Verteporfin treatment confers survival benefit in two independent PDX models: as monotherapy in de-novo GBM and in combination with standard-of-care chemoradiation in recurrent GBM. These findings indicate potential therapeutic value of this FDA-approved drug if repurposed for GBM patients.

The glutamine antagonist prodrug JHU-083 slows malignant glioma growth and disrupts mTOR signaling

BackgroundMetabolic reprogramming is a common feature in cancer, and it is critical to facilitate cancer cell growth. Isocitrate Dehydrogenase 1/2 (IDH1 and IDH2) mutations (IDHmut) are the most common genetic alteration in glioma grade II and III and secondary glioblastoma and these mutations increase reliance on glutamine metabolism, suggesting a potential vulnerability. In this study, we tested the hypothesis that the brain penetrant glutamine antagonist prodrug JHU-083 reduces glioma cell growth.Material and MethodsWe performed cell growth, cell cycle, and protein expression in glutamine deprived or Glutaminase (GLS) gene silenced glioma cells. We tested the effect of JHU-083 on cell proliferation, metabolism, and mTOR signaling in cancer cell lines. An orthotopic IDH1R132H glioma model was used to test the efficacy of JHU-083 in vivo.ResultsGlutamine deprivation and GLS gene silencing reduced glioma cell proliferation in vitro in glioma cells. JHU-083 reduced glioma cell growth in vitro, modulated cell metabolism, and disrupted mTOR signaling and downregulated Cyclin D1 protein expression, through a mechanism independent of TSC2 modulation and glutaminolysis. IDH1R132H isogenic cells preferentially reduced cell growth and mTOR signaling downregulation. In addition, guanine supplementation partially rescued IDHmut glioma cell growth, mTOR signaling, and Cyclin D1 protein expression in vitro. Finally, JHU-083 extended survival in an intracranial IDH1 mut glioma model and reduced intracranial pS6 protein expression.ConclusionTargeting glutamine metabolism with JHU-083 showed efficacy in preclinical models of IDHmut glioma and measurably decreased mTOR signaling.

Improved radiosynthesis of 123I-MAPi, an auger theranostic agent

Purpose 123I-MAPi, a novel PARP1-targeted Auger radiotherapeutic has shown promising results in pre-clinical glioma model. Currently, 123I-MAPi is synthesized using multistep synthesis that results in modest yields and low molar activities (MA) that limits the ability to translate this technology for human studies where high doses are administered. Therefore, new methods are needed to synthesize 123I-MAPi in high activity yields (AY) and improved MA to facilitate clinical translation and multicenter trials. Materials and methods 123I-MAPi was prepared in a single step via 123I-iododetannylation of the corresponding tributylstannane precursor. In vitro internalization assay, subcellular fractionation and confocal microscopy where used to evaluate the performance of 123I-MAPi in a small cell lung cancer model. Results 123I-MAPi was synthesized in a single step from the corresponding stannane precursor in AY of 45 ± 2% and MA of 11.8 ± 4.8 GBq µmol−1. In vitro in LX22 cells showed rapid internalization (5 min) with accumulation found predominantly in the membrane, nucleus and chromatin of the cell as determined by subcellular fractionation. Conclusions Here, we have developed an improved radiosynthesis of 123I-MAPi, an Auger theranostic agent. This process was achieved using a single step, 123I-iododestannylation reaction from the corresponding stannane precursor in good AY and MA. 123I-MAPi was evaluated in vitro in a small cell lung cancer model with high PARP expression, rapid internalization and high nuclear uptake shown.