GL261 Glioma Research Articles

Differentiating Radiation Necrosis from Brain Tumor Using Hyperpolarized Carbon-13 MR Metabolic Imaging.

Differentiation between radiation-induced necrosis and tumor recurrence is crucial to determine proper management strategies but continues to be one of the central challenges in neuro-oncology. We hypothesized that hyperpolarized 13C MRI, a unique technique to measure real-time in vivo metabolism, would distinguish radiation necrosis from tumor on the basis of cell-intrinsic metabolic differences. The purpose of this study was to explore the feasibility of using hyperpolarized [1-13C]pyruvate for differentiating radiation necrosis from brain tumors. Radiation necrosis was initiated by employing a CT-guided 80-Gy single-dose irradiation of a half cerebrum in mice (n = 7). Intracerebral tumor was modeled with two orthotopic mouse models: GL261 glioma (n = 6) and Lewis lung carcinoma (LLC) metastasis (n = 7). 13C 3D MR spectroscopic imaging data were acquired following hyperpolarized [1-13C]pyruvate injection approximately 89 and 14days after treatment for irradiated and tumor-bearing mice, respectively. The ratio of lactate to pyruvate (Lac/Pyr), normalized lactate, and pyruvate in contrast-enhancing lesion was compared between the radiation-induced necrosis and brain tumors. Histopathological analysis was performed from resected brains. Conventional MRI exhibited typical radiographic features of radiation necrosis and brain tumor with large areas of contrast enhancement and T2 hyperintensity in all animals. Normalized lactate in radiation necrosis (0.10) was significantly lower than that in glioma (0.26, P =.004) and LLC metastatic tissue (0.25, P =.00007). Similarly, Lac/Pyr in radiation necrosis (0.18) was significantly lower than that in glioma (0.55, P =.00008) and LLC metastasis (0.46, P =.000008). These results were consistent with histological findings where tumor-bearing brains were highly cellular, while irradiated brains exhibited pathological markers consistent with reparative changes from radiation necrosis. Hyperpolarized 13C MR metabolic imaging of pyruvate is a noninvasive imaging method that differentiates between radiation necrosis and brain tumors, providing a groundwork for further clinical investigation and translation for the improved management of patients with brain tumors.

Radiation-induced bystander effects may contribute to radiation-induced cognitive impairment

Purpose Despite being a major treatment modality for brain cancer due to its efficiency in achieving cancer control, radiotherapy has long been known to cause long-term side effects, including radiation-induced cognitive impairment (RICI). Neurogenesis inhibition due to radiation-induced damage in neural stem cells (NSCs) has been demonstrated to be an important mechanism underlying RICI. Radiation-induced bystander effects (RIBEs) denote the biological responses in non-targeted cells after their neighboring cells are irradiated. We have previously demonstrated that RIBEs could play an important role in the skin wound healing process. Therefore, we aimed to investigate whether RIBEs contribute to RICI in this study. Materials and methods The transwell co-culture method was used to investigate bystander effects in mouse NSCs induced by irradiated GL261 mouse glioma cells in vitro. The proliferation, neurosphere-forming capacity and differentiation potential of NSCs were determined as the bystander endpoints. The exosomes were extracted from the media used to culture GL261 cells and were injected into the hippocampus of C57BL/6 mice. Two months later, the neurogenesis of mice was assessed using BrdU incorporation and immunofluorescence microscopy, and cognitive function was evaluated by the Morris Water Maze. Results After co-culture with GL261 glioma cells, mouse NSCs displayed inhibited proliferation and reduced neurosphere-forming capacity and differentiation potential. The irradiated GL261 cells caused greater inhibition and reduction in NSCs than unirradiated GL261 cells. Moreover, adding the exosomes secreted by GL261 cells into the culture of NSCs inhibited NSC proliferation, suggesting that the cancer cell-derived exosomes may be critical intercellular signals. Furthermore, injection of the exosomes from GL261 cells into the hippocampus of mice caused significant neurogenesis inhibition and cognitive impairment two month later, and the exosomes from irradiated GL261 cells induced greater inhibitory effects. Conclusion RIBEs mediated by the exosomes from irradiated cancer cells could contribute to RICI and, therefore, could be a novel mechanism underlying RICI.

Closed-loop trans-skull ultrasound hyperthermia leads to improved drug delivery from thermosensitive drugs and promotes changes in vascular transport dynamics in brain tumors.

Effective drug delivery in brain tumors remains a major challenge in oncology. Although local hyperthermia and stimuli-responsive delivery systems, such as thermosensitive liposomes, represent promising strategies to locally enhance drug delivery in solid tumors and improve outcomes, their application in intracranial malignancies remains unexplored. We hypothesized that the combined abilities of closed-loop trans-skull Magnetic Resonance Imaging guided Focused Ultrasound (MRgFUS) hyperthermia with those of thermosensitive drugs can alleviate challenges in drug delivery and improve survival in gliomas.Methods: To conduct our investigations, we first designed a closed loop MR-guided Focused Ultrasound (MRgFUS) system for localized trans-skull hyperthermia (ΔT < 0.5 °C) in rodents and established safety thresholds in healthy mice. To assess the abilities of the developed system and proposed therapeutic strategy for FUS-triggered chemotherapy release we employed thermosensitive liposomal Dox (TSL-Dox) and tested it in two different glioma tumor models (F98 in rats and GL261 in mice). To quantify Dox delivery and changes in the transvascular transport dynamics in the tumor microenvironment we combined fluorescent microscopy, dynamic contrast enhanced MRI (DCE-MRI), and physiologically based pharmacokinetic (PBPK) modeling. Lastly, to assess the therapeutic efficacy of the system and of the proposed therapeutic strategy we performed a survival study in the GL261 glioma bearing mice.Results: The developed closed-loop trans-skull MRgFUS-hyperthermia system that operated at 1.7 MHz, a frequency that maximized the brain (FUS-focus) to skull temperature ratio in mice, was able to attain and maintain the desired focal temperature within a narrow range. Histological evidence (H&E and Nissl) suggests that focal temperature at 41.5 ± 0.5 °C for 10 min is below the threshold for tissue damage. Quantitative analysis of doxorubicin delivery from TSLs with MRgFUS-hyperthermia demonstrated 3.5-fold improvement in cellular uptake in GL261 glioma mouse tumors (p < 0.001) and 5-fold increase in delivery in F98 glioma rat tumors (p < 0.05), as compared to controls (TSL-Dox-only). Moreover, PBPK modeling of drug transport that was calibrated using the experimental data indicated that thermal stress could lead to significant improvement in the transvascular transport (2.3-fold increase in the vessel diffusion coefficient; P < 0.001), in addition to promoting targeted Dox release. Prospective experimental investigations with DCE-MRI during FUS-hyperthermia, supported these findings and provided evidence that moderate thermal stress (≈41 °C for up to 10 min) can promote acute changes in the vascular transport dynamics in the brain tumor microenvironment (Ktrans value for control vs. FUS was 0.0097 and 0.0148 min-1, respectively; p = 0.026). Crucially, survival analysis demonstrated significant improvement in the survival in the TSL-Dox-FUS group as compared to TSL-Dox-only group (p < 0.05), providing supporting evidence on the therapeutic potential of the proposed strategy.Conclusions: Our investigations demonstrated that spatially controlled thermal stress can be attained and sustained in the mouse brain, using a trans-skull closed-loop MRgFUS system, and used to promote the effective delivery of chemotherapy in gliomas from thermosensitive drugs. This system also allowed us to conduct mechanistic investigations that resulted in the refinement of our understanding on the role of thermal stress in augmenting mass and drug transport in brain tumors. Overall, our study established a new paradigm for effective drug delivery in brain tumors based on closed-loop ultrasound-mediated thermal stress and thermosensitive drugs.

Reprogramming the immunosuppressive microenvironment of IDH1 wild-type glioblastoma by blocking Wnt signaling between microglia and cancer cells

ABSTRACT The vast majority (>90%) of glioblastoma (GBM) patients belong to the isocitrate dehydrogenase 1 wild type (IDH1WT) group which exhibits a poor prognosis with a median survival of less than 15 months. This study demonstrated numerous immunosuppressive genes as well as β-catenin gene, pivotal for Wnt/β-catenin signaling, were upregulated in 206 IDH1WT glioma patients using the Chinese Glioma Genome Atlas (CGGA) database. The increase in microglia with an immunosuppressive phenotype and the overexpression of β-catenin protein were further verified in IDH1WT GBM patients and IDH1WT GL261 glioma allografts. Subsequently, we found that IDH1WT GL261 cell-derived conditioned medium activated Wnt/β-catenin signaling in primary microglia and triggered their transition to an immunosuppressive phenotype. Blocking Wnt/β-catenin signaling not only attenuated microglial polarization to the immunosuppressive subtype but also reactivated immune responses in IDH1WT GBM allografts by simultaneously enhancing cytotoxic CD8+ T cell infiltration and downregulating regulatory T cells. Positron emission tomography imaging demonstrated enhanced proinflammatory activities in IDH1WT GBM allografts after the blockade of Wnt signaling. Finally, gavage administration of a Wnt signaling inhibitor significantly restrained tumor proliferation and improved the survival of model mice bearing IDH1WT GBM allografts. Depletion of CD8+ T cells remarkably abrogated the therapeutic efficacy induced by the Wnt signaling inhibitor. Overall, the present work indicates that the crosstalk between IDH1WT glioma cells and immunosuppressive microglia is important in maintaining the immunosuppressive glioma microenvironment. Blocking Wnt/β-catenin signaling is a promising complement for IDH1WT GBM treatment by improving the hostile immunosuppressive microenvironment.

Intrinsic Autofluorescent Phenotype: A Novel Biomarker to Identify and Isolate Glioma Stem-Like Cells

Although defined markers such as prominin-1 play a successful role in isolating glioma stem-like cells, their expression is susceptible to environmental alteration. Therefore, we determine the efficiency of the riboflavin-addicted autofluorescence method in isolating glioma stem-like cells. GL261 glioma stem-like cells were selected and the agent of riboflavin was added to the culture medium. Then, glioma stemlike cells with autofluorescence were identified and isolated through flow cytometry. Subsequently, cell counting kit-8 was used to detect the proliferation of targeted cells and we developed a mouse model of xenograft to record the overall survival rate and tumor size and weight per day. Additionally, the expressions of Ki67, proliferating cell nuclear antigen and nestin in the tumor mass were explored by immunohistochemical assay. The feature of autofluorescence in glioma stem-like cells was identified and isolated by flow cytometry. The proliferation and malignancy of the autofluorescence glioma stem-like cells were stronger than glioma stem GL261 cells screened by prominin-1 labeling. Cell counting kit-8 assay demonstrated a significantly stronger proliferation of glioma stem-like cells with autofluorescence (p<0.01). In vitro, the Kaplan-Meier curve indicated mice bearing fluorescent stem cells had significantly decreased overall survival (p<0.01) and increased tumor volume (p<0.01) and weight (p<0.01). Then, the expression of Ki67, proliferating cell nuclear antigen and nestin in the tumor-derived from fluorescent stem cells was significantly increased by immunohistochemical assay (p<0.05). Intrinsic autofluorescent phenotype in glioma stem-like cells were identified and characterized and the marker was used to isolate these cells with high malignancy.

Anti-PD-1 checkpoint blockade monotherapy in the orthotopic GL261 glioma model: the devil is in the detail.

The GL261 cell line, syngeneic on the C57BL/6 background, has, since its establishment half a century ago in 1970, become the most commonly used immunocompetent murine model of glioblastoma. As immunotherapy has entered the mainstream of clinical discourse in the past decade, this model has proved its worth as a formidable opponent against various immunotherapeutic combinations. Although advances in surgical, radiological, and chemotherapeutic interventions have extended mean glioblastoma patient survival by several months, 5-year survival postdiagnosis remains below 5%. Immunotherapeutic interventions, such as the ones explored in the murine GL261 model, may prove beneficial for patients with glioblastoma. However, even common immunotherapeutic interventions in the GL261 model still have unclear efficacy, with wildly discrepant conclusions being made in the literature regarding this topic. Here, we focus on anti-PD-1 checkpoint blockade monotherapy as an example of this pattern. We contend that a fine-grained analysis of how biological variables (age, sex, tumor location, etc.) predict treatment responsiveness in this preclinical model will better enable researchers to identify glioblastoma patients most likely to benefit from checkpoint blockade immunotherapy moving forward.

Anesthesia triggers drug delivery to experimental glioma in mice by hijacking caveolar transport.

BackgroundPharmaceutical intervention in the CNS is hampered by the shielding function of the blood–brain barrier (BBB). To induce clinical anesthesia, general anesthetics such as isoflurane readily penetrate the BBB. Here, we investigated whether isoflurane can be utilized for therapeutic drug delivery.MethodsBarrier function in primary endothelial cells was evaluated by transepithelial/transendothelial electrical resistance, and nanoscale STED and SRRF microscopy. In mice, BBB permeability was quantified by extravasation of several fluorescent tracers. Mouse models including the GL261 glioma model were evaluated by MRI, immunohistochemistry, electron microscopy, western blot, and expression analysis.ResultsIsoflurane enhances BBB permeability in a time- and concentration-dependent manner. We demonstrate that, mechanistically, isoflurane disturbs the organization of membrane lipid nanodomains and triggers caveolar transport in brain endothelial cells. BBB tightness re-establishes directly after termination of anesthesia, providing a defined window for drug delivery. In a therapeutic glioblastoma trial in mice, simultaneous exposure to isoflurane and cytotoxic agent improves efficacy of chemotherapy.ConclusionsCombination therapy, involving isoflurane-mediated BBB permeation with drug administration has far-reaching therapeutic implications for CNS malignancies.

Sustained localized delivery of immunotherapy to lymph nodes reverses immunosuppression and increases long-term survival in murine glioblastoma

ABSTRACT Introduction Despite the advent of immunotherapy as a promising therapeutic, glioblastoma (GBM) remains resistant to using checkpoint blockade due to its highly immunosuppressive tumor milieu. Moreover, current anti-PD-1 treatment requires multiple infusions with adverse systemic effects. Therefore, we used a PCL:PEG:PCL polymer gel loaded with anti-PD-1 and implanted at the site of lymph nodes in an attempt to maximize targeting of inactivated T cells as well as mitigate unnecessary systemic exposure. Methods Mice orthotopically implanted with GL261 glioma cells were injected with hydrogels loaded with anti-PD-1 in one of the following locations: cervical lymph nodes, inguinal lymph nodes, and the tumor site. Mice treated systemically with anti-PD-1 were used as comparative controls. Kaplan–Meier curves were generated for all arms, with ex vivo flow cytometric staining for L/D, CD45, CD3, CD4, CD8, TNF-α and IFN-y and co-culture ELISpots were done for immune cell activation assays. Results Mice implanted with PCL:PEG:PCL hydrogels carrying anti-PD-1 at the site of their lymph nodes showed significantly improved survival outcomes compared to mice systemically treated with anti-PD-1 (P = .0185). Flow cytometric analysis of brain tissue and co-culture of lymph node T cells from mice implanted with gels demonstrated increased levels of IFN-y and TNF-α compared to mice treated with systemic anti-PD-1, indicating greater reversal of immunosuppression compared to systemic treatment. Conclusions Our data demonstrate proof of principle for using localized therapy that targets lymph nodes for GBM. We propose an alternative treatment paradigm for developing new sustained local treatments with immunotherapy that are able to eliminate the need for multiple systemic infusions and their off-target effects.

Comparative evaluation of T-cell receptors in experimental glioma-draining lymph nodes.

BackgroundGlioblastomas, the most common primary malignant brain tumors, are considered immunologically cold malignancies due to growth in an immune sanctuary site. While peptide vaccines have shown to generate intra-tumoral antigen-specific T cells, the identification of these tumor-specific T cells is challenging and requires detailed analyses of tumor tissue. Several studies have shown that CNS antigens may be transported via lymphatic drainage to cervical lymph nodes, where antigen-specific T-cell responses can be generated. Therefore, we investigated whether glioma-draining lymph nodes (TDLN) may constitute a reservoir of tumor-reactive T cells.MethodsWe addressed our hypothesis by flow cytometric analyses of chicken ovalbumin (OVA)-specific CD8+ T cells as well as T-cell receptor beta (TCRβ) next-generation-sequencing (TCRβ-NGS) of T cells from tumor tissue, TDLN, spleen, and inguinal lymph nodes harvested from experimental mouse GL261 glioma models.ResultsLongitudinal dextramer-based assessment of specific CD8+ T cells from TDLN did not show tumor model antigen reactivity. Unbiased immunogenomic analysis revealed a low overlap of TCRβ sequences from glioma-infiltrating CD8+ T cells between mice. Enrichment scores, calculated by the ratio of productive frequencies of the different TCRβ-CDR3 amino-acid (aa) rearrangements of CD8+ T cells derived from tumor, TDLN, inguinal lymph nodes, and spleen demonstrated a higher proportion of tumor-associated TCR in the spleen compared to TDLN.ConclusionsIn experimental glioblastoma, our data did not provide evidence that glioma-draining cervical lymph nodes are a robust reservoir for spontaneous glioma-specific T cells highlighting the requirement for detailed analyses of glioma-infiltrating T cells for the discovery of tumor-specific TCR.

IMMU-05. B7-H3-SPECIFIC CAR T CELLS HAVE POTENT ANTI-TUMOR ACTIVITY IN THE GL261 IMMUNE-COMPETENT MURINE BRAIN TUMOR MODEL

BACKGROUNDWe and others have identified B7-H3 (CD276) as a promising target for CAR-based immunotherapies for pediatric brain tumors. So far, B7-H3-CAR T cells have only been studied in xenograft models for brain tumors, which do not recapitulate the immunosuppressive tumor microenvironment (TME). To overcome this obstacle, we decided to adapt the immune-competent GL261 murine glioma model which mimics human disease and host immune barriers.METHODSTo evaluate the safety and efficacy of antigen-specific CAR T cells, murine B7-H3-CAR T cells were generated using retroviral particles encoding 2nd generation B7-H3-specific CD28.z CAR. Expansion, persistence, and anti-tumor activity were evaluated in vitro and in vivo. Components of the brain TME were then evaluated using flow cytometry and immunostaining.RESULTSB7-H3-CAR T cells only killed B7-H3+ tumor cells, secreted significant levels of IFNγ and IL-2 in an antigen-dependent manner and expanded an average of 33-fold in repeat stimulation assay with B7-H3+ tumor cells in contrast to control CAR T cells. In vivo, intratumoral injection of B7-H3-CAR T cells into orthotopic GL261 glioma induced complete regression in 60% of treated mice. Preliminary studies show numerous infiltration of suppressive tumor-associated macrophages within the tumor and its periphery.CONCLUSIONSIn summary, we successfully generated murine B7-H3-CAR T cells and have demonstrated that these cells have potent anti-tumor activity in the immune-competent GL261 glioma model. However, it is likely that the tumor-associated macrophages are mediating immunosuppressive effects on B7-H3-CAR T cells. Therefore, studies focusing on TME/CAR T cell interactions are in progress.

Doxorubicin-loaded nanoparticle coated with endothelial cells-derived exosomes for immunogenic chemotherapy of glioblastoma.

Treatments of glioblastoma (GBM) have not been very effective, largely due to the inefficiency of drugs in penetrating the blood brain barrier (BBB). In this study, we investigated the potential of exosome‐coated doxorubicin (DOX)‐loaded nanoparticles (ENPDOX) in BBB penetration, inducing immunogenic cell death (ICD) and promoting survival of GBM‐bearing mice. DOX‐loaded nanoparticles (NPDOX) were coated with exosomes prepared from mouse brain endothelial bEnd.3 cells. ENPDOX cellular uptake was examined. Penetration of ENPDOX through the BBB was tested in an in vitro transwell system and a GBM mouse model. The effects of ENPDOX in inducing apoptosis and ICD were assessed. Finally, the efficacy of ENPDOX in the treatment of GBM‐bearing mice was assessed. ENPDOX was taken up by bEnd.3 cells and could penetrate the BBB both in vitro and in vivo. In vitro, ENDDOX induced apoptosis and ICD of glioma GL261 cells. Systemic administration of ENPDOX resulted in maturation of dendritic cells, activation of cytotoxic cells, altered production of cytokines, suppressed proliferation and increased apoptosis of GBM cells in vivo and prolonged survival of GBM‐bearing mice. Our findings indicate that ENPDOX may be a potent therapeutic strategy for GBM which warrants further investigation in clinical application.

Brain cancer induces systemic immunosuppression through release of non-steroid soluble mediators.

Immunosuppression of unknown aetiology is a hallmark feature of glioblastoma and is characterized by decreased CD4 T-cell counts and downregulation of major histocompatibility complex class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for glioblastoma. We recapitulated the immunosuppression observed in glioblastoma patients in the C57BL/6 mouse and investigated the aetiology of low CD4 T-cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of brain cancer, including mice harbouring GL261 glioma, B16 melanoma, and in a spontaneous model of diffuse intrinsic pontine glioma. In addition to thymic involution, we determined that tumour growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC II expression on blood leucocytes, and a modest increase in bone marrow resident CD4 T cells. Using parabiosis we report that thymic involution, declines in peripheral T-cell counts, and reduced major histocompatibility complex class II expression levels were mediated through circulating blood-derived factors. Conversely, T-cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is non-steroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the immunosuppression was not unique to cancer itself, but rather occurs in response to brain injury. Non-cancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that brain cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.

Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression.

Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo.Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment.Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 × g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 × g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between the two groups, while shRab27a tumors showed lower proliferation at the tumor borders. Decreased infiltration of IBA1 positive macrophages and microglia, but not FoxP3 positive regulatory T cells was observed.Conclusion: Rab27a plays an important role in the release of small EVs from glioma cells, and also in their viability and expression of CCL2 in vitro. As interference in Rab27a expression influences glioma cell viability and expression profiles, future studies should be cautious in using the knockdown of Rab27a as a means of studying the role of small EVs in glioma growth.

EXTH-20. SYNGENEIC B7-H3-SPECIFIC CAR T-CELLS HAVE POTENT ANTI-BRAIN TUMOR ACTIVITY VIA LOCAL OR SYSTEMIC DELIVERY

Abstract BACKGROUND We and others have identified B7-H3 (CD276) as a promising target for CAR T-cell-based immunotherapies for pediatric brain tumors. So far, B7-H3-CAR T cells have only been studied in xenograft models for brain tumors, which do not recapitulate the immunosuppressive tumor microenvironment (TME). To overcome this obstacle, we decided to adapt the immune competent GL261 murine glioma model which mimics human disease and host immune barriers. METHODS To evaluate their safety and efficacy, murine B7-H3-CAR T-cells were generated using retroviral particles encoding a 2nd generation B7-H3-CAR with a CD28.z signaling domain. Expansion, persistence, and anti-tumor activity were evaluated in vitro and in vivo. Components of the brain TME were then evaluated using flow cytometry and immunostaining. RESULTS B7-H3-CAR T cells only killed B7-H3+ tumor cells, secreted significant levels of IFNγ and IL-2 in an antigen-dependent manner and expanded an average of 85-fold in repeat stimulation assay with B7-H3+ tumor cells in contrast to control CAR T-cells. In vivo, intratumoral (2x106) or systemic (3x106) injection of syngeneic B7-H3-CAR T-cells into mice with orthotopic GL261 glioma induced complete regression in 60% of treated mice resulting in a significant survival advantage. Mice showed no evidence of acute or long-term toxicities related to CAR T-cell infusions. We confirmed this encouraging safety profile by systemic administration of a high dose (1x107) B7-H3-CAR T-cells and performing histological analyses of all major organs on day 14 post T-cell injection, which showed no notable signs of injury or on-target/off-tumor toxicities. CONCLUSIONS We successfully generated syngeneic B7-H3-CAR T-cells and have demonstrated that these cells have potent anti-tumor activity in the immune competent GL261 glioma model via local or systemic delivery without apparent toxicities. Our study paves the way for future testing of B7-H3-CAR T-cells in early phase clinical studies.

IMMU-43. ZIKA VIRUS TO TREAT GLIOMA: TURNING COLD TUMORS HOT

Abstract GBM is a hallmark example of an immunotherapy-resistant tumor. Previously, we discovered that Zika virus (ZIKV) has a natural honing capacity to specifically kill GBM stem cells (GSCs). Using two syngeneic glioma models, GL261 and CT2A, we found that in vivo intratumoral ZIKV treatment reduces tumor size and extends median survival far beyond that expected for its anti-GSC effects; additionally, approximately 50% of mice were long-term survivors. We now report that ZIKV treatment is associated with an increase in tumor-specific CD8+ T cells and activated myeloid cells expressing MHC-II and NOS2. Treatment with CD8-depleting antibody abrogates the survival advantage conferred by ZIKV, demonstrating that CD8+ T cells are required for ZIKV efficacy. Remarkably, ZIKV treatment also confers protection against tumor re-challenge 1.5 years after the original treatment, and this too is dependent on CD8+ T cells. Lastly, in a bilateral tumor implantation model, intratumoral ZIKV treatment of one tumor increases overall survival, suggesting ZIKV is efficacious because it generates a global anti-tumor response. Building on the observation that ZIKV treatment increases CD8+ T cells in the tumor bed, we combined ZIKV with α-PD1 immunotherapy. We found survival was dramatically increased compared to either treatment alone. Finally, to improve safety of potential ZIKV treatment for patients, we generated an immune-sensitized virus. This strain remains efficacious as a single agent and in combination with immunotherapy in vivo. Overall, our results suggest ZIKV treatment addresses two key problems in our current treatment of GBM: it specifically targets highly resistant GSC and initiates a potent anti-tumor immune response against the remaining tumor. Our work reveals the mechanistic basis of ZIKV efficacy and demonstrates the potential for ZIKV as a therapy for GBM.

IMMU-25. SEVERE AND MULTIFACETED SYSTEMIC IMMUNOSUPPRESSION CAUSED BY EXPERIMENTAL CANCERS OF THE CENTRAL NERVOUS SYSTEM REQUIRES RELEASE OF NON-STEROID SOLUBLE MEDIATORS

Abstract Immunosuppression of unknown etiology is a hallmark feature of glioblastoma (GBM) and is characterized by decreased CD4 T cell counts and down regulation of MHC class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for GBM. We recapitulated the immunosuppression observed in GBM patients in the C57BL/6 mouse and investigated the etiology of low CD4 T cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of CNS cancer, including mice harboring GL261 glioma, B16 melanoma, and in a spontaneous model of Diffuse Intrinsic Pontine Glioma (DIPG). In addition to thymic involution, we determined that tumor growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC class II expression on hematopoietic cells, and a modest increase in bone marrow resident CD4 T cells with a naïve phenotype. Using parabiosis we report that thymic involution, declines in peripheral T cell counts, and reduced MHC class II expression levels were mediated through circulating blood-derived factors. Conversely, T cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is nonsteroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the aforementioned immunosuppression was not unique to cancer itself, but rather occurs in response to CNS injury. Noncancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that CNS cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.

Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity

BackgroundImmunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term...

Down-regulation of Aquaporin-1 mediates a microglial phenotype switch affecting glioma growth

Aquaporin 1 (AQP1), a transmembrane protein that forms water channels, has previously been shown to facilitate growth and progression of many types of tumors by modulating tumor cell migration, proliferation and angiogenesis. Here, we determined the impact of AQP1 expression in the tumor environment on the progression of brain tumors. Primary microglia from wild type(WT) and AQP1 knockout(KO) mice were used to test AQP1 effect on microglia function by using Western blot, quantative PCR, in an experimental in vivo mouse glioma model and organotypic brain slice culture. Deletion of AQP1 in the host tissue significantly reduced the survival of the mice implanted with GL261 glioma cells. The density of glioma-associated microglia/macrophages was almost doubled in AQP1KO mice. We found that factors secreted from GL261 cells decrease microglial AQP1 expression via the MEK/ERK pathway, and that inhibition of this pathway with Trametinib reduced tumor growth and prolonged the survival of tumor bearing mice, an effect which required the presence of microglia. Deletion of AQP1 in cultured microglia resulted in an increase in migratory activity and a decrease in TLR4-dependent innate immune responses. Our study demonstrates a functional relevance of AQP1 expression in microglia and hints to AQP1 as a potential novel target for glioma therapy.

Advanced Age Increases Immunosuppression in the Brain and Decreases Immunotherapeutic Efficacy in Subjects with Glioblastoma.

Wild-type isocitrate dehydrogenase-expressing glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median age at diagnosis of ≥65 years. It accounts for approximately 90% of all GBMs and has a median overall survival (OS) of <15 months. Although immune checkpoint blockade (ICB) therapy has achieved remarkable survival benefits in a variety of aggressive malignancies, similar success has yet to be achieved for GBM among phase III clinical trials to date. Our study aimed to understand the relationship between subject age and immunotherapeutic efficacy as it relates to survival from glioma. (i) Clinical data: GBM patient datasets from The Cancer Genome Atlas, Northwestern Medicine Enterprise Data Warehouse, and clinical studies evaluating ICB were stratified by age and compared for OS. (ii) Animal models: young, middle-aged, and older adult wild-type and indoleamine 2,3 dioxygenase (IDO)-knockout syngeneic mice were intracranially engrafted with CT-2A or GL261 glioma cell lines and treated with or without CTLA-4/PD-L1 mAbs, or radiation, anti-PD-1 mAb, and/or a pharmacologic IDO enzyme inhibitor. Advanced age was associated with decreased GBM patient survival regardless of treatment with ICB. The advanced age-associated increase of brain IDO expression was linked to the suppression of immunotherapeutic efficacy and was not reversed by IDO enzyme inhibitor treatment. Immunosuppression increases in the brain during advanced age and inhibits antiglioma immunity in older adults. Going forward, it will be important to fully understand the factors and mechanisms in the elderly brain that contribute to the decreased survival of older patients with GBM during treatment with ICB.

Radiation with STAT3 Blockade Triggers Dendritic Cell-T cell Interactions in the Glioma Microenvironment and Therapeutic Efficacy.

Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood-brain barrier-penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.