Murine Glioma Cells Research Articles

Primary murine high-grade glioma cells derived from RCAS/tv-a diffuse glioma model reprogram naive T cells into immunosuppressive regulatory T lymphocytes

High-grade gliomas (HGGs) and glioblastomas (GBMs) are the most aggressive and lethal brain tumors. The current standard of care (SOC) includes gross safe surgical resection followed by chemoradiotherapy. The main chemotherapeutic agents are the DNA-alkylating agent temozolomide (TMZ) and adjuvants. Due to the outdated therapeutic protocols and lack of specific treatments, there is an urgent and rising need to improve ourunderstanding of tumor biology and design more effective therapeutic strategies. Invitro models are essential for investigating glioma biology and testing novel therapeutic approaches. While using commercially available and patient-derived glioma cell lines for invitro studies is common practice, they exhibit several limitations, including failing to maintain the genetic and phenotypic diversity of primary tumors, undergo genetic drift over time, and often lacking the invasive and stem-like characteristics of patient tumors. These limitations can lead to inconsistent and non-reproducible results, hampering translational research progress. In this study, we established a novel primary murine HGG cell line, isolated from an immunocompetent HGG-bearing RCAS/T-va mouse. We characterized the transcriptome and phenotype to ensure that this cell line resembles the nature of HGGs and retains the ability to reprogram primary murine T lymphocytes.

LGG-49. EVALUATING THE EFFECTS OF MIRDAMETINIB IN NF1-MUTANT LOW-GRADE GLIOMAS USING BOTH CULTURED CELLS AND A NOVEL ZEBRAFISH XENOGRAFT MODEL

Abstract BACKGROUND Pediatric low-grade gliomas (LGGs) with NF1 mutations represent a significant portion of childhood brain tumors. NF1 mutations lead to the dysregulation of the MAPK kinase and mTOR pathways, contributing to tumor growth. However, obtaining cell lines and xenograft animal models for NF1-mutated LGGs remains challenging due to their slow growth and less aggressive nature. Methods and RESULTS We examined the therapeutic efficacy of a novel MEK inhibitor mirdametinib in targeting the MAPK pathway within JHH-NF1-PA1 cells, a cell line derived from a low-grade optic pathway glioma originating from a patient with type 1 neurofibromatosis. Mirdametinib decreased p-ERK protein expression levels on western blot within four hours. We confirmed that mirdametinib significantly suppressed tumor cell proliferation by approximately 60% (BrdU staining (P<0.05), CellTiter Blue assay). β-galactosidase staining revealed approximately a 50% induction of cellular senescence in JHH-NF1-PA1 cells following mirdametinib treatment (P<0.05). We confirmed our findings using murine Nf1-null low grade glioma cell lines 1861 and 1491. To establish an in vivo model for drug testing, we utilized zebrafish due to their transparency, rapid reproduction, and short development time. Injecting RFP-labeled JHH-NF1-PA1 cells into the zebrafish at the 1000-cell stage, we monitored tumor growth using TECAN fluorescence detection from the third to eighth days post-injection. The fluorescence signals obtained from TECAN were validated by fluorescence microscopy. Western blot analysis confirmed a decrease in p-ERK levels within the zebrafish following mirdametinib treatment. We are evaluating the effect of mirdametinib on NF1 low grade glioma cell survival, migration and senescence in the zebrafish brain. CONCLUSION Our findings show that mirdametinib inhibits cell proliferation and induces senescence in NF1-mutated LGGs. Moreover, our zebrafish model provides a valuable tool for evaluating the efficacy of MEK inhibitors in vivo, particularly in cases where traditional xenograft models are difficult to establish.

Abstract LB116: Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors

Abstract In solid cancers, an immunosuppressive tumor microenvironment (TME) restrains immunotherapy. Induction of immunogenic cell death in tumor cells and simultaneously programming the TME from immunosuppressive to inflammatory employing dsRNA agonists for endosomal Toll-like receptor 3 (TLR3) represents an appealing in situ immunomodulation strategy. However, uptake of dsRNA by tumor cells is limited and systemic administration is linked to adverse side effects. Here we describe an exceptional therapeutically effect of systemically delivered TLR3 agonist RIBOXXOL using a recently developed antibody-guided nanoparticle-system designated “Rapid Inducer of Cellular Inflammation and Apoptosis” (RICIA) for targeting epidermal growth factor variant III (EGFRvIII) on tumor cells. We suggest that systemically applied monotherapy with RICIA might be effective in experimental solid tumors. This includes, (i) preferential enrichment of the immune-stimulatory RIBOXXOL and (ii) subsequent induction of a type-I interferon response in tumor cells expressing TLR3, and (iii) synergistic inflammation of the TME through TLR3 agonist thereby expediting anti-tumor immune responses. The effects of RICIA-treatment were comprehensively assessed in vitro and in murine syngeneic epidermal growth factor receptor variant III (EGFRvIII)-positive SMAvIII gliomas. Our in vitro studies confirm uptake of RICIA by EGFRvIII-mediated endocytosis in human and murine glioma cells and furthermore demonstrate TLR3-triggered type-I interferon release and mRNA signatures indicative of an innate immune response against virus. We show in the SMAvIII tumor model that monotherapy with picomolar amounts of RICIA induces regression of subcutaneous (s.c.) tumors leading to increase in survival time and results in complete tumor clearance in a significant fraction of mice accompanied by immune memory when compared to RICIAs equipped with an unspecific control antibody. Mechanistically, RICIA treatment reshapes the immunosuppressive tumor environment by mimicking viral infection leading to increased expression of the interferon beta (IFN-β) gene (Ifnb1), by subsequent induction of an inflammatory state characterized by RNA-expression signatures indicating induction of apoptosis, interleukin 1beta (IL-1β) signaling, and programming immunosuppressive M2 TAMs to activated pro-inflammatory M1 TAMs. In addition, a single stereotactic injection of RICIA into established intracranial glioma significantly improves mean survival time of treated mice and is able to induce complete tumor clearance thus showing therapeutic potency of RICIA in a relevant pre-clinical setting for glioma. Thus, we show that targeted activation of TLR3 represents a promising therapeutic strategy for immune-modulation of tumors with the prospect to elicit long-lasting anti-tumor immune responses. Citation Format: Achim Temme, Alexander Hagstotz, Isabell Schau, Michael Seifert, Duran Sürün, Stefan Zimmermann, Frank Buchholz, Luise Rupp, Rebekka Wehner, Marc Schmitz, Gabriele Schackert, Ilker Y. Eyüpoglu. Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB116.

Tumor associated microglia/macrophages utilize GPNMB to promote tumor growth and alter immune cell infiltration in glioma

Tumor-associated microglia and blood-derived macrophages (TAMs) play a central role in modulating the immune suppressive microenvironment in glioma. Here, we show that GPNMB is predominantly expressed by TAMs in human glioblastoma multiforme and the murine RCAS-PDGFb high grade glioma model. Loss of GPNMB in the in vivo tumor microenvironment results in significantly smaller tumor volumes and generates a pro-inflammatory innate and adaptive immune cell microenvironment. The impact of host-derived GPNMB on tumor growth was confirmed in two distinct murine glioma cell lines in organotypic brain slices from GPNMB-KO and control mice. Using published data bases of human glioma, the elevated levels in TAMs could be confirmed and the GPNMB expression correlated with a poorer survival.

EZH2 Inhibition Sensitizes IDH1R132H-Mutant Gliomas to Histone Deacetylase Inhibitor.

Isocitrate Dehydrogenase-1 (IDH1) is commonly mutated in lower-grade diffuse gliomas. The IDH1R132H mutation is an important diagnostic tool for tumor diagnosis and prognosis; however, its role in glioma development, and its impact on response to therapy, is not fully understood. We developed a murine model of proneural IDH1R132H-mutated glioma that shows elevated production of 2-hydroxyglutarate (2-HG) and increased trimethylation of lysine residue K27 on histone H3 (H3K27me3) compared to IDH1 wild-type tumors. We found that using Tazemetostat to inhibit the methyltransferase for H3K27, Enhancer of Zeste 2 (EZH2), reduced H3K27me3 levels and increased acetylation on H3K27. We also found that, although the histone deacetylase inhibitor (HDACi) Panobinostat was less cytotoxic in IDH1R132H-mutated cells (either isolated from murine glioma or oligodendrocyte progenitor cells infected in vitro with a retrovirus expressing IDH1R132H) compared to IDH1-wild-type cells, combination treatment with Tazemetostat is synergistic in both mutant and wild-type models. These findings indicate a novel therapeutic strategy for IDH1-mutated gliomas that targets the specific epigenetic alteration in these tumors.

TMIC-24. FERROPTOSIS INDUCES IMMUNOGENIC CELL DEATH AND SYNERGIZES WITH CD47 BLOCKADE TO REPROGRAM THE GLIOMA MICROENVIRONMENT

Abstract Immunotherapy has thus far proven ineffective in glioblastoma, in part due to the immune cold tumor microenvironment. Here we demonstrate that ferroptosis specifically targets a quiescent, immunosuppressive astrocyte-like glioma population and can therefore be used as a novel inducer of immunogenic cell death to repolarize the immune microenvironment by promoting tumor cell phagocytosis and antigen presentation. First, we characterized the immunogenic effects of ferroptosis by using a glioma cell-myeloid cell co-culture system and patient-derived organotypic slice cultures and performed phagocytosis assays, flow-cytometry, immunohistochemistry, cytokine arrays and single cell RNAseq. GPX4-dependent ferroptosis induced translocation of the pro-phagocytic eat-me signal calreticulin in both murine glioma cells and human tissue samples. Ferroptotic-driven tumor cell death reprogramed local myeloid populations to promote glioma phagocytosis, MHCII-driven antigen presentation, and paracrine cytokine signaling involved in T-cell recruitment. Local delivery of RSL3, a GPX4 inhibitor, in a murine glioma model polarized myeloid cells to a pro-phagocytic phenotype and significantly increased intra-tumoral levels of CD4+ and CD8+ T-cells. Astrocytic-like tumor cells upregulate CD47 as a mechanism to evade phagocytosis. Therefore, we combined ferroptosis with an anti-CD47 monoclonal antibody (aCD47) to enhance reprogramming of the immune cold microenvironment. In an orthotopic murine glioma model, adding aCD47 to RSL3 led to significantly increased tumor phagocytosis, antigen presentation and prolonged survival. Thus, we demonstrate that local delivery of ferroptosis-inducing agents in conjunction with CD47 blockade is an effective multi-modality therapeutic regimen capable of reversing the immunosuppressive glioma microenvironment.

EXTH-53. VEGFR2-SPECIFIC CAR-T CELLS SHOW IMPROVED TUMOR INFILTRATION IN SYNGENEIC MOUSE GLIOMA MODELS

Abstract BACKGROUND Chimeric antigen receptor (CAR-)T cell therapy, a powerful immunotherapy against hematological malignancies, has so far limited efficiency in solid tumors. Also in glioblastoma, a major obstacle remains limited infiltration of CAR-T cells into the tumor. Vessel-targeting CAR-T cells have been shown to infiltrate solid tumors more efficiently. Vascular endothelial growth factor receptor 2 (VEGFR2) is expressed in the glioma vasculature and VEGFR2-specific CAR-T cells prolong the survival of xenograft glioma-bearing mice. In this study, we investigated the efficacy of murine VEGFR2-specific CAR-T cells against syngeneic experimental gliomas and assessed the CAR-T cell infiltration of this approach. METHODS VEGFR2-CAR-T cells were generated by retroviral transduction to express a second generation CAR construct. Their activity was assessed in co-culture assays in vitro against murine endothelial and glioma cells, respectively. Syngeneic orthotopic mouse glioma models were used to explore the infiltration of intravenously administered CAR-T cells by in vivo fluorescence molecular tomography (FMT), ex vivo flow cytometry as well as ex vivo 3D light-sheet fluorescence microscopy (LSFM). RESULTS VEGFR2-CAR-T cells showed anti-endothelial and anti-glioma activity in vitro. Intravenous treatment with fluorescently labeled CAR-T or mock-transduced T cells of tumor-bearing mice resulted in a higher FMT signal at the tumor site. This was confirmed by ex vivo flow cytometry, wherein CAR-T cells represented a larger fraction of all T cells within the tumor (26.5% CAR versus 5.4% Mock, p = 0.0013) resulting in a higher donor T cell to tumor cell ratio. Similarly, significantly more VEGFR2-CAR-T cells were detected at the tumor site compared to tumor-targeting (but not vessel-targeting) control CAR-T cells. Currently, VEGFR2-CAR-T cell infiltration patterns as well as vasculature density are further investigated and visualized by LSFM. CONCLUSION VEGFR2-CAR-T cells show improved glioma infiltration in syngeneic mouse models. Therefore, VEGFR2 might be a relevant target that could improve CAR-T cell infiltration in glioblastoma.

NIMG-43. A 57-YEAR-OLD MALE WITH PRIMARY GLIOBLASTOMA TREATED WITH LASER INTERSTITIAL THERMAL THERAPY: IMAGING AND AUTOPSY FINDINGS

Abstract INTRODUCTION: Glioblastoma (GBM) is an aggressive primary brain cancer with significant resistance to the current therapeutic approach of chemotherapy and radiotherapy, jointly known as chemoradiation (CRT). Magnetic hyperthermia therapy (MHT) is a promising therapy for GBM that can be used to perform multiple sessions of non-invasive, localized hyperthermia by activating locally delivered magnetic iron oxide nanoparticles (MIONPs) with an external alternating magnetic field (AMF). In this study, MHT-mediated enhancement of CRT was evaluated in murine and human glioma cell lines both in cell culture and in rodents.

EXTH-75. A MACROPHAGE-BASED DRUG DELIVERY PLATFORM FOR GLIOMA TREATMENT

Abstract There is an urgent need for more effective treatment strategies against gliomas. Even though various drugs have potent anti-tumor activity in vitro, their application in vivo is limited by ineffective delivery and systemic toxicity. Therefore, novel strategies are needed to deliver these drugs effectively and safely to the tumor site. Here, we developed an adoptive transfer strategy against malignant brain tumors utilizing monocyte-derived macrophages that are loaded with ferritin-protein cages containing drugs or other proteins and transfer these nanocarriers effectively to cancer cells for intracellular release and cancer cell killing. Using flow cytometry and microscopy, we observed a high transfer efficiency of ferritin-cages from loaded macrophages into human and mouse glioma cells in vitro in co-culture assays. We also confirmed the transfer of ferritin-cages from macrophages to glioma cells in vivo upon intravenous or intratumoral treatment of GL-261 or CT-2A glioma-bearing mice using in vivo imaging and ex vivo flow cytometry and microscopy. To study the anti-glioma activity with therapeutically active payloads, we loaded murine/human macrophages with ferritin cages carrying cytotoxic auristatin. Co-culture of these loaded macrophages with murine or human glioma cells in vitro revealed a time- and concentration-dependent cytotoxicity to glioma cells. In vivo, intravenous or intratumoral administration of ferritin-drug protein cages was tolerated without toxicities and conferred a survival benefit in two orthotopic murine glioma models (GL-261 and CT-2A) leading to a large fraction of long-term surviving mice. Interactome studies of ferritin-cage-binding proteins revealed phagocytic and cytoskeleton re-arrangement pathways to be involved in uptake and transfer of ferritin-cages from macrophages to glioma cells. This ‘Trojan Horse’ approach based on macrophages-ferritin-protein cages constitutes a promising platform to deliver cytotoxic drugs effectively and safely to gliomas and provides a rationale for clinical translation.

MODL-06. THE GUT MICROBIOTA OF BRAIN TUMOR PATIENTS CAN IMPACT IMMUNOTHERAPY EFFICACY IN A PRECLINICAL MODEL OF GLIOMA

Abstract Combination immune checkpoint inhibitors (ICI) can affect durable response in melanoma brain metastases (MBM) but have largely been ineffective for the treatment of glioblastoma (GBM), a primary brain tumor. Recent studies in animal models and humans have suggested that the microbe composition of the gut can influence the effectiveness of ICI in melanoma and other cancers. Our central hypothesis is that the gut microbiome can influence immunotherapy responsiveness for GBM patients. We obtained microbiome (stool) samples from GBM and MBM patients prior to beginning any treatment or surgical interventions. We then transplanted these microbiome samples into gnotobiotic mice to create humanized microbiome mice. We transplanted two GBM patients and two MBM patients microbiome samples to create 4 unique patient-derived humanized microbiome mouse lines (GBM1, GBM2, MBM1, and MBM2). Using 16s ribosomal RNA microbiome sequencing, we found that each patient-derived microbiome mouse line had a unique microbiome composition as measured by relative abundance and diversity of species. Mice from each line were then intracranially injected with GL261 murine glioma cells and treated with the ICI anti-PD-1 or isotype control. We found that MBM1 and GBM2 mice were resistant to anti-PD-1 and did not display prolonged survival with anti-PD-1 treatment. However, GBM1 and MBM2 mice were positive responders to anti-PD-1 and displayed significantly prolonged survival compared to isotype control. Additional microbiome sequencing analyses are ongoing that will reveal positive microbiome profiles among these patient-derived lines, and additional GBM and MBM patient-derived microbiome lines are being created and tested for immunotherapy efficacy. Ultimately, understanding how certain microbial communities can enhance the effectiveness of immunotherapy may lead to the development of new approaches that include modification or transplantation of microbe communities in GBM patients for increased therapeutic impact.

IMMU-12. T CELL SEQUESTRATION IN POSTERIOR FOSSA TUMORS

Abstract INTRODUCTION T cell dysfunction contributes to immune escape in patients with cancer and is particularly severe in CNS tumors. T cell sequestration is a contributing mechanism of T cell lymphopenia and lymphoid organ contraction occurring in treatment naive patients and mice with Glioblastoma. Our group demonstrated that this phenomenon is mediated by loss of S1P1 surface expression on T-Cells within the bone marrow. We demonstrated in murine models that reversing T cell sequestration via stabilization of surface S1P1 licenses T cell activating therapies. As pediatric brain tumors much more commonly involve the posterior fossa and brainstem, the present work set out to assess whether T cell sequestration also occurs in tumors located in the cerebellum (medulloblastoma, high grade glioma). METHODS CT2A murine glioma cells were orthotopically implanted into the posterior fossa of C57BL/6 mice. Blood, spleen, and bone marrow were analyzed when tumors became sizable (Day 18-21). Flow cytometry was utilized to measure total, CD4+, and CD8+ T cell counts, as well as the proportion of T cells expressing surface S1P1. RESULTS Compared with mice injected with methylcellulose, mice bearing cerebellar CT2A tumors demonstrated significantly decreased splenic weight, increased total, CD4, and CD8 T cell counts in the bone marrow, and a decreased proportion of surface S1P1 expressing T cells within the bone marrow. CONCLUSIONS T cell sequestration within the bone marrow associated with loss of surface S1P1, as previously demonstrated in high grade tumors of the forebrain, also occurs with tumors implanted into the cerebellum, suggesting that the genesis T cell sequestration is independent of location within the brain. Future work on this project will add additional tumor histology to our analysis as well as measuring the effects of S1P1 stabilization on response to T cell activating therapies in these models of cerebellar tumors.

Efficacy of function-enhanced, re-activatable, dual-specific CAR T cells pre-loaded with oncolytic virus for immunotherapy of high-grade glioma.

2062 Background: While cancer therapeutics have made tremendous progress within the past several decades, this benefit has not been seen in many primary cancers of the brain. Particularly confounding have been high-grade gliomas (HGG), which retain a dismal prognosis. Currently, novel therapies are being explored to rise to this unmet, critical need. One such therapy are CAR T cells, immune cells which have been engineered to target malignancy-specific antigens. Unfortunately, the efficacy of CAR T cell therapies against solid tumors is significantly limited, in large part due to impaired expansion/persistence in the immune suppressive tumor microenvironment (TME). Here we show that in vivo reactivation of CAR T cells through their native T Cell Receptor (TCR) by an oncolytic virus (OV) has therapeutic benefit in HGG. Methods: An EGFRvIII third-generation MSGV1 retroviral CAR construct containing the CD28, 4-1BB, and CD3z moieties, in tandem with the scFv derived from the human monoclonal antibody 139 and the marker Thy1.1 (38) was used to generate our CAR T cells. C57Bl/6 mice were used for in vivo experiments and both B16- and CT2A-EGFRvIII murine glioma cell lines were injected in the brain to model HGG. OVs and CAR T cells were given systemically by tail vein. OVs used include reovirus, vesicular stomatitis virus, and adenovirus. Results: By using OV in combination with EGFRvIII CAR T cells, we were able to generate a CD8 CAR population with TCR specificity for both the EGFRvIII and OV epitopes. These dual-specific (DS) CAR T expressed a memory phenotype and persisted for much longer than conventional CAR T cells. Further, we showed that these DS CAR T cells are more cytotoxic and can respond more rapidly than their conventional counterparts. We created a novel delivery mechanism for this combination OV + CAR T therapy using virus-loaded CAR T cells to bypass initial antiviral clearance from the immune system. Treatment with these OV-loaded CAR T cells lead to significant benefit in mice with HGG tumors which could be further enhanced by a systemic boost with OV, which rapidly re-activated DS CAR T cells against tumor and resulted in long-term cures of greater than 80% of treated animals. Conclusions: These promising results show that DS CAR T cells can overcome the critical therapeutic challenge of CAR T as a treatment for solid tumors. Given these promising results, we will go on to develop a clinical trial in which CAR T cells will be pre-loaded with OV and administered intravenously to patients with HGG, followed by systemic boosting with virus to re-activate DS CAR T cells against their tumor.

Internalization of PLGA nanoparticles coated with poloxamer 188 in glioma cells: A confocal laser scanning microscopy study

Internalization of poloxamer 188-coated PLGA nanoparticles (NPs) in GL261 murine glioma cells was studied using confocal laser scanning microscopy. For visualization, both poloxamer 188 (P188) and PLGA were labeled covalently with fluorescent dyes Rhodamine B and Cyanine5, respectively. The results indicated that the PLGA NPs coated with poloxamer 188 enter a cell as an integral core–shell structure, which can be helpful for gaining further insight into the in vivo performance of surfactant-coated polymeric NPs as core–shell delivery systems

Abstract 2497: Glioma-induced neuronal remodeling promotes regional immunosuppression

Abstract Recent studies have elucidated that gliomas remodel neuronal circuits, and distinct intratumoral regions maintain functional connectivity through a subpopulation of synaptogenic malignant cells expressing Thrombospondin-1 (TSP-1, encoded by the THBS1 gene). Single-cell RNA sequencing analyses of our primary glioblastoma patient samples identified a significant downregulation of immune response signatures in myeloid cells and lymphoid cells in functionally connected intratumoral regions characterized by upregulated THBS1. Understanding the biological significance of immunosuppression within functionally connected intratumoral regions may uncover therapeutic vulnerabilities. Here, we investigate glioma-neuronal-immune crosstalk across clinical tumor specimens and preclinical syngeneic models through bulk and single-cell RNA sequencing (13,670 cells), flow cytometry, and spatial transcriptomics. Using an SB28 murine glioma cell line that endogenously expresses Thbs1 at high levels, we generated a CRISPR Thbs1-knock-out (KO) cell line. In bulk RNA-sequencing data performed on orthotopic tumor models, Thbs1-KO tumors were characterized by the downregulated expression of synapse-associated genes and synaptogenic factors and the recovered expression of genes related to immune response. It highlighted the important role of Thrombospondin-1 in synaptogenesis and immunosuppression consistent with the observation from our primary patient tumor samples. Flow cytometry revealed that tumor-associated macrophages isolated from Thbs1-KO tumors were more polarized toward the pro-inflammatory “M1-like” phenotype (median M1/M2 ratio = 0.6 [WT] vs. 1.34 [KO], p < 0.006). Unbiased gene expression program analysis using spatial transcriptomics for in vivo tumor-harboring mouse brains demonstrated a significant negative correlation between signatures of synaptogenesis (represented by Ntng1 and Nlgn3 genes) and those of immune response (represented by Nfkb1 and Cd83 genes). SB28-Thbs1-KO syngeneic models demonstrated slower tumor growth and significantly longer survival compared to Thbs1-WT counterparts (19 days [WT] vs. 25 days [KO], p < 4.5E-5). The survival difference was abrogated in B6-SCID immunodeficient mice, indicating the critical role of adaptive immunity in the survival advantage associated with Thrombospondin-1 downregulation. Our results identify previously unknown immunosuppression mechanisms in the context of glioma-induced intratumoral connectivity via Thrombospondin-1. Future therapeutic strategies targeting this glioma-neuronal-immune crosstalk may open up new avenues for glioblastoma immunotherapy. Citation Format: Takahide Nejo, Saritha Krishna, Christian Jimenez, Akane Yamamichi, Jacob S. Young, Tiffany Chen, Senthilnath Lakshmanachetty, Payal Watchmaker, Abrar Choudhury, Hirokazu Ogino, David R. Raleigh, Shawn L. Hervey-Jumper, Hideho Okada. Glioma-induced neuronal remodeling promotes regional immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2497.

467 Stimulator of Interferon Genes Protein (STING) Agonist Incited Sustained Antitumor Immunity in Murine Models of Glioblastoma

INTRODUCTION: Introduction: Stimulator of interferon genes (STING) is an innate pattern recognition receptor natively localized to the endoplasmic reticulum that detects both exogenous and endogenous pathogenic or damage-associated cytosolic cyclic dinucleotides (CDNs), activating IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce a potent type I interferon and proinflammatory cytokine responses. Activation of the STING pathway has been recognized as a critical component of the innate immune sensing of tumors that is required for optimal cytotoxic T-cell priming against tumor antigens. Recently a highly potent cyclic dinucleotide STING agonist, IACS-8803, showed robust activation of the STING pathway resulting in systemic anti-tumor response in the B16 murine model of melanoma. Additionally, the STING agonists have been shown to achieve robust tumor regression at both injected and distal lesions and are reported to facilitate generation of long-lived immunologic memory. METHODS: Methods: We established murine gliomas with stereotactic implantation of GL261, a murine glioma cell line. Mice were assessed with luciferase imaging to ensure baseline tumor size and then randomized into treatment arms (IgG, IACS-8803, CD47, PD-1 and combinations thereof). Treatment was initiated one week after the tumor implantation. RESULTS: Results: All mice that received IACS-8803 had no detectable luciferase activity. Tumor regression was additionally verified via MR Imaging. Utilization of IACS-8803 led to long-term tumor-free survival (p < 0.01). Re-challenge with GL261 tumors ensured long term memory was sustained with a single dose of agonist. CONCLUSIONS: Conclusion: Engaging the STING pathway may be an ideal targeting strategy for GBM. Ongoing studies are delineating the mechanisms of activating this pathway with specific attention being paid to reprogramming and education of innate immunity.

Co-delivery of paclitaxel and etoposide prodrug by human serum albumin and PLGA nanoparticles: synergistic cytotoxicity in brain tumour cells

The aims of this study were to develop co-delivery systems of paclitaxel (PTX) and etoposide prodrug (4’-O-benzyloxycarbonyl-etoposide, ETP-cbz) based on non-cross-linked human serum albumin (HSA) and poly(lactide-co-glycolide) nanoparticles and to evaluate the synergistic potential of these drugs in vitro. The nanoformulations were prepared by the high-pressure homogenisation technique and characterised using DLS, TEM, SEM, AFM, HPLC, CZE, in-vitro release, and cytotoxicity in human and murine glioma cells. All nanoparticles had 90-150 nm in size and negative ζ-potentials. The Neuro2A cells were the most sensitive to both HSA- and PLGA-based co-delivery systems (IC50 0.024 µM and 0.053 µM, respectively). The drugs’ synergistic effect (combination index < 0.9) was observed in the GL261 cells for both types of co-delivery formulations and in the Neuro2A cells for the HSA-based system. These nanodelivery systems may be useful to improve combination chemotherapy for brain tumour treatment. To our knowledge, this is the first report describing the non-cross-linked HSA-based co-delivery nanosuspension which was prepared using nab™ technology.

Radiotherapy combined with anti-CEACAM1 immunotherapy to induce survival advantage in glioma

BackgroundWe aimed to observe the effect of radiotherapy on the expression of immune checkpoint molecule CEACAM1 in patients with glioma and the therapeutical effect of radiotherapy combined with blockade of CEACAM1 in mice with intracranial gliomas.MethodsThe expression of CEACAM1 on T-lymphocytes in the peripheral blood of patients with glioma was detected before and after radiotherapy; GL261 murine glioma cells (stably transfected with the luciferase gene) were implanted in the right caudate nucleus of C57BL/6 mice, and tumour growth was observed using the small animal in vivo imaging system. Mice were divided into 4 groups: (1) the isotype control; (2) the radiotherapy; (3) the anti-CEACAM1 treatment; and (4) the combination therapy. The survival of mice after treatment was recorded; the expression of CEACAM1 on murine glioma cells was detected by immunohistochemistry before and after radiotherapy; flow cytometry was adopted to detect CD8+ T-cells (Treg) (CD4+FoxP3+CD25+) among mouse brain-infiltrating T-cells; serum levels of IFN-γ and IL-10 were detected by ELISA; proliferation and apoptosis were observed by immunohistochemistry; Retrospective RNA-seq data analysis was conducted in a cohort of 325 patients with glioma in the Chinese Glioma Genome Atlas (CGGA) database and 702 patients in The Cancer Genome Atlas (TCGA) database.ResultsThe expression of CEACAM1 on CD4+ and CD8+ T-cells in the peripheral blood of patients with glioma was significantly higher 1 week after radiotherapy than before radiotherapy and was further increased 1 month after radiotherapy. Combined therapy notably inhibited the growth of intracranial tumours in mice and prolonged their survival time, with some mice being capable of surviving long-term (> 90 d). Immunohistochemistry revealed that the expression of CEACAM1 in murine glioma tissues after radiotherapy was elevated in a time-dependent manner. Flow cytometry analysis showed an increase in mouse brain-infiltrating CD8+ T-lymphocytes, a decrease in Treg cells, and an increase in CD8+ T/Treg cells after treatment. ELISA demonstrated the elevated levels of IFN and decreased levels of IL-10 in the serum of mice in the combination therapy group.ConclusionsRadiotherapy combined with CEACAM1 inhibitors resulted in strong and durable anti-tumour immune responses against murine glioma and long-term survival of some mice. Hence, this study is expected to offer new effective immunotherapy strategies against glioma.

Immunological and tumor-intrinsic mechanisms mediate the synergistic growth suppression of experimental glioblastoma by radiotherapy and MET inhibition

The hepatocyte growth factor (HGF)/MET signaling pathway has been proposed to be involved in the resistance to radiotherapy of glioblastoma via proinvasive and DNA damage response pathways.Here we assessed the role of the MET pathway in the response to radiotherapy in vitro and in vivo in syngeneic mouse glioma models. We find that the murine glioma cell lines GL-261, SMA-497, SMA-540 and SMA-560 express HGF and its receptor MET and respond to exogenous HGF with MET phosphorylation. Glioma cell viability or proliferation are unaffected by genetic or pharmacological MET inhibition using tepotinib or CRISPR/Cas9-engineered Met gene knockout and MET inhibition fails to sensitize glioma cells to irradiation in vitro. In contrast, the combination of tepotinib with radiotherapy prolongs survival of orthotopic SMA-560 or GL-261 glioma-bearing mice compared with radiotherapy or tepotinib treatment alone. Synergy is lost when such experiments are conducted in immunodeficient Rag1−/− mice, and, importantly, also when Met gene expression is disrupted in the tumor cells. Combination therapy suppresses a set of pro-inflammatory mediators including matrix metalloproteases that are upregulated by radiotherapy alone and that have been linked to poor outcome in glioblastoma. Several of these mediators are positively regulated by transforming growth factor (TGF)-β, and pSMAD2 levels as a surrogate marker of TGF-β pathway activity are suppressed by combination treatment. We conclude that synergistic suppression of experimental syngeneic glioma growth by irradiation and MET inhibition requires MET expression in the tumor as well as an intact immune system. Clinical evaluation of this combined strategy in newly diagnosed glioblastoma is warranted.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism

Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.