Glioblastoma Model Research Articles

EXTH-52. SIMULTANEOUS TARGETING OF TUMOR CELLS AND TUMOR-ASSOCIATED MACROPHAGES TO REPROGRAM GLIOBLASTOMA BY TUMOR-SUPPRESSIVE MICRORNA

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumors due in part to cell-intrinsic and cell-extrinsic mechanisms. The ability of GBM tumor to not only resist conventional therapies but also induce an immunosuppressive tumor microenvironment (TME) is thought to underlie the failure of immunotherapies. Thus, strategies to target both tumor cells and immunosuppressive immune cells, such as tumor-associated macrophages (TAMs), are of great interest. The ability of microRNA (miRNA) to target multiple genes is favorable to alter the character of the TME and cancer cells. Previously, we have shown that tumor suppressive miRNA, miR-138, can effectively regulate the genes in GBM cells relating to cell proliferation and viability. The exogenous delivery of miRNAs, however, has been limited due to the lack of delivery mechanisms that minimize off target effects. We argue that extracellular vesicles (EVs) are an ideal modality of targeted delivery for therapeutic miRNA, which we can accomplish by decorating the EV surface with cancer specific ligands, such as folate. Here, we report that trypsin digestion of the EV surface can increase the surface labeling efficiency for cancer targeting ligands. Folate-labeled trypsinized EVs effectively targeted folate receptor (FR) positive GBM cells and TAMs, delivering miR-138 in in vitro and in vivo orthotopic intracranial murine models of GBM. This simultaneous delivery decreased cancer cell proliferation and altered macrophage polarization through the inhibition of known target genes, phenotypically reprogramming both cancer cells and TAMs. To our knowledge, this is the first preclinical study attempting to target both tumor cells and innate immunity by delivering tumor suppressive miR-138 using a trypsinized EV-based RNA delivery system. These findings can be rapidly translated into a novel therapeutic option for GBM patients.

IMMU-25. B-CELL BASED THERAPY (BVAX) PRODUCES ANTIBODIES TO SUPPRESS GLIOBLASTOMA TUMOR MIGRATION AND INVASION

Abstract Glioblastoma (GBM) presents as a highly aggressive and malignant brain tumor with limited treatment options. Despite the availability of existing therapies, the invasive characteristics of GBM frequently result in tumor recurrence and poor prognosis. We previously developed a B-cell-based therapy (BVax) that has shown promising results in its ability to induce therapeutic responses in preclinical models of GBM. Our research revealed potency of BVax can be attributed to its robust antigen-presenting capability, cognate antigen specificity with T cells, and ability to generate tumor-reactive antibodies. The present study aims to characterize the unique antigenic reactivity of BVax-derived antibodies by evaluating their immunological effects and therapeutic potential in both murine models and GBM patient-specific samples. Leveraging CD45.1 and CD45.2 congenic mouse models, we first revealed that BVax preferentially migrated to glioma-bearing brains upon intravenous injection, resulting in a CD38+CD20-CD19+ plasmablast phenotype. To further characterize the reactivity of BVax-derived immunoglobulins (Ig), we utilized proteomic analysis and identified preferential targets of Gelsolin, Fibronectin, Versican, Fibrinogen, and collagen type IV alpha, commonly associated with cell motility and the extracellular matrix. Microdialysis data confirmed the increased presence of many of these BVax-derived Igs-recognized antigens in tumor regions compared to normal brain regions in GBM patients. Utilizing purified BVax-derived antibodies from patient specimen, in vitro cell motility assays demonstrated a significant prevention of the invasion and migration of cell lines from patient derived xenograft (PDX). In this study, we concluded that BVax elicits antitumor activity through targeted migration to tumor-bearing regions, differentiation into plasmablasts, and robust secretion of specific Igs that retain the ability to inhibit functionalities associated with cell invasion and migration. Here, we present the first in-depth study that characterizes GBM patients’ antibody reactivity, specifically a subset of inflammatory B cells (BVax) with therapeutic effects that offer an indication for a new direction of immunotherapies.

IMMU-36. MULTI-OMIC CHARACTERIZATION OF A SYNGENEIC MOUSE CANCER STEM CELL MODEL OF GBM FOR PRECLINICAL APPLICATIONS

Abstract Glioblastoma (GBM) is resistant to many therapies including immunotherapies. There is an urgent need to explore the role of the microenvironment in resistance to therapy with tailored pre-clinical models. Using multi-omic approaches we describe a syngeneic cancer stem cell mouse model of GBM, with a spontaneous amplification of Igf2. We investigate whether Igf2 influences tumour and microenvironment cells to promote immunosuppression in GBM. Three cell lines were previously established from spontaneous brain tumours in C57Bl/6 Trp53+/-/Nf1+/- mice and maintained under neural stem cell culture conditions. Whole genome sequencing (WGS) was used to determine single nucleotide, copy number, and structural variation, revealing loss of both copies of Trp53 and Nf1 in all three cell lines. However only one cell line, mBT0309, developed tumors when orthotopically allografted. Analysis of genomic alterations and transcriptomics revealed that mBT309 exhibits amplification of the Igf2 loci and overexpression was confirmed at the RNA and protein level. Spatial and single cell transcriptomics showed that Igf2 is overexpressed in mBT0309 allografted tumours and correlated with specific transcriptomic programs. A high-parameter Imaging Mass Cytometry (IMCTM) panel was used for spatial proteomic analysis, to monitor the development of tumours in a time-course experiment. In vitro growth characteristics and stem marker expression in both transcriptomic and spatial IMC data suggest that high levels of Igf2 may regulate GBM stemness features. Analysis of human GBM datasets revealed that Igf2 amplified tumours have reduced CXCL11 expression. These findings suggest that high levels of Igf2 may influence immunosuppression by reducing T-cell recruitment. This syngeneic immunocompetent GBM stem cell model harbouring Igf2 amplification adds to the suite of models to study the GBM microenvironment and its role in immunosuppression and resistance to therapy.

STEM-14. INTEGRATED ANALYSIS OF GLIOBLASTOMA PROGENITOR SUBTYPES REVEALS A TUMOR-DERIVED STEM CELL POPULATION WITH NEURAL AND VASCULAR POTENTIAL

Abstract Glioblastoma (GBM) exhibits a vast heterogeneity in cell types and states. Recent research highlights the reactivation of neurodevelopmental programs in GBM, contributing to this heterogeneity and, ultimately, therapy resistance. To characterize the landscape of progenitor cell types in human primary GBM, we compiled a meta-atlas of single-cell transcriptomes from seven published studies. Using a novel bioinformatic method for gene program analysis, we derived GBM gene expression meta-modules represented across multiple tumors. Our analyses validated previously described GBM gene programs and revealed novel tumor progenitor subtypes. Among these subtypes, we identified a tumor-derived stem cell population exhibiting a mixed vascular identity and transcriptional programs reminiscent of neural stem cells, which we identified as a putative neurovascular progenitor (NVP). To our knowledge, this population has not been previously described in GBM. We validated the existence of NVPs in situ using immunofluorescence from primary patient GBM samples. We then performed an enrichment for NVPs using fluorescence-activated cell sorting direct from patient tumors, followed by single-cell RNA sequencing. Examination of data from traditional model systems, including gliomaspheres and mouse GBM models, confirmed the preservation of NVP identity across systems. In vivo analysis of NVPs in multiple mouse models of GBM elucidate the influence of NVPs on GBM cell type composition, highlighting their role as multipotent progenitors. Finally, we utilized single-cell lineage barcoding to trace the progeny of patient-derived NVP cells in a novel organoid transplantation model, revealing NVP differentiation into vascular-like and neural-like cells. These data indicate that NVP cells play crucial roles in tumor plasticity and may represent a valuable therapeutic target for future exploration.

STEM-17. THE UROKINASE RECEPTOR AS A NOVEL IMMUNOTHERAPEUTIC TARGET IN BRAIN CANCERS

Abstract Glioblastoma (GBM) is the common malignant brain tumor in adults, accounting for approximately 15% of all CNS tumors, and 48.6% of malignant brain tumors, with a median survival of approximately 15 months. GBM is characterized by extensive inter- and intra-tumoral heterogeneity and an extremely immunosuppressive tumor microenvironment. Following Standard-of-Care surgical resection and chemoradiotherapy, patients inevitably relapse, at which point few therapeutic avenues exist, owing in part due to a lack of clinically relevant targets. Data from our target identification pipeline shows the urokinase plasminogen activator receptor (uPAR) is significantly upregulated at recurrence on putative GBM brain tumor initiating cells (BTICs), which are believed to drive de novo tumor formation, recurrence, and therapeutic resistance. uPAR plays an important role in the plasminogen activation system, and in the context of cancer, has been implicated in numerous pro-tumorigenic processes such as invasion, proliferation, and therapy resistance. We used CRISPR-Cas9 to genetically delete uPAR from our in-house patient-derived GBM cell lines, and found that knockout of uPAR in recurrent GBM cells significantly reduces various functional characteristics of BTICs in vitro. In our patient-derived mouse model of GBM, we found that knockout of uPAR significantly increases survival, and decreases tumor burden. Further, we generated novel anti-uPAR single domain antibodies (sdAbs) which specifically and efficiently bind uPAR at low nanomolar concentrations in vitro. We developed anti-uPAR CAR Ts using the binder sequence of the sdAbs, which showed potent cytotoxicity in vitro, and drastically reduced tumor burden and extended survival in vivo. Further, we identified uPAR as being highly expressed on brain metastases from multiple origins (lung-to-brain, melanoma-to-brain, etc.), and demonstrate that anti-uPAR CAR Ts effectively kill brain metastases in vitro, and significantly extend survival using in vivo brain metastasis models. From this work we believe uPAR to be a clinically relevant target in both recurrent GBM and brain metastases, and investigation into therapeutic strategies targeting uPAR-positive brain cancers should be explored further.

NIMG-75. SIMILARITY DISTANCES-BASED DELTA RADIOMICS (SDDR) ENABLES LONGITUDINAL MRI COMPARISONS FOR SURVIVAL PREDICTION IN GLIOMAS: A MULTI-INSTITUTIONAL VALIDATION

Abstract Radiomics (computerized feature analysis) on treatment-naive MRI scans has demonstrated great value in outcome prediction for Glioblastoma (GBM). However, delta radiomics (analysis of radiomic feature variation between different events, e.g., before and after treatment) has not been explored on account of challenges with precise spatial correspondences between pre- and post-operative scans. This work presents a survival prediction model for GBM, Similarity Distances-based delta radiomics (SDDR), based on the hypothesis that similarity distance-measures between radiomic features on pre-, and post-treatment images can accurately capture temporal changes within-and-around the tumor, and provide improved prediction of overall survival, compared to models based on a single timepoint. 150 patients from 2 publicly-available Glioma repositories (University of California San Francisco “UCSF” (n=48), LUMIERE (n=67)) and 2 two proprietary datasets (from xCures (n=13), and Cleveland Clinic (CCF) (n=74)) were obtained. UCSF and LUMIERE cohorts were used as separate hold-out sets, in each of which the remaining collections were used as discovery set. Following pre-processing, tumor segmentation into Edema (ED) and Enhancing tumor (ET) were performed. SDDR was then computed, which is the distance between probability densities functions from 156 gray co-occurrence (GLCM) and 52 gradient-based co-occurrences statistics (COLLAGE) features extracted from pre and-post-treatment MRI. Six statistical distances (e.g., Earth mover distance) normalized by the scanning interval (in weeks) yielded n=2496 SDDR measurements per tumor region (1248/region, 936 delta-GLCM, and 312 delta-COLLAGE). The same set of features were computed from pretreatment MRI (Pre-R) for comparison. Features were fed to LASSO-Cox regression for survival analysis. SDDR for GLCM features extracted from ET outperformed Pre-R, and demonstrated significant differences between high and low risk groups for both hold out tests: UCSF (p=0.0069, HR =2.8, C-index=0.69) and LUMIERE (p=0.0061, HR = 1.9, C-index 0.65). SDDR-based multi-institutional survival analysis demonstrated promise in reliable longitudinal risk-stratification in GBM.

DDDR-25. IBUDILAST AND ANTI-PD-L1 COMBINATION TREATMENT SIGNIFICANTLY IMPROVES SURVIVAL IN A SEX-DEPENDENT MANNER IN A MURINE MODEL OF GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) is the most common primary malignant brain tumor, and there remains a significant need for new treatments. Previous trials studying immune checkpoint inhibitors (ICI) as single agents have been negative. Ibudilast is a brain penetrant inhibitor of the macrophage migration inhibitory factor (MIF)-CD74 interaction that sensitizes GBM to temozolomide in vitro and alters myeloid-derived suppressor cells in vitro and in vivo. The purpose of this study was to investigate whether ibudilast increases ICI efficacy in a murine model of GBM. METHODS 6-week-old male and female C57BL/6 mice were implanted with SB28 cells intracranially. One week after implantation, each group was randomized to receive intraperitoneal treatment with either: ibudilast (50 mg/kg), anti-PD-L1 (1mg/kg), combination, or vehicle control. Mice were euthanized at neurological endpoint. Survival was modeled with Cox proportional hazards regression. RESULTS Among females, there was a significant difference in survival among treatment groups (p=0.0002). Specifically, combination treatment led to significantly longer survival compared to ibudilast (p=0.004) and control (p=0.03), but not anti-PD-L1 (p=0.12). Among males, there was no significant difference in survival among groups (p=0.40). Males had significantly shorter median survival compared to females (overall: 18 versus 22 days, combination treatment: 20 days versus 32 days, anti-PD-L1: 14 versus 22 days, ibudilast: 17 versus 17 days, control: 18 versus 22 days, p=0.02). CONCLUSIONS Ibudilast with anti-PD-L1 is a promising combination treatment against GBM. Future research is needed to identify the mechanisms underlying this treatment regimen and its sex-dependent effect. These results may inform the development of a combination new clinical trial, as ibudilast is currently being evaluated in the newly-diagnosed and recurrent GBM setting with temozolomide.

CNSC-29. CIRCADIAN SIGNALING IN GLIOBLASTOMA SYNCHRONIZES TUMOR DAILY RHYTHMS AND REGULATES TUMOR PROGRESSION

Abstract Glioblastoma (GBM) is the most common primary brain tumor in adults with a poor prognosis despite aggressive therapy. A recent, retrospective clinical study found that administering Temozolomide in the morning increased patient overall survival by 6 months compared to evening. Previous work has shown that murine and human models of GBM have cell-intrinsic circadian rhythms in expression of the core clock genes Bmal1 and Per2. This suggests that GBM has circadian rhythms that entrain to the hosts’ central clock. Understanding the mechanisms underlying circadian entrainment and tumor growth in GBM is essential to determine whether regulation of tumor biology and susceptibility to therapies varies with time of day. Here, we tested the hypothesis that daily host signaling regulates tumor growth and synchronizes circadian rhythms in GBM. We found that GBM cells transduced with luciferase reporters to record clock gene expression have intrinsic circadian rhythms, with Bmal1 peaking 8-12 h before Per2 in murine and human cell lines in vitro and in vivo. Further, intrinsic circadian rhythms in clock gene expression in GBM entrain to the host through glucocorticoid signaling, regardless of tumor type or host immune status. We found daily glucocorticoids (i.e., Dexamethasone) promoted or suppressed GBM growth depending on time of day of administration and on the clock gene, Bmal1. Blocking circadian signals, like VIP or glucocorticoids, dramatically slowed GBM growth and disease progression. Our results suggest that GBM tumors entrain to the circadian circuit of the brain and depend on clock-controlled cues like VIP and glucocorticoids to grow at specific times of day. This work may inform personalized circadian medicine to improve individual GBM patient outcomes.

EXTH-78. A NOVEL BISPECIFIC S100A4/TFR ANTIBODY TO REPROGRAM THE GLIOMA TUMOR MICROENVIRONMENT AND TARGET GLIOMA STEM CELLS

Abstract GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. While immunotherapy is a promising treatment modality for GBM, most clinical trials have thus far only provided a modest benefit to GBM patients. We propose that enhancing the efficacy of immunotherapy for GBM requires approaches that increase T cell infiltration and reprogram the myeloid cells to a more pro-inflammatory state. S100A4 is a major regulator of stemness, epithelial-mesenchymal transition, and a critical regulator of immune suppressive myeloid and T cell phenotypes in GBM. Targeting S100A4 in either glioma or stromal cells is sufficient to reprogram the tumor immune landscape and extend survival of glioma bearing mice, indicating that S100A4 is a promising immunotherapy target. We recently developed a S100A4 blocking monoclonal antibody; however, this antibody does not cross the blood brain barrier (BBB) efficiently to confer significant therapeutic benefit, limiting its efficacy in treating GBM. To overcome this challenge, we generated a bispecific S100A4-TfR antibody (BsA) that allows robust BBB penetration, and tested this in a pre-clinical syngeneic, orthotopic mouse model of GBM. Using ELISA, confocal immunofluorescent microscopy, flow cytometry, and bulk RNA sequencing, we report that systemic administration of the S100A4-TfR BsA results in increased immune cell infiltration with a preferential increase in CD4 T cell infiltration, and potent immune remodeling of the tumor microenvironment to a more pro-inflammatory state. Furthermore, the BsA is taken up by glioma cells intracellularly, which is correlated with depletion of intracellular S100A4 and decreased nuclear SOX2 localization, which would reduce stemness of glioma cells. Functionally, in vitro BsA treatment of primary glioma cell cultures shows decreased S100A4 expression, proliferation, and self-renewal. Our results suggest that S100A4-TfR BsA is a novel dual activity inhibitor to simultaneously reprogram the GBM immune landscape and reduce glioma stemness.

DDDR-23. DECODING GLIOBLASTOMA DRUG RESISTANCE DYNAMICS USING NOVEL MICROFLUIDIC EX VIVO MODELS

Abstract Glioblastoma (GBM) is clinically challenging due to its aggressive nature and limited treatment options. Current standard of care involves surgical resection and chemoradiation with temozolomide (TMZ). GBM, however, often returns post-treatment fueled by its complex biology and the presence of resilient GBM stem cells. Compounding the issue is the blood-brain barrier, which poses a formidable obstacle to drug delivery into the brain. Understanding the molecular dynamics post-therapy within the brain, influenced by the tissue microenvironment, is key for developing more effective therapeutic strategies. Unfortunately, existing research models have fallen short in capturing the complexity of GBM and its response to treatment. While animal models have been a mainstay in GBM research, their ability to predict human responses has been called into question, highlighted by the disappointing results seen in clinical trials. We are therefore advancing bioprinting-based approaches for modeling various components of the tumor microenvironment to gain insight into mechanisms of drug resistance. We report on the development of three novel microfluidic platforms to investigate and interrogate GBM biology. First, we have developed a novel microfluidic model of GBM, termed GlioFlow-3D, produced through a combination of 3D printing methods. GlioFlow-3D mimics specific aspects of the perivascular niche, including protection from circulating TMZ. Second, we have adapted GlioFlow-3D as part of a platform to investigate advanced ultrasound techniques for delivering novel therapeutics into the human brain. Third, we are developing a novel approach aimed at exploring the relationship between the brain extracellular matrix and tumoral cells using bioprinting of living tumor organoids obtained from patients with a history of recurrence. By integrating bioprinting technologies with patient-derived models to create novel ex vivo platforms, we aim to obtain new insights into the intricate dynamics of drug resistance in GBM, ultimately paving the way for more effective therapeutic strategies. Funding: The Brain Tumor Center Research Award (UCGNI) The ARC Project - CCHMC and UC joint Research Award

TMOD-33. DOES PRE-CLINICAL DRUG ACTIVITY PREDICT PHASE II CLINICAL EFFICACY IN GLIOBLASTOMA? A DETAILED ANALYSIS

Abstract Despite decades of research, there are fewer than six FDA-approved therapies for glioblastoma (GBM). The drug approval process relies on studies establishing pre-clinical activity, most commonly using murine models, followed by in human studies establishing safety and then efficacy. Although murine models for GBM have allowed for insights into tumor biology, their utility in assessing the efficacy of novel anti-cancer therapies remains highly debated. Here, we sought to establish whether there was any relationship between drug activity in preclinical models and phase 2 clinical trial outcomes. Using ClinicalTrials.gov (NIH), all phase 2 trials in glioblastoma up to December 2023 were queried and downloaded. Trials that were never started, had not yet started, were withdrawn, not completed, or contained indications beyond glioblastoma were excluded. Studies examining anti-tumor effects of an investigational agent with or without bevacizumab, temozolomide, or radiation therapy in glioblastoma were included, and studies examining alternative dosing strategies, various medical devices, or drug combinations, were excluded. The corresponding preclinical studies were identified via literature review for each clinical trial. Data including the progression free and overall survival benefit, mouse models used, and sample sizes were extracted from each study. 659 trials between 1994 and 2023 were included for analysis. Of these, 272 (41%) were for monotherapies, examining 90 unique small molecules. Preclinical endpoints included murine survival and tumor weight. In comparing mouse survival and tumor size to progression free and overall survival in humans, our preliminary analysis did not reveal any clear correlation. The correlation between preclinical and clinical drug response in GBM remains uncertain. Future work will examine whether mouse genotype, immune status, or GBM cell line used affected the degree of concordance with human clinical trial data. Our analysis suggests that further investigation into preclinical models of GBM, and standardized reporting of preclinical models, are needed.

TMOD-15. TOWARD BRAIN CANCER ORGANOID-INFORMED PRECISION MEDICINE FOR GLIOBLASTOMA

Abstract BACKGROUND Improved preclinical models for glioblastoma are urgently needed to enhance the translation of research to clinical applications. Patient-Derived Tumor Organoids (PDTOs) offer a promising model that can be generated quickly. Here we explored their feasibility. METHODS Primary (n=16) and recurrent (n=2) PDTOs were established using the method described in Jacob et al. Cell. 2020 by dissecting tumours into small fragments, preserving their cytoarchitecture. Histological features of the parent tumour were compared to the matched PDTOs by a neuropathologist. To assess the molecular fidelity, comprehensive whole genome sequencing compared matched tumor tissue, PDTOs, and corresponding normal blood samples (n=11), along with RNA sequencing of matched tumor tissue and PDTOs (n=10). We assessed PDTO responses to regorafenib (n=18) and conventional TMZ (n=9), lomustine (n=9) and 10 Gy of ionizing radiation (IR) (n=6) using the automated imaging system IncuCyte S3® to evaluate whole spheroid brightfield and invasion areas following drug/IR treatment and assess viability. RESULTS We achieved a 100% establishment rate of primary GBMs using same-day procedures and an 80% rate if held under 3 days. Histological features were retained in the PDTO. Molecular overlap was greater than 90%, along with a high concordance in gene expression. PDTOs exhibited notable resistance to temozolomide and lomustine, however, showed a very modest response to IR and a moderate response to regorafenib. IncuCyte S3® analysis revealed discernible changes in whole spheroid brightfield and invasion area for regorafenib-treated PDTOs. Clinically relevant concentrations of regorafenib reduced GBM cell proliferation (Ki67 staining). Beyond this concentration, regorafenib exhibited efficacy in eliminating GBM cells. CONCLUSION PDTOs recapitulate the molecular and histological features of the original tumor and may enable the prediction of individual patient responses to therapies and guide more effective treatment selection. We are now applying our established methodology to assess a new set of promising drug candidates.

IMMU-09. REVERSING SYSTEMIC T CELL DYSFUNCTION TO LICENSE IMMUNOTHERAPY IN INTRACRANIAL TUMORS

Abstract Intracranial tumors present unique challenges for immunotherapy. These can include both local and systemic modes of immune suppression, the mechanistic underpinnings of which are incompletely understood. Our work reveals that intracranial tumors elicit systemic increases in circulating catecholamine levels, and that this chronic sympathetic hyperactivity results in T cell dysfunction that impedes immunotherapeutic efficacy. We show that treatment with b-adrenergic blockade can partially overcome the negative impacts of chronic sympathetic hyperactivity by increasing NF-kB activity in immune cells, restoring T cell polyfunctionality, favorably modifying the tumor microenvironment, and extending survival in murine models of glioblastoma treated with immune-based therapies. Furthermore, we demonstrate that extended survival is also observed in glioblastoma patients receiving b-adrenergic blockade, as well as in patients with melanoma and lung cancer brain metastases who received b-blockade alongside concomitant immune checkpoint inhibition. Importantly, while local sympathetic hyperactivity in the tumor microenvironment and b-blockade also impact the anti-tumor immune response in the context of extracranial disease, these effects are markedly more pronounced in intracranial disease. Taken together, these data reveal that sympathetic hyperactivity facilitates systemic immune dysfunction in the setting of intracranial tumors and highlight a novel role for the application of b-adrenergic blockade to license immunotherapy in intracranial malignancies.

IMMU-19. PRECLINICAL THERAPEUTIC ACTIVITY AND PET IMAGING OF STING AGONIST 8803 IN GLIOBLASTOMA

Abstract STING (stimulator of interferon genes) activation triggers interferon (IFN) release within the tumor microenvironment from myeloid cells and other cell types, inducing proinflammatory anti-tumor immune responses. The small molecule STING agonist, 8803, elicits long-term survival in 56% (QPP4; p=0.0003) to 100% (QPP8; p<0.0001) of mice with orthotopically implanted immune checkpoint-resistant glioblastoma models after 2-3 doses. In humanized mice, in which glioblastoma-cell STING is epigenetically silenced, 8803 therapeutic activity is maintained. The combination therapy of 8803 with anti-PD-1 relatively further enhances survival in immune checkpoint-resistant models. Ex vivo flow cytometry and proteomic sequential multiplex profiling during the therapeutic window demonstrate global immunological reprogramming, specific tumor-antigen immune responses, increased tumor immune effector trafficking, and release of IFN. It has been shown that IFN signaling augments intracellular nucleotide metabolism and increases [18F]-FLT uptake in pancreatic cancer. We evaluated the longitudinal [18F]-FLT uptake in CT-2A-bearing mice using MRI/PET/CT at days 0, 2, and 4 after intratumoral 8803 delivery. Brain images were acquired before and after [18F]-FLT tracer and 8803 administrations. The CT was used for PET attenuation/correction, and the MRI was used for tumor segmentation. Regional analysis of PET-derived [18F]-FLT SUV maps used advanced image processing approaches as a function of time to enhance the voxel-by-voxel interpretation of the acquired images. [18F]-FLT uptake increased from baseline in all mice treated with 8803 compared to control mice with a peak at 48-96 hours. Concordant sequential multiplex immunofluorescence demonstrated that the STING expression was prominent along the tumor vasculature, myeloid, and tumor cells. p-IRF-3 expression, reflective of STING pathway activation, showed a diffuse pattern after treatment. Based on the preclinical data, PET imaging at 48-96 hours post-treatment represents the appropriate imaging modality and timepoint to maximize the ability to detect STING-dependent changes for a therapeutic strategy that reprograms the glioblastoma microenvironment and improves survival across multiple preclinical models of glioblastoma.

NIMG-32. ORAL ADMINISTRATION OF DEUTERATED CHOLINE AS A NEW APPROACH TO PROVIDE HIGH TUMOR-TO-BRAIN IMAGE CONTRAST IN DEUTERIUM METABOLIC IMAGING (DMI)

Abstract BACKGROUND Metabolic imaging using FDG-PET is a key component of disease management in many cancers outside of the brain, but in brain tumors it often shows low tumor-to-brain contrast because of high background signal from normal brain. We explore generating cancer-specific metabolic image contrast using Deuterium Metabolic Imaging (DMI), which is a novel 2H magnetic resonance spectroscopic imaging-based technique. DMI was applied after oral administration of deuterated choline, an essential nutrient needed for membrane synthesis whose uptake is often increased in tumors. METHODS Total choline (tCho) DMI was performed in an orthotopic model of glioblastoma, F344 rats (n=11) implanted with RG2 cells, on an 11.7 tesla MRI scanner, with isotropic spatial resolution of 2.5 mm. tCho-DMI data were collected for 36 min during intravenous infusion (IV, n=6) or after 3 days of oral administration (PO, n=5) of [2H9]-choline chloride dissolved in sterile water. At a dose of 285 mg/kg body weight, bolus-continuous IV infusion via the tail vein required 1.57 ml for a 250-gram rat. PO administration via gavage on 3 consecutive days used the max daily dose recommended for human adults, 50 mg/kg. Results - In the PO group, average tumor tCho concentration was 0.78±0.14 mM and 0.20±0.05 mM in contralateral normal brain. In the IV group, the tCho concentration was 0.88±0.18 mM in the tumor, and 0.18±0.03 mM in normal brain (PO vs. IV: p=0.52). Tumor-to-brain contrast was 5.9±1.2 in PO-administered animals, and 7.1±3.3 for animals receiving IV 2H-choline, (p=0.54). CONCLUSIONS - In a preclinical GBM model oral administration of [2H9]-choline resulted in comparably high tumor-to-brain image contrast on tCho-DMI-based maps as during IV infusion. Work is ongoing to identify the different choline metabolites that contribute to the tCho signal. Because oral administration of choline is very safe, this approach could facilitate the translation of tCho-DMI to human subjects.

EXTH-80. A NOVEL BISAMINOQUINOLINE DERIVATIVE NANOPARTICLE (BAQ13-NP) FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Aminoquinolines, such as hydroxycholoquine, are known to inhibit autophagy and slow cancer cell growth, improving survival in preclinical models of glioblastoma. Unfortunately, they failed to demonstrate benefit in clinical trials due to a lack of sufficient potency and BBB penetration. We have developed a novel bisaminoquinoline derivative packaged in a nanoparticle (BAQ13-NP) with improved potency and intratumoral accumulation after systemic administration, confirmed by distribution and toxicity studies in animals. In this study, we evaluated the impact of BAQ13-NP on tumor cell autophagy and survival using patient-derived glioma cell lines and murine models. GBM patient-derived cell lines (n=4) and murine glioma cells (GL261, CT-2A) were treated with BAQ13-NP (1-4 µM) in culture, demonstrating growth restriction to less than 50% of control at 5-7 days (p<0.01) by MTT assay. Cells treated with BAQ13-NP also demonstrated reduced autophagy with significant increases in accumulation of LC3B and p62 (p<0.01) and reduced IL-6 dependent STAT3 phosphorylation in tumor cells, T cells, and myeloid cells (p<0.01). In addition, treatment of tumor cells with BAQ13-NP reduced IL-6 production over 50% (p<0.01). Mice with orthotopic GL261 or CT2A tumors were treated with BAQ13-NP by tail vein injection (25 mg/kg q2 days) starting 7 days post implantation. Significantly increased survival was observed in BAQ13-NP treated GL261 (HR 0.75, p=0.030) and CT2A (HR 0.78, p=0.033) bearing mice compared to vehicle control treated animals. Reduced tumor size at specific intervals was also observed with BAQ13-NP treatment, corelating with improved survival. BAQ13-NP can inhibit autophagy and slow glioma tumor growth, improving survival in preclinical models. Given the excellent distribution and low toxicity of this novel agent when delivered intravenously, we believe it has great potential to improve outcomes for GBM patients. Our drug has recently received IND approval and will soon be tested in a phase I clinical trial.

NIMG-81. PREDICTING TUMOR PROGRESSION WITH FIBER DENSITY-WEIGHTED WHITE MATTER PATHLENGTH MAPS IN PATIENTS WITH GLIOBLASTOMA

Abstract PURPOSE Although histopathological evidence indicates that glioma cells preferentially migrate along large white-matter bundles, standard-of-care (SOC) radiation therapy (RT) planning remains an isotropic expansion of the anatomical lesion. We hypothesize that anisotropic expansion of RT target volumes along white-matter pathways using fiber density-weighted, white-matter pathlength maps (DW-WMPLMs) derived from diffusion tensor imaging (DTI) could improve the prediction of tumor cell migration beyond surgical margins by a deep learning model compared to SOC clinical target volumes (CTVs). METHODS DTI and anatomical MRI from 118 patients newly-diagnosed with glioblastoma were retrospectively analyzed with anatomical imaging from post-surgical resection and latest progression scan before intervention. Parallel-transport tractography seeded from the pre-surgery T2-lesion was used to estimate white-matter fiber propagation. Fibers that intersected with the resection cavity were weighted by their density to control the length of expansion and create a DW-WMPLM that was used together with T2-FLAIR and T1-post-contrast images as inputs to a novel 3D-Swin-UNetR network, trained to predict the recurrence region using 4-fold cross-validation and evaluated in a holdout test-set (95/23 CV/test). New weighted-overlap, coverage, and sparing indices were introduced to evaluate coverage of the progressed lesion and healthy brain excluded. RESULTS 52% of patients had progression outside the standard 2cm-isotropic CTV margin, motivating the need for anisotropic CTVs. Our novel DW-WMPLMs had 87±20% and 83±18% overlap with contrast-enhancing and T2-lesion progression, respectively. The deep-learning model trained with DW-WMPLMs and anatomical images achieved 19% and 56% higher Dice and weighted-overlap indices compared to the isotropic 2cm-CTV(p<0.02), with less normal brain included. CONCLUSION This study demonstrates the feasibility and benefit of incorporating a novel metric of white-matter track characterization into deep learning progression prediction models of glioblastoma for RT-planning. Current work is validating findings in a prospective cohort acquired immediately prior to RT and incorporating other imaging metrics.

IMMU-43. GUT MICROBIOTA SHAPE IMMUNE PATTERNS IN NAÏVE AND GLIOMA-BEARING HUMANIZED MICROBIOME MICE

Abstract Immunotherapy increases survival in mouse models of glioblastoma, but this is not observed in clinical trials. To address this issue, we employed a humanized microbiome (HuM) mouse model where fecal matter is transplanted from healthy human donors into gnotobiotic mice. Our previous research found that in the GL261 model of glioma, HuM mouse lines could be classified as either responders or nonresponders to anti-PD-1 therapy based solely on the gut microbiota unique to each HuM line. Here, we sought to determine the immune differences between responder and nonresponder HuM lines in both naïve (tumor-free) and glioma conditions. Single-cell RNA-sequencing (scRNA-seq) analysis of naïve colonic CD45+ cells showed that responder colons had higher frequencies of neutrophils and naïve B cells and lower frequencies of plasmablast and plasma cells. Notably, colonic neutrophils in naïve responders upregulated H2-Eb1 and Il1a compared to nonresponders. To validate the scRNA-seq data, flow cytometry was also performed on the naïve lymph nodes (LNs) and brains. The gut-draining LNs of responders had a higher frequency of CD45+ immune cells and CD4+CD8+ T cells and a lower frequency of Tregs than nonresponders. In naïve brains, responders had a higher frequency of CD45+ cells and macrophages than nonresponders and trended toward having fewer CD206+ macrophages. When mice were injected with GL261 tumor cells and treated with one dose of anti-PD-1, there was a trend for an increase in Nos2+ CD11b+ cells in both frequency and count compared to nonresponders, indicating an early and robust anti-tumor myeloid response in the responders. In summary, these results show how variations in the microbiota of HuM mice impact the immune environment across multiple organs in the baseline and tumor states, which potentially accounts for the positive response to anti-PD-1 therapy in the glioma model.

IMMU-40. INVESTIGATING THE ROLE OF MUTATIONAL BURDEN IN TUMOR IMMUNOGENICITY USING A PRE-CLINICALMLH1-DEFICIENT GLIOBLASTOMA MODEL

Abstract BACKGROUND Although there is an established correlation between tumor mutational burden (TMB) and response to immune checkpoint inhibition (ICI) in many cancers, such an association has not been definitively shown in high-grade gliomas. Most studies of ICI therapy in gliomas, including glioblastoma (GBM), have failed to demonstrate efficacy, with the exception of hypermutated cases resulting from rare inherited defects in mismatch repair (MMR) genes. We sought to investigate the association of TMB to immune response using a syngeneic murine model with a spectrum of mutational burdens. METHODS We used CRISPR-Cas9 to knockout the MMR gene Mlh1 in the murine glioma model SB28, an ICI-resistant cell line with a very low mutational burden (108 non-synonymous mutations). RESULTS Single-cell sorted Mlh1 knockout SB28 clones revealed a two- to six-fold increase in TMB. Importantly, loss of Mlh1 also brought about global changes in immune- and inflammatory-related gene expression in vitro, without correlation to increasing TMB. While all clones had the same rate of in vitro proliferation, flank injection of a high TMB vs a low TMB SB28 Mlh1 knockout clone resulted in similar survival benefits compared to Mlh1 wildtype SB28. CONCLUSIONS These preliminary results suggest that, although disruption of the MMR system may result in heightened tumor immunogenicity, elevated TMB may not be the sole significant determinant. We are currently investigating tumor proliferation after flank injection in immunocompromised mice to confirm that slowed growth is immune-related, as well as clone-specific response to ICI treatment. We are also exploring the role of specific cytokines/chemokines in conferring this survival advantage and potential immune response. By generating a GBM model of varied mutational burdens and immune profiles and comparing to the baseline SB28 cell line, we can interrogate the relative significance of TMB in tumor immunogenicity and ICI response.

IMMU-41. GBM PLATELET HYPERACTIVITY DRIVES THE IMMUNOSUPPRESSIVE TME IN A SEX-DEPENDENT MANNER

Abstract Cancer-related thrombosis is the second leading cause of death for cancer patients, including those with glioblastoma (GBM), the most common primary malignant brain tumor. Patients with GBM have a 30% risk of venous thromboembolism. Moreover, sex biases are well established in GBM, with men 1.6-fold more likely to develop GBM and exhibiting a poorer prognosis than females. GBM is extremely difficult to treat due to its highly immunosuppressive tumor microenvironment (TME), often infiltrated with suppressive myeloid populations and a reduction in T-cells intratumorally and systemically. Our group has recently demonstrated these sex-biases in survival are due to differences in the immune cell populations making up the TME. Despite these findings, there is limited information as to how a hyper-thrombotic state contribute to the immunosuppressive TME. Additionally, the role that sex biases play in these mechanisms has not been explored. Our previous work demonstrated that thrombin, a major platelet activator through PAR1 and PAR4 receptor signaling, is secreted from cancer stem cells into the tumor microenvironment. We now show that GBM patient have elevated levels of the thrombin-antithrombin complex and have increased PAR4 receptor mediated platelet activation. Similarly, we show that tumor bearing mice have elevated PAR4 receptor mediated platelet activation relative to non-tumor bearing mice. We further demonstrate pharmacologically targeting the thrombin-PAR4 signaling axis in murine GBM models prolongs survival in females but has not affect in males by preventing female platelet hyperactivity and subsequently increasing the number and function of tumor infiltrating CD8+ T-cells. These findings demonstrate how activated platelets interact and regulate immune cell populations in GBM. These results suggest that the hyper-thrombotic state seen in many GBM patients simultaneously contributes to the immunosuppressive TME. In addition, these results identify therapeutic strategies to leverage the platelet hyperactivity seen in GBM by hyperactive thrombin-PAR4 for sex-dependent therapeutic purposes.