Murine Glioma Model Research Articles

EXTH-29. DUAL TGFB AND PD1 BLOCKADE PROMOTES GERMINAL-CENTER B-CELL IMMUNE RESPONSES AGAINST GLIOBLASTOMA

Abstract In contrast to other malignancies such as melanoma and sarcoma, Glioblastoma (GBM) remains difficult to treat with immunotherapies. Recent studies have shown that positive immunotherapeutic responses are mediated by the accumulation of germinal-center-like B cells which are predictive of survival in patients treated with neoadjuvant PD1 blockade. In contrast, GBM-associated B-cells are scarce and the establishment of germinal-center like cells have not been observed. This study seeks to identify how B-cells are driven towards their immunosuppressive phenotypes in GBM and how this prevents immunotherapeutic efficacy. Utilizing single-cell RNA sequencing (scRNA-seq) in a CT2A murine glioma model, TGFb receptors 1 and 3 were identified as the most highly expressed inhibitory receptors on GBM-associated B cells. Furthermore, using scRNA-seq, TGFb1 was identified as the most highly expressed immunosuppressive cytokine in the TME, which was produced principally by tumor-associated myeloid cells (TAMCs). Inhibiting the myeloid compartment using intracranial anti-Gr1 antibody in combination with PD1 blockade resulted in B-cells exhibiting greater proliferation and differentiation into memory B-cells in addition to germinal-center-like B-cells. Further demonstrating B-cell functional reprogramming, autologous T cells isolated from spleens exhibited greater proliferation and robust anti-tumor cytotoxicity when cocultured with tumor-associated B-cells from the dual treatment group. Finally, inhibiting a5b8 integrin, a key complex in releasing active TGFb, increased tumor-infiltrating proliferating B-cells and conferred a long-term survival benefit in the CT2A murine model. Our results demonstrate that the immunosuppressive TME of GBM is influenced by the vital interplay between B-cells and the TME through TGFb signaling. This study highlights the potential therapeutic benefits of targeting the TGFb signaling pathway in conjunction with the current standard of care for GBM.

Large Animal Models of Glioma: Current Status and Future Prospects.

Enhanced understanding of the molecular features of glioma has led to an expansion of murine glioma models and successful preclinical studies. However, clinical trials continue to have a high cost, extended production time, and low proportion of success. Studies in large-animal models of various cancer types have emerged to bridge the translational gap between in vitro and in vivo animal studies and human clinical trials. The anatomy and physiology of large animals are of more direct relevance to human disease, allowing for more rigorous testing of treatments such as surgical resection and adjuvant therapy in glioma. The recent generation of multiple porcine glioma models supports their use in high-throughput preclinical studies. The demonstration of spontaneous glioblastoma formation in canines further provides a unique avenue for the study of de novo glioma. The aim of this review was to outline the current status of large animal models of glioma and their value as a transitional step between rodent models and human clinical trials.

Focused ultrasound-enhanced delivery of intranasally administered anti-programmed cell death-ligand 1 antibody to an intracranial murine glioma model

Immune checkpoint inhibitors have great potential for treating the gliomas; however, their therapeutic efficacy has been partially limited by their inability to cross the blood–brain barrier (BBB). The objective of this study was to evaluate the capability of focused-ultrasound-mediated intranasal brain drug delivery (FUSIN) in achieving the locally enhanced delivery of anti-programmed cell death-ligand 1 antibody (aPD-L1) to the brain. aPD-L1 was labeled with a near-infrared fluorescence dye (IRDye 800CW) and administered to mice through the nasal route to the brain, followed by focused ultrasound sonication in the presence of systemically injected microbubbles. FUSIN enhanced the accumulation of aPD-L1 at the FUS-targeted brainstem by an average of 4.03- and 3.74-fold compared with intranasal (IN) administration alone in the non-tumor mice and glioma mice, respectively. Immunohistochemistry staining found that aPD-L1 was mainly located within the perivascular spaces after IN delivery, while FUSIN further enhanced the penetration depth and delivery efficiency of aPD-L1 to the brain parenchyma. The delivered aPD-L1 was found to be colocalized with the tumor cells after FUSIN delivery to the brainstem glioma. These findings suggest that FUSIN is a promising technique to enhance the delivery of immune checkpoint inhibitors to gliomas.

P16.05 Implementation of a novel ex-vivo brain slice model to study human glioblastoma and glioma-associated microglia

Abstract BACKGROUND Glioblastoma multiforme is a highly malignant brain tumor with a devastating prognosis. Resection followed by radio-chemotherapy leads to an overall survival of only 15 months. Up to 40% of the tumor mass consist of tumor-associated microglia and macrophages (TAMs). These cells were shown to promote tumor growth and invasiveness in many murine glioma models. The interaction between TAMs and tumor cells is crucial for tumor progression and includes several known pathways. Still, murine glioma models only partially mirror the human tumor microenvironment. Several known genes, which are highly upregulated in human glioma and TAMs are only expressed in human tissue and not in mice. To further investigate some of these genes, we aimed at establishing a humanized ex-vivo brain slice model, in which human TAMs and human glioma cells can be studied in a standardized manner. MATERIAL AND METHODS We used 250 micrometer thick murine brain slices, which were depleted of intrinsic microglia by applying clodoronated liposomes. Next, we inoculated human glioma cells (originating from the cell lines mCherryU87, mCherryU251MG, mCherryLN229 and several patient derived cells lines) with or without human microglia derived from induced pluripotent stem cells (iPSCs). Slices were cultivated for 7 to 14 days. Next, we performed a detailed analysis of microglia morphology (sphericity, cell body volume, process length and branching pattern) and tumor volume. RESULTS Clodronation efficacy was high, depending on duration of treatment and length of cultivation. iPSCs and tumor cells integrated into the slice very well. The presence of tumor cells led to an increased sphericity of iPSC-dervied microglia and to an increased cell body volume. Branching pattern and process length did not differ between both conditions. Tumor volume was significantly larger when iPSC-derived microglia were present. This was found in various glioma cells lines and also in patient derived cells. CONCLUSION The newly established humanized ex-vivo brain slice system was shown to be feasible. The method successfully allows to study the interaction between human TAMs and tumor cells. Microglia foster tumor growth not only in murine glioma models, but also in a human paradigm. The humanized ex-vivo brain slice model therefore is the optimal basis to study the role human-specific genes in TAM-glioma interaction.

P16.09 Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

Abstract BACKGROUND Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most aggressive type of primary brain tumor. GBM has the poorest prognosis with no efficient cure to date due to diffusive growth into the brain, resistance to treatments and the immunosuppressive tumor microenvironment (TME). The growth and invasiveness of GBM is supported by the heterogeneous TME including local microglia and bone-marrow-derived macrophages (collectively known as glioma-associated microglia and macrophages, GAMs). In addition, tumor hypoxia is a key factor in the progression of GBM, as it can globally and rapidly alter gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can influence the pro-tumorigenic function of GAMs by inducing the expression of cytokines and cell surface receptors. However, little is known on the hypoxia-imposed chromatin changes of GAMs and GBM cells, which can in turn impact the interaction between these cell populations. Here we analyze these changes using a single-cell method, which preserves in situ hypoxia within the TME of GBM. MATERIAL AND METHODS Single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing) method in a GL261 murine glioma model was used to simultaneously assess genome-wide chromatin accessibility and expression of intracellular protein markers in single cells, enabling accurate selection of hypoxic and non-hypoxic tumor cells and GAMs. Pi-ATAC-seq is used on paraformaldehyde-perfused tumors and therefore allows capturing unaltered hypoxia-dependent cellular states, that often become distorted during dissociation and preparation of fresh material in most common single-cell methods. RESULTS We optimized Pi-ATAC method in a GL261 GBM mouse model, with specific sorting of GAMs using CD11b+ immunosorting followed by separation of microglia and macrophages, based on intensity of CD45 staining. HIF-1α induction and binding of pimonidazole were used to mark hypoxic populations. Currently, we are investigating the chromatin accessibility profiles of cancer cells and GAMs within the hypoxic tumor microenvironment of GBM. Exploring open chromatin profiles in GAMs and glioma-microglia co-cultures will allow to unravel the mechanisms of chromatin accessibility modulation in the oxygen-dependent manner. CONCLUSION In summary, we optimized the Pi-ATAC method in a mouse GBM model to characterize the chromatin openness changes in GAMs and cancer cells in response to hypoxic stress. Further validation of these results will provide the potential to identify novel markers for GAMs/glioma interactions in hypoxic GBMs and develop novel therapeutic targets.

Mass spectrometry imaging-based multi-modal technique: Next-generation of biochemical analysis strategy

Mass spectrometry imaging-based multi-modal technique: Next-generation of biochemical analysis strategy

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

Immune Microenvironment Landscape in CNS Tumors and Role in Responses to Immunotherapy.

Despite the important evolution of immunotherapeutic agents, brain tumors remain, in general, refractory to immune therapeutics. Recent discoveries have revealed that the glioma microenvironment includes a wide variety of immune cells in various states that play an important role in the process of tumorigenesis. Anti-tumor immune activity may be occurring or induced in immunogenic hot spots or at the invasive edge of central nervous system (CNS) tumors. Understanding the complex heterogeneity of the immune microenvironment in gliomas will likely be the key to unlocking the full potential of immunotherapeutic strategies. An essential consideration will be the induction of immunological effector responses in the setting of the numerous aspects of immunosuppression and evasion. As such, immune therapeutic combinations are a fundamental objective for clinical studies in gliomas. Through immune profiling conducted on immune competent murine models of glioma and ex vivo human glioma tissue, we will discuss how the frequency, distribution of immune cells within the microenvironment, and immune modulatory processes, may be therapeutically modulated to lead to clinical benefits.

Treatment of glioblastoma with current oHSV variants reveals differences in efficacy and immune cell recruitment

Oncolytic herpes simplex viruses (oHSVs) have demonstrated efficient lytic replication in human glioblastoma tumors using immunodeficient mouse models, but early-phase clinical trials have reported few complete responses. Potential reasons for the lack of efficacy are limited vector potency and the suppressive glioma tumor microenvironment (TME). Here we compare the oncolytic activity of two HSV-1 vectors, a KOS-strain derivative KG4:T124 and an F-strain derivative rQNestin34.5v.1, in the CT2A and GL261N4 murine syngeneic glioma models. rQNestin34.5v1 generally demonstrated a greater in vivo viral burden compared to KG4:T124. However, both vectors were rapidly cleared from CT2A tumors, while virus remained ensconced in GL261N4 tumors. Immunological evaluation revealed that the two vectors induced similar changes in immune cell recruitment to either tumor type at 2 days after infection. However, at 7 days after infection, the CT2A microenvironment displayed the phenotype of an untreated tumor, while GL261N4 tumors exhibited macrophage and CD4+/CD8+ T cell accumulation. Furthermore, the CT2A model was completely resistant to virus therapy, while in the GL261N4 model rQNestin34.5v1 treatment resulted in enhanced macrophage recruitment, impaired tumor progression, and long-term survival of a few animals. We conclude that prolonged intratumoral viral presence correlates with immune cell recruitment, and both are needed to enhance anti-tumor immunity.

CD137 and PD-L1 targeting with immunovirotherapy induces a potent and durable antitumor immune response in glioblastoma models

BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our...

Abstract 59: CAR T cell therapy reshapes the tumor microenvironment to promote host antitumor immune repsonses in glioblastoma

Abstract CAR T cell therapy is emerging as a promising strategy to treat cancer and may offer new therapeutic options for individuals diagnosed with glioblastoma (GBM) and other solid tumors. While early clinical studies evaluating CAR T cell therapy in GBM have established evidence of safety and bioactivity, objective clinical responses have been limited. It remains unclear whether productive CAR T cell therapy for solid tumors requires solely CAR T cell engagement with tumor antigen, or if it also necessitates the stimulation of a patient's endogenous immune response. Focusing on our preclinical and clinical program evaluating IL13Rα2-targeted CAR-T cells for the treatment of IL13Rα2-positive glioblastoma (GBM), we set out to mechanistically interrogate the interplay between CAR T cell therapy and the host tumor microenvironment. We designed a murine CAR T cell syngeneic platform in C57BL/6 immunocompetent mice and demonstrate that single intratumoral infusion of IL13Rα2-CAR T cells mediate potent antitumor activity against established KR158 tumors, a highly invasive and poorly immunogenic murine glioma model. We demonstrate that CAR T cell treatment of mouse syngeneic GBM alters the tumor immune landscape, activates intratumoral myeloid cells and induces endogenous T cell memory responses coupled with feed forward propagation of CAR T responses. IFNγ production by CAR T cells and IFNγ-responsiveness of host immune cells is critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these findings was explored in patient samples from our on-going IL13Rα2-CAR T cell phase I clinical trial [NCT02208362]. Consistent with our preclinical findings, we show that locoregional CAR T cell infusions result in spikes in inflammatory cytokines and an influx of endogenous immune cells into the cerebrospinal fluid (CSF) and resected tumor cavity. Single cell RNA-sequencing revealed unique genes upregulated in immune cells from blood and CSF samples after treatment. One patient of particular interest, who presented with recurrent multifocal GBM, remarkably achieved a complete response (CR) following locoregional delivery of IL13Rα2-CAR T cells, despite heterogeneous IL13Rα2 tumor expression (PMID: 28029927). In this responding patient, we now show the induction of endogenous tumor-specific T cell reactivity and T cell clones whose dynamics contracted with tumor volume following IL13Rα2-targeted CAR T therapy. These studies establish that CAR T cell therapy has the potential to re-shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity and emphasize the importance of the host innate and adaptive immunity in productive CAR T cell therapy of solid tumors. Citation Format: Christine E. Brown, Darya Alizadeh, Vanessa Jonsson, Jonathan Hibbard, Stephanie Yahn, Robyn A. Wong, Xin Yang, Rachel Ng, Natalie Dullerud, Madeleine Maker, Sharahreh Gholamin, Renate Starr, Nicholas Banovich, Stephen J. Forman, Behnam Badie. CAR T cell therapy reshapes the tumor microenvironment to promote host antitumor immune repsonses in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 59.

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

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

Abstract 3072: Clinically relevant MAPK pathway inhibition reverses stem cell fate defects and sensitize BRAF mutant glioma to immune modulatory therapies

Abstract Pediatric low-grade gliomas (pLGG) are the most common central nervous system (CNS) tumor in children. Although the majority of pLGG has unregulated MAPK pathway activity, and clinical MAPK pathway inhibitors are available, pLGG treatment remains challenging. Tumor progression, rebound, and therapy resistance limit the success of MAPK pathway inhibitors, especially in high risk subgroups, such as BRAF V600E mutant CDKN2A deleted gliomas.To define mechanisms for progression and overcome resistance, we generated BRAF V600E mutant, CDKN2A deleted murine glioma models of different grades and drug sensitivities. Here, we use genetically engineered mice to target expression of BRAF V600E and deletion of CDKN2A to various germinal and non-germinal areas, assessing neuro-anatomical aspects of oncogene sensitivity. Using neural stem cell (NSC)- and astrocyte-, and oligodendrocyte progenitor cell (OPC)-selective driver line, we investigate the likely origin for pLGG and therapy resistant glioma stem cells, developing models that faithfully recapitulate human tumor development. Our numerous immunocompetent, orthotopic BRAF V600E mutant glioma model derived from endogenous tumors exhibit a range of sensitivities to MAPK inhibitor therapies, allowing us to define mechanisms for therapy resistance. In syngeneic, orthotopic BRAF V600E mutant CDKN2A deleted gliomas, we define MAPK pathway inhibitor-induced-changes in the tumor microenvironment, including the immune infiltrate, and investigate immune modulatory therapy as novel treatment strategy for children with BRAF mutant gliomas. Our numerous models for BRAF mutant glioma will facilitate research on tumor development, progression and the tumor-immune interactions. Collectively, our results describe genetic alteration- and microenvironment-based mechanisms for BRAF mutant tumor initiation and progression, and suggest therapeutic combination therapies for glioma. Citation Format: Claudia K. Petritsch, Stefan Grossauer, Wei Wang, Anne Marie Barrette, Jongwhi Park. Clinically relevant MAPK pathway inhibition reverses stem cell fate defects and sensitize BRAF mutant glioma to immune modulatory therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3072.

Lack of GD3 synthase (St8sia1) attenuates malignant properties of gliomas in genetically engineered mouse model.

High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet‐derived growth factor B‐driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma‐bearing mice and low‐grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S‐deficient gliomas. Re‐expression of GD3S restored expression of MMP9 in primary‐cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α‐MMP9 axis. This is the first report to describe the tumor‐enhancing functions of GD3S in vivo.

Modeling of Glioma Growth With Mass Effect by Longitudinal Magnetic Resonance Imaging.

It is well-known that expanding glioblastomas typically induce significant deformations of the surrounding parenchyma (i.e., the so-called "mass effect"). In this study, we evaluate the performance of three mathematical models of tumor growth: 1) a reaction-diffusion-advection model which accounts for mass effect (RDAM), 2) a reaction-diffusion model with mass effect that is consistent only in the case of small deformations (RDM), and 3) a reaction-diffusion model that does not include the mass effect (RD). The models were calibrated with magnetic resonance imaging (MRI) data obtained during tumor development in a murine model of glioma (n = 9). We obtained T2-weighted and contrast-enhanced T1-weighted MRI at 6 time points over 10 days to determine the spatiotemporal variation in the mass effect and the volume fraction of tumor cells, respectively. We calibrated the three models using data 1) at the first four, 2) only at the first and fourth, and 3) only at the third and fourth time points. Each of these calibrations were run forward in time to predict the volume fraction of tumor cells at the conclusion of the experiment. The diffusion coefficient for the RDAM model (median of 10.65 × 10 -3 mm 2· d -1) is significantly less than those for the RD and RDM models (17.46 × 10 -3 mm 2· d -1 and 19.38 × 10 -3 mm 2· d -1, respectively). The error in the tumor volume fraction for the RD, RDM, and RDAM models have medians of 40.2%, 32.1%, and 44.7%, respectively, for the calibration using data from the first four time points. The RDM model most accurately predicts tumor growth, while the RDAM model presents the least variation in its estimates of the diffusion coefficient and proliferation rate. This study demonstrates that the mathematical models capture both tumor development and mass effect observed in experiments.

LGG-01. NF1 MUTATION DRIVES NEURONAL ACTIVITY-DEPENDENT OPTIC GLIOMA INITIATION

Neurons have recently emerged as essential cellular constituents of the tumor microenvironment, where their activity increases the growth of a diverse number of solid tumors. While the role of neurons in tumor progression has been previously demonstrated, the importance of neuronal activity to tumor initiation is less clear, particularly in the setting of cancer predisposition syndromes. In the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome, in which tumors arise in close association with nerves, 15% of individuals develop low-grade neoplasms of the optic pathway (optic gliomas) during early childhood, raising the intriguing possibility that postnatal light-induced optic nerve activity drives tumor initiation. Here, we employ an authenticated murine model of Nf1 optic glioma to demonstrate that stimulation of optic nerve activity increases optic glioma growth, while decreasing optic nerve activity via light deprivation prevents tumor formation and maintenance. By manipulating environmental light to modulate optic pathway (retinal) neuron activity, we show that Nf1 optic glioma initiation depends on neuronal activity during a developmental period susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly high optic nerve neuroligin-3 (Nlgn3) shedding in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacologic inhibition of Nlgn3 shedding blocks murine Nf1 optic gliomagenesis and progression. Collectively, these studies establish an obligate role for neuronal activity in the development of certain brain tumors, elucidate a therapeutic strategy to reduce optic glioma incidence or mitigate tumor progression, and underscore the role of Nf1 mutation-mediated dysregulation of neuronal signaling pathways in the NF1 cancer predisposition syndrome.

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

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

RNA sequence analysis reveals ITGAL/CD11A as a stromal regulator of murine low-grade glioma growth.

Emerging insights from numerous laboratories have revealed important roles for nonneoplastic cells in the development and progression of brain tumors. One of these nonneoplastic cellular constituents, glioma-associated microglia (GAM), represents a unique population of brain monocytes within the tumor microenvironment that have been reported to both promote and inhibit glioma proliferation. To elucidate the role of GAM in the setting of low-grade glioma (LGG), we leveraged RNA sequencing meta-analysis, genetically engineered mouse strains, and human biospecimens. Publicly available disease-associated microglia (DAM) RNA-seq datasets were used, followed by immunohistochemistry and RNAScope validation. CD11a-deficient mouse microglia were used for in vitro functional studies, while LGG growth in mice was assessed using anti-CD11a neutralizing antibody treatment of Neurofibromatosis type 1 (Nf1) optic glioma mice in vivo. We identified Itgal/CD11a enrichment in GAM relative to other DAM populations, which was confirmed in several independently generated murine models of Nf1 optic glioma. Moreover, ITGAL/CD11A expression was similarly increased in human LGG (pilocytic astrocytoma) specimens from several different datasets, specifically in microglia from these tumors. Using CD11a-knockout mice, CD11a expression was shown to be critical for murine microglia CX3CL1 receptor (Cx3cr1) expression and CX3CL1-directed motility, as well as glioma mitogen (Ccl5) production. Consistent with an instructive role for CD11a+ microglia in stromal control of LGG growth, antibody-mediated CD11a inhibition reduced mouse Nf1 LGG growth in vivo. Collectively, these findings establish ITGAL/CD11A as a critical microglia regulator of LGG biology relevant to future stroma-targeted brain tumor treatment strategies.

A phase I/II study to evaluate the safety and efficacy of a novel long-acting interleukin-7, NT-I7, for patients with newly diagnosed high-grade gliomas after chemoradiotherapy: The interim result of the phase I data.

2040 Background: High-grade gliomas (HGG) patients can develop prolonged lymphopenia after standard radiation therapy (RT) and temozolomide (TMZ), which has been shown to correlate with worse survival. Interleukin-7 (IL-7) level, a cytokine that stimulates T-cell homeostasis and proliferation, is disproportionally low in HGG patients with lymphopenia. NT-I7 (efineptakin alfa) is the first-in-class long-acting recombinant human IL-7 that supports proliferation and survival of CD4+ and CD8+ T-cells in humans and mice. Our previous study demonstrated that NT-I7 could correct lymphopenia and improve the survival of orthotopic murine glioma models. The current study aims to examine the safety of administering NT-17 after chemoradiotherapy to HGG patients and its effect on systemic absolute lymphocyte count (ALC). Methods: All patients with newly diagnosed HGG who have completed concurrent RT/TMZ were considered eligible, regardless of ALC. NT-I7 was initially administered intramuscularly within 1 week after completion of RT/TMZ and then every 12 weeks for up to 4 doses. Patients also received adjuvant TMZ 4 weeks after RT/TMZ. The phase I study tested 6 dose levels of NT-I7, including 60, 120, 240, 540, 720, and 960 mcg/kg, adopting an accelerated phase for the first two doses followed by the standard 3+3 design. The primary endpoint was the safety of NT-I7 in HGG. The Phase II study is a double-blinded randomized study with 10 patients per arm to evaluate the effect of NT-I7 on ALC compared to placebo controls. Blood samples at baseline and during the NT-I7 administrations will be collected for immune profiling by CyTOF, single-cell RNA-sequencing, and cytokine analysis. Results: Phase I was completed with 19 patients (2 anaplastic oligodendrogliomas and 17 glioblastomas), with a median age of 58 years (range: 25-78). Median baseline ALC was 1000 cells/mm3 before NT-I7 administration, and the median baseline dexamethasone use was 0 mg/day (range 0-12). The median number of NT-I7 doses given was 2 (range: 2-4). Treatment-related adverse events (TRAEs) were dose-dependent. The most common TRAEs were grade 1/2 injection site reactions (50%), flu-like symptoms (26%), rash (21%), and fatigue (21%). Two patients had dose-limiting toxicities at 960 mcg/kg (a grade 3 elevated alanine aminotransferase and a grade 3 muscle pain). ALC was increased in a dose-dependent manner with a range of 1.3 – 4.1 fold at week 4 after NT-I7 injection and lasted up to 12 weeks. Thus, 720 mcg/kg was identified as the recommended phase II dose (RP2D). Conclusions: NT-I7 is well tolerated for HGG patients after chemoradiotherapy and has a RP2D of 720 mcg/kg. Immune profiling and cytokine analysis are ongoing and will be updated. The Phase II randomized study to evaluate the effect of NT-I7 vs placebo on ALC and survival is ongoing. Clinical trial information: NCT03687957.

High grade gliomas impact cell fate differentiation of hematopoietic stem and progenitor cells in the bone marrow

Abstract Glioblastoma (GBM) remains a deadly disease with an overall survival of 18 months. Despite advances in cancer immunotherapy, especially in the context of solid tumors derived outside of the central nervous system, GBM remains difficult to treat. This may be due, in part, to reduced T cell function and expansion of suppressive myeloid cells within the periphery. Here we sought to determine if GBM impacts the phenotype and gene expression of progenitor populations within the bone marrow where hematopoietic stem and progenitor cells (HSPCs) originate. Using RNAseq, we found HSPCs derived from intracranial glioma-bearing mice possess altered gene expression relative to HSPCs derived from non-tumor-bearing mice. In addition, we found glioma-bearing mice possess an expansion of myeloid-derived suppressor cells (MDSCs) within their bone marrow and are significantly more suppressive on T cell proliferation and T cell-mediated tumor cell killing than MDSCs isolated from non-tumor-bearing mice. We also determined HSPCs derived from glioma-bearing mice are more likely to differentiate into MDSCs than HSPCs derived from non-tumor-bearing mice. Interestingly, we found components of immunotherapy are capable of redirecting cell fate differentiation of glioma-bearing HSPCs. We determined HSPCs cultured in T cell supernatants are capable redirecting their differentiation from suppressive MDSCs towards stimulatory DCs. Finally, using two murine glioma models, we found adoptive cellular therapy using HSPCs derived from glioma-bearing mice is capable of providing a similar survival benefit as adoptive cellular therapy using HSPCs derived from non-tumor-bearing mice.