Murine Glioblastoma Research Articles

An oncolytic HSV-1 vector induces a therapeutic adaptive immune response against glioblastoma

BackgroundGlioblastoma (GBM) is the most frequent and aggressive brain tumor in adults with the lowest survival rates five years post-diagnosis. Oncolytic viruses (OVs) selectively target and damage cancer cells, and for this reason they are being investigated as new therapeutic tools also against GBM.MethodsAn oncolytic herpes simplex virus type 1 (oHSV-1) with deletions in the γ34.5 neurovirulence gene and the US12 gene, expressing enhanced green fluorescent protein (EGFP-oHSV-1) as reporter gene was generated and tested for its capacity to infect and kill the murine GL261 glioblastoma (GBM) cell line. Syngeneic mice were orthotopically injected with GL261cells. Seven days post-implantation, EGFP-oHSV-1 was administered intratumorally. Twenty-one days after parental tumor challenge in the opposite brain hemisphere, mice were sacrified and their brains were analysed by immunohistochemistry to assess tumor presence and cell infiltrate.ResultsoHSV-1 replicates and induces cell death of GL261 cells in vitro. A single intracranial injection of EGFP-oHSV-1 in established GL261 tumors significantly prolongs survival in all treated mice compared to placebo treatment. Notably, 45% of treated mice became long-term survivors, and rejected GL261 cells upon rechallenge in the contralateral brain hemisphere, indicating an anamnestic antitumoral immune response. Post-mortem analysis revealed a profound modification of the tumor microenvironment with increased infiltration of CD4 + and CD8 + T lymphocytes, intertumoral vascular collapse and activation and redistribution of macrophage, microglia, and astroglia in the tumor area, with the formation of intense fibrotic tissue suggestive of complete rejection in long-term survivor mice.ConclusionsEGFP-oHSV1 demonstrates potent antitumoral activity in an immunocompetent GBM model as a monotherapy, resulting from direct cell killing combined with the stimulation of a protective adaptive immune response. These results open the way to possible application of our strategy in clinical setting.

An ILK/STAT3 pathway controls glioblastoma stem cell plasticity.

Glioblastoma (GBM) is driven by malignant neural stem-like cells that display extensive heterogeneity and phenotypic plasticity, which drive tumor progression and therapeutic resistance. Here, we show that the extracellular matrix-cell adhesion protein integrin-linked kinase (ILK) stimulates phenotypic plasticity and mesenchymal-like, invasive behavior in a murine GBM stem cell model. ILK is required for the interconversion of GBM stem cells between malignancy-associated glial-like states, and its loss produces cells that are unresponsive to multiple cell state transition cues. We further show that an ILK/STAT3 signaling pathway controls the plasticity that enables transition of GBM stem cells to an astrocyte-like state invitro and invivo. Finally, we find that ILK expression correlates with expression of STAT3-regulated proteins and protein signatures describing astrocyte-like and mesenchymal states in patient tumors. This work identifies ILK as a pivotal regulator of multiple malignancy-associated GBM phenotypes, including phenotypic plasticity and mesenchymal state.

Desert Hedgehog Down-regulation Mediates Inhibition of Proliferation by γ-Glutamylcyclotransferase Knockdown in Murine Glioblastoma Stem Cells.

Glioblastoma is the most frequent type of adult-onset malignant brain tumor and has a very poor prognosis. Glioblastoma stem cells have been shown to be one of the mechanisms by which glioblastoma acquires therapy resistance. Therefore, there is a need to establish novel therapeutic strategies useful for inhibiting this cell population. γ-Glutamylcyclotransferase (GGCT) is an enzyme involved in the synthesis and metabolism of glutathione, which is highly expressed in a wide range of cancer types, including glioblastoma, and inhibition of its expression has been reported to have antitumor effects on various cancer types. The aim of this study was to clarify the function of GGCT in glioblastoma stem cells. We searched for pathways affected by GGCT overexpression in mouse embryonic fibroblasts NIH-3T3 by comprehensive gene expression analysis. Knockdown of GGCT and overexpression of desert hedgehog (DHH), a representative ligand of the pathway, were performed in glioblastoma stem cells derived from a mouse glioblastoma model. GGCT overexpression activated the hedgehog pathway. Knockdown of GGCT inhibited proliferation of glioblastoma stem cells and reduced expression of DHH and the downstream target GLI family zinc finger 1 (GLI1). DHH overexpression significantly restored the growth-suppressive effect of GGCT knockdown. High GGCT expression is important for expression of DHH and activation of the hedgehog pathway, which is required to maintain glioblastoma stem cell proliferation. Therefore, inhibition of GGCT function may be useful in suppressing stemness of glioblastoma stem cells accompanied by activation of the hedgehog pathway.

Single-cell dissection of the human blood-brain barrier and glioma blood-tumor barrier

The blood-brain barrier (BBB) serves as a crucial vascular specialization, shielding and nourishing brain neurons and glia while impeding drug delivery. Here, we conducted single-cell mRNA sequencing of human cerebrovascular cells from 13 surgically resected glioma samples and adjacent normal brain tissue. The transcriptomes of 103,230 cells were mapped, including 57,324 endothelial cells (ECs) and 27,703 mural cells (MCs). Both EC and MC transcriptomes originating from lower-grade glioma were indistinguishable from those of normal brain tissue, whereas transcriptomes from glioblastoma (GBM) displayed a range of abnormalities. Among these, we identified LOXL2-dependent collagen modification as a common GBM-dependent trait and demonstrated that inhibiting LOXL2 enhanced chemotherapy efficacy in both murine and human patient-derived xenograft (PDX) GBM models. Our comprehensive single-cell RNA sequencing-based molecular atlas of the human BBB, coupled with insights into its perturbations in GBM, holds promise for guiding future investigations into brain health, pathology, and therapeutic strategies.

Machine Learning-Directed Conversion of Glioblastoma Cells to Dendritic Cell-Like Antigen-Presenting Cells as Cancer Immunotherapy.

Immunotherapy has limited efficacy in glioblastoma (GBM) due to the blood-brain barrier and the immunosuppressed or "cold" tumor microenvironment (TME) of GBM, which is dominated by immune-inhibitory cells and depleted of CTL and dendritic cells (DC). Here, we report the development and application of a machine learning precision method to identify cell fate determinants (CFD) that specifically reprogram GBM cells into induced antigen-presenting cells with DC-like functions (iDC-APC). In murine GBM models, iDC-APCs acquired DC-like morphology, regulatory gene expression profile, and functions comparable to natural DCs. Among these acquired functions were phagocytosis, direct presentation of endogenous antigens, and cross-presentation of exogenous antigens. The latter endowed the iDC-APCs with the ability to prime naïve CD8+ CTLs, a hallmark DC function critical for antitumor immunity. Intratumor iDC-APCs reduced tumor growth and improved survival only in immunocompetent animals, which coincided with extensive infiltration of CD4+ T cells and activated CD8+ CTLs in the TME. The reactivated TME synergized with an intratumor soluble PD1 decoy immunotherapy and a DC-based GBM vaccine, resulting in robust killing of highly resistant GBM cells by tumor-specific CD8+ CTLs and significantly extended survival. Lastly, we defined a unique CFD combination specifically for the human GBM to iDC-APC conversion of both glioma stem-like cells and non-stem-like cell GBM cells, confirming the clinical utility of a computationally directed, tumor-specific conversion immunotherapy for GBM and potentially other solid tumors.

Astrocytes and the tumor microenvironment inflammatory state dictate the killing of glioblastoma cells by Smac mimetic compounds

Smac mimetic compounds (SMCs) are small molecule drugs that sensitize cancer cells to TNF-α-induced cell death and have multiple immunostimulatory effects through alterations in NF-κB signaling. The combination of SMCs with immunotherapies has been reported to result in durable cures of up to 40% in syngeneic, orthotopic murine glioblastoma (GBM) models. Herein, we find that SMC resistance is not due to a cell-intrinsic mechanism of resistance. We thus evaluated the contribution of GBM and brain stromal components to identify parameters leading to SMC efficacy and resistance. The common physiological features of GBM tumors, such as hypoxia, hyaluronic acid, and glucose deprivation were found not to play a significant role in SMC efficacy. SMCs induced the death of microglia and macrophages, which are the major immune infiltrates in the tumor microenvironment. This death of microglia and macrophages then enhances the ability of SMCs to induce GBM cell death. Conversely, astrocytes promoted GBM cell growth and abrogated the ability of SMCs to induce death of GBM cells. The astrocyte-mediated resistance can be overcome in the presence of exogenous TNF-α. Overall, our results highlight that SMCs can induce death of microglia and macrophages, which then provides a source of death ligands for GBM cells, and that the targeting of astrocytes is a potential mechanism for overcoming SMC resistance for the treatment of GBM.

The role of progranulin in macrophages of a glioblastoma model.

Glioblastoma (GBM), characterized by astrocytic tumorigenesis, remains one of the most prognostically challenging tumor types. Targeting entire GBM microenvironment using novel therapeutic factors is currently desired investigation approach. In this study, we focused on progranulin (PGRN), a regulator of diverse cellular functions. Recent studies implicated PGRN in the poor prognostics of GBM patients. However, the specific role of PGRN in the GBM microenvironment remains elusive. We utilized public databases of GBM patient and previous single-cell RNA sequence to examine association between PGRN expression and patient survival/grade, and expression levels of PGRN in each cell constituting the tumor microenvironment. To clarify the role of PGRN in Tumor-associated macrophage (TAM), we examined cell proliferation and expression of some proteins in murine GBM cells when cell supernatants derived from TAM of PGRN knockout (Grn-/-) or wild type mice were treated with murine GBM cells. Our results reveal significant PGRN expression in macrophages within the GBM environment, suggesting an association between increased PGRN expression in macrophages and tumor malignancy. TAM induction led to PGRN expression enhancement. Treatment with Grn-/- mouse -derived bone marrow-derived macrophage (BMDM) supernatant resulted in diminished GBM cell proliferation and cell cycle- and mesenchymal GBM subtype-associated reduced protein expression. Furthermore, the Grn-/- mouse-derived BMDM supernatant treatment reduced the phosphorylated STAT3 expression in GBM cells, while the expression of IL-6 and IL-10, known STAT3 pathway activators, diminished in Grn-/- mouse-derived BMDMs. Our results suggest that macrophage-derived PGRN is pivotal for fostering malignant transformations within the tumor microenvironment.

Immunomodulatory R848-Loaded Anti-PD-L1-Conjugated Reduced Graphene Oxide Quantum Dots for Photothermal Immunotherapy of Glioblastoma.

Glioblastoma multiforme (GBM) is the most severe form of brain cancer and presents unique challenges to developing novel treatments due to its immunosuppressive milieu where receptors like programmed death ligand 1 (PD-L1) are frequently elevated to prevent an effective anti-tumor immune response. To potentially shift the GBM environment from being immunosuppressive to immune-enhancing, we engineered a novel nanovehicle from reduced graphene oxide quantum dot (rGOQD), which are loaded with the immunomodulatory drug resiquimod (R848) and conjugated with an anti-PD-L1 antibody (aPD-L1). The immunomodulatory rGOQD/R8/aPDL1 nanoparticles can actively target the PD-L1 on the surface of ALTS1C1 murine glioblastoma cells and release R848 to enhance the T-cell-driven anti-tumor response. From in vitro experiments, the PD-L1-mediated intracellular uptake and the rGOQD-induced photothermal response after irradiation with near-infrared laser light led to the death of cancer cells and the release of damage-associated molecular patterns (DAMPs). The combinational effect of R848 and released DAMPs synergistically produces antigens to activate dendritic cells, which can prime T lymphocytes to infiltrate the tumor in vivo. As a result, T cells effectively target and attack the PD-L1-suppressed glioma cells and foster a robust photothermal therapy elicited anti-tumor immune response from a syngeneic mouse model of GBM with subcutaneously implanted ALTS1C1 cells.

IRF8-driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma.

Glioblastoma (GBM) has a highly immunosuppressive tumor immune microenvironment (TIME), largely mediated by myeloid-derived suppressor cells (MDSCs). Here, we utilized a retroviral replicating vector (RRV) to deliver Interferon Regulatory Factor 8 (IRF8), a master regulator of type 1 conventional dendritic cell (cDC1) development, in a syngeneic murine GBM model. We hypothesized that RRV-mediated delivery of IRF8 could "reprogram" intratumoral MDSCs into antigen-presenting cells (APCs) and thereby restore T-cell responses. Effects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. We assayed functional immunosuppression and antigen presentation by ex vivo T-cell-myeloid co-culture. Intratumoral injection of RRV-IRF8 in mice bearing intracerebral SB28 glioma significantly suppressed the tumor growth and prolonged survival. RRV-IRF8 treated tumors exhibited significant enrichment of cDC1s and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Arg1 and IDO1 and demonstrated reduced suppression of naïve T-cell proliferation in ex vivo co-culture, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDC1- and CD8 T-cell-enriched TIME. Our results indicate that reprogramming of glioma-infiltrating myeloid cells by in vivo expression of IRF8 may reduce immunosuppression and enhance antigen presentation, achieving improved tumor control.

Endocannabinoid Receptor 2 Function is Associated with Tumor-Associated Macrophage Accumulation and Increases in T Cell Number to Initiate a Potent Antitumor Response in a Syngeneic Murine Model of Glioblastoma.

Introduction: Glioblastoma patients have a highly immunosuppressive tumor microenvironment and systemic immunosuppression that comprise a major barrier to immune checkpoint therapy. Based on the production of endocannabinoids by glioblastomas, we explored involvement of endocannabinoid receptor 2 (CB2R), encoded by the CNR2 gene, which is predominantly expressed by immune cells, in glioblastoma-related immunosuppression. Materials & Methods: Bioinformatics of human glioblastoma databases was used to correlate enzymes involved in the synthesis and degradation of endocannabinoids, as well as CB2Rs, with patient overall survival. Intrastriatal administration of luciferase-expressing, murine GL261 glioblastoma cells was used to establish in in vivo glioblastoma model for characterization of tumor growth and intratumoral immune cell infiltration, as well as provide immune cells for in vitro co-culture experiments. Involvement of CB2Rs was determined by treatment with CB2R agonist (GW405833) or CB2R antagonist (AM630). ELISA, FACS, and immunocytochemistry were used to determine perforin, granzyme B, and surface marker levels. Results: Bioinformatics of human glioblastoma databases showed high expression of CB2R and elevated endocannabinoid production correlated with poorer prognosis, and involved immune-associated pathways. AM630treatment of GL261 glioblastoma-bearing mice induced a potent antitumor response, with survival plateauing at 50% on Day 40, when all control mice (median survival 28 days) and mice treated with GW405833 (median survival 21 days) had died. Luciferase tumor imaging revealed accelerated tumor growth by GW405833 treatment, but stable or regressing tumors in AM630-treated mice. Notably, in spleens, AM630 treatment caused an 83% decrease in monocytes/macrophages, and 1.8- and 1.6-fold increases in CD8+ and CD4+ cells, respectively. Within tumors, there was a corresponding decrease in tumor-associated macrophages (TAMs) and increase in CD8+ T cells. In vitro, lymphocytes from AM630-treated mice showed greater cytotoxic function (increased percentage of perforin- and granzyme B-positive CD8+ T cells). Discussion: These results suggest that inhibition of CB2R enhances both immunosuppressive TAM infiltration and systemic T-cell suppression through CB2R activation, and that inhibition of CB2Rs can potently counter both the immunosuppressive tumor microenvironment, as well as systemic immunosuppression in glioblastoma.

IMMU-22. B7-H3 NANOBODY CAR-T AND SOLUBLE TREM2 GENETICALLY ENGINEERED MYELOID CELL THERAPY IN MURINE SYNGENEIC GLIOBLASTOMA

Abstract BACKGROUND Myeloid immunosuppression can be a major impediment to CAR-T cell therapy for CNS tumors. Importantly, CAR-T cells are often evaluated in immunocompromised mice which cannot accurately recapitulate the tumor immune response or tumor microenvironment. CNS tumor macrophages express TREM2, an important driver of immunosuppression. This study combines B7-H3 targeting nanobody CAR-T cells which are cross reactive in humans and mice (currently being evaluated for clinical development) with genetically engineered myeloid cells (GEMys) expressing soluble TREM2 (sTREM2) in a syngeneic mouse model of glioblastoma (GBM), with the aim of blocking TREM2-mediated immunosuppression to enhance CAR-T cell function. METHODS Syngeneic murine GBM, designed to mimic human GBM and generated at the Center for Advanced Preclinical Research (CAPR), was used for all studies. B7-H3 nanobody CAR-T cells were generated from mouse splenic T cells. In vitro tumor killing assays were performed by xCELLigence. sTREM2 GEMys were generated from bone marrow progenitor cells using lentiviral transduction. For in vivo studies, mice underwent orthotopic implantation of tumors and placement of an MRI-compatible, permanent catheter-port system for serial intracerebroventricular (ICV) administration of cell therapies. Mice were treated with CAR-T and/or GEMy therapies, evaluated weekly for tumor burden by MRI, and were monitored for survival. RESULTS Syngeneic GBM expression of B7-H3 was confirmed by flow cytometry, immunofluorescence, and western blot. B7-H3 nanobody CAR-T cells demonstrated effective killing of GBM by xCELLigence. sTREM2 GEMys were successfully generated and confirmed to produce sTREM2. Cell therapies were successfully infused ICV via the implanted catheter-port system, demonstrating safety and feasibility of this model for serial ICV cellular therapy. CONCLUSIONS B7-H3 nanobody CAR-T cells were validated against syngeneic GBM, and we have demonstrated safety and feasibility of ICV catheter administration of cellular therapies in vivo. Future studies will continue to evaluate in vivo timing and efficacy of CAR-T and GEMy therapy.

KR158 Spheres Harboring Slow-Cycling Cells Recapitulate High-Grade Glioma Features in an Immunocompetent System.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

Single cell RNA sequencing analysis of tumor infiltrating γδ T cells in murine and human glioblastoma.

Abstract Glioblastoma multiforme (GBM) is the most commonly occurring malignant brain tumor. The anatomical location and the immunosuppressive TME have brought disappointing results for multiple therapeutics approaches, including immune checkpoint inhibitors (ICIs). γδ T cells are an attractive target for adoptive cell therapies (ACTs) due to their MHCI-independent tumor killing mechanism and GVHD-Free nature. Thus, investigating GBM infiltrating γδ T cells can provide insight into the pro-tumoral and anti-tumoral roles of γδ T cells and provide potential targets for gene engineering prior to ACT. Our Single-cell RNA sequencing (scRNA-seq) and flow cytometric data demonstrate that GBM harbors subtypes of γδ T cells of different origins and functions. γδ T cells from the meninges produce IL-17A and are enriched for genes related to Rorγt programme and immune checkpoint molecules. On the contrary, blood-borne γδ T cells produce IFN-γ and express genes related to cytotoxicity, co-stimulation and activating NK receptors. Functional dichotomy of γδ T cell is also observed in human GBM, with Areg producing tumor-promoting subset and Gzmb producing cytotoxic subset. Interaction analysis reveals that these subtypes of γδ T cell strongly interact with myeloid cells through unique receptor-ligand interaction. These finding reveal the comprehensive role of γδ T cells during cancer progression and provide insights into potential gene-editing targets for effective ACTs.

Linagliptin decreased the tumor progression on glioblastoma model

PurposeDipeptidyl peptidase-4 (DPP-4) inhibitors are oral hypoglycemic drugs and are used for type II diabetes. Previous studies showed that DPP-4 expression is observed in several tumor types and DPP-4 inhibitors suppress the tumor progression on murine tumor models. In this study, we evaluated the role of DPP-4 and the antitumor effect of a DPP-4 inhibitor, linagliptin, on glioblastoma (GBM). MethodsWe analyzed DPP-4 expression in glioma patients by the public database. We also analyzed DPP-4 expression in GBM cells and the murine GBM model. Then, we evaluated the cell viability, cell proliferation, cell migration, and expression of some proteins on GBM cells with linagliptin. Furthermore, we evaluated the antitumor effect of linagliptin in the murine GBM model. ResultsThe upregulation of DPP-4 expression were observed in human GBM tissue and murine GBM model. In addition, DPP-4 expression levels were found to positively correlate with the grade of glioma patients. Linagliptin suppressed cell viability, cell proliferation, and cell migration in GBM cells. Linagliptin changed the expression of phosphorylated NF-kB, cell cycle, and cell adhesion-related proteins. Furthermore, oral administration of linagliptin decreases the tumor progression in the murine GBM model. ConclusionInhibition of DPP-4 by linagliptin showed the antitumor effect on GBM cells and the murine GBM model. The antitumor effects of linagliptin is suggested to be based on the changes in the expression of several proteins related to cell cycle and cell adhesion via the regulation of phosphorylated NF-kB. This study suggested that DPP-4 inhibitors could be a new therapeutic strategy for GBM.

1241 Sustained Localized Delivery of Anti-PD-1 to Tumor Draining Lymph Nodes Increases Survival and Reverses Immunosuppression in Murine Glioblastoma Models

INTRODUCTION: Despite the advent of immunotherapy as a promising therapeutic, glioblastoma (GBM) remains resistant to using checkpoint blockade against programmed cell death protein 1 (PD-1) on T cells. The highly immunosuppressive tumor milieu of GBM prevents rescue of inactivated T cells due to decreased numbers of infiltrating T cells at the tumor site as well as increased recruitment of myeloid cells. Moreover, the current strategy of using antibodies against PD-1 (anti-PD-1) requires multiple intravenous infusions every two or three weeks with debilitating systemic effects. METHODS: Mice orthotopically implanted with GL261 glioma cells were injected with PCL:PEG:PCL hydrogel polymers loaded with anti-PD-1 in one of the following locations: cervical lymph nodes, inguinal lymph nodes, and the tumor site. Mice treated systemically with anti-PD-1 were used as comparative controls. Kaplan-Meier curves were generated for all arms, with subsequent ex vivo flow cytometric staining for CD4, CD8, IFN-y, TNF-a, Foxp3, and PD-1. RESULTS: Mice implanted with PCL:PEG:PCL hydrogels carrying anti-PD-1 at the site of their lymph nodes showed significantly improved survival outcomes compared to mice systemically treated with three doses of anti-PD-1 (p < 0.001). Flow cytometric analysis of lymph nodes and brain tissue in mice injected with this gel demonstrated increased levels of IFN-y, indicating greater reversal of immunosuppression compared to standard treatment. CONCLUSIONS: Our data demonstrates proof of principle of the advantages of using localized therapy that targets lymph nodes for GBM. We propose a paradigm shift for developing new sustained local treatments with immunotherapy that are able to eliminate the need for multiple systemic infusions and their off-target effects.

Abstract 1417: IRF8 driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma

Abstract Glioblastoma (GBM) has an extremely immunosuppressive microenvironment, primarily mediated by the recruitment of immature myeloid cells (IMCs) to the tumor. IMCs suppress T cell function and dampen the anti-tumor response. In this study, we sought to “reprogram” IMCs into antigen-presenting cells (APCs) to enhance anti-tumor T cell responses. To induce IMC differentiation into APCs, we utilized a retroviral replicating vector (RRV) expressing IRF8, a master regulator of type 1 conventional dendritic cell (cDC1) development. We evaluated the impact of IRF8 RRV on in vivo tumor growth kinetics, survival, immunosuppression, and immune microenvironment. Because RRV transduction requires the proliferation of target cells, we assessed the proliferative capacity of myeloid cells in murine and human GBM samples. We utilized the SB28 cell line, a poorly immunogenic murine GBM model. We pre-transduced SB28 cells with IRF8 RRV and implanted them intracranially into syngeneic C57BL/6 mice; controls expressed an empty vector. Intratumoral myeloid cells were proliferative in both murine and human samples. In vivo, tumor and myeloid cells are infected proportionately to their respective levels of proliferation. Mice with IRF8 RRV pre-transduced intracranial tumors had significantly longer survival (median 28 days) compared to controls (median 19 days) (p <0.0001). Additionally, tumor growth was delayed in IRF8 RRV mice. IRF8 RRV mice had significant enrichment of dendritic cells (p = 0.0145), cDC1s (p < 0.0001), T cells (p = 0.0025) and CD8+ T cells (p < 0.0001). Both monocytic and granulocytic IMCs had decreased expression of the immunosuppressive markers Arginase 1 and IDO1. Further, ex vivo co-culture of intra-tumoral myeloid cells with naïve T cells showed a functional decrease in immunosuppression in IRF8 RRV tumors compared to controls (p < 0.0001). To study the mechanism of action, we assessed whether IRF8 transduction of tumor cells alone or both tumor and immune cells, was necessary for the observed survival benefit. To inhibit RRV spread from infected cells to other cells, we treated mice with azidothymidine, a reverse transcription inhibitor. A robust survival benefit and tumor growth inhibition were seen only in mice where the RRV could spread freely (p = 0.0005). This finding shows that both tumor and immune cells must be infected to cause the phenotype and suggests that reprogramming immune cells is required for therapeutic efficacy. Our novel approach used RRV to deliver a myeloid transcriptional regulator in intracerebral GBM. This therapy achieved significant survival benefits and increased intratumoral cytotoxic T cells and APCs, while decreasing myeloid immunosuppression. Further studies will expand upon the mechanism of action and test combination therapies. Citation Format: Megan Montoya, Sara A. Collins, Pavlina Chuntova, Noriyuki Kasahara, Hideho Okada. IRF8 driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1417.

Abstract 2777: A unique sub-population of PDGFRB+ glioma-associated mesenchymal stem cells constitute the perivascular niche in murine glioblastoma

Abstract Microvascular proliferation (MVP) and spatial heterogeneity are defining histological features of glioblastoma (GBM). Using ex vivo culture method to analyze human GBMs we have previously identified mesenchymal stem cell (MSC)-like cells, called glioma-associated-MSCs (GA-MSCs) in the heterogenous tumor-microenvironment (TME) of GBMs. Like pericytes, fibroblastic cells and smooth muscle cells (SMCs), these GA-MSCs reside in the perivascular niche (PVN) within GBM, and their presence correlates with MVP, decrease in immune cell counts, and poor prognosis. However, the impact of GA-MSCs on the TME remains correlative, and to date, a causative role of GA-MSCs in MVP and immune evasion has not been established in vivo. We hypothesize that recruited GA-MSCs promote MVP and enhance immune evasion in the GBM-TME. To begin to address this hypothesis, we first identified and characterized GA-MSCs at single-cell resolution in an immunocompetent murine GBM model. We sorted tumor, immune, and non-tumor-non-immune (NTNI) cells from GFP-labelled GSC005 tumors in C57Bl/6 mice and performed scRNA-seq. Tumor cells (GFP+ cells) clustered together into 13 distinct cell states. Of the immune cells (CD45+), the most abundant cell types were microglia (33.9%), macrophages (31.9%), T cells (10.3%), dendritic cells (7.5%) and B cells (1.4%), mimicking human GBM. In the NTNI compartment, we identified 17 clusters, which mapped to endothelial cells, oligodendrocytes, astrocytes and other cell types. Interestingly, there were 4 distinct cell clusters that exclusively expressed platelet-derived growth factor receptor beta (PDGFRB). Three of these clusters mapped to pericytes, SMCs and fibroblastic cells. The fourth cluster expressed both adipose tissue and bone marrow MSC markers, identifying for the first time, GA-MSCs in murine GBM. Next, to define the spatial organization of GA-MSCs and their relationship to immune cells in the GBM TME, we conducted spatial proteomics. GA-MSC counts were significantly higher in the PVN compared with the avascular niche (AN) (p= 0.009) and GA-MSCs were absent in contralateral non-tumor regions. Interestingly, GA-MSCs co-localized with SMCs, fibroblastic cells and pericytes as well as pro-tumorigenic macrophages/microglia (Iba1+/CD163+) in the PVN indicating potential interactions between these cell populations. In conclusion, we have identified for the first time, MSC-like cells in the PVN of murine GBM. Our results suggest that these GA-MSCs are recruited to the TME where they may promote MVP, a hallmark of GBM, and interact with immunosuppressive immune cells. Building on these observations, future experiments will investigate the extent to which GA-MSCs drive MVP formation and immune evasion in GBM. Citation Format: Mohammad F. Zaman, Sanjay K. Singh, Anwar Hossain, Lynette M. Phillips, Joy Gumin, Daniel Ledbetter, Bojana Milutinovic. A unique sub-population of PDGFRB+ glioma-associated mesenchymal stem cells constitute the perivascular niche in murine glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2777.

Abstract 55: CD70-specific CAR-T cell therapy for the treatment of glioblastoma

Abstract Despite the remarkable success of chimeric antigen receptor (CAR)-T cell treatment for patients with hematologic malignancies, this method has yet failed to confer meaningful survival benefits to patients suffering from glioblastoma (GB), the most lethal type of brain tumor. This highlights the need to develop novel CAR-T cell approaches for this disease. CD70 is a member of the tumor necrosis factor receptor (TNFR) superfamily. Although absent on normal brain tissue, it is ectopically expressed in a substantial fraction of GB patients, indicating its suitability as a CAR-T cell therapy candidate. In this study, we generated CD70-targeting CAR-T cells and tested their cytotoxicity in vitro and in vivo. First, we detected CD70 in a panel of primary GB cell lines and to investigate its role, it was overexpressed in human and murine GB cells. In a syngeneic glioma model, C57BL/6J mice injected with CD70-overexpressing GL261 cells developed significantly larger tumors compared to the control counterparts. Additionally, RNA-sequencing revealed that CD70-overexpression in the same cells led to higher expression levels of immunosuppressive marker CD200 and lower levels of tumor inhibition genes Prkg2 and Sh3bgrl2, suggesting a tumor-promoting role in GB. For our immunotherapeutic intervention, we designed CD70-targeting CAR-T cell constructs featuring different co-stimulatory domains (CD27, CD28 or 4-1BB) and successfully transduced primary T-Cells from healthy donors. In an in vitro co-culture, all CAR-T cells recognized and eliminated primary cancer cells in a target-dependent and donor-independent manner, while secreting high levels of Granzyme B. The killing capacity of these cells was further highlighted in a 3D system in which in vitro-generated cortical organoids were treated with CAR-T cells after being infiltrated by CD70-expressing or control cells. Immunofluorescence (IF) staining and enzyme-linked immunosorbent assay (ELISA) revealed increased levels of Granzyme B and IFN-γ in all treated organoids previously infiltrated by CD70+ tumor cells. Importantly, generated CAR-T cells showed high specificity and efficiency in killing CD70+ tumors in vivo in the brains of immunodeficient mice. Namely, 80% of NSG mice orthotopically implanted with CD70+ GB cells and subsequently treated by CARs but not mock-transduced T-Cells showed complete tumor remission by the end of the experimental endpoint, determined by bioluminescence imaging (BLI). IF analysis of these brains showed high levels of apoptotic marker cleaved caspase-3, enhanced effector cell presence and significantly lower tumor cell occupancy compared to control-treated animals. In conclusion, we provide evidence in favor of utilization of CAR-T cells against CD70-expressing gliomas. Based on these findings, a phase-I clinical trial to assess the safety and efficacy of autologous CD70-specific CAR-T cells for relapsed CD70+ GB is being planned. Citation Format: Alexandros Kourtesakis, Hiu Nam Hannah Chow, Dennis Alexander Agardy, Eileen Bailey, Sandra Horschitz, Ammar Jabali, Rainer Will, Denise Reibold, Sonja Pusch, Christoph Schifflers, Manuel Fischer, Ling Hai, Dirk C. Hoffmann, Yu-Chan Chih, Robin Wagener, Leon Kaulen, Philipp Koch, Michael Breckwoldt, Michael Schmitt, Wolfgang Wick, Tim Sauer, Tobias Kessler. CD70-specific CAR-T cell therapy for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 55.

Abstract 2508: EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies

Abstract Background: Glioblastoma (GBM) is characterized as the most aggressive and lethal primary brain tumor. One of the hallmarks of GBM tumor growth and progression lies within the proangiogenic tumor microenvironment. Current anti-angiogenic drugs such as the anti-VEGF antibody take effect by inducing tumor cell starvation, leading to a functional normalization of the tumor vasculature. However, the survival benefits of anti-angiogenic drugs have been very limited. To this end, we hypothesized that the tetraspan Epithelial Membrane Protein-2 (EMP2) may help mediate resistance. Objective: We hypothesize that Epithelial Membrane Protein-2 may serve as a targetable biomarker for anti-VEGF resistance. We further explored the effectiveness of combination therapy in SB28 and GL261 GBM mouse models using anti-VEGF drug, Aflibercept, with a novel anti-EMP2 monoclonal antibody (mAb). Results: Tumor tissue was collected prospectively from patients undergoing surgery for suspected glioblastoma and stained for EMP2 and HIF2a using standard immunohistochemistry. Patients had banked tumor tissue both prior to and after bevacizumab treatment with 3 months of clinical and radiologic follow-up available. Bevacizumab treatment increased EMP2 protein expression. This increase in EMP2 correlated with reduced mean survival time post-bevacizumab therapy. Similarly, HIF-2a showed increased expression post-bevacizumab treatment and showed expression in both tumor and stromal cells. Limited animal models exist for anti-VEGF therapy for GBM. To establish syngeneic models, 10 week-old mice were subcutaneously implanted with murine SB28 or GL261 GBM cells. Treatment with either polyclonal sera to VEGF or Aflibercept failed to reduce tumor load or provide a therapeutic benefit. To next investigate if this model could recapitulate the response obtained patients tumors, formalin fixed paraffin embedded tissue was analyzed for both EMP2 and HIF-2a expression. Both markers were significantly upregulated in Aflibercept treated samples compared to the control.To next investigate if combination treatment could produce a significant reduction in tumor load, animals were treated with a combination treatment with Aflibercept and anti-EMP2 mAb. The combination therapy significantly reduced tumor volume and weight in comparison to control-treated tumors. Combination treatment in SB28 tumors resulted in a two fold reduction in tumor load and 50% reduction in tumor weight. Moreover, tumors showed reduced Ki67 expression. Similarly, treated GL261 tumors produced a 2.3 fold reduction in tumor size and ~2.7 fold reduction in tumor weight. Our results suggest that EMP2 can be a promising new target therapeutic target that can be combined with anti-VEGF treatment to potentially increase overall survival in GBM patients. Citation Format: Brian Aguirre, Mahlet Mekonnen, Riddhi Mehta, Meera Suresh, Anubhav Chandla, Karam Han, Isaac Yang, Madhuri Wadehra. EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2508.

Abstract 4159: Noninvasive measurement of murine glioblastoma in vivo with a benchtop ultrasound instrument

Abstract The purpose of this study was to evaluate a new in vivo approach for rapidly measuring tumor volumes in rodent models for glioblastoma (GBM). GBM is the most common primary brain tumor with <5% of patients surviving >5 years post diagnosis. These poor outcomes underscore the importance of researching new treatment options, and murine models play a vital role in this. Noninvasive measurements of murine GBM tumors are commonly performed using CT and MRI but these tools are costly and time-consuming to use. BLI can quickly detect the presence of GBM cells in vivo, but image data can be challenging to correlate to tumor volume. In this study we demonstrate the feasibility of imaging GBM in vivo through the intact mouse skull using a benchtop robotic ultrasound scanner. This approach could offer researchers a means to quickly size tumors in the murine brain noninvasively. To test feasibility of this approach, a female athymic nude mouse aged 16 weeks was implanted with 2e5 U87 GBM cells in the brain. The tumor was given 25 days to develop prior to ultrasound imaging. After imaging, the brain was harvested for ex vivo validation of tumor presence and volume. Imaging was performed using a Vega ultrasound system (Revvity, Waltham, MA, USA) with the mouse in supine position and fur removed from head with depilatory cream. In vivo imaging consisted of a contrast-enhanced acoustic angiography (AA) scan to assess tumor perfusion. An ex vivo B-mode scan of the brain was collected after sacrificing the animal to validate the location and size of the tumor without interference from the skull. The tumor volumes were measured in each image and compared. 3D segmentations in the ex vivo and in vivo images yielded volumes of 43.1 mm3 and 44.6 mm3, respectively, a 3.5% variance. In the in vivo AA image, the borders of the tumor were adequately well-defined for volume estimation, despite some shadowing from the sagittal suture of the skull. In the B-mode image of the excised brain, the tumor appeared hyperechoic compared to the contralateral hemisphere. Images were acquired in < 2 min. This pilot study demonstrates a potential alternative to CT and MRI for monitoring of GBM volumes in vivo. Citation Format: Thomas M. Kierski, Juan D. Rojas, Morrent Thang, Shawn D. Hingtgen, Phillip G. Durham, Paul A. Dayton, Tomek J. Czernuszewicz, Ryan C. Gessner. Noninvasive measurement of murine glioblastoma in vivo with a benchtop ultrasound instrument [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4159.