Murine Glioblastoma Cells Research Articles

Abstract P005: Fibroblast Activation Protein (FAP)-targeted radioligand therapy as a promising treatment for glioblastoma

Abstract Introduction: Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The 5-year survival rate is only 4% despite the current standard treatment approaches of surgery, chemotherapy, and radiation. Fibroblast Activation Protein (FAP)-targeted radioligand therapy (RLT) has emerged as a potential alternative to traditional radiation methods. FAP is a protein highly expressed in Cancer-Associated Fibroblasts (CAFs) and on GBM cells. It can be targeted by radiolabeled molecules for selective tumor irradiation, to enhance treatment precision and reduce damage to healthy tissue. Here we evaluated the relevance of FAP inhibitor 46 (FAPi-46) as a molecular probe for Radioligand Therapy (RLT) in GBM models. Methods: U87MG, a human GBM cell line shown to express human FAP and SB28, a murine GBM cell line engineered to express murine FAP were used as GBM models. NSG and C57BL6J mice were, respectively, inoculated with U87MG and SB28 cells subcutaneously. Tumor growth was evaluated by computed tomography (CT). FAP expression was assessed by 68Ga-FAPi-46 PET imaging when tumors reached around 100 mm3 prior to treatment. Mice were subsequently randomized into 4 groups: (1) vehicle, (2) 5 mg/kg temozolomide (TMZ), (3) 60 kBq 225Ac-FAPi-46, and (4) 5 mg/kg TMZ and 60 kBq 225Ac-FAPi-46. Dose of TMZ and 225Ac-FAPi-46 were increased for the SB28 study. Overall survival was tracked. Blood was collected 24h before, after, and 7 days after RLT treatment to assess blood toxicity. Tumor were resected 24h after RLT treatment to further evaluate DNA-damage. Results: Our results show that combining TMZ with 225Ac-FAPi-46 successfully delays tumor progression and extends survival in immunocompromised mice bearing subcutaneous human U87MG GBM tumors. In the SB28 GBM model neither TMZ nor a single dose of FAPi-RLT alone affected tumor growth or survival, confirming its in vivo resistance to both chemotherapy and radiation. However, by adapting the 225Ac-FAPi-46-RLT regimen, we observed a significant reduction in tumor volume and an increase in survival in immunocompetent mice. These results suggest that higher doses of 225Ac-FAPi-46-RLT can overcome resistance in this model. Conclusion: In conclusion, our study shows that 225Ac-FAPi-46-RLT could be an effective treatment for GBM, with positive results across different tumor models. To further investigate the efficacy of FAP-targeted RLT for GBM, we have initiated an evaluation of immune cell infiltration within subcutaneous SB28 tumors. Using immunohistochemistry, RNA sequencing, and flow cytometry, ongoing studies will characterize the immunogenic response induced by RLT in this GBM model. Citation Format: Pauline Jeanjean, Rachel Dove, Ines Camille Azrour, Samantha Kwock, Sarah Taylor, Sarina Smolev, Johannes Czernin, Giuseppe Carlucci, David Nathanson, Christine Mona, Elie Besserey-Offroy. Fibroblast Activation Protein (FAP)-targeted radioligand therapy as a promising treatment for glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P005

BIOM-74. IDENTIFICATION OF EMP2 AS A TARGETABLE BIOMARKER IN GLIOBLASTOMA TUMORS RESISTANT TO ANTI-ANGIOGENIC THERAPIES

Abstract BACKGROUND The role of Epithelial Membrane Protein-2 (EMP2) in mediating resistance to current anti-angiogenic drugs in the treatment of glioblastoma (GBM) is an intriguing hypothesis that may provide insights into improving the limited survival benefits observed in current therapies. Investigating the interaction between EMP2 and anti-angiogenic drugs such as bevacizumab or Aflibercept could lead to a better understanding of the mechanisms underlying resistance, potentially opening up new avenues for more effective treatment strategies. OBJECTIVE To use EMP2 as a targetable biomarker for anti-VEGF resistance and test combination therapy with anti-VEGF (Aflibercept or bevacizumab), and a novel anti-EMP2 mAb in SB28 and GL261 GBM mouse models. METHODS Tumor tissue was collected prospectively from patients undergoing surgery for suspected glioblastoma and stained for EMP2 and HIF-2a using standard immunohistochemistry. Patients had banked tumor tissue both before and after bevacizumab treatment with 3 months of clinical and radiologic follow-up available. To establish syngeneic models, 10-week-old mice were subcutaneously implanted with murine SB28 or GL261 GBM cells. RESULTS Immunohistochemistry conducted on pre- and post-bevacizumab-treated patient samples revealed an increase in EMP2 and HIF-2a protein expression post-treatment. This increase in EMP2 expression correlated with reduced survival time. Similarly, the in vivo models showed a significant upregulation in both markers in the Aflibercept-treated cohort compared to the control. Treatment with either polyclonal sera to VEGF or Aflibercept failed to reduce tumor load or provide a therapeutic benefit. However, combination therapy with Aflibercept and anti-EMP2 mAb was found to significantly reduce tumor volume and weight in both SB28 and GL261 tumors. These findings highlight the potential effectiveness of combination therapies targeting EMP2 and VEGF in reducing tumor load in GBM. CONCLUSION Our results suggest that EMP2 can be a promising new therapeutic target that can be combined with anti-VEGF treatment to potentially increase overall survival in GBM patients.

SURG-27. DIRECTIONALLY NON-ROTATING ELECTRIC FIELD THERAPY (DNEFT) DELIVERED THROUGH IMPLANTED ELECTRODES: A NOVEL SURGICAL PLATFORM FOR GLIOBLASTOMA IMMUNOTHERAPY

Abstract BACKGROUND While directionally rotating Tumor Treating Fields (TTF) therapy has garnered considerable clinical interest in recent years, there has been comparatively less focus on directionally non-rotating electric field therapy (dnEFT). OBJECTIVE Here, we explore dnEFT generated through implanted electrodes as a glioblastoma therapeutic platform, with the goal of facilitating clinical translation. METHODS Using custom-designed electrode arrays to study the effect of dnEFT using in vitro and in vivo glioblastoma models. RESULTS In vitro, dnEFT generated using a clinical grade spinal cord stimulator showed anti-neoplastic activity against independent glioblastoma cell lines. In support of the results obtained using the clinical grade electrode, dnEFT delivered through a customized, two-electrode array induced glioblastoma apoptosis. To characterize this effect in vivo, a custom-designed four-electrode array was fabricated such that tumor cells can be implanted into murine cerebrum through a center channel equidistant from the electrodes. After implantation with this array and luciferase expressing murine GL261 glioblastoma cells, mice were randomized to dnEFT or placebo. Relative to placebo treated mice, dnEFT reduced tumor growth (measured by bioluminescence) and prolonged survival (median survival gain of 6.5 days). Analysis of brain sections following dnEFT showed a notable increase in the accumulation of peri-tumoral macrophage/microglia with increased expression of M1 genes (IFNγ, TNFα, IL-6) and decreased expression of M2 genes (CD206, Arg, IL-10) relative to placebo tumors. CONCLUSIONS Our results suggest therapeutic potential in glioblastoma for dnEFT delivered through implanted electrodes as a novel immunotherapy platform, supporting the concept of a proof-of-principle clinical trial using commercially available deep brain stimulator electrodes.

IMMU-72. TYPE I INTERFERON SIGNALING WITHIN GLIOBLASTOMA CELLS PROMOTES T-CELL EXHAUSTION AND RESISTANCE TO CHECKPOINT INHIBITORS

Abstract Glioblastoma (GBM) remains among the most fatal cancers with few treatment modalities and resistance to modern immunotherapeutic treatment paradigms including immune checkpoint inhibitors (ICI). ICIs neutralize the regulatory signals that disable T cells and have demonstrated remarkable efficacy against a variety of cancers, though not in GBM. The impotence of ICI in GBM has been linked to the tumor’s remarkable capacity to elicit T cell exhaustion, though the mechanism remains poorly understood. Chronic interferon (IFN) cytokine signaling is known to promote immunosuppression and Tex in several cancer types as well as resistance to ICI. However, the role of IFN in GBM has not been investigated despite evidence that IFN signatures exist within clinical samples of GBM. Syngeneic murine GBM cell lines CT2A and SB28 were utilized for in vivo and in vitro studies. Anti-mouse PD-1 and CD8 antibodies or PBS control were injected intraperitoneally. CRISPR editing was used to create validated knockouts (KO) of the type I IFN receptor (IFNAR1) in CT2A. Mice bearing IFNAR1 KO intracranial tumors have improved median survival (p < 0.05) and are exquisitely sensitive to checkpoint anti-PD-1 blockade (p < 0.05) with the majority of mice surviving. Furthermore, surviving mice reject the subsequent intracranial re-challenge with parental CT2A tumors compared to controls (p <0.05) suggesting central memory responses mediating rejection. We conclude that abrogation of Type I IFN signaling is sufficient to extend survival which is further enhanced by ICI. Work is ongoing to further elucidate the precise mechanism that mediates this response and to identify a therapeutic intervention that enables tumor-specific inhibition of the IFN pathway with preliminary work suggesting a CD8 T cell mediated response and reversal of the T cell exhaustion phenotype.

Targeting Retinaldehyde Dehydrogenases to Enhance Temozolomide Therapy in Glioblastoma.

Glioblastoma (GB) is an aggressive malignant central nervous system tumor that is currently incurable. One of the main pitfalls of GB treatment is resistance to the chemotherapeutic standard of care, temozolomide (TMZ). The role of aldehyde dehydrogenases (ALDHs) in the glioma stem cell (GSC) subpopulation has been related to chemoresistance. ALDHs take part in processes such as cell proliferation, differentiation, invasiveness or metastasis and have been studied as pharmacological targets in cancer treatment. In the present work, three novel α,β-acetylenic amino thiolester compounds, with demonstrated efficacy as ALDH inhibitors, were tested in vitro on a panel of six human GB cell lines and one murine GB cell line. Firstly, the expression of the ALDH1A isoforms was assessed, and then inhibitors were tested for their cytotoxicity and their ability to inhibit cellular ALDH activity. Drug combination assays with TMZ were performed, as well as an assessment of the cell death mechanism and generation of ROS. A knockout of several ALDH genes was carried out in one of the human GB cell lines, allowing us to discuss their role in cell proliferation, migration capacity and resistance to treatment. Our results strongly suggest that ALDH inhibitors could be an interesting approach in the treatment of GB, with EC50 values in the order of micromolar, decreasing ALDH activity in GB cell lines to 40-50%.

Adipose-Derived Stem Cells as Carrier of Pro-Apoptotic Oncolytic Myxoma Virus: To Cross the Blood–Brain Barrier and Treat Murine Glioma

Treatment of glioblastoma is ineffective. Myx-M011L-KO/EGFP, a myxoma virus actively inducing apoptosis in BTICs linked to recurrence, offers innovative treatment. We loaded this construct into adipose-derived stem cells (ADSCs) to mitigate antiviral host responses and enable systemic delivery. The apoptotic and cytotoxic effects of the construct were studied using murine and human glioblastoma cell lines. Before implementing systemic delivery, we delivered the construct locally using ADSC to verify elimination of orthotopic murine glioma lesions. vMyx-M011L-KO/EGFP was cytotoxic to a murine cell line, preventing effective virus multiplication. In three human glioma cell lines, viral replication did occur, coupled with cell killing. The knock-out construct induced apoptotic cell death in these cultures. ADSCs infected ex vivo were shown to be sufficiently migratory to assure transfer of the therapeutic cargo to murine glioma lesions. Virus-loaded ADSCs applied to the artificial blood–brain barrier (BBB) yielded viral infection of glioma cells grown distally in the wells. Two rounds of local administration of this therapeutic platform starting 6 days post tumor implantation slowed down growth of orthotopic lesions and improved survival (total recovery < 20%). ADSCs infected ex vivo with vMyx-M011L-KO/EGFP show promise as a therapeutic tool in systemic elimination of glioma lesions.

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.

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.

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.

Abstract 2887: Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain

Abstract This study aims to analyze irradiation-induced metabolic alterations in glioblastoma cells and astrocytes to enhance understanding of cellular responses and facilitate the development of more effective radiotherapeutic strategies. Metabolic reprogramming of tumor cells is considered one of the hallmarks of cancer. Studies have shown that abnormal activation of oncogenes and cancer-related signaling pathways, as well as the inactivation of tumor suppressor genes can induce metabolic reprogramming. These metabolic aberrations facilitate rapid proliferation, continuous growth, invasion, metastasis, and immune evasion. Following the application of radiotherapy, the activity of several metabolic pathways significantly changes, potentially leading to the development of radioresistance. However, a differential effect of ionizing radiation on tumor and normal cells is not well understood. Hence, quantifying irradiation-induced metabolic changes in glioblastoma cells and astrocytes is of high importance. We have developed a novel approach to measure dynamic metabolite changes upon irradiation, in vivo, in situ, and in real-time. This tool combines imaging of recently developed fluorescent biosensors via 2-photon microscopy and millimeter-scaled irradiation. Murine glioblastoma cells and astrocytes were generated with stable expression of biosensors for the quantification of intracellular concentrations of various metabolites, including lactate. Cytosolic concentrations of lactate depend on the balance between glycolytic production, mitochondrial consumption of pyruvate and lactate, and the exchange with the extracellular lactate pool via monocarboxylate transporters. Many cancer cells are characterized by an excessive conversion of glucose to lactate even under normoxic conditions. We quantified this so-called Warburg Effect in glioblastoma cells and astrocytes as the ratio between basal lactate production and the lactate increase after OXPHOS inhibition and determined irradiation-induced metabolic changes on the single-cell level. Complementarily, we used metabolomics and Seahorse analyses before and after irradiation. Our data indicate that irradiation increases oxygen consumption in glioblastoma cells but not in astrocytes. Also, we established an orthotopic glioblastoma mouse model via injection of sensor-expressing tumor cells for in vivo measurements of metabolite changes during and after irradiation. These data are expected to significantly advance the understanding of the cellular response to irradiation on a molecular level in situ and in real-time. This knowledge will help to develop combination treatments and increase the efficiency of radiotherapy. Citation Format: Marvin Kreuzer, Pascal Imseng, Bruno Weber, Martin Pruschy. Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain [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 2887.

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.

Directionally non-rotating electric field therapy delivered through implanted electrodes as a glioblastoma treatment platform: A proof-of-principle study.

While directionally rotating tumor-treating fields (TTF) therapy has garnered considerable clinical interest in recent years, there has been comparatively less focus on directionally non-rotating electric field therapy (dnEFT). We explored dnEFT generated through customized electrodes as a glioblastoma therapy in in vitro and in vivo preclinical models. The effects of dnEFT on tumor apoptosis and microglia/macrophages in the tumor microenvironment were tested using flow-cytometric and qPCR assays. In vitro, dnEFT generated using a clinical-grade spinal cord stimulator showed antineoplastic activity against independent glioblastoma cell lines. In support of the results obtained using the clinical-grade electrode, dnEFT delivered through a customized, 2-electrode array induced glioblastoma apoptosis. To characterize this effect in vivo, a custom-designed 4-electrode array was fabricated such that tumor cells can be implanted into murine cerebrum through a center channel equidistant from the electrodes. After implantation with this array and luciferase-expressing murine GL261 glioblastoma cells, mice were randomized to dnEFT or placebo. Relative to placebo-treated mice, dnEFT reduced tumor growth (measured by bioluminescence) and prolonged survival (median survival gain of 6.5 days). Analysis of brain sections following dnEFT showed a notable increase in the accumulation of peritumoral macrophage/microglia with increased expression of M1 genes (IFNγ, TNFα, and IL-6) and decreased expression of M2 genes (CD206, Arg, and IL-10) relative to placebo-treated tumors. Our results suggest therapeutic potential in glioblastoma for dnEFT delivered through implanted electrodes, supporting the development of a proof-of-principle clinical trial using commercially available deep brain stimulator electrodes.

The Putative S1PR1 Modulator ACT-209905 Impairs Growth and Migration of Glioblastoma Cells In Vitro.

Glioblastoma (GBM) is still a deadly tumor due to its highly infiltrative growth behavior and its resistance to therapy. Evidence is accumulating that sphingosine-1-phosphate (S1P) acts as an important tumor-promoting molecule that is involved in the activation of the S1P receptor subtype 1 (S1PR1). Therefore, we investigated the effect of ACT-209905 (a putative S1PR1 modulator) on the growth of human (primary cells, LN-18) and murine (GL261) GBM cells. The viability and migration of GBM cells were both reduced by ACT-209905. Furthermore, co-culture with monocytic THP-1 cells or conditioned medium enhanced the viability and migration of GBM cells, suggesting that THP-1 cells secrete factors which stimulate GBM cell growth. ACT-209905 inhibited the THP-1-induced enhancement of GBM cell growth and migration. Immunoblot analyses showed that ACT-209905 reduced the activation of growth-promoting kinases (p38, AKT1 and ERK1/2), whereas THP-1 cells and conditioned medium caused an activation of these kinases. In addition, ACT-209905 diminished the surface expression of pro-migratory molecules and reduced CD62P-positive GBM cells. In contrast, THP-1 cells increased the ICAM-1 and P-Selectin content of GBM cells which was reversed by ACT-209905. In conclusion, our study suggests the role of S1PR1 signaling in the growth of GBM cells and gives a partial explanation for the pro-tumorigenic effects that macrophages might have on GBM cells.

Local administration of shikonin improved the overall survival in orthotopic murine glioblastoma models with temozolomide resistance

BackgroundGlioblastoma is a type of intracranial malignancy. Shikonin, a Chinese traditional medicine, has been shown to have anti-tumor efficacy toward human glioblastoma cells in vitro. However, shikonin cannot easily cross the blood–brain barrier. To address this issue, we evaluated the anti-tumor effects of direct intracranial infusion of shikonin in in vivo orthotopic syngeneic murine glioblastoma models using C57BL/6 mice. Materials and methodsThe cytotoxic effects of shikonin against murine glioblastoma cells, SB28 and CT-2A, were reported resistance to temozolomide, were evaluated using an allophycocyanin-conjugated annexin V and propidium iodide assay with flow cytometry. Impedance-based real-time cell analysis (RTCA) was used to analyze the inhibitory effects of shikonin on growth and proliferation. To evaluate the anti-tumor activity of shikonin in vivo, we used orthotopic syngeneic murine glioblastoma models with SB28 and CT-2A cells. ResultsIn flow cytometry-based cytotoxic assays, shikonin induced apoptosis. RTCA indicated that shikonin decreased the cell index of murine glioblastoma cells, SB28 and CT-2A, in a dose-dependent manner (p < 0.0001 for both cell lines), while temozolomide did not (p = 0.91 and 0.82, respectively). In murine glioblastoma models, SB28 and CT-2A, direct intracranial infusion of shikonin, as a local chemotherapy, improved the overall survival of mice in a dose-dependent manner compared with control groups (p < 0.0001 and p = 0.02, respectively). While temozolomide did not (p = 0.48 and 0.52, respectively). ConclusionsThe direct intracranial infusion of shikonin has potential as a local therapy for patients with glioblastoma.

TCF12 Deficiency Impairs the Proliferation of Glioblastoma Tumor Cells and Improves Survival

Isocitrate dehydrogenase (IDH)-wild-type glioblastoma (GBM) is the most common and aggressive primary brain tumor which carries a very poor overall prognosis and is universally fatal. Understanding the transcriptional regulation of the proliferation of GBM tumor cells is critical for developing novel and effective treatments. In this study, we investigate the role of the transcription factor TCF12 in the regulation of GBM proliferation using human and murine GBM cell lines and an in vivo GBM xenograft model. Our study shows that TCF12 deficiency severely impairs proliferation of tumor cells in vitro by disrupting/blocking the G1 to S phase transition. We also discover that TCF12 loss significantly improves animal survival and that TCF12-deficient tumors grow much slower in vivo. Overexpression of TCF12, on the other hand, leads to an increase in the proliferation of tumor cells in vitro and more aggressive tumor progression in vivo. Interestingly, loss of TCF12 leads to upregulation of signature genes of the oligodendrocytic lineage in GBM stem cells, suggesting a role for TCF12 in inhibiting differentiation along the oligodendrocytic lineage. Transcriptomic data also reveals that loss of TCF12 leads to dysregulation of the expression of key genes in the cell cycle. Our work demonstrates critical roles of TCF12 in GBM tumor progression.

Myeloperoxidase exerts anti-tumor activity in glioma after radiotherapy.

BackgroundHost immune response is a critical component in tumorigenesis and immune escape. Radiation is widely used for glioblastoma (GBM) and can induce marked tissue inflammation and substantially alter host immune response. However, the role of myeloperoxidase (MPO), a key enzyme in inflammation and host immune response, in tumorigenesis after radiotherapy is unclear. In this study, we aimed to determine how post-radiation MPO activity influences GBM and outcome.MethodsWe injected C57BL/6J or MPO-knockout mice with 005 mouse GBM stem cells intracranially. To observe MPO's effects on post-radiation tumor progression, we then irradiated the head with 10 Gy unfractionated and treated the mice with a specific MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), or vehicle as control. We performed semi-quantitative longitudinal molecular MRI, enzymatic assays and flow cytometry to assess changes in inflammatory response and tumor size, and tracked survival. We also performed cell culture experiments in murine and human GBM cells to determine the effect of MPO on these cells.ResultsBrain irradiation increased the number of monocytes/macrophages and neutrophils, and boosted MPO activity by ten-fold in the glioma microenvironment. However, MPO inhibition dampened radiation-induced inflammation, demonstrating decreased MPO-specific signal on molecular MRI and attenuated neutrophil and inflammatory monocyte/macrophage recruitment to the glioma. Compared to saline-treated mice, both ABAH-treated and MPO-knockout mice had accelerated tumor growth and reduced survival. We further confirmed that MPO decreased tumor cell viability and proliferation in cell cultures.ConclusionLocal radiation to the brain initiated an acute systemic inflammatory response with increased MPO-carrying cells both in the periphery and the GBM, resulting in increased MPO activity in the tumor microenvironment. Inhibition or absence of MPO activity increased tumor growth and decreased host survival, revealing that elevated MPO activity after radiation has an anti-tumor role.

JCI-20679 suppresses autophagy and enhances temozolomide-mediated growth inhibition of glioblastoma cells

Glioblastoma, a type of brain cancer, is one of the most aggressive and lethal types of malignancy. The present study shows that JCI-20679, an originally synthesized mitochondrial complex I inhibitor, enhances the anti-proliferative effects of suboptimal concentrations of the clinically used chemotherapeutic drug temozolomide in glioblastoma cells. Analysis of the effects of temozolomide combined with JCI-20679 using isobologram and combination index methods demonstrated that the combination had synergistic effects in murine and human glioblastoma cells. We found that JCI-20679 inhibited the temozolomide-mediated induction of autophagy that facilitates cellular survival. The autophagy induced by temozolomide increased ATP production, which confers temozolomide resistance in glioblastoma cells. JCI-20679 blocked temozolomide-mediated increases in ATP levels and increased the AMP/ATP ratio. Furthermore, JCI-20679 enhanced the therapeutic effects of temozolomide in an orthotopic transplantation model of glioblastoma. These results indicate that JCI-20679 may be promising as a novel agent for enhancing the efficacy of temozolomide against glioblastoma.

Endothelial cell-specific reduction of heparan sulfate suppresses glioma growth in mice

PurposeHeparan sulfate (HS) is one of the factors that has been suggested to be associated with angiogenesis and invasion of glioblastoma (GBM), an aggressive and fast-growing brain tumor. However, it remains unclear how HS of endothelial cells is involved in angiogenesis in glioblastoma and its prognosis. Thus, we investigated the effect of endothelial cell HS on GBM development.MethodsWe generated endothelial cell-specific knockout of Ext1, a gene encoding a glycosyltransferase and essential for HS synthesis, and murine GL261 glioblastoma cells were orthotopically transplanted. Two weeks after transplantation, we examined the tumor progression and underlying mechanisms.ResultsThe endothelial cell-specific Ext1 knockout (Ext1CKO) mice exhibited reduced HS expression specifically in the vascular endothelium of the brain capillaries compared with the control wild-type (WT) mice. GBM growth was significantly suppressed in Ext1CKO mice compared with that in WT mice. After GBM transplantation, the survival rate was significantly higher in Ext1CKO mice than in WT mice. We investigated how the effect of fibroblast growth factor 2 (FGF2), which is known as an angiogenesis-promoting factor, differs between Ext1CKO and WT mice by using an in vivo Matrigel assay and demonstrated that endothelial cell-specific HS reduction attenuated the effect of FGF2 on angiogenesis.ConclusionsHS reduction in the vascular endothelium of the brain suppressed GBM growth and neovascularization in mice.