Animal Models Of Glioblastoma Research Articles

Preparation of a nanoemulsion containing active ingredients of cannabis extract and its application for glioblastoma: in vitro and in vivo studies

Recently, the anti-tumor effects of cannabis extract on various cancers have attracted the attention of researchers. Here, we report a nanoemulsion (NE) composition designed to enhance the delivery of two active components in cannabis extracts (∆9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD)) in an animal model of glioblastoma. The efficacy of the NE containing the two drugs (NED) was compared with the bulk drugs and carrier (NE without the drugs) using the C6 tumor model in rats. Hemocompatibility factors (RBC, MCV, MCH, MCHC, RDW, PPP, PT and PTT) were studied to determine the potential in vivo toxicity of NED. The optimized NED with mean ± SD diameter 29 ± 6 nm was obtained. It was shown that by administering the drugs in the form of NED, the hemocompatibility increased. Cytotoxicity studies indicated that the NE without the active components (i.e. mixture of surfactants and oil) was the most cytotoxic group, while the bulk group had no toxicity. From the in vivo MRI and survival studies, the NED group had maximum efficacy (with ~4 times smaller tumor volume on day 7 of treatment, compared with the control. Also, survival time of the control, bulk drug, NE and NED were 9, 4, 12.5 and 51 days, respectively) with no important adverse effects. In conclusion, the NE containing cannabis extract could be introduced as an effective treatment in reducing brain glioblastoma tumor progression.Graphical abstract

Synthesis and Evaluation of the Anticancer Effects of Iron Oxide-Chitosan Magnetic Nanoparticles Loaded Atorvastatin on the Invasion and Tumorigenicity of C6 Cell Line in Glioblastoma Animal Model

Synthesis and Evaluation of the Anticancer Effects of Iron Oxide-Chitosan Magnetic Nanoparticles Loaded Atorvastatin on the Invasion and Tumorigenicity of C6 Cell Line in Glioblastoma Animal Model

Radiosynthesis and preclinical evaluation of a 68Ga-labeled tetrahydroisoquinoline-based ligand for PET imaging of C-X-C chemokine receptor type 4 in an animal model of glioblastoma

BackgroundThis study aimed to develop a novel positron emission tomography (PET) tracer, [68Ga]Ga-TD-01, for CXCR4 imaging. To achieve this goal, the molecular scaffold of TIQ15 was tuned by conjugation with the DOTA chelator to make it suitable for 68Ga radiolabeling.MethodsA bifunctional chelator was prepared by conjugating the amine group of TIQ15 with p-NCS-Bz-DOTA, yielding TD-01, with a high yield (68.92%). TD-01 was then radiolabeled with 68Ga using 0.1 M ammonium acetate at 60 °C for 10 min. A 1-h dynamic small animal PET/MRI study of the labeled compound in GL261-luc2 tumor-bearing mice was performed, and brain tumor uptake was assessed. Blocking studies involved pre-administration of TIQ15 (10 mg/kg) 10 min before the PET procedure started.Results[68Ga]Ga-TD-01 exhibited a radiochemical yield (RCY) of 36.33 ± 1.50% (EOS), with a radiochemical purity > 99% and a molar activity of 55.79 ± 1.96 GBq/µmol (EOS). The radiotracer showed in vitro stability in PBS and human plasma for over 4 h. Biodistribution studies in healthy animals revealed favorable kinetics for subsequent PET pharmacokinetic modeling with low uptake in the brain and moderate uptake in lungs, intestines and spleen. Elimination could be assigned to a renal-hepatic pathway as showed by high uptake in kidneys, liver, and urinary bladder. Importantly, [68Ga]Ga-TD-01 uptake in glioblastoma (GBM)-bearing mice significantly decreased upon competition with TIQ15, with a baseline tumor-to-background ratios > 2.5 (20 min p.i.), indicating high specificity.ConclusionThe newly developed CXCR4 PET tracer, [68Ga]Ga-TD-01, exhibited a high binding inhibition for CXCR4, excellent in vitro stability, and favorable pharmacokinetics, suggesting that the compound is a promising candidate for full in vivo characterization of CXCR4 expression in GBM, with potential for further development as a tool in cancer diagnosis.

A novel L-shaped ortho-quinone analog suppresses glioblastoma progression by targeting acceleration of AR degradation and regulating PI3K/AKT pathway

Glioblastoma (GBM) is a primary intracranial malignant tumor with the highest mortality and morbidity among all malignant central nervous system tumors. Tanshinone IIA is a fat-soluble active ingredient obtained from Salvia miltiorrhiza, which has an inhibitory effect against various cancers. We designed and synthesized a novel L-shaped ortho-quinone analog TE5 with tanshinone IIA as the lead compound and tested its antitumor activity against GBM. The results indicated that TE5 effectively inhibited the proliferation, migration, and invasion of GBM cells, and demonstrated low toxicity in vitro. We found that TE5 may bind to androgen receptors and promote their degradation through the proteasome. Inhibition of the PI3K/AKT signaling pathway was also observed in TE5 treated GBM cells. Additionally, TE5 arrested the cell cycle at the G2/M phase and induced mitochondria-dependent apoptosis. In vivo experiments further confirmed the anti-tumor activity, safety, and effect on androgen receptor level of TE5 in animal models of GBM. Our results suggest that TE5 may be a potential therapeutic drug to treat GBM.

Targeting the molecular chaperone CCT2 inhibits GBM progression by influencing KRAS stability

Proper protein folding relies on the assistance of molecular chaperones post-translation. Dysfunctions in chaperones can cause diseases associated with protein misfolding, including cancer. While previous studies have identified CCT2 as a chaperone subunit and an autophagy receptor, its specific involvement in glioblastoma remains unknown. Here, we identified CCT2 promote glioblastoma progression. Using approaches of coimmunoprecipitation, mass spectrometry and surface plasmon resonance, we found CCT2 directly bound to KRAS leading to increased stability and upregulated downstream signaling of KRAS. Interestingly, we found that dihydroartemisinin, a derivative of artemisinin, exhibited therapeutic effects in a glioblastoma animal model. We further demonstrated direct binding between dihydroartemisinin and CCT2. Treatment with dihydroartemisinin resulted in decreased KRAS expression and downstream signaling. Highlighting the significance of CCT2, CCT2 overexpression rescued the inhibitory effect of dihydroartemisinin on glioblastoma. In conclusion, the study demonstrates that CCT2 promotes glioblastoma progression by directly binding to and enhancing the stability of the KRAS protein. Additionally, dihydroartemisinin inhibits glioblastoma by targeting the CCT2 and the following KRAS signaling. Our findings overcome the challenge posed by the undruggable nature of KRAS and offer potential therapeutic strategies for glioblastoma treatment.

Abstract 5808: Designing arrays for delivering tumor treating fields (TTFields) to the mouse head

Abstract Introduction: Tumor Treating Fields (TTFields) therapy is an approved treatment for glioblastoma (GBM), the most abundant malignant brain tumor. TTFields therapy is delivered to patients suffering from GBM continuously by two array pairs placed on their scalp. Since arrays for TTFields application to the mouse head are lacking, and as most GBM animal models are based on mice, in vivo studies of GBM treatment with TTFields are limited. The development of such arrays is challenging due to the small dimensions and specific geometries of the mouse head, but is needed to allow continued preclinical studies to broaden the understanding of the effects of TTFields on GBM. Methods: We tested various array layouts to identify one that will be suited to the specific geometries of the mouse head. For animal well-being, the selected array was required to minimally restrict head movement. Additionally, we investigated adhesive tapes to allow on one hand good adherence of the arrays to the skin, and on the other hand easy array removal. To examine the feasibility of treating mice with these arrays, they were applied to mice for 11 days, while recording the electric currents and the actual treatment time (from which the percent usage could be calculated). To validate that the selected array layout delivers meaningful field intensity to the desired region, we performed field measurements using a floating scope. Results: To tackle the limitation of the small head size, we developed an array layout in which only two arrays were situated on the head, while the opposing arrays were located on the mouse torso. Additionally, the arrays on the head were divided into two smaller disks. We also identified a thin and transparent adhesive tape (facilitating correct arrays positioning), that offered good tackiness and allowed easy removal without leaving residual adhesive on the skin. The arrays met the minimum treatment requirements: current ≥50 mA, usage ≥75%, field intensity ≥1 V/cm RMS. At the posterior side of the mouse head, at a location more directly underneath the arrays, field intensity was higher than at the anterior position. Conclusion: TTFields could be delivered at sufficient intensities to mice heads by utilizing specifically engineered arrays. Our newly developed arrays are flexible and strongly adhering to enable efficient electric field delivery. By allowing TTFields delivery to mice heads, we will facilitate further advancing this research field. Citation Format: Sewar Zbidat, Roni Blatt, Mariell Sellevoll, Martin Gabay, Inbar Schlachet, Shay Cahal, Shiri Davidi, Itai Tzchori, Adi Haber, Moshe Giladi, Yoram Palti. Designing arrays for delivering tumor treating fields (TTFields) to the mouse head [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 5808.

Abstract 4046: Orally active PD-L1 inhibitor MAX-10181 in combination with temozolomide effectively prolonged survival in GBM animal model study

Abstract Treatment of glioblastoma (GBM) with PD-1/PD-L1 immuno therapy is highly challenging due to the limited brain blood barrier (BBB) penetration of the marketed antibody drugs. During the past several years, we have successfully developed orally active, BBB penetrating, small molecule PD-L1 inhibitor MAX-10181 which is in the phase II clinical development. To demonstrate the utility of PD-1/L1 immuno therapy for the treatment of GBM, we completed the study of MAX-10181 in combination with Temozolomide (TMZ) in GL-261 animal model study. In this study, the combination of MAX-10181 with TMZ effectively prolonged the survival by 50% in comparison with TMZ single therapy. Details of this study will be reported in this meeting. Citation Format: Yuguang Wang, Zhenhua Feng, Qiang Zhao, Wu Zhang, Yiwei Jiang, Yingqi Huang, Chenxing Qi, Cheng Zhang. Orally active PD-L1 inhibitor MAX-10181 in combination with temozolomide effectively prolonged survival in GBM animal model study [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 4046.

Abstract A004: GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

Abstract OBJECTIVE: We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS: Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION: The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. Citation Format: Weihua Zhou, Zitong Zhao, Angelica Lin, John Yang, Jie Xu, Kari Wilder-Romans, Annabel Yang, Andrew J. Scott, Jing Li, Sumeet Solanki, Jennifer Speth, Natalie Walker, Ayesha U. Kothari, Yangyang Yao, Erik R. Peterson, Navyateja Korimerla, Christian K. Werner, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra M Obrien, Ameer L Elaimy, Sean P. Ferris, Shuang G. Zhao, Jann N. Sarkaria, Balázs Győrffy, Shuqun Zhang, Wajd N. Al-Holou, Yoshie Umemura, Meredith A Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Marc Peters-Golden, Yatrik M. Shah, Daniel R. Wahl. GTP signaling links metabolism, DNA repair, and responses to genotoxic stress [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A004.

A new antiviral hypothesis and radioactive iodine therapy to other cancers, such as breast cancer, lung cancer, and glioblastoma multiforme (GBM)?

Radioactive iodine therapy (RIT) is an effective, safe, and cheap method in benign and malignant thyroid diseases. There is still an unresolved question of whether RIT treatment also plays a role in the treatment of, for example, breast cancer, lung cancer, or glioblastoma multiforme (GBM). These studies are currently being carried out in rats in combination with genes, but it may be an interesting challenge to assess "pure" RIT alone, thanks to the expression of sodium iodide symporter (NIS), is effective in other organ nodules, both benign and malignant. Cloning of the NIS in 1996 provided an opportunity to use NIS as a powerful theranostic transgene. In addition, NIS is a sensitive reporter gene that can be monitored by high-resolution PET imaging using the radiolabels [¹²⁴I]sodium iodide ([¹²⁴I]NaI) or [18F] tetrafluoroborate ([¹⁸F]TFB). Based on published positron emission tomography (PET) results, [¹²⁴I]sodium iodide and internally synthesized [18F]TFB were compared in an orthotopic animal model of NIS-expressing glioblastoma. The results showed improved image quality using [¹⁸F]TFB. Based on these results, we will be able to extend the NIS gene therapy approach using non-viral gene delivery vehicles to target orthotopic tumour models with low-volume disease such as GBM. Is it possible to treat RIT alone without using the NIS gene in GBM? After all, the NIS symporter was detected not only in the thyroid gland, but also in different tumours. The administration of RIT is completely harmless; the only complication is hypothyroidism. Indeed, recently it has been shown that, for example, in the case of thyroid cancer, the maximum RIT is 37000 MBq (1000 mCi). When beneficial effects of therapy in GBM are not possible (e.g. neurosurgery, modulated electro-hyperthermia, chemotherapy, immunotherapy, cancer vaccines, or oncolytic viruses), could RIT provide a "revolution" using NIS?

Syngeneic Mouse Model of Glioblastoma: Intracranial Implantation of GL261 Cells.

Glioblastoma (GBM) is the most aggressive and prevalent primary brain malignancy in adults. Current treatments provide limited benefit, and thus, the median overall survival of GBM patients is only 15months. GBM progression is highly dependent on its ability to evade immune response, so understanding the mechanisms behind GBM-driven immunosuppression seems crucial for designing more efficient therapies. Animal models of GBM constitute a convenient tool in glioma research, and several different approaches have been already developed to model this disease in vivo, including genetic and xenograft models. Here, we describe a murine syngeneic model of glioma which recapitulates many of the key features of human disease, including complex tumor microenvironment. We present an optimized protocol for stereotactic intracranial implantation of GL261 cells into C57BL/6 mice which results in tumor growth in the striatum. This model has been widely used to get insight into glioma biology, as well as in the studies aiming at the development and validation of new therapeutic approaches.

RBIO-08. GTP SIGNALING LINKS ALTERED METABOLISM, DNA REPAIR AND TREATMENT RESISTANCE IN GLIOBLASTOMA

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.

BIOM-05. FIRST-IN-HUMAN PROSPECTIVE TRIAL OF SONOBIOPSY IN GLIOBLASTOMA PATIENTS USING NEURONAVIGATION-GUIDED FOCUSED ULTRASOUND

Abstract INTRODUCTION Blood-based liquid biopsy for brain cancer offers a noninvasive alternative to invasive surgical biopsies. However, the presence of the blood-brain barrier (BBB) limits the release of brain tumor-derived molecular biomarkers into the bloodstream for sensitive diagnosis. Focused ultrasound (FUS)-enabled blood-based liquid biopsy (sonobiopsy) locally disrupts the BBB and releases tumor-derived biomarkers into blood circulation. Our previous studies have demonstrated that sonobiopsy improved the detection sensitivity of glioblastoma (GBM)-specific cirulating tumor DNA (ctDNA) in animal models of GBM. This study aimed to evaluate the feasibility and safety of sonobiopsy in enriching circulating tumor biomarkers in a first-in-human, prospective clinical trial. METHODS Five GBM patients (G01-05) were enrolled in the initial study. The sonobiopy procedures were performed prior to tumor resection, using a nimble FUS device integrated with a clinical neuronavigation system. The plasma level of glial fibrillary acidic protein (GFAP) was quantified using ultrasensitive single-molecule array (Simoa), while the cell-free DNA (cfDNA) levels were quantified using Bioanalyzer. Patient-specific tumor variants were comprehensively analyzed using a personalized tumor-informed ctDNA assay (Invitae Personalized Cancer Monitoring assay). Additionally, the two most common GBM-specific tumor variants were analyzed using customized ddPCR assays. RESULTS Analysis of plasmas collected before and after FUS sonication showed sonobiopsy enhanced plasma circulating tumor biomarker levels, including a maximum increase of 1.3-fold for GFAP, 1.6-fold for the concentration of mono-nucleosomal cfDNA fragment (120–280 bp), 1.9-fold increase in patient-specific tumor variant levels, and 5.6-fold increase in the TERT mutation level. Histological analysis of surgically resected tumors confirmed the safety of the procedure. Transcriptome analysis of sonicated and nonsonicated tumor tissues found that FUS sonication mainly modulated cell physical structure-related genes. CONCLUSIONS These feasibility and safety data support the continued investigation of sonobiopsy for noninvasive molecular diagnosis of brain diseases.

Development and validation of a GC-MS method for determination of metformin in normal brain and in glioblastoma tissues

Metformin hydrochloride (MH) has recently been repurposed as an anticancer agent, showing antiproliferative activity in vitro and in vivo. In particular, experimental evidence has suggested its potential clinical efficacy in glioblastoma (GBM), a very aggressive tumor frequently characterized by gloomy prognosis. Unfortunately, the published literature concerning experimental applications of MH in glioblastoma animal models report no data on metformin levels reached in the brain, which, considering the high hydrophilicity of the drug, are likely very low. Therefore, new sensitive analytical methods to be applied on biological tissues are necessary to improve our knowledge of MH in vivo biodistribution and biological effects on tumors. In this research work, a GC-MS method for MH quantification in brain tissues is proposed. MH has been derivatized using N-methyl-bis(trifluoroacetamide), as already described in the literature, but the derivatization conditions have been optimized; moreover, deuterated MH has been selected as the best internal standard, after a comparative evaluation including other internal standards employed in published methods. After ascertaining method linearity, its accuracy, precision, specificity, repeatability, LOD and LOQ (0.373 µM and 1.242 µM, respectively, corresponding to 0.887 and 2.958 pmol/mg of wet tissue) have been evaluated on mouse brain tissue samples, obtained through a straightforward preparation procedure involving methanolic extraction from lyophilized brain homogenates and solid phase purification. The method has been validated on brain samples obtained from mice, either healthy or xenografted with GBM cells, receiving metformin dissolved in the drinking water. This analytical method can be usefully applied in preclinical studies aiming at clarifying MH mechanism of action in brain tumors.

Establishment of a Rodent Glioblastoma Partial Resection Model for Chemotherapy by Local Drug Carriers—Sharing Experience

Local drug delivery systems (LDDS) represent a promising therapy strategy concerning the most common and malignant primary brain tumor glioblastoma (GBM). Nevertheless, to date, only a few systems have been clinically applied, and their success is very limited. Still, numerous new LDDS approaches are currently being developed. Here, (partial resection) GBM animal models play a key role, as such models are needed to evaluate the therapy prior to any human application. However, such models are complex to establish, and only a few reports detail the process. Here, we report our results of establishing a partial resection glioma model in rats suitable for evaluating LDDS. C6-bearing Wistar rats and U87MG-spheroids- and patient-derived glioma stem-like cells-bearing athymic rats underwent tumor resection followed by the implantation of an exemplary LDDS. Inoculation, tumor growth, residual tumor tissue, and GBM recurrence were reliably imaged using high-resolution Magnetic Resonance Imaging. The release from an exemplary LDDS was verified in vitro and in vivo using Fluorescence Molecular Tomography. The presented GBM partial resection model appears to be well suited to determine the efficiency of LDDS. By sharing our expertise, we intend to provide a powerful tool for the future testing of these very promising systems, paving their way into clinical application.

Sex-specific T cell exhaustion drives differential immune responses in glioblastoma

Abstract Sex differences in glioblastoma (GBM) incidence and outcome are well recognized; males show higher incidence and worse prognosis than females. Emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood. Using orthotopic GBM animal models, we recapitulated these sex differences in immune-competent B6 mice but not in immune-deficient mouse strains (NSG and RAG1KO). By depleting CD8+ T cells, we further confirm that CD8+ T cells play a critical role in driving sex differences in survival. Flow cytometry analysis revealed that male CD8+ T cells from tumor expressed higher levels of exhaustion markers such as PD-1, CTLA-4 and Tox, with higher frequency of progenitor exhausted T cell subset whereas female tumor contained more effector-like CD8+ T cells expressing elevated cytokine production. Anti-PD-1 antibody treatment exclusively extended the survival of male mice via inducing decreased exhausted CD8+ T cell subsets and increased effector function and proliferation. Survival analysis on bone marrow chimera model and adoptive transfer model indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, as the sex of donor cells was a critical contributing factor. Finally, we confirmed these observations in GBM patient tumor samples with male tumors having a higher frequency of progenitor exhausted T cells with increased TOX expression. Taken together, these findings demonstrate sex-specific pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy responses. Supported by grants from NIH (R35 NS127083, P01 CA245705).

Mapping of exogenous choline uptake and metabolism in rat glioblastoma using deuterium metabolic imaging (DMI).

There is a lack of robust metabolic imaging techniques that can be routinely applied to characterize lesions in patients with brain tumors. Here we explore in an animal model of glioblastoma the feasibility to detect uptake and metabolism of deuterated choline and describe the tumor-to-brain image contrast. RG2 cells were incubated with choline and the level of intracellular choline and its metabolites measured in cell extracts using high resolution 1H NMR. In rats with orthotopically implanted RG2 tumors deuterium metabolic imaging (DMI) was applied in vivo during, as well as 1 day after, intravenous infusion of 2H9-choline. In parallel experiments, RG2-bearing rats were infused with [1,1',2,2'-2H4]-choline and tissue metabolite extracts analyzed with high resolution 2H NMR to identify molecule-specific 2H-labeling in choline and its metabolites. In vitro experiments indicated high uptake and fast phosphorylation of exogenous choline in RG2 cells. In vivo DMI studies revealed a high signal from the 2H-labeled pool of choline + metabolites (total choline, 2H-tCho) in the tumor lesion but not in normal brain. Quantitative DMI-based metabolic maps of 2H-tCho showed high tumor-to-brain image contrast in maps acquired both during, and 24 h after deuterated choline infusion. High resolution 2H NMR revealed that DMI data acquired during 2H-choline infusion consists of free choline and phosphocholine, while the data acquired 24 h later represent phosphocholine and glycerophosphocholine. Uptake and metabolism of exogenous choline was high in RG2 tumors compared to normal brain, resulting in high tumor-to-brain image contrast on DMI-based metabolic maps. By varying the timing of DMI data acquisition relative to the start of the deuterated choline infusion, the metabolic maps can be weighted toward detection of choline uptake or choline metabolism. These proof-of-principle experiments highlight the potential of using deuterated choline combined with DMI to metabolically characterize brain tumors.

Abstract 3498: GTP-dependent signaling links metabolism, non-homologous end joining and treatment responses in cancer and normal tissues

Abstract Introduction/Objectives: Purines regulate DNA repair in brain tumors through uncertain mechanisms. We sought to define the mechanisms by which purines regulate DNA repair and therapy response. Methods: Crispr/Cas9, siRNA, and CDNA overexpression and immunoblot were used to modulate and confirm protein levels. γ-H2AX and Rad51 foci were enumerated using immunofluorescence. Fluorescent reporter assays were performed to detect DNA repair activity. Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after RT and antibodies generated against novel sites. Celltiter-glo or clonogenic assay were performed to evaluate drug/RT responses. IHC was used to detect protein expression. Animal models of glioblastoma and normal tissue (irradiation of GI system) were used to assess DNA repair and treatment responses in vivo. Results: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT, slowed the repair of RT-induced DSBs and inhibited the activity of non-homologous end joining (NHEJ), but not homologous recombination (HR). These effects could be rescued by GTP (but not ATP) supplementation. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced DSB repair and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to the small GTP-activated protein Rac1. Expression of constitutively active Rac1 promoted the dephosphorylation of Abi-1 (S323) and DSB repair while these effects were blocked by dominant negative Rac1. Pharmacogenomic inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. These findings have therapeutic relevance. In GBM PDX samples, levels of p-Abi-1 (S323) negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibition of Rac1 enhanced RT responses and suppressed Abi-1 S323 dephosphorylation. Abi-1 knockout enhanced efficacy of numerous genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in normal astrocytes and enteroids and protected mice from RT-mediated gastrointestinal injury. Conclusions: GTP promotes DNA repair by activating Rac1 and PP5 to dephosphorylate Abi-1 and stimulate NHEJ. Disrupting this regulation could improve brain tumor treatment responses while augmenting it could help protect normal tissues from genotoxic injury. Citation Format: Weihua Zhou, Angelica Lin, Zitong Zhao, John Yang, Kari Wilder-Romans, Jie Xu, Sumeet Solanki, Jing Li, Annabel Yang, Andrew Scott, Ayesha Kothari, Erik Peterson, Navyateja Korimerla, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra Obrien, Sean Ferris, Shuang Zhao, Jann N. Sarkaria, Meredith Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Yatrik Shah, Daniel R. Wahl. GTP-dependent signaling links metabolism, non-homologous end joining and treatment responses in cancer and normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3498.

Abstract 705: Potentiation of immunotherapy by LSD1 modulation

Abstract LSD1 has emerged as a potential therapeutic target to increase the effectiveness of cancer immunotherapy. We have developed a series of novel small molecules, exemplified by the lead substance BEA-17, that modulates LSD1 via binding to an allosteric site, without directly inhibiting its enzymatic activity. In cells, BEA-17 induces a reduction of LSD1 levels. In addition, BEA-17 upregulates the expression of endogenous retroviral genes and T cell-attractant chemokines and does so in an LSD1-dependent manner. In a co-culture of HeLa and PBMCs, BEA-17 increases cell kill of cancer cells by immune effector T cells, also in an LSD1-dependent manner. In a CT26 syngeneic animal model of colon cancer, BEA-17 potentiates the activity of anti-PD1 inhibitors. Finally, in a syngeneic GL261 animal model of glioblastoma, BEA-17 increases the effectiveness of standard-of-care temozolomide + radiation. Citation Format: Wei B. Emond, Rajiv Sawant, Matthis Geitmann, Johan Winquist, Peter Brandt, Ulf Bremberg, Per Källblad, Vendela Parrow, Claes Andersson, Kristin Blom, Nasrin Najafi, Tobias Bergström, Fredrik J. Swartling, Mats Hellström, Konrad F. Koehler. Potentiation of immunotherapy by LSD1 modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 705.

Protein disulfide-isomerase A4 confers glioblastoma angiogenesis promotion capacity and resistance to anti-angiogenic therapy

IntroductionIncreasing evidence has revealed the key activity of protein disulfide isomerase A4 (PDIA4) in the endoplasmic reticulum stress (ERS) response. However, the role of PDIA4 in regulating glioblastoma (GBM)-specific pro-angiogenesis is still unknown.MethodsThe expression and prognostic role of PDIA4 were analyzed using a bioinformatics approach and were validated in 32 clinical samples and follow-up data. RNA-sequencing was used to search for PDIA4-associated biological processes in GBM cells, and proteomic mass spectrum (MS) analysis was used to screen for potential PDIA4 substrates. Western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assays (ELISA) were used to measure the levels of the involved factors. Cell migration and tube formation assays determined the pro-angiogenesis activity of PDIA4 in vitro. An intracranial U87 xenograft GBM animal model was constructed to evaluate the pro-angiogenesis role of PDIA4 in vivo.ResultsAberrant overexpression of PDIA4 was associated with a poor prognosis in patients with GBM, although PDIA4 could also functionally regulate intrinsic GBM secretion of vascular endothelial growth factor-A (VEGF-A) through its active domains of Cys-X-X-Cys (CXXC) oxidoreductase. Functionally, PDIA4 exhibits pro-angiogenesis activity both in vitro and in vivo, and can be upregulated by ERS through transcriptional regulation of X-box binding protein 1 (XBP1). The XBP1/PDIA4/VEGFA axis partially supports the mechanism underlying GBM cell survival under ER stress. Further, GBM cells with higher expression of PDIA4 showed resistance to antiangiogenic therapy in vivo.ConclusionsOur findings revealed the pro-angiogenesis role of PDIA4 in GBM progression and its potential impact on GBM survival under a harsh microenvironment. Targeting PDIA4 might help to improve the efficacy of antiangiogenic therapy in patients with GBM.

GD2 CAR-T cells in combination with Nivolumab exhibit enhanced antitumor efficacy

Glioblastoma (GBM) is a common primary brain tumor with poor clinical prognosis. Although CAR-T therapy has been trialed for treatment of GBM, the outcomes are sub-optimal possibly due to exhaustion of T cells and life-threatening neurotoxicity. To address these issues, a combined therapeutic strategy was tested in the current study using GD2 CAR-T together with Nivolumab - an anti-PD-1 monoclonal antibody. An effector-to-target co-culture system was established to evaluate the short-term and long-term cytotoxicity of CAR-T, as well as to investigate the inhibitory activity and T cell exhaustion associated with the PD-1/PD-L1 signaling pathway. Orthotopic NOD/SCID GBM animal models were generated to evaluate the safety and efficacy of the combined therapeutic strategy at various dosages of GD2 CAR-T with Nivolumab. GD2 CAR-T exhibited significant antigen-specific cytotoxicity in a dose-dependent manner in vitro. The persistence of cytotoxicity of GD2 CAR-T could be enhanced by addition of Nivolumab in the co-culture system. Animal studies suggested that GD2 CAR-T effectively infiltrated into tumor tissue and significantly hampered tumor progression. The optimal therapeutic outcome was obtained via using the medium dosage of CAR-T with Nivolumab, which displayed the highest efficacy in extending the survival up to 60 days. Further investigation of toxicity revealed that high-dosage of GD2 CAR-T could induce tumor apoptosis through p53/caspase-3/PARP signaling pathway. This study suggests that GD2 CAR-T in combination with Nivolumab may offer an improved therapeutic strategy for treatment of GBM.