Brain Tumor Model Research Articles

Abstract P45: REDUCED IMMUNOGENICITY DRIVES SUPERIORITY OF THE NANOLUCIFERASE (NLUC) REPORTER IN AN IMMUNOCOMPETENT GL261 MOUSE GLIOMA MODEL

Abstract Bioluminescence imaging (BLI) with luciferase-based reporters is a robust approach for monitoring tumour growth and treatment response in preclinical in vivo cancer models. Luciferases are considered foreign molecules that could elicit immune responses when introduced in immunocompetent live animal models [1, 2]. The immunogenicity of luciferases and other transgenic imaging reporters, though often overlooked, was recently discussed in relation to mouse cancer models [3-5]. Herein, we investigated the immunogenicity of two luciferase reporters: (1) red-shifted firefly luciferase (RFLuc, 61 kDa) and (2) deep-sea shrimp nanoluciferase (nLuc, 19 kDa) in an orthotopic brain tumour model using GL261 mouse glioma cells in a syngeneic C57BL/6J mouse. We evaluated their suitability for monitoring of brain tumour growth kinetics by BLI. Mice were implanted with 2 × 104 either GL261, GL261-NLuc or GL261-RFLuc cells in the right cerebrum using a stereotaxic device. GL261-RFLuc tumours could only be detected by BLI for 2 weeks, and the mice did not die of disease (median overall survival; mOS > 35 days). In contrast, GL261-nLuc cells formed lethal brain tumours that grew in volume as determined by BLI. GL261-NLuc implanted mice exhibited mOS comparable to mice implanted with GL261 cells (24.5 days vs. 23 days). These observations indicated that nLuc is not significantly immunogenic in mouse brains and did not cause rejection of the tumour cells as observed with RFluc. Cytokine expression profiles and immune cell profiles of the tumour-bearing brain hemispheres were comparable between tumours from GL261 and GL261-NLuc. In contrast, GL261-RFLuc tumours exhibited elevated CD8+ T cells, particularly effector (CD62L¯CD44+) and granzyme B-expressing (GZB+) CD8+ T cells. GL261-nLuc formed stable glioma tumors comparable to the original GL261 mouse model and is a suitable in vivo preclinical model for evaluating experimental therapeutic interventions against gliomas, when paired with Promega’s proprietary fluoro-furimazine substrate. Funding: This research was supported by Singapore’s Health and Biomedical Sciences (HBMS) Industry Alignment Fund Pre-Positioning (IAF-PP) grant H18/01/a0/018 (to AMC) administered by A*STAR and Singapore-MIT Alliance for Research and Technology (SMART) Innovation Grant (ING-000913 BIO) (to AMC). Citation Format: Carla Bianca Luena Victorio, Wisna Novera, Arun Ganasarajah, Joanne Ong, Peter Li, Sven Hans Petersen, Rasha Msallam, Ann-Marie Chacko. REDUCED IMMUNOGENICITY DRIVES SUPERIORITY OF THE NANOLUCIFERASE (NLUC) REPORTER IN AN IMMUNOCOMPETENT GL261 MOUSE GLIOMA MODEL [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P45.

Optimised ResNet50 for Multi-Class Classification of Brain Tumors

Categorizing brain cancers, including glioma, meningioma, and pituitary tumors, based on magnetic resonance imaging (MRI) images presents a significant challenge. Deep learning and machine learning techniques have shown promise in enhancing image categorization. To address this challenge, we leverage the power of the optimized ResNet50 model. Our approach involves classifying medical images using Convolutional Neural Network (CNN) features, which are then compared with the ResNet50 model. The primary goal is to detect brain tumors at an early stage using an advanced deep-learning model. We utilize an accessible dataset from Figshare, containing MRI images of the three distinct categories of brain tumors. Existing brain tumor models face limitations in handling multi-class problems and early-stage diagnosis. Therefore, we propose a fully automated approach employing Convolutional Neural Networks (CNN) to extract diverse properties from brain MRI scans. This method aims to provide accurate tumor diagnosis, even with a high number of classes and limited information in MRI data. Our proposed model involves the creation of identification blocks within a four-layered primary architecture, followed by testing and assessment of the interconnected layers. The results demonstrate that our model outperforms existing methods, achieving an impressive overall classification accuracy of 99.03%,

Abstract CT056: Development of a novel next-generation RET inhibitor

Abstract Oncogenic RET alterations confer constitutive activation in multiple types of human cancers. Two first-generation selective RET inhibitors selpercatinib and pralsetinib have been approved for patients with RET-driven solid tumors including non-small cell lung cancer (NSCLC) and thyroid cancer. However, critical unmet medical needs remain due to drug resistance caused by RET mutations in patients treated with the first-generation RET inhibitors. To address this emerging clinical issue, we discovered a highly potent and selective RET inhibitor with novel chemical scaffold through structure-based drug design, APS03118. Preclinical studies showed APS03118 had stronger in vitro and in vivo biological activity than the first-generation RET inhibitors targeting a broad range of RET fusions and mutations. Importantly, APS03118 was highly potent against a series of resistant RET mutations including the solvent front mutations RET G810C/R/S (IC50 0.04-5.72 nM) and gatekeeper mutations RET V804E/L/M (IC50 0.04-1.36 nM). Off-target screen analysis using the 468-kinase panel demonstrated APS03118 was highly selective for RET. APS03118 was orally bioavailable with good ADME and PK properties and markedly suppressed tumor growth in various RET-driven mouse xenograft tumor models (tumor growth inhibition 87-108% at 10-30 mg/kg BID). In addition, APS03118 exhibited excellent brain penetrating properties with strong efficacy in the in vivo orthotopic brain tumor model. GLP toxicity studies showed acceptable safety profiles. APS03118 received IND approval from both US FDA and China NMPA, and was granted the fast-track designation by FDA. We recently achieved First-in-Human milestone for APS03118 and the Ph 1 clinical trial is ongoing. As of January 5, 2024, three dose levels have been tested. Plasma exposure of APS03118 increased proportionally following single and repeated administration of APS03118 at different doses. APS03118 was well tolerated in NSCLC and thyroid cancer patients harboring RET fusions or mutations relapsed on the chemotherapy, multi-kinase inhibitor and first-generation RET inhibitor therapy. In addition to the favorable safety profiles, we observed sign of efficacy in the RET altered cancer patients in response to APS03118 treatment even in the early clinical trial testing. Partial response and stable disease were observed in patients 2-4 months after administration of APS03118 and dose limiting toxicity (DLT) dose has not been reached. Dose escalation for APS03118 is continuing. Taken together, APS03118 is a novel highly potent and selective next-generation RET inhibitor overcoming resistance following treatment with the first-generation RET inhibitors. APS03118 warrants further clinical development. Citation Format: Jun Zhong, Ruxue Xiao, Wenya Xie, Minchun Chen, Xiaohu Chen, Hao Wang, Yongbo Liu, Qin Gao, Xiupeng Wang, Zongbao Wang, Qian Chen, Jing Yang, Yizhe Ji, Yuxun Wang. Development of a novel next-generation RET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT056.

Abstract LB021: BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation

Abstract The excessive expression of antiapoptotic members in the B-cell leukemia/lymphoma-2 (BCL-2) family proteins emerges as a noteworthy oncogenic target in cancer cells. Employing a multistep virtual screening and filtering approach that integrates structure and ligand-based drug design, we aimed to identify novel and potent BCL-2 inhibitors from a sizable small molecule database, comprising over 210,000 compounds. Subsequently, seven selected molecules underwent rigorous biological activity tests in human cancer cell lines and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays. Four of these compounds exhibited notable inhibitory effects on cancer cell proliferation and led to an increase in apoptotic cell fractions. Among the identified compounds, BAU-243 displayed a substantial affinity for Bcl-2, demonstrating efficacy in reducing glioblastoma (GBM) cell proliferation, including cancer-initiating cells. Notably, in contrast to the selective Bcl-2 inhibitor ABT-199, BAU-243 induced autophagic cell death in GBM cells. In vivo investigations using orthotopic brain tumor models revealed a significant reduction in tumor growth with BAU-243 compared to both the vehicle group and animals treated with ABT-199. Computational modeling simulations, in concordance with in vitro findings, proposed that BAU-243 activates autophagic cell death by disrupting the Beclin 1:Bcl-2 complex. This positions BAU-243 as a promising small molecule for the treatment of GBM. Citation Format: Seyma Calis, Berna Dogan, Asuman Celebi, Ozlem Yapicier, Turker Kilic, Eda Tahir Turanli, Timucin Avsar, Serdar Durdagi. BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB021.

Tumor suppressor role of the complement inhibitor CSMD1 and its role in TNF-induced neuroinflammation in gliomas

BackgroundThe complement inhibitor CSMD1 acts as a tumor suppressor in various types of solid cancers. Despite its high level of expression in the brain, its function in gliomas, malignant brain tumors originating from glial cells, has not been investigated.MethodsThree cohorts of glioma patients comprising 1500 patients were analyzed in our study along with their clinical data. H4, U-118 and U-87 cell lines were used to investigate the tumor suppressor function of CSMD1 in gliomas. PDGFB-induced brain tumor model was utilized for the validation of in vitro data.ResultsThe downregulation of CSMD1 expression correlated with reduced overall and disease-free survival, elevated tumor grade, wild-type IDH genotype, and intact 1p/19q status. Moreover, enhanced activity was noted in the neuroinflammation pathway. Importantly, ectopic expression of CSMD1 in glioma cell lines led to decreased aggressiveness in vitro. Mechanically, CSMD1 obstructed the TNF-induced NF-kB and STAT3 signaling pathways, effectively suppressing the secretion of IL-6 and IL-8. There was also reduced survival in PDGFB-induced brain tumors in mice when Csmd1 was downregulated.ConclusionsOur study has identified CSMD1 as a tumor suppressor in gliomas and elucidated its role in TNF-induced neuroinflammation, contributing to a deeper understanding of glioma pathogenesis.

492 Novel BH3 Mimetic Bcl-2 Inhibitor Promotes Autophagic Cell Death and Reduces in Vivo Glioblastoma Tumor Growth

INTRODUCTION: Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma, the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. METHODS: In silico studies resulted in 500,000 molecules being screened with computer-aided drug design methods and the molecule with the highest binding affinity was selected and named BAU-243. Cell viability, IC50 value, tumor formation potential, tumor spheroid formation potential, tumor invasion studies were investigated following BAU-243 application. The molecular changes caused by Bcl-2 inhibition were demonstrated by quantitative PCR, western blot and immunofluorescence methods. In in vivo studies, glioma tumors were transplanted intracranially into Nod Scid Gamma(NSG) mice using the mouse xenograft model. RESULTS: Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall glioblastoma cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. CONCLUSIONS: Results indicated that BAU-243 activates autophagic cell death by disrupting Beclin 1:Bcl-2 complex, and may serve as a potential small molecule for the treatment of glioblastoma.

Abstract 1961: Discovery of a highly potent and brain penetrant fourth generation EGFR tyrosine kinase inhibitor

Abstract Third generation EGFR tyrosine kinase inhibitors (TKIs) such as Osimertinib have emerged as first-line standard of care therapy to treat non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (del19, L858R or T790M). However, within 12 to 18 months following treatment, 15-20% of the patients develop C797S mutation, which drives resistance to these drugs; with the progression of disease brain metastasis is observed in 40-70% of the EGFRm+ NSCLC patients. Here, we report a highly potent and brain penetrant fourth generation EGFR-TKI ACE-34237307, designed to overcome C797S mutation (del19/C797S and L858R/C797S), and common single point mutations (del19 and L858R). ACE-34237307 is also designed to potently inhibit the more prevalent non-canonical EGFR mutations such as G719S and L861Q. ACE-34237307 showed robust anti-proliferative activity against EGFR double mutations in Ba/F3 (EGFR-del19/C797S and EGFR-L858R/C797S) cell lines with IC50 values of 0.1 and 0.23 nM, respectively. In phospho-EGFR assay, ACE-34237307 demonstrates over 79-fold selectivity against A431 (EGFR-WT) cell line when compared to PC-9 (EGFR-del 19) cell line. ACE-34237307 is highly brain penetrant with B/P ratio of 2.3 in rat and 1.2 in mouse. It has an excellent pharmacokinetic profile in rat, mouse and dog with good bioavailability, a flat PK curve and reduced Cmax, which will translate to a better safety profile. The in vivo anti-tumor efficacy was evaluated in Osimertinib resistant double mutant Ba/F3-EGFR-del19/C797S and L858R/C797S as well as del19 and L858R single mutant xenograft models. It has been shown that ACE-34237307 at doses of ≥ 0.6 mg/kg induces complete tumor regression or eradication in all tumor models tested. In brain tumor models with Ba/F3 (EGFR-del19/C797S) and PC-9 (EGFR-del19), ACE-34237307 showed complete inhibition or eradication of brain tumors and drastically extended animal survival at ≥ 0.6 mg/kg. In summary, ACE-34237307 is a potent fourth generation EGFR-TKI with high WT selectivity, excellent brain penetration, highly favorable DMPK profile, outstanding in vivo efficacy in subcutaneous and brain tumor models and low projected human dose amenable to QD dosing. Citation Format: sanjeev kumar, junmei wang, wenqian li, weitao pan, zhiming wen, Kuo-Long yu, Genshi Zhao, tinggui yin. Discovery of a highly potent and brain penetrant fourth generation EGFR tyrosine kinase inhibitor [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 1961.

Abstract 4526: Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302

Abstract The inhibition of ribosome biogenesis through pharmacological means has drawn attention as a potential strategy in cancer therapy. In our previous study we highlighted a novel property of the FDA-approved antimalarial drug amodiaquine, demonstrating its ability to dose-dependently inhibit ribosomal RNA (rRNA) transcription, a critical step in ribosome biogenesis (Espinoza JA, et al Cell Death Diff, 2020). Amodiaquine was found to induce the degradation of the catalytic subunit of RNA polymerase I, known as POLR1A (RPA194), and resulting in nucleolar stress and the stabilization of p53. Notably, RNA Pol I shutdown occurred independently of DNA damage. Motivated by these findings, we sought to find additional inhibitors of ribosome biogenesis. Acridine derivatives display anti-cancer properties. The activity of acridines is mainly attributed to the planarity of these structures, which can intercalate within the DNA structure, and inhibition of chromatin associated protein targets including topoisomerases. JP-1302 is an experimental drug, on a 9-aminoacridine structural scaffold, acting as a selective alpha-2 adrenergic receptor antagonist. It was recently shown that JP-1302 inhibits transcription, blocking RNA Pol II phosphorylation and inhibiting p21 expression at a concentration of 10 μM (Mitchell DC, et al Nat Biotech, 2023). Given JP-1302's structural similarity to other acridine derivatives such as aminacrine, ethacridine, and the experimental compound BMH-21, all known to inhibit RNA Pol I transcription, we hypothesized that JP-1302 might share similar properties. The purpose of our new study was to evaluate JP-1302´s effect on ribosome biogenesis, p53, and cancer cell growth. We show here that JP-1302 acts as an inhibitor of RNA Pol I transcription. Treatment with JP-1302 induced a rapid decrease in the levels of POLR1A, cessation of rRNA synthesis and nucleolar stress already within six hours and at a concentration of 0.5 μM. JP-1302 treatment stabilized wild type p53 and enhanced p21 expression in the osteosarcoma line U2OS. RNA Pol I shutdown occurred in the absence of DNA damage response, as determined by gH2A.X staining. JP-1302 impaired the growth of both p53 wild type and p53 null HCT116 cells with GI50 values of around 0.6 μM, as well as in U2OS cells at 0.8 μM, as determined after 48 hours. Paradoxically, at very low concentrations JP-1302 appeared to transiently stimulate RNA synthesis, a phenomenon that will require further investigation. In conclusion, we identified JP-1302 as a novel inhibitor of ribosomal RNA synthesis, exerting this effect at concentrations that do not induce DNA damage, and independently of p53. Given that JP-1302 passes the blood-brain barrier in rodents, and accumulating in different brain regions, it is of future interest to evaluate this drug in brain tumor models. Citation Format: Sheetanshu Saproo, Styliani Papadaki, Jaime A. Espinoza, Jiri Bartek, Mikael S. Lindström. Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302 [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 4526.

Abstract 4137: Feasibility of a novel image guided injection tool for non-surgical implantation of orthotopic tumors

Abstract The purpose of this study was to evaluate a novel device for non-surgical implantation of tumor cells using ultrasonic (US) image guidance. There are a variety of mouse model cancer types (e.g. sub-cu, orthotopic, genetically engineered). Orthotopic tumors are increasingly prevalent but usually require surgery, which reduces throughput and injures the surrounding anatomy. Injections are an alternative to surgery, but usually require image guidance or extensive training. US guided injection systems exist, but are suboptimal because they require significant training, as the US beam is not necessarily coplanar with needle location or trajectory, reducing accuracy and throughput. We tested a new approach to image guided injection which ensures that the US beam is coplanar with the needle. To test the efficacy of the image guided injection device across a variety of common orthotopic tumors, we delivered luc-tagged cells to the prostate (2.5e5 cells in 40 µL, PC3, N=8), liver (2e6 cells in 100 µL, Hep G2, N=5), and brain (1.75e5 cells in 100 µL, U87-MG, N=3). The brain tumor model was introduced via intracardiac injection to simulate metastases. Between 3 and 8 mice were injected for each tumor model. For injection, animals were anesthetized and positioned on motion stage. A 1 mL syringe was mounted to the adjustable injection gantry with the needle co-planar with the US beam, and the target organ was visualized with US. Cells were delivered to the targets using the US live stream as a guide. Between 1 to 3 weeks after the injections, tumors were confirmed via BLI with an IVIS (Revvity, Waltham, MA, USA). For each of the three tumor cell lines, animal preparation and injections could be performed in 10 mins by recently-trained novice users. All three cell lines could be effectively delivered to the orthotopic host tissues, and this was confirmed by using BLI of the cells. This study represents the first time a co-planar US beam and needle delivery mechanism was used for the purpose of non-surgical implantation of orthotopic tumors. Future developments of this platform could include improvements to automation of needle placement and trajectory to ensure successful payload delivery by novice users. Citation Format: Thomas M. Kierski, Juan D. Rojas, Nathan Beaumont, Theresa Charity, Robert D. Marek, Kenneth H. Young, Gregory M. Palmer, Ryan C. Gessner, Tomek J. Czernuszewicz. Feasibility of a novel image guided injection tool for non-surgical implantation of orthotopic tumors [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 4137.

Abstract 7458: Inhibition of brain cancer progression via resolution of inflammation

Abstract While inflammation is a hallmark of cancer, the mechanisms that disrupt its resolution in cancer are not well understood. A paradigm shift is emerging in our understanding that the resolution of inflammation is an active process controlled by specialized pro-resolving mediators (SPMs), including protectins and resolvins, which are endogenous lipid autacoids present in multiple tissues including the brain and cerebrospinal fluid. SPMs counter-regulate pro-inflammatory cytokine production and stimulate phagocytosis of cellular debris (“pro-resolution”) without being immunosuppressive. We hypothesized treatment with pro-resolving agonists such as SPMs presents a novel debris-clearing and resolution-enhancing therapeutic approach to complement cytotoxic brain cancer therapies. Here, we demonstrate the failure of resolution of inflammation via dysregulated SPMs may be a critical risk factor for aggressive brain cancer growth. Moreover, chemotherapy and targeted therapy further disrupt inflammation resolution via a tumor cell death (“debris”)-induced cytokine storm by macrophages. The SPM receptors GPR37 (protectin D1), GPR18/DRV2 (resolvin (Rv) D2), GPR32 (RvD1), and ChemR23/ERV (RvE1) are expressed in tumor-infiltrating macrophages and endothelial cells in the brain tumor microenvironment. Plasma levels of SPMs were significantly reduced in the medulloblastoma-bearing mice, suggesting that brain cancer progression is associated with a failure of endogenous resolution of inflammation. To determine whether failed resolution in brain cancer can be rescued by lipid mediator agonists, we systemically administered SPMs to subcutaneous and orthotopic glioblastoma and medulloblastoma murine brain tumor models. Significantly, RvD1 and RvD2 each inhibited the growth of human glioblastoma tumors by 15-fold at nanogram concentrations without toxicity. Likewise, RvD2 inhibited the growth of human medulloblastoma tumors by 3-fold. Protectins, PDX and PD1n-3, each inhibited growth of human medulloblastoma in mice by approximately 5-fold. Systemic administration of PDX or RvD2 prolonged survival of mice injected with orthotopic human D556 medulloblastoma and also glioblastoma models. Flow cytometry analysis of orthotopic glioblastoma tumors revealed increased immune infiltration into the brain tumor microenvironment in the PCTR1-treatment group when compared to the tumor-bearing non-treated group. At nanogram per day doses, protectins and resolvins suppressed orthotopic debris-stimulated brain tumor growth by activating macrophage and microglial phagocytosis of tumor cell debris and counter-regulating pro-inflammatory cytokines, including TNF-α, CCL2, CXCL1, and CCL4. Thus, stimulating the resolution of inflammation via the clearance of cellular debris with pro-resolving agonists represents a novel therapeutic approach to brain cancer, including glioblastoma and medulloblastoma. Citation Format: Jacqueline Capuano, Kimberly Vasquez, Katherine Quinlivan, Rachel Bayer, Sarina Virani, Michael Gillespie, Mark Kieran, Charles Serhan, Diane Bielenberg, Dipak Panigrahy. Inhibition of brain cancer progression via resolution of inflammation [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 7458.

Abstract 657: Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects

Abstract KRAS is the most frequently dysregulated oncogene with high prevalence in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). Currently, FDA-approved sotorasib and adagrasib provide ground breaking through therapies for cancer patients harboring KRAS G12C mutation. However, there is still high unmet medical needs for inhibitors targeting broader KRAS-driven tumor, especially a substantial unmet medical need for agents that can penetrate BBB to control the brain metastases. SHP2, a key activator of KRAS located in the upstream of RAS pathway, facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state serving as a key mechanism of KRAS activation. Therefore, SHP2 could be a promising therapeutic target to shut down the oncogenic RAS pathway as a broad combination partner with RAS/RAF/MEK/ERK pathway inhibitors. Here we report a highly potent and selective brain penetrant SHP2 inhibitor that is designed to target solid tumors metastazied to brain. KT-01766 showed excellent potency against SHP2 protein in various biochemical and cellular assays and showed the robust synergistic effect with RAS pathway inhibitors. KT-01766 as a mono therapy and in combination with KRAS G12C inhibitors demonstrated great anti-tumor efficacy with no apparent toxicity in mouse xenograft models harboring KRAS mutations. Furthermore, robust anti-tumor acitivty was confirmed in intracranial brain tumor model, supporting its potential to control brain-metastasized tumors. The present data suggest that KT-01766 is a potential candidate for combination therapy with agents targeting RAS pathway, as it is able to effectively suppress tumor growth both systemically and within the brain in KRAS mutated cancers. Citation Format: Miyeon Kim, Dongsu Kim, Yeejin Jeon, Kyeong Jin Yoon, Anna Jang, Mijung Lee, Dahye Jeon, Soyeon Jang, Jinhwan Kim, Eunji Kim, Jihoon Park, Victor Hong, Hayoun Jung, Sungpil Choi. Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects [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 657.

Abstract 4140: Enhanced microglial phagocytosis of metastatic brain tumor cells with Cd24a/Cd47 depletion

Abstract Despite the robust homeostasis of the brain, its high prevalence of tumor metastasis is intriguing and the underlying mechanism may contribute to the development of novel therapies. Microglia have emerged as promising therapeutic targets due to their role in brain parenchymal homeostasis. However, with evidence for both pro- and anti-tumorigenic functions, their role in brain metastasis (BrM) remains unclear. Here, we show that the microglial response to tumor cells within a single host is heterogeneous and can alter tumor fate. Single-cell RNA sequencing was performed on five surgically resected metastatic brain tumors, and the transcriptome of tumor-associated microglia was extracted by single-cell variational inference (scVI). Evidence for the coexistence of cytokine-secreting and angiogenesis-supporting subsets, as well as subsets associated with phagocytosis and antigen presentation, within each tumor sample was determined. To demonstrate the intra-tumoral functional heterogeneity of tumor-associated microglia, we established a metastatic brain tumor model that allows us to visualize the metastatic process and the dynamic microglial response in vivo by injecting mCherry-tagged allogeneic cancer cells into the internal carotid artery of CX3CR1-EGFP mice. Metastasis formation was suppressed in microglia-depleted mice, identifying a dominant microglial response that promotes tumor growth. However, continuous in vivo imaging of the BrM model revealed tumor-eradicating anti-tumor microglia coexisting with pro-tumor microglia in the same host. The transcriptome of anti-tumor microglia supported that they are enriched for phagocytosis, antigen processing and antigen presentation genes. The subset was identified in microglia extracted from human samples. We then examined the immune checkpoint of microglial phagocytosis in the tumor transcriptome and identified Cd24a and Cd47. Simultaneous deletion of both genes in cancer cells enhanced microglial phagocytosis, reduced tumor volume and improved survival, and rescue of both genes reversed these anti-tumor effects. The microglial dependency was addressed by microglial depletion experiments, and the anti-tumor effects were reproducible in nude mice lacking mature T cells. These results suggest that there are distinct immune cell subtypes that respond to lung tumor metastases in the brain and either promote tumor growth or inhibit metastasis through phagocytosis. Furthermore, anti-tumor activity in mouse models can be enhanced by genetic deletion of Cd24a and Cd47 in tumor cells, which regulate phagocytosis by microglia. Citation Format: Takahiro Tsuji, Haruka Hirose, Mariko Shindo, Rahadian Yudo Hartantyo, Yutaro Saito, Kazutaka Hosoya, Hiroshi Yoshida, Daisuke Kato, Akihiko Yoshizawa, Yoshiki Arakawa, Hiroaki Ozasa, Toyohiro Hirai, Hiroaki Wake. Enhanced microglial phagocytosis of metastatic brain tumor cells with Cd24a/Cd47 depletion [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 4140.

Enhancing Photothermal/Photodynamic Therapy for Glioblastoma by Tumor Hypoxia Alleviation and Heat Shock Protein Inhibition Using IR820-Conjugated Reduced Graphene Oxide Quantum Dots.

We use low-molecular-weight branched polyethylenimine (PEI) to produce cytocompatible reduced graphene oxide quantum dots (rGOQD) as a photothermal agent and covalently bind it with the photosensitizer IR-820. The rGOQD/IR820 shows high photothermal conversion efficiency and produces reactive oxygen species (ROS) after irradiation with near-infrared (NIR) light for photothermal/photodynamic therapy (PTT/PDT). To improve suspension stability, rGOQD/IR820 was PEGylated by anchoring with the DSPE hydrophobic tails in DSPE-PEG-Mal, leaving the maleimide (Mal) end group for covalent binding with manganese dioxide/bovine serum albumin (MnO2/BSA) and targeting ligand cell-penetrating peptide (CPP) to synthesize rGOQD/IR820/MnO2/CPP. As MnO2 can react with intracellular hydrogen peroxide to produce oxygen for alleviating the hypoxia condition in the acidic tumor microenvironment, the efficacy of PDT could be enhanced by generating more cytotoxic ROS with NIR light. Furthermore, quercetin (Q) was loaded to rGOQD through π-π interaction, which can be released in the endosomes and act as an inhibitor of heat shock protein 70 (HSP70). This sensitizes tumor cells to thermal stress and increases the efficacy of mild-temperature PTT with NIR irradiation. By simultaneously incorporating the HSP70 inhibitor (Q) and the in situ hypoxia alleviating agent (MnO2), the rGOQD/IR820/MnO2/Q/CPP can overcome the limitation of PTT/PDT and enhance the efficacy of targeted phototherapy in vitro. From in vivo study with an orthotopic brain tumor model, rGOQD/IR820/MnO2/Q/CPP administered through tail vein injection can cross the blood-brain barrier and accumulate in the intracranial tumor, after which NIR laser light irradiation can shrink the tumor and prolong the survival times of animals by simultaneously enhancing the efficacy of PTT/PDT to treat glioblastoma.

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Quasi-steady-state (QUASS) reconstruction enhances T1 normalization in apparent exchange-dependent relaxation (AREX) analysis: A reevaluation of T1 correction in quantitative CEST MRI of rodent brain tumor models.

The apparent exchange-dependent relaxation (AREX) analysis has been proposed as an effective means to correct T1 contribution in CEST quantification. However, it has been recognized that AREX T1 correction is not straightforward if CEST scans are not performed under the equilibrium condition. Our study aimed to test if quasi-steady-state (QUASS) reconstruction could boost the accuracy of the AREX metric under common non-equilibrium scan conditions. Numerical simulation and in vivo scans were performed to assess the AREX metric accuracy. The CEST signal was simulated under different relaxation delays, RF saturation amplitudes, and durations. The AREX was evaluated as a function of the bulk water T1 and labile proton concentration using the multiple linear regression model. AREX MRI was also assessed in brain tumor rodent models, with both apparent CEST scans and QUASS reconstruction. Simulation showed that the AREX calculation from apparent CEST scans, under non-equilibrium conditions, had significant dependence on labile proton fraction ratio, RF saturation time, and T1. In comparison, QUASS-boosted AREX depended on the labile proton fraction ratio without significant dependence on T1 and RF saturation time. Whereas the apparent (2.7 ± 0.8%) and QUASS MTR asymmetry (2.8 ± 0.8%) contrast between normal and tumor regions of interest (ROIs) were significant, the difference was small. In comparison, AREX contrast between normal and tumor ROIs calculated from the apparent CEST scan and QUASS reconstruction was 3.8 ± 1.1%/s and 4.4 ± 1.2%/s, respectively, statistically different from each other. AREX analysis benefits from the QUASS-reconstructed equilibrium CEST effect for improved T1 correction and quantitative CEST analysis.

A clinically relevant computed tomography (CT) radiomics strategy for intracranial rodent brain tumour monitoring

Here, we establish a CT-radiomics based method for application in invasive, orthotopic rodent brain tumour models. Twenty four NOD/SCID mice were implanted with U87R-Luc2 GBM cells and longitudinally imaged via contrast enhanced (CE-CT) imaging. Pyradiomics was employed to extract CT-radiomic features from the tumour-implanted hemisphere and non-tumour-implanted hemisphere of acquired CT-scans. Inter-correlated features were removed (Spearman correlation > 0.85) and remaining features underwent predictive analysis (recursive feature elimination or Boruta algorithm). An area under the curve of the receiver operating characteristic curve was implemented to evaluate radiomic features for their capacity to predict defined outcomes. Firstly, we identified a subset of radiomic features which distinguish the tumour-implanted hemisphere and non- tumour-implanted hemisphere (i.e, tumour presence from normal tissue). Secondly, we successfully translate preclinical CT-radiomic pipelines to GBM patient CT scans (n = 10), identifying similar trends in tumour-specific feature intensities (E.g. ‘glszm Zone Entropy’), thereby suggesting a mouse-to-human species conservation (a conservation of radiomic features across species). Thirdly, comparison of features across timepoints identify features which support preclinical tumour detection earlier than is possible by visual assessment of CT scans. This work establishes robust, preclinical CT-radiomic pipelines and describes the application of CE-CT for in-depth orthotopic brain tumour monitoring. Overall we provide evidence for the role of pre-clinical ‘discovery’ radiomics in the neuro-oncology space.

MRNA-based precision targeting of neoantigens and tumor-associated antigens in malignant brain tumors

BackgroundDespite advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized antigens expressed within brain tumors that can mediate tumor rejection; the low mutational burden of these tumors that limits the abundance of targetable neoantigens; and the immunologically “cold” tumor microenvironment that hampers the generation of sustained and productive immunologic responses. The field of mRNA-based therapeutics has experienced a boon following the universal approval of COVID-19 mRNA vaccines. mRNA-based immunotherapeutics have also garnered widespread interest for their potential to revolutionize cancer treatment. In this study, we developed a novel and scalable approach for the production of personalized mRNA-based therapeutics that target multiple tumor rejection antigens in a single therapy for the treatment of refractory brain tumors.MethodsTumor-specific neoantigens and aberrantly overexpressed tumor-associated antigens were identified for glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (O.R.A.N). Personalized tumor antigen-specific mRNA vaccine was developed for each individual tumor model using selective gene capture and enrichment strategy. The immunogenicity and efficacy of the personalized mRNA vaccines was evaluated in combination with anti-PD-1 immune checkpoint blockade therapy or adoptive cellular therapy with ex vivo expanded tumor antigen-specific lymphocytes in highly aggressive murine GBM models.ResultsOur results demonstrate the effectiveness of the antigen-specific mRNA vaccines in eliciting robust anti-tumor immune responses in GBM hosts. Our findings substantiate an increase in tumor-infiltrating lymphocytes characterized by enhanced effector function, both intratumorally and systemically, after antigen-specific mRNA-directed immunotherapy, resulting in a favorable shift in the tumor microenvironment from immunologically cold to hot. Capacity to generate personalized mRNA vaccines targeting human GBM antigens was also demonstrated.ConclusionsWe have established a personalized and customizable mRNA-therapeutic approach that effectively targets a plurality of tumor antigens and demonstrated potent anti-tumor response in preclinical brain tumor models. This platform mRNA technology uniquely addresses the challenge of tumor heterogeneity and low antigen burden, two key deficiencies in targeting the classically immunotherapy-resistant CNS malignancies, and possibly other cold tumor types.

HoloBrain: 3D low-cost mobile augmented reality rendering of brain tumour using the GVF snake model segmentation

ABSTRACT Augmented reality (AR) is a dynamic field intersecting various disciplines, with applications spanning medicine and diagnostics. Our focus on advancing medical imaging has led to groundbreaking progress in brain tumor diagnosis, fostering optimism in treatment. In this pursuit, we introduce HoloBrain, an accessible AR application designed to transform medical education. Utilizing smartphones, HoloBrain enables automatic segmentation, AR visualization, and 3D interaction without the need for head-mounted devices. Emphasizing practicality over hyper-realism, our innovation allows medical students to seamlessly integrate a virtual brain tumor model into their surroundings. This immersive approach facilitates exploration of the 3D representation of an afflicted brain, enhancing educational experiences. Tests in a controlled environment using a phantom head yielded promising results, with our solution demonstrating average errors of 2 mm to 4.5 mm on smartphone displays. These outcomes highlight the precision and effectiveness of our contribution, underlining its potential in transforming medical education and training. HoloBrain represents a step forward in making AR accessible and impactful for aspiring medical professionals, offering a valuable tool for enhancing their understanding of complex medical scenarios.

Chitosan-coated magnetic graphene oxide for targeted delivery of doxorubicin as a nanomedicine approach to treat glioblastoma

In this study, magnetic graphene oxide (mGO) was first prepared and modified with chitosan to prepare chitosan-coated mGO (mGOC). Gastrin-releasing peptide (GRP)-conjugated mGOC (mGOCG) was then prepared from mGOC. The chemo drug doxorubicin (DOX) was adsorbed to mGOCG surface for dual active/magnetic targeted drug delivery. The DOX loading to mGOCG is 1.71 mg/mg, and drug release is pH-sensitive to facilitate drug delivery in endosomes. In vitro studies confirmed enhanced mGOCG endocytosis by U87 glioblastoma cells, with which enhanced cytotoxicity towards cancer cells could be achieved. This could be revealed from the drastically reduced half-maximal inhibitory concentration of mGOCG/DOX compared with DOX and mGOC/DOX. Furthermore, mGOCG/DOX can be localized under the influence of a magnetic field (MF) to exert this cytotoxic effect. An orthotopic brain tumor model by implanting U87 cells in the intracranial area of BALB/c nude mice was used to study the in vivo anti-tumor efficacy by intravenous injection of different samples and followed with bioluminescence imaging. The tumor size in the mGOCG/DOX + MF group demonstrated the best potency to suppress tumor growth and prolong animal survival time compared with mGOCG/DOX, mGOC/DOX, or DOX groups, indicating this new dual-targeting delivery system for DOX can effectively treat glioblastoma.

Evaluation the Effect of Sonodynamic Therapy with 5-Aminolevulinic Acid and Sodium Fluorescein by Preclinical Animal Study.

Sonodynamic therapy (SDT) is a novel tumor treatment that combines biosafe sonosensitizers and noninvasive focused ultrasound to eradicate solid tumors. Sonosensitizers such as 5-aminolevulinic acid and fluorescein have great potential in tumor treatment. Here, rodent subcutaneous and brain tumor models were used to evaluate the treatment effect of both 5-ALA- and fluorescein-mediated SDT. The subcutaneous tumor growth rates of both SDT groups were significantly inhibited compared with that of the control groups. For intracranial tumors, 5-ALA-SDT treatment significantly inhibited brain tumor growth, while fluorescein-SDT exerted no therapeutic effect in animals. The distribution of fluorescein in the brain tumor region underwent further assessment. Seven days post tumor implantation, experimental animals received fluorescein and were sacrificed for brain specimen collection. Analysis of the dissected brains revealed no fluorescence signals, indicating an absence of fluorescein accumulation in the early-stage glioma tissue. These data suggest that the fluorescein-SDT treatment response is closely related to the amount of accumulated fluorescein. This study reports the equivalent effects of 5-ALA and fluorescein on the treatment of somatic tumors. For orthotopic brain tumor models, tumor vascular permeability should be considered when choosing fluorescein as a sonosensitizer. In conclusion, both fluorescein and 5-ALA are safe and effective SDT sonosensitizers, and the tumor microenvironment and pathologic type should be considered in the selection of adequate sonosensitizers.