Orthotopic Glioblastoma Multiforme Research Articles

MSOR4 Presentation Time: 5:15 PM: Feasibility of Diffusing Alpha-Emitting Radiotherapy in Orthotopic Glioblastoma Multiforme Murine Tumors and Its Combination with 'Standard-of-Care' Drugs in Mice-Bearing Human Xenografts

MSOR4 Presentation Time: 5:15 PM: Feasibility of Diffusing Alpha-Emitting Radiotherapy in Orthotopic Glioblastoma Multiforme Murine Tumors and Its Combination with 'Standard-of-Care' Drugs in Mice-Bearing Human Xenografts

Deguelin inhibits the glioblastoma progression through suppressing CCL2/NFκB signaling pathway

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor with characteristics of high aggressiveness and poor prognosis. Deguelin, a component from the bark of Leguminosae Mundulea sericea (African plant), displays antiproliferative effects in some tumors, however, the inhibitory effect and mechanism of deguelin on GBM were still poorly understood. At first, we found that deguelin reduced the viability of GBM cells by causing cell cycle arrest in G2/M phase and inducing their apoptosis. Secondly, deguelin inhibited the migration of GBM cells. Next, RNA-seq analysis identified that CCL2 (encoding chemokine CCL2) was downregulated significantly in deguelin-treated GBM cells. As reported, CCL2 promoted the cell growth, and CCL2 was associated with regulating NFκB signaling pathway, as well as involved in modulating tumor microenvironment (TME). Furthermore, we found that deguelin inactivated CCL2/NFκB signaling pathway, and exougous CCL2 could rescue the anti-inhibitory effect of deguelin on GBM cells via upregulating NFκB. Finally, we established a syngeneic intracranial orthotopic GBM model and found that deguelin regressed the tumor growth, contributed to an anti-tumorigenic TME and inhibited angiogenesis of GBM by suppressing CCL2/NFκB in vivo. Taken together, these results suggest the anti-GBM effect of deguelin via inhibiting CCL2/NFκB pathway, which may provide a new strategy for the treatment of GBM.

An Engineered Nanoplatform with Tropism Toward Irradiated Glioblastoma Augments Its Radioimmunotherapy Efficacy.

Combining radiotherapy with immune checkpoint blockade therapy offers a promising approach to treat glioblastoma multiforme (GBM), yet challenges such as limited effectiveness and immune-related adverse events (irAEs) persist. These issues are largely due to the failure in targeting immunomodulators directly to the tumor microenvironment. To address this, a biomimetic nanoplatform that combines a genetically modified mesenchymal stem cell (MSC) membrane with a bioactive nanoparticle core for chemokine-directed radioimmunotherapy of GBM is developed. The CC chemokine receptor 2 (CCR2)-overexpressing MSC membrane acts as a tactical tentacle to achieve radiation-induced tropism toward the abundant chemokine (CC motif)ligand 2 (CCL2) in irradiated gliomas. The nanoparticle core, comprising diselenide-bridged mesoporous silica nanoparticles (MSNs) and PD-L1 antibodies (αPD-L1), enables X-ray-responsive drug release and radiosensitization. In two murine models with orthotopic GBM tumors, this nanoplatform reinvigorated immunogenic cell death, and augmented the efficacy and specificity of GBM radioimmunotherapy, with reduced occurrence of irAEs. This study suggests a promising radiation-induced tropism strategy for targeted drug delivery, and presents a potent nanoplatform that enhances the efficacy and safety of radio-immunotherapy.

Deciphering the potential of a novel cytostatic autophagy modulator for the treatment of brain cancer along with reduction in brain inflammation

Background and Purpose: Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, with significant failure of current treatment options, with the five-year survival rate of GBM being less than 7%. Temozolomide (TMZ), the standard chemotherapy, leads to severe toxicity, whereas radiation therapy leads to necrosis. Moreover, GBM leads to severe neuroinflammation, leading to severe fatalities and, thus, poor quality of life in patients. Therefore, owing to the pressing need for safer treatment alternatives, we repurposed a novel FDA-approved acetamide, BRZ, which not only exhibited a significant curbing of GBM progression but also reduced neuroinflammation. Thus, our study provides a treatment option that can also lead to better quality of life in GBM patients. Methods: Sulfarhodamine B cytotoxicity assay was performed on human (SF268, SF295, SF188) and murine (CT2A-LUC) GBM cell lines for both BRZ and TMZ. In the quest for figuring out the mode of cell-growth suppression, we performed annexin and acridine orange flow cytometry assays along with b-galactosidase cellular senescence staining assay. Cellular staining was also confirmed using confocal microscopy. Oncogenic modulation due to BRZ was studied by performing western blotting. In order to study the effect of BRZ on neuroinflammation, we examined the modulation of different neuroinflammatory genes on LPS-stimulated human and murine microglia using RT-qPCR and western blotting. We corroborated our in vitro findings by investigating the tumor progression in intracranially-injected GBM tumors in C57/BL6 mice. Mice weight and organ weights were recorded for both vehicle and BRZ-treated mice to assess toxicity profiles. Results: BRZ exhibited ~300 times more cytotoxicity in GBM cells as compared to TMZ, making it a more potent treatment option. BRZ lead to an increase in acridine orange in a concentration-dependent manner, hinting towards autophagic cell death. b-galactosidase staining assay and confocal microscopy exhibited a dose-dependent increase in b-galactosidase, showcasing a probable cytostatic effect. Expression of autophagic markers such as Atg5, Atg101, LC3A/B, and senescence markers b-galactosidase, p16, and p21 were increased due to BRZ confirming a cytostatic autophagic modulation in GBM cells. Immunoblotting revealed oncogenic modulation due to BRZ wherein Notch-1, Gli-1, Akt, and p-STAT3 were significantly downregulated, as observed in western blotting. Neuroinflammatory genes such as CXCL10, IL6, and ISG15 were downregulated in LPS-stimulated microglia using BRZ in a concentration-dependent manner, hinting towards the potential of BRZ in brain inflammation. BRZ significantly reduced the progression of orthotopic GBM tumors in C57/BL6 mice. There was no significant difference observed in body and organ weights in the vehicle and BRZ-treated mice, hinting toward the safety profile of BRZ. Conclusions and future direction: Our study provides a novel and safer treatment option for GBM in the form of a cytostatic autophagy modulator, BRZ, which also reduces neuroinflammation with the potential to increase patient’s quality of life. In the future, we aim to perform ex vivo analyses of mice brain tumors and perform the neuroinflammation study in GBM mice. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A comparison study of dynamic [18F]Alfatide II imaging and [11C]MET in orthotopic rat models of glioblastoma

PurposeTo investigate and compare the dynamic positron emission tomography (PET) imaging with [18F]Alfatide II Imaging and [11C]Methionine ([11C]MET) in orthotopic rat models of glioblastoma multiforme (GBM), and to assess the utility of [18F]Alfatide II in detecting and evaluating neoangiogenesis in GBM.Methods[18F]Alfatide II and [11C]MET were injected into the orthotopic GBM rat models (n = 20, C6 glioma cells), followed by dynamic PET/MR scans 21 days after surgery of tumor implantation. On the PET image with both radiotracers, the MRI-based volume-of-interest (VOI) was manually delineated encompassing glioblastoma. Time-activity curves were expressed as tumor-to-normal brain ratio (TNR) parameters and PET pharmacokinetic modeling (PKM) performed using 2-tissue-compartment models (2TCM). Immunofluorescent staining (IFS), western blotting and blocking experiment of tumor tissue were performed for the validation.ResultsCompared to 11C-MET, [18F]Alfatide II presented a persistent accumulation in the tumor, albeit with a slightly lower SUVmean of 0.79 ± 0.25, and a reduced uptake in the contralateral normal brain tissue, respectively. This resulted in a markedly higher tumor-to-normal brain ratio (TNR) of 18.22 ± 1.91. The time–activity curve (TACs) showed a significant increase in radioactive uptake in tumor tissue, followed by a plateau phase up to 60 min for [18F]Alfatide II (time to peak:255 s) and 40 min for [11C]MET (time to peak:135 s) post injection. PKM confirmed significantly higher K1 (0.23/0.07) and K3 (0.26/0.09) in the tumor region compared to the normal brain with [18F]Alfatide II. Compared to [11C]MET imaging, PKM confirmed both significantly higher K1/K2 (1.24 ± 0.79/1.05 ± 0.39) and K3/K4 (11.93 ± 4.28/3.89 ± 1.29) in the tumor region with [18F]Alfatide II. IFS confirmed significant expression of integrin and tumor vascularization in tumor region.Conclusion[18F]Alfatide II demonstrates potential in imaging tumor-associated neovascularization in the context of glioblastoma multiforme (GBM), suggesting its utility as a tool for further exploration in neovascular characterization.

Abstract 6908: Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies

Abstract Glioblastoma poses a significant challenge in oncology, necessitating improved translational models that faithfully replicate the intricate tumor microenvironment. Patient-derived xenograft (PDX) models, are key in glioblastoma multiforme (GBM) preclinical research. PDX models (orthotopic and subcutaneous) provide a more clinically relevant representation compared to CDX xenografts. Analyzing PDX tumors in their native environment allows for a nuanced understanding of GBM biology, tumor-stroma interactions, and treatment responses. This abstract explores the rationale and advantages of utilizing PDX GBM in vivo models coupled with patient diagnosis and treatment history, empowered by tumor whole exome sequencing (WES), to enhance the relevance of preclinical studies in evaluating GBM-specific treatments. TD2 has access to over 100 GBM PDX tumors models with fully annotated patient details and treatment history. Moreover, 68 of these PDXs also have WES data, that allows most appropriate model selection to ensure alignment with the therapeutic development strategy. Below is a detailed analysis of two GBM PDX models, GBM6 and GBM46, using patient history and WES data to define criteria for success in assessing novel preclinical therapeutic agents. GBM6, was obtained prior to treatment from a 65-year-old man with glioblastoma multiforme, that contains 642 mutations, including EGFR VIII amplification. GBM46, was obtained from a 55-year-old man with prior treatment, contained 646 mutations, including RAS pathway alterations and EGFR (MET and FLT3) amplification. When grown subcutaneously the anti-tumor activity of cetuximab against GBM6 and GBM46 resulted in 41% and 48% growth inhibition, respectively. Traditional treatments have also been evaluated in the orthotopic setting, with temozolomide (TMZ) showing a nominal improvement in lifespan (11%) as expected for the unmethylated MGMT found in the GBM6 model. By contrast, GBM46 from a patient with prior treatment with TMZ, OSI-774, radiation, and BCNU, exhibited a 58% increase in overall lifespan when treated with TMZ alone. Orthotopic PDX GBM models preserve many molecular characteristics, enabling the study of intrinsic GBM mutations for developing novel therapeutic strategies. While orthotopic models better capture GBM's dynamic nature, traditional xenograft systems remain valuable as rapid screening tools. Models with defined clinical treatment history and WES data allows for testing new agents in models that more closely resemble the clinical development path for a given new agent and aids in deciphering factors influencing therapeutic resistance. These models have the potential to reshape preclinical research paradigms, accelerating the translation of promising therapeutic agents increasing the likelihood of clinical benefit for patients with glioblastoma multiforme. Citation Format: Erin E. Trachet, Paul M. Gonzales, Stephen Gately. Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies [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 6908.

Abstract 4179: Magnetic resonance imaging and bioluminescence imaging for evaluating tumor burden in orthotopic glioblastoma

Abstract Accurate monitoring of orthotopic tumors in oncological research is critical. While conventional surgical exposure method or imaging methods such as bioluminescence imaging (BLI), ultrasound imaging (US) and computed tomography (CT) have different advantages and limitations. Magnetic Resonance Imaging (MRI) offers a non-invasive modality with high sensitivity. This study compares BLI and MRI in tracking tumor progression, focusing on the human glioblastoma multiforme (GBM) orthotopic xenograft mouse model. To establish the GBM model, human glioblastoma cells U87 MG-luc, expressing firefly luciferase, were intracranially implanted in female BALB/c nude mice. In-life tumor growth was assessed longitudinally every 3-4 days using BLI (IVIS Lumina S5, PerkinElmer, US) and MRI (Bruker BioSpec 117/16, 11.7T ultra-high field scanner, Germany) with a T2-weighted sequence. Mice were randomized into two groups (n=8) based on the MRI-derived tumor volume and BLI signal on the 3rd day post inoculation. One day after grouping, each cohort received specific treatments: one with the standard vehicle control and the other with Temozolomide (TMZ) (10mg/kg, 5 days/week, oral gavage). This treatment lasted until day 21, after which whole brains were removed and tumors excised from surrounding normal tissue and weighed. The results from both BLI and MRI showed that the control group exhibited consistent tumor growth, while TMZ treatment significantly inhibited tumor growth by day 21. However, some differences were observed, particularly for small tumor size, with more accurate measurement with MRI compared to BLI. By day 7 (3 days post treatment), MRI indicated a Tumor Growth Inhibition (TGI) of 32.63%, whereas BLI indicated a 5.04% increase in the TMZ-treated group compared to the control group. From day 11 onwards, both methods demonstrated a TGI exceeding 70% with TMZ treatment. Moreover, there was a pronounced difference in tumor weight correlations: MRI assessments showed a strong correlation (R2=0.9967) with extracted tumor weights, whereas in contrast to BLI showed a weaker correlation (R2=0.5371). The lower correlation for BLI, could be explained by the hypoxia and necrosis commonly observed in larger tumors or after treatment leading to decrease of ATP and reduction of bioluminescence. BLI offers rapid 2D imaging but lacks precision in determining location and depth of GBM tumor. Moreover, it requires luciferase engineered cell lines. In contrast, MRI provides high-resolution 3D imaging without genetic modifications, providing precise anatomical evaluations. Our study highlights the advantages of MRI over BLI for longitudinal monitoring of orthotopic GBM models. MRI imaging modality opens the possibility to use more clinically relevant patient derived xenograft (PDX) models of GBM in an orthotopic manner, improving the clinical transability to evaluate new GBM treatment modalities. Citation Format: Mingrui Guo, Ruisong Su, Jeff W H Chor, Zheyan Zhang, Mandy Tan, Xing Qi Teo, Haosheng Feng, Kuan J. Lee, Li Hua, Longyun Zhang, Jie Cai, Guanping Mao, Jingjing Wang, Keefe Chng, Weiping Han, Colin Guo. Magnetic resonance imaging and bioluminescence imaging for evaluating tumor burden in orthotopic 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 4179.

Sophocarpine inhibits the progression of glioblastoma via PTEN/PI3K/Akt signaling pathway.

Glioblastoma multiforme (GBM) is the most fatal primary brain tumor which lacks effective treatment drugs. Alkaloids are known as a class of potential anti-tumor agents. Sophocarpine, a tetracyclic quinazoline alkaloid derived from Sophora alopecuroides L., possesses several pharmacological effects including anti-tumor effects in some malignancies. However, the effect and mechanism of sophocarpine on GBM remains to be explored. In this study, based on in vitro experiments, we found that sophocarpine significantly inhibited the viability, proliferation and migration of GBM cells including U251 and C6 cells in a dose- and time-dependent manner. Besides, sophocarpine arrested GBM cell cycle in G0/G1 phase and induced their apoptosis. Subsequently, we found that sophocarpine upregulated the expression of PTEN, a GBM tumor suppressor, and downregulated PI3K/Akt signaling in GBM cells. Moreover, inactivating of PTEN with bpV(phen) trihydrate partially restored the anti-GBM effects of sophocarpine via PI3K/Akt signaling. Finally, sophocarpine significantly inhibited the growth of tumor both in subcutaneous and orthotopic U251 xenograft GBM model in nude mice via PTEN/PI3K/Akt axis. Taken together, these results suggested that sophocarpine impeded GBM progression via PTEN/PI3K/Akt axis both in vitro and in vivo, providing with a promising therapy for treating GBM.

Intracavitary Spraying of Nanoregulator-Encased Hydrogel Modulates Cholesterol Metabolism of Glioma-Supportive Macrophage for Postoperative Glioblastoma Immunotherapy.

Glioblastoma multiforme (GBM) is notoriously resistant to immunotherapy due to its intricate immunosuppressive tumor microenvironment (TME). Dysregulated cholesterol metabolism is implicated in the TME and promotes tumor progression. Here, it is found that cholesterol levels in GBM tissues are abnormally high, and glioma-supportive macrophages (GSMs), an essential "cholesterol factory", demonstrate aberrantly hyperactive cholesterol metabolism and efflux, providing cholesterol to fuel GBM growth and induce CD8+ T cells exhaustion. Bioinformatics analysis confirms that high 7-dehydrocholesterol reductase (DHCR7) level in GBM tissues associates with increased cholesterol biosynthesis, suppressed tumoricidal immune response, and poor patient survival, and DHCR7 expression level is significantly elevated in GSMs. Therefore, an intracavitary sprayable nanoregulator (NR)-encased hydrogel system to modulate cholesterol metabolism of GSMs is reported. The degradable NR-mediated ablation of DHCR7 in GSMs effectively suppresses cholesterol supply and activates T-cell immunity. Moreover, the combination of Toll-like receptor 7/8 (TLR7/8) agonists significantly promotes GSM polarization to antitumor phenotypes and ameliorates the TME. Treatment with the hybrid system exhibits superior antitumor effects in the orthotopic GBM model and postsurgical recurrence model. Altogether, the findings unravel the role of GSMs DHCR7/cholesterol signaling in the regulation of TME, presenting a potential treatment strategy that warrants further clinical trials.

Bioinspired Lipoproteins of Furoxans-Gemcitabine Preferentially Targets Glioblastoma and Overcomes Radiotherapy Resistance.

Radiotherapy (RT) resistance is an enormous challenge in glioblastoma multiforme (GBM) treatment, which is largely associated with DNA repair, poor distribution of reactive radicals in tumors, and limited delivery of radiosensitizers to the tumor sites. Inspired by the aberrant upregulation of RAD51 (a critical protein of DNA repair), scavenger receptor B type 1 (SR-B1), and C-C motif chemokine ligand 5 (CCL5) in GBM patients, a reduction-sensitive nitric oxide (NO) donor conjugate of gemcitabine (RAD51 inhibitor) (NG) is synthesized as radio-sensitizer and a CCL5 peptide-modified bioinspired lipoprotein system of NG (C-LNG) is rationally designed, aiming to preferentially target the tumor sites and overcome the RT resistance. C-LNG can preferentially accumulate at the orthotopic GBM tumor sites with considerable intratumor permeation, responsively release the gemcitabine and NO, and then generate abundant peroxynitrite (ONOO- ) upon X-ray radiation, thereby producing a 99.64% inhibition of tumor growth and a 71.44% survival rate at 120 days in GL261-induced orthotopic GBM tumor model. Therefore, the rationally designed bioinspired lipoprotein of NG provides an essential strategy to target GBM and overcome RT resistance.

Study of DNA Damage-Induced MSI1 Serine-347 Phosphorylation in Regulation of Radiosensitivity in Glioblastoma Multiforme

Despite the growing therapeutic techniques, Glioblastoma Multiforme (GBM) patients have limited survival due to the location and malignancies of tumor burdens. Radiotherapy is one of the common treatments with maximal surgery and chemotherapy. However, the often radioresistance occurred in GBM results in recurrence and marginal disease control. Therefore, the medical need remains unmet to treat GBM efficiently. Musashi-1 (MSI1) is one of the RNA-binding proteins known for its high oncogenicity in many cancers, including GBM. MSI1 is highly expressed in tumors and abundant in tumorigenic cancer cells. However, the relationship between MSI1 and GBM radioresistance is still largely unknown. Three human GBM cell lines, U87-MG, 05MG, and S1R1, were used in vitro, and an orthotopic nude mouse GBM model was employed in vivo. Phos-Tag blotting and phospho-MSI1 (Ser 347) antibody helped to detect the phosphorylation level of MSI1. Ultra-low attachment dishes and 3D cell culture were performed for the tumor initiation study. Flow cytometry, immunohistochemistry staining, and confocal microscope with immunofluorescent staining provide respective biomarker expressions. We initiated our study by inducing DNA damage on GBM cell lines using ionizing radiation and an alkylating agent. With Phos-Tag and NetPhos3.1 prediction, we hypothesized that a novel phosphorylation site on MSI1 serine-347 (S347) may sense DNA damage events. To unveil the molecular mechanism, we found DNA-dependent protein kinase catalytic subunits (DNA-PKcs), one of the DNA damage repair components, physically interacted with MSI1 in the role of a kinase. We suggest that activated DNA-PKcs may phosphorylate MSI1-S347, leading to enhanced oncogenicity and subsequent malignancies. Wild-type MSI1-overexpressing GBM cells (MSI1-WT) showed better colony and sphere-forming ability, whereas loss-of-function mutation S347A deprived the oncogenic behaviors. Besides, S347A limited the cancer initiation in the orthotopic animal model compared with MSI1-WT, suggesting that phosphorylation of MSI1-S347 promotes GBM tumorigenicity both in vitro and in vivo. In addition, we noticed that CD133 (Prominin-1), a well-characterized cancer stem-like cell marker, accompanied MSI1-WT introduction but was not seen in S347A cells. Intriguingly, we identified a few members of the let-7 microRNA family, including let-7b, 7e, and 7i, who were rescued by the S347A mutation and correspondingly expressed with CD133 levels and tumorigenic abilities. This study explains a vital relationship between GBM radioresistance and a novel oncogenic post-translational modification on MSI1 S347. IR-induced DNA damage triggers DNA-PKcs activation that subsequently phosphorylates MSI1-S347, enhancing GBM tumorigenicity via let-7 pathway. Further studies on the potential regulators, either inhibiting DNA-PKcs or antagonizing agents, may provide GBM patients with an optimistic outcome.

GRAPHENE NANOMATERIAL REPROGRAMMING OF IMMUNE CELLS TO UNLOCK THE EffCACY OF IMMUNOTHERAPY IN GLIOBLASTOMA

Abstract AIMS Immunotherapy has substantiated its promise and achieved striking clinical effcacy in at least a subset of patients in several cancer types. However, in patients with glioblastoma multiforme (GBM), immunotherapies have lacked effcacy as a consequence of the highly immunosuppressive “cold” tumour microenvironment. Small, thin graphene oxide (GO) nanosheets possess notable pharmacokinetic properties in GBM including deep translocation throughout the whole tumour volume and selective internalization by tumour associated macrophages and microglia (TAMMs) following local administration. The current study aimed to investigate whether this selective nanomaterial-TAMM interaction could be exploited to reprogram the immune- microenvironment of GBM. METHOD GO nanosheets were non-covalently complexed with a TLR7/8 agonist to form a stable immunomodulatory nanosystem (imodGO) which was tested in primary macrophages in vitro, and an orthotopic syngeneic GBM model in vivo. RESULTS ImodGO enhanced the polarization of macrophages toward a pro-inflammatory M1-like phenotype in vitro without disrupting their phagocytic or cytokine producing capacity. A single intratumoral administration of the same nanocomplex in an intracranial GL261 model was also effective in increasing M1-like TAMMs (CD86+, MHCII+, TNFα+) while decreasing immunosuppressive M2-like TAMMs (Ym1+, Arg1+) which resulted in a wider reprogramming of the brain tumour immune-microenvironment. Repeat imodGO administration was able to significantly inhibit tumour progression highlighting the therapeutic potential of this nanomaterial enabled immunomodulatory strategy. CONCLUSIONS Based on these results, imodGO has emerged as a novel strategy to ameliorate the immunosuppressive char- acter of GBM. Further work is warranted to assess whether imodGO could enhance the effcacy of systemic immunotherapies used in combination.

Engineering and Characterization of an Artificial Drug-Carrying Vesicles Nanoplatform for Enhanced Specifically Targeted Therapy of Glioblastoma.

Glioblastoma multiforme (GBM) treatment is hindered by complex pathologies and the need to cross the blood-brain barrier (BBB) during drug delivery. Although exosomes have great potential for GBM treatment, these alone cannot fully meet the therapeutic requirements, owing to their limitations in targeting and delivery. Herein, engineered artificial vesicles (EAVs), ANG-TRP-PK1@EAVs, which areconstructed using a liposome extruder from HEK293T cells expressing ANG-TRP-PK1 peptides, is developed. ANG-TRP-PK1 is a fusion peptide of Angiopep-2 fused to the N-terminus of TRP-PK1, to present Angiopep-2 on the EAVs. ANG-TRP-PK1@EAVs have similar characteristics to the secreted exosomes, but a much higher yield. ANG-TRP-PK1@EAVs have efficient BBB-penetration and GBM-targeting abilities in a mock BBB model in in vitro and orthotopic GBM mouse models in vivo. Doxorubicin loading EAVs (ANG-TRP-PK1@DOX) do not alter the characteristics of the EAVs, which can cross the BBB, reach the GBM, and kill tumor cells in orthotopic GBM mouse models. These engineered drug-loaded artificial vesicles show better therapeutic effects on GBM than temozolomide in mice, with very few side effects. In conclusion, EAVs can be inserted into different targeting ligands and packed into different drugs, and they may serve as unique and efficient nanoplatforms for drug delivery and tumor promise therapy.

P06.13.B GRAPHENE NANOSHEET ENABLED REPROGRAMMING OF GLIOBLASTOMA MYELOID CELLS TO UNLOCK THE EFFICACY OF IMMUNOTHERAPY

Abstract Immunotherapy has substantiated its promise and achieved striking clinical efficacy in at least a subset of patients in several cancer types. However, in patients with glioblastoma multiforme (GBM), immunotherapies have lacked efficacy as a consequence of the highly immunosuppressive “cold” tumour microenvironment. Small, thin graphene oxide (GO) nanosheets possess notable pharmacokinetic properties in GBM including deep translocation throughout the whole tumour volume and selective internalization by tumour associated macrophages and microglia (TAMMs) following local administration. The current study aimed to investigate whether this selective nanomaterial-TAMM interaction could be exploited to reprogram the immune-microenvironment of GBM. GO nanosheets were non-covalently complexed with a TLR7/8 agonist to form a stable immunomodulatory nanosystem (imodGO) which was tested in primary macrophages in vitro, and an orthotopic syngeneic GBM model in vivo. imodGO enhanced the polarization of macrophages toward a pro-inflammatory M1-like phenotype in vitro without disrupting their phagocytic or cytokine producing capacity. A single intratumoral administration of the same nanocomplex in an intracranial GL261 model was also effective in increasing M1-like TAMMs (CD86+, MHCII+, TNFα+) while decreasing immunosuppressive M2-like TAMMs (Ym1+, Arg1+) which resulted in a wider reprogramming of the brain tumour immune-microenvironment. Repeat imodGO administration was able to significantly inhibit tumour progression highlighting the therapeutic potential of this nanomaterial enabled immunomodulatory strategy. Based on these RESULTS , imodGO has emerged as a novel strategy to ameliorate the immunosuppressive character of GBM. Further work is warranted to assess whether imodGO could enhance the efficacy of systemic immunotherapies used in combination.

SYST-31 PROBIOTIC DELIVERY TO ORTHOTOPIC GLIOBLASTOMA MULTIFORME MODELS AS AN IMMUNOTHERAPY

Abstract Therapeutic bacteria have been shown to be effective as an immunotherapy in several types of solid tumors because of their inherent immunogenicity and ability to target tumor tissue with systemic administration. Furthermore, advances in synthetic biology enable production and release of cancer therapeutics from bacteria. However, there is little work describing therapeutic probiotics in brain cancers such as glioblastoma multiforme (GBM). To bridge this gap, we tested different routes of administration for delivery of engineered strains of E. coli Nissle (EcN), and observed the colonization of orthotopic GBM tumors in immunocompetent mouse models. In these models, we successfully colonized tumors using both intratumoral and intravenous injection with little to no observable off-target colonization. This specificity in both delivery methods demonstrates that systemic delivery of wild-type EcN is sufficient to achieve targeted colonization of the tumor. After systemic delivery of bacteria was established in GBM, we sought to reduce edema reactions to the bacteria that occurred after colonization. To reduce the inflammatory response, we treated the edema with antibodies against vascular endothelial growth factor (VEGF), which are a clinical standard treatment for high intracranial pressure. Injecting anti-VEGF antibodies prior to bacteria administration substantially increased the survival rate compared to the mice not given anti-VEGF, suggesting that anti-VEGF significantly reduces the toxicity resulting from the bacteria that has been observed in our work and that of other works. This work therefore opens up the possibility of further work in optimizing the engineered strains for treating GBM.

Ionizable polymeric nanocarriers for the codelivery of bi-adjuvant and neoantigens in combination tumor immunotherapy.

Ionizable lipid nanocarriers have made historical contribution to COVID-19 mRNA vaccines. Here, we report ionizable polymeric nanoparticles that co-deliver bi-adjuvant and neoantigen peptides for cancer immunotherapy in combination with immune checkpoint blockade (ICB). Current cancer ICB benefits only a small subset of patients, largely due to a lack of pre-existing target cells and checkpoint targets for ICB, tumor antigenic heterogeneity, and tumor immunosuppression. Therapeutic vaccines hold the potential to enhance ICB therapeutic efficacy by expanding antitumor cell repertoires, upregulating immune checkpoint levels and hence sensitizing ICB, and reducing tumor immunosuppression. Chemically defined peptide vaccines are attractive, but their current therapeutic efficacy has been limited due to 1) poor vaccine delivery to immunomodulatory lymph nodes (LNs) and antigen (Ag)-presenting cells (APCs), 2) poor immunostimulant adjuvant efficacy with restricted target cell subsets in humans, 3) limited adjuvant/Ag codelivery to enhance Ag immunogenicity, and 4) limited ability to overcome tumor antigenic heterogeneity. Here, we developed nanovaccines (NVs) using pH-responsive polymeric micellular nanoparticles (NPs) for the codelivery of bi-adjuvant [Toll-like receptor (TLR) 7/8 agonist R848 and TLR9 agonist CpG] and peptide neoantigens (neoAgs) to draining LNs for efficient Ag presentation in a broad range of APC subsets. These NVs potentiated the immunogenicity of peptide Ags and elicits robust antitumor T cell responses with memory, and remodeled the tumor immune milium with reduced tumor immunosuppression. As a result, NVs significantly enhanced ICB therapeutic efficacy for murine colorectal tumors and orthotopic glioblastoma multiforme (GBM). These results suggest marked potential of bi-adjuvant/neoAg-codelivering NVs for combination cancer immunotherapy.

Camouflaging multifunctional nanoparticles with bacterial outer membrane for augmented chemodynamic/photothermal/starvation/chemo multimodal synergistic therapy of orthotopic glioblastoma

Innovative therapeutic strategy and effective drug delivery are significant challenges in current glioblastoma multiforme (GBM) therapy. The development of efficient combination therapy based on chemodynamic therapy (CDT) has attracted widespread attention in recent years. Herein, a lipopolysaccharide-free bacterial outer membrane vesicles (dOMV) camouflaged biomimetic nanoreactor was constructed for quadruple synergistic therapy of orthotopic GBM. Benefiting from the immune escaping property and blood–brain barrier (BBB) penetration capacity of dOMV, nanoreactors can target cancer cells and accumulate in the tumor site. Firstly, for quadruple synergistic therapy, Glucose oxidase (GOx) can rapidly consume intratumoral glucose to generate H2O2 and gluconic acid for starvation therapy. Secondly, the abundant H2O2 produced by the enzyme-catalyzed reaction can continuously supply Cu9S8-mediated Fenton-like reaction to enhance CDT. Meanwhile, tumor hypoxic microenvironment exacerbated by GOx-induced oxygen depletion activated prodrug AQ4N for chemotherapy. Finally, the second near-infrared (NIR-II) absorbed Cu9S8 increased the temperature in local tumor site upon NIR-II laser irradiation, which can achieve photothermal-enhanced CDT, elevate GOx activity, and accelerate drug release. In vitro and in vivo results demonstrated that the camouflaging multifunctional nanoparticles with bacterial outer membrane (AG@Cu9S8@dOMV) was a promising multifunctional therapeutic nanomedicine platform for highly efficient treatment of GBM, which may be extended to as a treatment option for clinical cancer theranostics.

Recruiting T-Cells toward the Brain for Enhanced Glioblastoma Immunotherapeutic Efficacy by Co-Delivery of Cytokines and Immune Checkpoint Antibodies with Macrophage-Membrane-Camouflaged Nanovesicles.

Immunotherapy with immune checkpoint inhibitors (CPIs) shows promising prospects for glioblastoma multiforme (GBM) but with restricted results, mainly attributed to the immunosuppressive tumor microenvironment (TME) and the limited antibody permeability of the blood-tumor barrier (BTB) in GBM. Here, nanovesicles with amacrophage-mimicking membrane are described, that co-deliverchemotactic CXC chemokine ligand 10 (CXCL10), to pre-activate the immune microenvironment, and anti-programmed death ligand 1 antibody (aPD-L1), to interrupt the immune checkpoint, aiming to enhance the effect of GBM immunotherapy. Consequently, the tumor tropism of the macrophage membrane and the receptor-mediated transcytosis of the angiopep-2 peptide allowthe nanovesicle to effectively cross the BTB and target the GBM region, with 19.75-fold higher accumulation of antibodies compared to the free aPD-L1 group. The CPI therapeutic efficacy is greatly enhanced by CXCL10-induced T-cells recruitment with significant expansion of CD8+ T-cells and effector memory T-cells, leading to the elimination of tumor, prolonged survival time, and long-term immune memory in orthotopic GBM mice. The nanovesicles, that relieve the tumor immunosuppressive microenvironment by CXCL10 to enhance aPD-L1 efficacy, may present a promising strategy for brain-tumor immunotherapy.

Abstract LB313: EGFRvIII-targeted alpha therapy shows significant therapeutic efficacy as both a single-agent and in combination with standard of care against preclinical GBM models

Abstract Targeted alpha therapy (TAT) is a rapidly advancing class of radiotherapeutics that can effectively deliver potent and local radiation to cancer cells while sparing the surrounding normal cells. TATs hold great promise for treatment-resistant tumors such as glioblastoma multiforme (GBM) due to the extensive DNA damage and cell death induced by alpha particles. GBM is an aggressive and lethal primary adult brain tumor that is highly resistant to external beam radiation and chemotherapy. Herein, we present the preclinical evaluation of a novel TAT for treatment of GBM targeting the most common tumour-specific mutant, epidermal growth factor receptor variant 3 (EGFRvIII). Our EGFRvIII TAT consists of a humanized EGFRvIII monoclonal antibody, a proprietary bifunctional chelate, and the alpha-emitting radionuclide, actinium-225 (225Ac). In vivo biodistribution and efficacy of our EGFRvIII TAT was evaluated in two aggressive orthotopic EGFRvIII-expressing GBM patient-derived xenograft models (PDXs; G06 and G39) with varying degrees of blood-brain-tumor barrier (BBTB) permeability. Imaging biodistribution studies were performed using an [111In]-anti-EGFRvIII agent analogous to our [225Ac] therapeutic candidate. SPECT/BLT imaging 96 h after intravenous administration of the imaging agent revealed high tumor-specific uptake of 50.2 %ID/cc in G39 tumors with a disrupted BBTB, and 10.4% ID/cc in G06 tumors with a relatively intact BBTB. Normal brain showed very low uptake (1.6-2.1% ID/cc), and all other normal organs were <10 % ID/cc except for highly perfused organs such as heart and liver, indicating no significant off-target uptake. [225Ac]-anti-EGFRvIII single-dose and fractionated multi-dose efficacy studies were conducted in G06 and G39 orthotopic PDX models, respectively. Survival analysis showed a dose-dependent increase in survival in response to treatment with [225Ac]-anti-EGFRvIII therapy, with a greater than 3-fold increase in survival compared to vehicle controls at doses of 200-400 nCi. Furthermore, the survival benefit of the 400 nCi single-dose or fractionated equivalent dose groups were comparable, demonstrating that efficacy was dependent upon the total administered radioactivity dose. Finally, we show that combined treatment of [225Ac]-anti-EGFRvIII with the standard of care (SoC), external beam radiation plus temozolomide, resulted in a significant survival advantage (>1.7-fold) compared to TAT or SoC therapy alone. Collectively, these results demonstrate that [225Ac]-anti-EGFRvIII is a highly selective and potent therapeutic for GBM which holds great potential as both a single-agent and in combination with SoC. Citation Format: Julie Metcalf, Alexander Nielsen, Suma Prabhu, Nicole Robinson, Sarah Marinacci, Natalie Grinshtein, Brigitte L. Theriault, Anne Marcil, Maria L. Jaramillo, Traian Sulea, Ivan A. Lessard, Maria Moreno, Christopher P. Leamon, Eric Burak, John Valliant. EGFRvIII-targeted alpha therapy shows significant therapeutic efficacy as both a single-agent and in combination with standard of care against preclinical GBM models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB313.

Abstract 3576: Molecular imaging of CD8 infiltration following combination immunotherapy in preclinical glioblastoma

Abstract Background: Novel immune-promoting therapeutics for glioblastoma multiforme (GBM), such as oncolytic herpes simplex viruses (oHSV) and immune checkpoint inhibitors (ICI), have the potential to improve overall survival and lead to long-term remission, however their clinical benefit remains inconsistent. Under standard of care imaging, assessment of immunotherapeutic response can be limited by apparent radiological tumor progression associated with treatment-induced inflammation and immune infiltration. This has led to a need for better understanding of immune cell dynamics and immunotherapy response in GBM. The objective of this study is to evaluate changes in CD8+ infiltration and its relation to therapy response, through positron emission tomography (PET) imaging, in preclinical GBM. Methods: GSC005-luc orthotopic GBM models (n= 40) were treated with saline, M002 oHSV, anti-PD1 or combination immunotherapy following three weeks of tumor growth. One-week post-treatment, [89Zr]-CD8 PET imaging was performed and biologically validated through ex vivo PET, autoradiography and staining for H&E and CD8 immunohistochemistry (IHC). Further, longitudinal changes in CD8 infiltration were evaluated via [89Zr]-CD8 PET imaging one- and three-weeks post-immunotherapy with responses monitored every three days via bioluminescence imaging (BLI). Statistical analysis involved one-way ANOVA and unpaired T-test, with p<0.05 considered significant. Results: Linear correlations were seen between in vivo PET signal and ex vivo uptake (r=0.61, p<0.01), autoradiography (r=0.46, p<0.01), and IHC tumor CD8+ cell density (r=0.55, p<0.01) one-week post-treatment. Immunotherapy efficacy, defined by decreased BLI signal, resembled clinical findings as only a subset of mice exhibited long-term positive response measured by BLI signal decrease (n=9/24). Response classification revealed increased CD8+ cell tumor localization, as measured by peak standardized uptake value (SUVpeak) tumor to background ratio (TBR), in non-responders (p<0.01) and decreased heterogeneity in signal distribution in responders (p<0.05) relative to controls early in the course of therapy. Conclusions: Early CD8 infiltration and uptake distribution from CD8-PET imaging provides potential imaging metrics of therapeutic response to oHSV immunotherapy in GBM. Development of these immune-focused imaging approaches for the assessment of positive immunotherapeutic effects in GBM is beneficial for the better understanding of immune cell dynamics and their relation to clinical outcomes. Citation Format: Carlos A. Gallegos, Yun Lu, Alessandro Mascioni, Fang Jia, Jennifer C. Clements, Patrick N. Song, Shannon E. Lynch, Jason M. Warram, James M. Markert, Anna Sorace. Molecular imaging of CD8 infiltration following combination immunotherapy in preclinical glioblastoma. [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 3576.