Murine Glioblastoma Model Research Articles

Mitochondria-Targeting Pyroptosis Amplifier of Lonidamine-Modified Black Phosphorus Nanosheets for Glioblastoma Treatments.

Pyroptosis is accompanied by immunogenic mediators' release and serves as an innovative strategy to reprogram tumor microenvironments. However, damaged mitochondria, the origin of pyroptosis, are frequently eliminated by mitophagy, which will severely impair pyroptosis-elicited immune activation. Herein, black phosphorus nanosheets (BP) are employed as a pyroptosis inducer delivery and mitophagy flux blocking system since the degradation of BP could impair lysosomal function by altering the pH within lysosomes. The pyroptosis inducer of lonidamine (LND) was precoupled with the mitochondrial target moiety of triphenylphosphonium to facilitate the occurrence of pyroptosis. The mitochondria-targeting LND-modified BP (BPTLD) were further encapsulated into the macrophage membrane to endow the BPTLD with blood-brain barrier penetration and tumor-targeting capability. The antitumor activities of membrane-encapsulated BPTLD (M@BPTLD) were investigated using a murine orthotopic glioblastoma model. The results demonstrated that the engineered nanosystem of M@BPTLD could target the mitochondria, and induce as well as reinforce pyroptosis via mitophagy flux blocking, thereby boosting the release of immune-activated factors to promote the maturation of dendritic cells. Furthermore, upon near-infrared (NIR) irradiation, M@BPTLD induced stronger mitochondrial oxidative stress, which further advanced robust immunogenic pyroptosis in glioblastoma cells. Thus, this study utilized the autophagy flux inhibition and phototherapy performance of BP to amplify LND-mediated pyroptosis, which might greatly contribute to the development of pyroptosis nanomodulators.

PPRX-1701, a nanoparticle formulation of 6′-bromoindirubin acetoxime, improves delivery and shows efficacy in preclinical GBM models

Derivatives of the Chinese traditional medicine indirubin have shown potential for the treatment of cancer through a range of mechanisms. This study investigates the impact of 6'-bromoindirubin-3'-acetoxime (BiA) on immunosuppressive mechanisms in glioblastoma (GBM) and evaluates the efficacy of a BiA nanoparticle formulation, PPRX-1701, in immunocompetent mouse GBM models. Transcriptomic studies reveal that BiA downregulates immune-related genes, including indoleamine 2,3-dioxygenase 1 (IDO1), a critical enzyme in the tryptophan-kynurenine-aryl hydrocarbon receptor (Trp-Kyn-AhR) immunosuppressive pathway in tumor cells. BiA blocks interferon-γ (IFNγ)-induced IDO1 protein expression invitro and enhances Tcell-mediated tumor cell killing in GBM stem-like cell co-culture models. PPRX-1701 reaches intracranial murine GBM and significantly improves survival in immunocompetent GBM models invivo. Our results indicate that BiA improves survival in murine GBM models via effects on important immunotherapeutic targets in GBM and that it can be delivered efficiently via PPRX-1701, a nanoparticle injectable formulation of BiA.

Maximum Emission Peak Over 1500nm of Organic Assembly for Blood-Brain Barrier-Crossing NIR-IIb Phototheranostics of Orthotopic Glioblastoma.

The development of blood-brain barrier (BBB)-crossing phototheranostic agents in second near-infrared window (NIR-II), especially in the range of 1500-1700nm (NIR-IIb), affords great opportunities for glioblastoma (GBM) management. Herein, an organic assembly (denoted as LET-12) with the maximum absorption peak at 1400nm and emission peak at 1512nm with trailing over 1700nm through the self-assembly of organic small molecule IR-1064 is designed and subsequently decorated with choline and acetylcholine analogs. The LET-12 can effectively cross BBB through the brain's choline-like receptors-mediated transcytosis and accumulated in tumor tissues, thus achieving fluorescence/photoacoustic (FL/PA) duplex imaging of orthotopic GBM with ≈3.0mm depth and a superior tumor-to-normal tissue signal ratio (20.93 ± 0.59 for FL imaging and 32.63 ± 1.16 for PA imaging, respectively). Owing to its good photothermal conversion ability, the LET-12 also can serve as a photothermal conversion agent, achieving obvious tumor repression of orthotopic murine GBM model after once treatment. The findings indicate that the LET-12 holds great potential for BBB-crossing NIR-IIb phototheranostics of orthotopic GBM. This self-assembly strategy of organic small molecules opens a new avenue for the construction of NIR-IIb phototheranostics.

Abstract 2713: Modulation of the blood-tumor barrier by 6-bromoindirubin acetoxime increases intra-tumoral drug accumulation and extends survival in murine glioblastoma models

Abstract Introduction: Glioblastoma (GBM) is the most frequent primary malignant adult brain tumor. Median survival is ~15 months after diagnosis. Therapies to effectively manage glioblastoma are hindered due to the presence of the blood-tumor barrier (BTB). Indirubins are 3,2’-bisindole compounds of the Indigo naturalis used in traditional Chinese medicine to treat proliferative diseases. Herein, we demonstrate that systemic delivery of 6-bromoindirubin-3’-acetoxime (BIA), an indirubin derivative, increases cisplatin delivery to GBM tumors through alteration of the BTB integrity and synergy of BIA with platinum therapeutics to potentiate anti-tumor killing. Methods: Pre-clinical studies involved G9-pCDH glioblastoma cell line implantation in naïve nude mice. Cisplatin uptake 24 hours after BIA tail-vein administration into healthy brain and GBM murine tumors was assessed via inductively-coupled plasma mass spectrometry (ICP-MS). DNA-damage was assessed by γH2AX immunofluorescence. BIA combination with cisplatin in vitro studies were performed via luminescent viability assays. Treatment of human cerebrovascular endothelial cells (HCMEC/D3) with BIA for BBB studies in vitro involved trans-endothelial electrical resistance (TEER) measurement, transcriptome analysis, VE-cadherin western blotting and immunofluorescence. Results: Combined addition of cisplatin and BIA showed enhanced anti-GBM killing effects than cisplatin alone in G9-pCDH (IC50 cisplatin=1, BIA+cisplatin=0.2), G34-pCDH (IC50 cisplatin=3, BIA+cisplatin=1.7) and G30-LRP (IC50 cisplatin=1, BIA+cisplatin=0.3) lines. Efficacy studies using the G9-pCDH xenograft murine model showed extended survival when using cisplatin in combination with BIA (median 27d, p=0.0016) when compared to cisplatin (18.5d), BIA (21.5d) and control (18d). Increased intra-tumoral accumulation of cisplatin was observed when used in combination with BIA (mean 100 pg/mg, p=0.012) rather than cisplatin alone (5 pg/mg). No increased platinum uptake was seen in healthy brain tissue under any conditions. γH2AX foci in tumor were more frequent in the BIA and cisplatin combination arm (p=0.0086) than each single agent and control groups. Evaluation of HCMEC/D3 cells treated with BIA showed a decrease in TEER levels (p<0.001) and transcriptomic dysregulation of members of the WNT pathway and downregulation of VE-cadherin (~4-fold). BIA exerted cytoskeleton changes in HCMEC/D3 cells and decreased VE-cadherin expression at the protein level and its distribution on cellular surface, suggesting loss of adherens junctions as a potential mechanism for BIA-induced permeability of the BTB. Conclusion: Overall, our data support the applicability of BIA for modulating the BTB permeability and increasing intra-tumoral drug accumulation for improving GBM therapeutic efficacy. Citation Format: Jorge L. Jimenez Macias. Modulation of the blood-tumor barrier by 6-bromoindirubin acetoxime increases intra-tumoral drug accumulation and extends survival in murine glioblastoma models [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 2713.

Abstract 3001: Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of glioblastoma

Abstract WHO grade 4 glioblastoma multiforme (GBM) has a very poor prognosis even after standard treatment with surgery, radiotherapy, and chemotherapy. Tumor recurrence is virtually inevitable when it is fatal. Chimeric Antigen Receptor (CAR)-T cell therapy is a promising new modality of therapy for GBM patients. Even though second-generation CAR-T cells may be considered to operate autonomously because co-stimulation is inherent to CAR design, the tumor microenvironment of GBM is particularly hostile to immune cells and their normal functions. In our recently published work (Gargett et al. J Immunother Cancer 2022; 10(9): e005187), CAR-T cells specific for the glioblastoma antigen, GD2, had improved antitumor activity if the CAR-T cells co-expressed IL-15. Although IL-15 co-expression prolonged control of intracranial tumor xenografts, tumors escaped immune control in most cases. Our early data suggest that a relative lack of intratumoral dendritic cells (DCs) may be a contributory factor. DCs are central to the initiation and maintenance of adaptive immune responses. Hence, we were interested to survey and characterize the DC content of glioblastoma tissues obtained either from human surgical samples or mice bearing orthotopic xenografts and syngrafts. We have used multi-colour flow cytometry to enumerate and evaluate the maturation status of subsets of DCs together with other types of immune cells among single cell suspensions of isolated glioblastoma tissues. Our preliminary findings are: Total DCs (mean +/- SD), which were defined as lineage-negative and HLA-DR (+), were significantly reduced among total viable cells of glioblastoma tissues (0.845% +/- 0.569) and peripheral blood mononuclear cells (PBMCs) (0.755% +/- 0.601) from GBM patients (n=12) compared to PBMCs (1.77% +/- 0.611) from healthy donors (n=12).No significant differences were detected in the percentages of total DCs among splenocytes of nontumor-bearing C57BL/6 (B6) mice (n=6) compared to B6 bearing othotopic syngrafts of the GL261 (n=3) or C2TA (n=3) glioblastoma cell lines. We will present more detailed analyses from human glioblastoma samples. In addition, we will present a more extensive characterization of DCs from the orthotopic murine models of glioblastoma: we will compare DCs and other types of immune cells in tumor and in the primary and secondary lymphoid tissues of both tumor-bearing and nontumor-bearing mice. We expect that the final data will fill a current gap in knowledge and indicate possible therapeutic strategies that might correct for the quantitative and qualitative defects in dendritic cells in glioblastoma. We hypothesize that adequate correction of these defects in vivo may augment the anti-glioblastoma activity of CAR-T cell therapies. Citation Format: Michael P. Brown, Tessa Gargett, Bryan J. Gardam. Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of 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 3001.

Abstract 6263: Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma

Abstract Background: Glioblastoma (GBM) patients face a dismal prognosis, with median survival of ~15 months and 5-year survival rate of 6.8%. Standard of care includes surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Unfortunately, all GBM patients succumb to their disease. Glioma stem cells (GSCs), a GBM subpopulation, possess enhanced DNA repair that contributes to chemoradiotherapy resistance. The epigenetic modifier lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in GBM and emerging studies show KDM1A is implicated in DNA damage response. This study aims to determine the significance of KDM1A to promote DNA repair in GSCs. We hypothesize KDM1A inhibition augments therapeutic efficacy of TMZ via attenuation of DNA repair pathways. Methods: Patient-derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination KDM1A knockdown or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR), and non-homologous end-joining (NHEJ) reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ was determined using orthotopic murine GBM models. Results: GBM patient data sets showed KDM1A is elevated in recurrent GBM versus primary tumors. KDM1A knockdown, knockout, or pharmacological inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies showed KDM1A is enriched at the promoters of DNA repair genes. RNA-seq studies confirmed that KDM1A inhibition reduced expression of DNA repair-related genes. RT-qPCR and Western blotting validated downregulation of DNA double-strand break repair genes. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay demonstrated increased TMZ-mediated DNA damage in NCD38-treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of mice bearing orthotopic GBM tumors. Conclusions: Our results provide compelling evidence KDM1A is essential for DNA repair in GSCs and KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination therapy of KDM1A inhibitor NCD38 with TMZ is a potential novel therapeutic strategy to improve GBM outcomes. Citation Format: Salvador Alejo, Bridgitte Palacios, Prabhakar Pitta Venkata, He Yi, Wenjing Li, Jessica Johnson, Sridharan Jayamohan, Weixing Zhao, Siyuan Zheng, Takayoshi Suzuki, Rajeshwar R. Tekmal, Andrew Brenner, Ratna K. Vadlamudi, Gangadhara R. Sareddy. Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in 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 6263.

467 Stimulator of Interferon Genes Protein (STING) Agonist Incited Sustained Antitumor Immunity in Murine Models of Glioblastoma

INTRODUCTION: Introduction: Stimulator of interferon genes (STING) is an innate pattern recognition receptor natively localized to the endoplasmic reticulum that detects both exogenous and endogenous pathogenic or damage-associated cytosolic cyclic dinucleotides (CDNs), activating IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce a potent type I interferon and proinflammatory cytokine responses. Activation of the STING pathway has been recognized as a critical component of the innate immune sensing of tumors that is required for optimal cytotoxic T-cell priming against tumor antigens. Recently a highly potent cyclic dinucleotide STING agonist, IACS-8803, showed robust activation of the STING pathway resulting in systemic anti-tumor response in the B16 murine model of melanoma. Additionally, the STING agonists have been shown to achieve robust tumor regression at both injected and distal lesions and are reported to facilitate generation of long-lived immunologic memory. METHODS: Methods: We established murine gliomas with stereotactic implantation of GL261, a murine glioma cell line. Mice were assessed with luciferase imaging to ensure baseline tumor size and then randomized into treatment arms (IgG, IACS-8803, CD47, PD-1 and combinations thereof). Treatment was initiated one week after the tumor implantation. RESULTS: Results: All mice that received IACS-8803 had no detectable luciferase activity. Tumor regression was additionally verified via MR Imaging. Utilization of IACS-8803 led to long-term tumor-free survival (p < 0.01). Re-challenge with GL261 tumors ensured long term memory was sustained with a single dose of agonist. CONCLUSIONS: Conclusion: Engaging the STING pathway may be an ideal targeting strategy for GBM. Ongoing studies are delineating the mechanisms of activating this pathway with specific attention being paid to reprogramming and education of innate immunity.

463 Tandem CAR T Cells Targeting EGFRvIII and IL-13R⍺2 are Effective Against Heterogeneous Glioblastoma

INTRODUCTION: Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogenous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape. METHODS: We generated a TanCART construct with dual specificity consisting of both the humanized anti-EGFRvIII scFv and IL-13 zetakine translated in tandem separated by a flexible glycine-serine linker. We also employed two monospecific CAR T cells, CART-EGFRvIII and CART-IL-13Rɑ2 as controls. We used both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogenous tumors. RESULTS: Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < 0.05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < 0.05). CONCLUSIONS: We demonstrate that TanCART is effective against heterogenous tumors in the brain. To our knowledge, this is the first report of a tandem CAR T cell targeting EGFRvIII and IL-13Rɑ2 simultaneously. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.

301 Inhibition of lysine-specific histone demethylase 1A (KDM1A/LSD1) attenuates DNA double strand break repair and enhances efficacy of temozolomide in glioblastoma

OBJECTIVES/GOALS: Glioblastoma (GBM) patients face a poor prognosis. Glioma stem cells (GSCs), a chemo resistant GBM subpopulation, possess enhanced DNA repair and elevated levels of epigenetic modifier KDM1A. This study aims to establish the significance of KDM1A in DNA repair and determine the potential of novel KDM1A inhibitor NCD38 to enhance TMZ efficacy in GSCs. METHODS/STUDY POPULATION: Patient derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination of KDM1A knockdown, knockout, or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR) or non-homologous end-joining (NHEJ) DNA repair reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ treatment was determined using orthotopic murine GBM models. RESULTS/ANTICIPATED RESULTS: KDM1A knockdown, knockout, or inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies revealed KDM1A is enriched at DNA repair gene promoters. RNA-seq studies suggest KDM1A inhibition reduces DNA double strand break repair gene expression, with these findings validated using RT-qPCR and Western blotting. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay support findings of increased DNA damage in NCD38/TMZ combination treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of orthotopic GBM tumor-bearing mice. DISCUSSION/SIGNIFICANCE: Our results show compelling evidence that KDM1A is essential for DNA repair in GSCs and that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination of KDM1A inhibitor NCD38 with TMZ could serve as a promising novel therapeutic strategy that can be translated to improve GBM patient outcomes.

525 Diffuse Microglia Inflammation is Associated with Anti-Tumor Efficacy in Polio Virotherapy for Glioblastoma

INTRODUCTION: Glioblastomas (GBM) harbor abundant inflammatory infiltrates with glioma-associated macrophages and microglia (GAMM) actively promoting tumor progression. Intratumor virotherapy with the highly attenuated rhino:poliovirus chimera, PVSRIPO, causes sublethal infection and activation of GAMM via the constitutively expressed human poliovirus receptor, CD155. METHODS: We optimized immune-competent CT2A murine glioblastoma models for blinded neuropathological analysis of therapy responses. Samples (n = 55) were collected in a time series after PVSRIPO or mock intratumor infusion for systemic review of histological features related to therapy. Immune cells were defined both by morphology and immunohistochemical (IHC) staining patterns. Multiplex IHC and RNA sequencing were performed to study the immune infiltrate and PD1/PD-L1 immune checkpoint expression. The role of the PD1:PD-L1 axis in restricting the antitumor efficacy of PVSRIPO was tested using survival studies. RESULTS: PVSRIPO treatment caused substantial, but transient tumor regression with intense engagement of the GAMM infiltrate. This was accompanied by diffuse microglia activation and inflammtion in the CNS surrounding the tumor, extending to the ipsilateral and even the contralateral hemispheres. PVSRIPO-instigated pervasive microglia activation occurred against a backdrop of sustained innate antiviral inflammation, associated with induction of the PD-L1 immune checkpoint on GAMM. Combining PVSRIPO with PD1/PD-L1 blockade led to more durable remissions in CT2A model. CONCLUSIONS: Our work implicates GAMM as active drivers of PVSRIPO induced antitumor inflammation and reveals profound and widespread neuroinflammatory activation of the CNS-resident myeloid compartment by PVSRIPO, which is closely associated with tumor regression.

Myb Repression Mediates Stat5b-knockdown-induced Apoptosis and Inhibits Proliferation of Glioblastoma Stem Cells.

Glioblastoma is the most common and aggressive malignant brain tumor in adults, and glioblastoma stem cells (GSCs) contribute to treatment resistance and recurrence. Inhibition of Stat5b in GSCs suppresses cell proliferation and induces apoptosis. Herein, we investigated the mechanisms of growth inhibition by Stat5b knockdown (KD) in GSCs. GSCs were established from a murine glioblastoma model in which shRNA-p53 and EGFR/Ras mutants were induced in vivo using a Sleeping Beauty transposon system. Microarray analyses were performed on Stat5b-KD GSCs to identify genes that are differentially expressed downstream of Stat5b. RT-qPCR and western blot analyses were used to determine Myb levels in GSCs. Myb-overexpressing GSCs were induced by electroporation. Proliferation and apoptosis were evaluated by a trypan blue dye exclusion test and annexin-V staining, respectively. MYB, which is involved in the Wnt pathway, was identified as a novel gene whose expression was down-regulated by Stat5b-KD in GSCs. Both MYB mRNA and protein levels were down-regulated by Stat5b-KD. Overexpression of Myb rescued cell proliferation that was suppressed by Stat5b-KD. Furthermore, Stat5b-KD-induced apoptosis in GSCs was significantly inhibited by Myb overexpression. Down-regulation of Myb mediates Stat5b-KD-induced inhibition of proliferation and induction of apoptosis in GSCs. This may represent a promising novel therapeutic strategy against glioblastoma.

Evaluation of cellular water exchange in a mouse glioma model using dynamic contrast-enhanced MRI with two flip angles

This manuscript aims to evaluate the robustness and significance of the water efflux rate constant (kio) parameter estimated using the two flip-angle Dynamic Contrast-Enhanced (DCE) MRI approach with a murine glioblastoma model at 7 T. The repeatability of contrast kinetic parameters and kio measurement was assessed by a test–retest experiment (n = 7). The association of kio with cellular metabolism was investigated through DCE-MRI and FDG-PET experiments (n = 7). Tumor response to a combination therapy of bevacizumab and fluorouracil (5FU) monitored by contrast kinetic parameters and kio (n = 10). Test–retest experiments demonstrated compartmental volume fractions (ve and vp) remained consistent between scans while the vascular functional measures (Fp and PS) and kio showed noticeable changes, most likely due to physiological changes of the tumor. The standardized uptake value (SUV) of tumors has a linear correlation with kio (R2 = 0.547), a positive correlation with Fp (R2 = 0.504), and weak correlations with ve (R2 = 0.150), vp (R2 = 0.077), PS (R2 = 0.117), Ktrans (R2 = 0.088) and whole tumor volume (R2 = 0.174). In the treatment study, the kio of the treated group was significantly lower than the control group one day after bevacizumab treatment and decreased significantly after 5FU treatment compared to the baseline. This study results support the feasibility of measuring kio using the two flip-angle DCE-MRI approach in cancer imaging.

IL15 modification enables CAR T cells to act as a dual targeting agent against tumor cells and myeloid-derived suppressor cells in GBM

IntroductionThe immunosuppressive tumor microenvironment (TME) is a major barrier to the efficacy of chimeric antigen receptor T cells (CAR-T cells) in glioblastoma (GBM). Transgenic expression of IL15 is one attractive...

TCR-engineered adoptive cell therapy effectively treats intracranial murine glioblastoma

BackgroundAdoptive cellular therapies with chimeric antigen receptor T cells have revolutionized the treatment of some malignancies but have shown limited efficacy in solid tumors such as glioblastoma and face a...

Novel murine glioblastoma models that reflect the immunotherapy resistance profile of a human disease.

The lack of murine glioblastoma models that mimic the immunobiology of human disease has impeded basic and translational immunology research. We, therefore, developed murine glioblastoma stem cell lines derived from Nestin-CreERT2QkL/L; Trp53L/L; PtenL/L (QPP) mice driven by clinically relevant genetic mutations common in human glioblastoma. This study aims to determine the immune sensitivities of these QPP lines in immunocompetent hosts and their underlying mechanisms. The differential responsiveness of QPP lines was assessed in the brain and flank in untreated, anti-PD-1, or anti-CTLA-4 treated mice. The impact of genomic landscape on the responsiveness of each tumor was measured through whole exome sequencing. The immune microenvironments of sensitive (QPP7) versus resistant (QPP8) lines were compared in the brain using flow cytometry. Drivers of flank sensitivity versus brain resistance were also measured for QPP8. QPP lines are syngeneic to C57BL/6J mice and demonstrate varied sensitivities to T cell immune checkpoint blockade ranging from curative responses to complete resistance. Infiltrating tumor immune analysis of QPP8 reveals improved T cell fitness and augmented effector-to-suppressor ratios when implanted subcutaneously (sensitive), which are absent on implantation in the brain (resistant). Upregulation of PD-L1 across the myeloid stroma acts to establish this state of immune privilege in the brain. In contrast, QPP7 responds to checkpoint immunotherapy even in the brain likely resulting from its elevated neoantigen burden. These syngeneic QPP models of glioblastoma demonstrate clinically relevant profiles of immunotherapeutic sensitivity and potential utility for both mechanistic discovery and evaluation of immune therapies.

Efficient PD-L1 imaging of murine glioblastoma with FUS-aided immunoPET by leveraging FcRn-antibody interaction.

Rationale: The passage of antibodies through the blood-brain barrier (BBB) and the blood-tumoral barrier (BTB) is determinant not only to increase the immune checkpoint inhibitors efficacy but also to monitor prognostic and predictive biomarkers such as the programmed death ligand 1 (PD-L1) via immunoPET. Although the involvement of neonatal Fc receptor (FcRn) in antibody distribution has been demonstrated, its function at the BBB remains controversial, while it is unknown at the BTB. In this context, we assessed FcRn's role by pharmacokinetic immunoPET imaging combined with focused ultrasounds (FUS) using unmodified and FcRn low-affinity IgGs targeting PD-L1 in a preclinical orthotopic glioblastoma model. Methods: Transcranial FUS were applied over the whole brain in mice shortly before injecting the anti-PD-L1 IgG 89Zr-DFO-C4 or its FcRn low-affinity mutant 89Zr-DFO-C4Fc-MUT in a syngeneic glioblastoma murine model (GL261-GFP). Brain uptake was measured from PET scans acquired up to 7 days post-injection. Kinetic modeling was performed to compare the brain kinetics of both C4 formats. Results: FUS efficiently enhanced the delivery of both C4 radioligands in the brain with high reproducibility. 89Zr-DFO-C4Fc-MUT mean concentrations in the brain reached a significant uptake of 3.75±0.41%ID/cc with FUS against 1.92±0.45%ID/cc without, at 1h post-injection. A substantial and similar entry of both C4 radioligands was observed at a rate of 0.163±0.071 mL/h/g of tissue during 10.4±4.6min. The impaired interaction with FcRn of 89Zr-DFO-C4Fc-MUT significantly decreased the efflux constant from the healthy brain tissue to plasma compared with non-mutated IgG. Abolishing FcRn interaction allows determining the target engagement related to the specific binding as soon as 12h post-injection. Conclusion: Abolishing Fc-FcRn interaction confers improved kinetic properties to 89Zr-DFO-C4Fc-MUT for immunoPET imaging. FUS-aided BBB/BTB disruption enables quantitative imaging of PD-L1 expression by glioblastoma tumors within the brain.

Tandem chimeric antigen receptor (CAR) T cells targeting EGFRvIII and IL-13Rα2 are effective against heterogeneous glioblastoma.

Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogeneous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape. Here we have developed a novel, dual-specific, tandem CAR T (TanCART) cell with the ability to simultaneously target both EGFRvIII and IL-13Rα2, two well-characterized tumor antigens that are frequently found on the surface of GBM cells but completely absent from normal brain tissues. We employed both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogeneous tumors. Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < .05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete, and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < .05). We demonstrate that TanCART is effective against heterogeneous tumors in the brain. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.

Photostable Small-Molecule NIR-II Fluorescent Scaffolds that Cross the Blood-Brain Barrier for Noninvasive Brain Imaging.

The second near-infrared (NIR-II, 1000-1700 nm) fluorescent probes have significant advantages over visible or NIR-I (600-900 nm) imaging for both depth of penetration and level of resolution. Since the blood-brain barrier (BBB) prevents most molecules from entering the central nervous system, NIR-II dyes with large molecular frameworks have limited applications for brain imaging. In this work, we developed a series of boron difluoride (BF2) formazanate NIR-II dyes, which had tunable photophysical properties, ultrahigh photostability, excellent biological stability, and strong brightness. Modulation of the aniline moiety of BF2 formazanate dyes significantly enhances their abilities to cross the BBB for noninvasive brain imaging. Furthermore, the intact mouse brain imaging and dynamic dye diffusion across the BBB were monitored using these BF2 formazanate dyes in the NIR-II region. In murine glioblastoma models, these dyes can differentiate tumors from normal brain tissues. We anticipate that this new type of small molecule will find potential applications in creating probes and drugs relevant to theranostic for brain pathologies.

TMET-10. FATTY ACID METABOLISM REGULATES IMMUNE REACTIVITY OF IRRADIATED GLIOBLASTOMA

Abstract Radiation therapy (RT) is the standard-of-care for glioblastoma (GBM) and is the only treatment for inoperable brain tumors. In multiple cancer, RT stimulates anti-tumor immunity by at least activating cancer cell-intrinsic type I interferon (IFN-I) responses. However, GBM inevitably recur, suggesting that RT promotes immune evasion mechanisms. Altered fatty acid (FA) metabolism is an emerging mechanism that can account for treatment resistance and immune escape of GBM. Therefore, we hypothesize that RT induces a metabolic shift toward the production of FAs to foster immunosuppression of GBM. Supporting this hypothesis, our data show that RT upregulates the expression of the fatty acid synthase (FASN) and the activity of the fatty acid desaturase 2 (FADS2) to generate FAs in murine GBM models, GL261 and CT2A. To determine whether FA metabolism was preventing RT-induced IFN-I, we impaired FA synthesis by inhibiting either FASN or FADS2 using genetic (CRISPR-Cas9) or pharmacological inhibitors (FASNi or FADS2i). As expected, protein quantification (ELISA) as well as gene expression (RT-qPCR) revealed that irradiated GBM cells released greater levels of IFN-beta 24hrs post RT; an effect that was more pronounced when FASN or FADS2 were blocked. Reinforcing the role of FA metabolism in regulating immune activation, in situ immunofluorescence of irradiated intracranial GL261 tumors showed that mice treated with FASNi improved the recruitment of CD8+ T cells into GBM. Along similar lines, flow cytometry of digested GL261 tumors revealed an increase infiltration of CD11c+ CD103+ dendritic cells as well as activated CD86+ CD11b+ cells when FAs synthesis was blocked with FADS2i. Altogether, our data suggest that RT-induced FA synthesis represents a mechanism of resistance by preventing IFN-I and promoting immunological evasion of GBM. Targeting FA metabolism is a promising strategy to promote anti-tumor immunity and sensitize irradiated GBM to immunotherapies.

EXTH-62. EXAMINATION OF THE TUMOR MICROENVIRONMENT OF GLIOMAS TREATED WITH VIROIMMUNOTHERAPY REVEALS THE PRODUCTION OF ONCOMETABOLITES LEADING TO IMPROVED THERAPEUTIC STRATEGIES

Abstract Oncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials in a small fraction of patients. To improve the response rate, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. We engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus to express the T-cell activator OX40L, which triggered, in murine glioblastoma models, a dramatic reshaping of the tumor microenvironment dominated by changes in immune processes in an RNA-seq analysis of ingenuity pathways. Paradoxically, network analyses also revealed that Delta-24-RGDOX induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of gliomas to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma-bearing mice. The combined therapy decreased the activation of the IDO network without affecting viral activity. Flow cytometry assays revealed a decrease in the presence of the immunosuppressive cell populations, MDSCs and Tregs, which presence was higher in Delta-24-RGDOX-treated tumors. These data were further confirmed in gene set enrichment analyses. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens and that this activation was reversed by Kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing the immunosuppression caused by the virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy.