Intracranial Injection Research Articles

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy demand. Therefore, mitochondrial dysfunction is a pathological hallmark of various neurological disorders, including Parkinson's disease. The shape and organization of the mitochondrial network is highly plastic, which allows the cell to respond to environmental cues and needs, and the structure of mitochondria is also tightly linked to their health. Here, we present a protocol to study mitochondrial morphology in situ based on immunostaining of the mitochondrial protein VDAC1 and subsequent image analysis. This tool could be particularly useful for the study of neurodegenerative disorders because it can detect subtle differences in mitochondrial counts and shape induced by aggregates of α-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson's disease. This method allows one to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions show mitochondrial fragmentation (as suggested by their reduced Aspect Ratio, AR) compared to their healthy neighboring neurons in a pre-formed fibril intracranial injection Parkinson model.

EPEN-06. CHARACTERISING THE DEVELOPMENTAL ORIGINS AND TRAJECTORIES OF ZFTA-RELA FUSION EPENDYMOMA IN A NOVEL MOUSE MODEL

Abstract Ependymomas driven by the ZFTA-RELA fusion account for >70% of all supratentorial ependymomas. These tumours are now recognised in the WHO classification of CNS tumours and have been associated with a poor prognosis. Seven ZFTA-RELA fusion variants have been described: around two thirds of cases are fusion 1. No spontaneous genetically modified mouse models (GEMMS) have been described and current models require invasive intracranial injection (of transduced cells or RCAS-TVA system). Here we describe the first spontaneous GEMM of ZFTA-RELA fusion-driven ependymoma. Nestin-Flx-STOP-Flx-ZFTA-RELA (Fusion 1) open reading frames were targeted together with luciferase to the Rosa-26 locus. Breeding these mice with Nestin-CreERT2 or Blbp-Cre lines that drive recombination in neural progenitor cells resulted in forebrain tumours that could be tracked with bioluminescence imaging from P20. Tumours displayed NF-kB and L1CAM expression and ZFTA-RELA protein was detected using a novel in-house antibody. Tumours display expression of a known ZFTA-RELA fusion ependymoma transcriptomic signature. ZFTA-RELA tumours can be grown as neurospheres and passaged as allografts in nude mice and respond to irradiation. We provide the first spontaneous GEMM of this important group of ependymomas. Here we present our histologic and transcriptomic developmental timecourse of these tumours, their comparison to the human disease and further understanding of the lineage origins and potential novel targets.

ATRT-17. CAR T CELL THERAPY TARGETING CLAUDIN-6 IS EFFECTIVE IN CELLULAR AND XENOGRAFT MODELS OF ATYPICAL TERATOID/RHABDOID TUMOR

Abstract Novel therapies are needed to for the treatment of atypical teratoid/rhabdoid tumor (ATRT), an aggressive brain tumor that predominantly affects young children and has an average 5-year survival under 50%. Claudin-6 (CLDN6) is a tight junction protein present during development and is expressed in up to 70% of ATRT specimens but not in normal tissue, making it a promising immunotherapeutic target. CLDN6-targeted chimeric antigen receptor (CAR) T cells in combination with a CAR T cell-amplifying mRNA vaccine have demonstrated antitumor activity against other CLDN6-expressing cancers in pre-clinical and a phase I adult trial (NCT04503278; Haanen J et al AACR, 2022). To assess the effectiveness of CLDN6-targeted CAR T cells against ATRT, we utilized a second-generation mRNA CAR with a 4-1BB costimulatory domain and single-chain variable fragment against CLDN6 (Reinhard et al, 2020). CLDN6 expression in patient-derived ATRT specimens was profiled by RNAseq (mean FPKM 11.4) and immunohistochemistry (positive staining in 53% of specimens). Tumor-derived cell lines were assessed for CLDN6 expression by flow cytometry. Co-culture of CLDN6-directed mRNA CAR T cells resulted in tumor-specific cytotoxicity compared to CD19-directed control CAR T cells in CLDN6-positive ATRT cell lines 7316-2187 (92% versus 15% at 10:1, p < 0.0001; 86% versus 0% at 5:1, p<0.0001) and 7316-2141 (75% versus 7% at 10:1, p<0.0001; 53% versus 0% at 5:1, p< 0.0001). Patient-derived, orthotopic xenograft ATRT models were created through intracranial injection of tumor cells into the cerebellum of NSG mice. Following engraftment with 7316-2141, repeated intratumoral administration of mRNA CLDN6 CAR T cells resulted in significant tumor regression (-1.9x108 vs. +2.4x109 p/sec/cm2/sr, p<0.001) and improved survival compared to mRNA CD19 CAR T cells (median OS not reached vs. 13 days, p=0.002). This work highlights the potential for targeting CLDN6 via CAR T cell therapy in patients with ATRT as a novel therapeutic strategy.

SURG-06. ENHANCED DELIVERY OF ANTI-PD1 FOR TREATMENT OF DIFFUSE MIDLINE GLIOMA USING FOCUSED ULTRASOUND-MEDIATED BLOOD-BRAIN BARRIER OPENING

Abstract Diffuse midline glioma with H3K27 mutation is a fatal central nervous system tumor, most commonly arising in the brainstem. Despite a multitude of clinical trials, prognosis remains poor with a median overall survival of 9-12 months. Immune-checkpoint inhibitors (ICIs) have transformed the treatment landscape of multiple solid tumors. Programmed cell death protein 1 (PD1) is an immune checkpoint protein expressed on the surface of activated T cells; interaction with its ligand, PDL1, is tumor-protective, dampening T cell response. Recent phase I clinical trials have shown that ICIs targeting proteins along the PD1/PDL1 axis are well tolerated in patients with DMG; however, efficacy remains low. The blood-brain barrier (BBB) poses a major challenge to the efficacious delivery of therapeutic agents with large molecular size, such as anti-PD1. Here we show that delivery of anti-PD1 can be enhanced over 3.5-fold using low intensity focused ultrasound (FUS) and concurrent microbubble administration to achieve reversible blood-brain barrier opening (BBBO) in a syngeneic mouse DMG model established with intracranial injection of cell line 4423 (PDGFB+, H3.3K27M, p53-/-). We measured this increased delivery of anti-PD1 after BBBO using Western Blot and 3D in vivo optical fluorescent imaging/CT (OI/CT) of Cy7 labeled anti-PD1. OI/CT revealed enhanced real-time antibody distribution peritumorally. Furthermore, we demonstrated that this combined treatment of FUS and anti-PD1 led to benefit in local control of tumor growth using volumetric analysis of MRI. Preliminary survival studies suggest a positive trend for overall survival. We consider these findings strong rationale for further investigation of the therapeutic effects of combinatorial treatment using FUS-mediated BBBO and ICIs for the treatment of DMG.

Single walled carbon nanotube optical reporter for amyloid beta detection

AbstractBackgroundAmyloid beta (Aß) extracellular deposits are one of the pathohistological hallmarks of Alzheimer’s Disease (AD). In recent years intracellular accumulation of Aß has been linked with early pathogenic mechanisms. Recent reports now associate intracellular Aβ accumulation, and lysosomal disfunction as an early event in the disease, preceding extracellular amyloid‐deposits. Today methods to visualize intracellular Aβ accumulation rely on antibody staining or pre‐labeled Aβ. There is an outstanding need for live‐imaging tools capable of monitoring Aβ dynamics at the cellular level.MethodWe have developed an optical sensor that can monitor Aß in live cells transiently. Single wall carbon nanotubes (SWCNT) present advantages as optical sensors owing to their near‐infrared emission, as well as photostability and high‐sensitivity. SWCNT emission spectra (wavelength ranges of 1100‐1150nm) is within the biological transparency window (1000‐1700nm) allowing for optimal imaging in live samples. SWCNT were non‐covalently biofunctionalized; by which we confer selectivity to Aβ.To obtain intracellular detection we used two cell lines: monocytes (THP‐1) and differentiated SH‐SY5Y. Cells were incubated with functionalized nanotubes. We utilized near‐infrared spectroscopy and hyper‐spectral microscopy to obtain spectral response from live‐cells.ResultWe developed and characterized a new class of bio‐functionalized intrinsically photoluminescent SWCNT sensor that can detect Aß via shifting of their intrinsic near‐infrared emission wavelength. Our biofunctionalized sensors specifically recognize Aβ in the solution phase, showing a monochromatic center wavelength decrease in a dose‐dependent fashion. The reporters show high specificity compared to competing analytes and in serum samples. In cell models, nanosensors accumulate intracellularly and probe Aβ introduced to the cells specifically. Finally, initial through‐skull near‐IR imaging in‐vivo in an AD model (5XFAD), following intracranial injection, show significant decrease in central wavelength in AD mice compared to wild type.ConclusionThe SWCNT sensor provide a framework for dynamically investigating Aβ in live samples. We believe utilizing the nanosensors can enhance the study of Aβ ‐pathogenic mechanism, structure and molecular function and consequently improve and accelerate effective amyloid‐targeted therapies.

P.013 Patient preferences for selection of preventive migraine therapies in Canada: results from a discrete choice experiment

Background: This study assessed the importance of mode of administration relative to other treatment attributes when selecting a preventive migraine therapy. Methods: Cross-sectional study among Canadian adults diagnosed with migraine with ≥5 monthly migraine days and tried ≥2 prescription migraine treatments (any kind/duration). Preferences for treatments varying in the following attributes were evaluated via a discrete choice experiment: speed of efficacy (effective in 24hr/1wk/3mo), duration of efficacy (wears off never/1wk/2 wks before next dose), mode of administration (infusion/auto-injection/cranial injections), administration setting (clinic/home), and administration frequency (1mo/3mo). Attribute-level preference weights were estimated using Hierarchical Bayes modeling. Results: Of 200 respondents, 142 experienced episodic migraine and 58 experienced chronic migraine. Preference weights confirmed that respondents’ most preferred treatments were those that provided fast and long-lasting efficacy (effective in 24hr = 0.59; wears off never = 1.07) and were offered via infusion (0.58) or auto-injection (0.47) over intracranial injection (-1.04). Respondents reported being moderately willing to receive infusions in either a home or clinic setting (1-6 Likert scale from “not at all” to extremely” willing). Conclusions: Second to speed and duration of efficacy, respondents were most concerned with mode of administration when selecting their preferred migraine preventive, suggesting that physicians should consider patient preferences in treatment decision-making.

Targeting squalene epoxidase in breast-to-brain metastasis: A new therapeutic target for brain extravasation and colonization using animal models.

1084 Background: Brain metastasis is the most common malignancy of the central nervous system which causes severe morbidity and mortality in multiple cancer types of patients and represents an unmet medical need. Several critical steps are required for a successful brain metastasis, including local invasion, intravasation, dissemination, extravasation, and colonization. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information toward how cancer cells extravasate and colonize. Methods: To understand the underlying molecular mechanisms and applications of molecular targets for brain metastasis therapy, murine models are employed for investigation of brain extravasation and colonization. To investigate the roles of SQLE in breast-to-brain metastasis in vivo, we silenced SQLE expression directly with lentiviral shRNA in the brain metastatic MDA-MB-231-BR cell line (231-BR/shSQLE) and used 231-BR cells expressing a scramble shRNA (231-BR/shScr) as the control. Brain metastases were induced by intracardiac, orthotopic, or direct intracranial injections of 231-BR/shSQLE or 231-BR/shScr cells into immune deficient mice. The essential roles of SQLE in the specific step(s) of breast-to-brain metastatic process were evaluated by ex vivo immunofluorescence analysis of brain slices from the animals. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast-to-brain metastasis, we evaluated the inhibitory effects of NB-598 (a SQLE inhibitor) in both the 231-BR orthotopic and intracardiac mouse models. Results: Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in the cholesterol biosynthesis, plays a critical role in the processes of breast cancer metastasized to the brain, especially in brain extravasation and colonization. Interestingly, the pharmacologic inhibition of SQLE has been widely used against fungal infections, and the next-generation SQLE inhibitors have been recently shown to exert an anticancer effect. Our data demonstrated that SQLE is essential for 231-BR cells to extravasate into the parenchyma as well as the formation of micro and macro-metastases in the brain. Interestingly, we found that astrocytes play a key role in supporting 231-BR-developed brain macro-metastasis. In vitro blood-brain barrier (BBB) models further demonstrated the critical roles of SQLE in promoting 231-BR cell invasion and penetration through BBB. Inhibition of SQLE by NB-598 demonstrated anti-tumor proliferation and anti-metastasis. Conclusions: Pharmacologic inhibition of SQLE by NB-598 suppressed 231-BR tumor growth at the mammary fat pad and distal metastases to organs, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer metastases.

Intra-lesion injection of activated Natural Killer (NK) cells in recurrent malignant brain tumors

Despite multi-modal therapies for patients with malignant brain tumors, their median survival is < 2 years. Recently, NK cells have provided cancer immune surveillance through their direct natural cytotoxicity and by modulating dendritic cells to enhance the presentation of tumor antigens and regulate T-cell-mediated antitumor responses. However, the success of this treatment modality in brain tumors is unclear. The main reasons are; the brain tumor microenvironment, the NK cell preparations and administration, and the donor selection. Our previous study showed that intracranial injection of activated haploidentical NK cells resulted in the eradication of glioblastoma tumor mass in the animal model without any evidence of tumor recurrence. Therefore, in the present study, we evaluated the safety of intra-surgical cavity or intra cerebrospinal fluid (CSF) Injectionofex vivoactivated haploidentical NK cells in six patients with recurrent glioblastoma multiform (GBM) and malignant brain tumors resistance to chemo/radiotherapy. Our results indicated that activated haploidentical NK cells express activator and inhibitor markers and can kill the tumor cells. However, their cytotoxic potential on patient-derived GBM (PD-GBM) was more than that of its cell line. Also, their infusion increased the overall disease control rate by about 33.3%, with a mean survival of 400 days. Moreover, we showed that local administration of the activated haploidentical NK cells in malignant brain tumors is safe, feasible, tolerated at higher doses, and cost-effective.

High fidelity sensory-evoked responses in neocortex after intravenous injection of genetically encoded calcium sensors.

Two-photon imaging of genetically-encoded calcium indicators (GECIs) has traditionally relied on intracranial injections of adeno-associated virus (AAV) or transgenic animals to achieve expression. Intracranial injections require an invasive surgery and result in a relatively small volume of tissue labeling. Transgenic animals, although they can have brain-wide GECI expression, often express GECIs in only a small subset of neurons, may have abnormal behavioral phenotypes, and are currently limited to older generations of GECIs. Inspired by recent developments in the synthesis of AAVs that readily cross the blood brain barrier, we tested whether an alternative strategy of intravenously injecting AAV-PHP.eB is suitable for two-photon calcium imaging of neurons over many months after injection. We injected C57BL/6 J mice with AAV-PHP.eB-Synapsin-jGCaMP7s via the retro-orbital sinus. After allowing 5 to 34 weeks for expression, we performed conventional and widefield two-photon imaging of layers 2/3, 4 and 5 of the primary visual cortex. We found reproducible trial-by-trial neural responses and tuning properties consistent with known feature selectivity in the visual cortex. Thus, intravenous injection of AAV-PHP.eB does not interfere with the normal processing in neural circuits. In vivo and histological images show no nuclear expression of jGCaMP7s for at least 34 weeks post-injection.

An AAV capsid increases transduction of striatum and a ChAT promoter allows selective cholinergic neuron transduction

Adeno-associated virus (AAV) vectors are currently the most efficient option for intracranial gene therapies to treat neurodegenerative disease. Increased efficacy and safety will depend upon robust and specific expression of therapeutic genes into target cell-types within the human brain. In this study, we set out with two objectives: (1) to identify capsids with broader transduction of the striatum upon intracranial injection in mice and (2) to test a truncated human choline acetyltransferase (ChAT) promoter that would allow efficient and selective transduction of cholinergic neurons. We compared AAV9 and an engineered capsid, AAV-S, to mediate widespread reporter gene expression throughout the striatum. We observed that AAV-S transduced a significantly greater area of the injected hemisphere primarily in the rostral direction compared with AAV9 (CAG promoter). We tested AAV9 vectors packaging a reporter gene expression cassette driven by either the ChAT or CAG promoter. Specificity of transgene expression of ChAT neurons over other cells was 7-fold higher, and efficiency was 3-fold higher for the ChAT promoter compared with the CAG promoter. The AAV-ChAT transgene expression cassette should be a useful tool for the study of cholinergic neurons in mice, and the broader transduction area of AAV-S warrants further evaluation of this capsid.

Intracranial injection of natural killer cells engineered with a HER2-targeted chimeric antigen receptor in patients with recurrent glioblastoma.

Glioblastoma (GB) is incurable at present without established treatment options for recurrent disease. In this phase I first-in-human clinical trial we investigated safety and feasibility of adoptive transfer of clonal chimeric antigen receptor (CAR)-NK cells (NK-92/5.28.z) targeting HER2, which is expressed at elevated levels by a subset of glioblastomas. Nine patients with recurrent HER2-positive GB were treated with single doses of 1 × 107, 3 × 107, or 1 × 108 irradiated CAR-NK cells injected into the margins of the surgical cavity during relapse surgery. Imaging at baseline and follow-up, peripheral blood lymphocyte phenotyping and analyses of the immune architecture by multiplex immunohistochemistry and spatial digital profiling were performed. There were no dose-limiting toxicities, and none of the patients developed a cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. Five patients showed stable disease after relapse surgery and CAR-NK injection that lasted 7 to 37 weeks. Four patients had progressive disease. Pseudoprogression was found at injection sites in 2 patients, suggestive of a treatment-induced immune response. For all patients, median progression-free survival was 7 weeks, and median overall survival was 31 weeks. Furthermore, the level of CD8+ T-cell infiltration in recurrent tumor tissue prior to CAR-NK cell injection positively correlated with time to progression. Intracranial injection of HER2-targeted CAR-NK cells is feasible and safe in patients with recurrent GB. 1 × 108 NK-92/5.28.z cells was determined as the maximum feasible dose for a subsequent expansion cohort with repetitive local injections of CAR-NK cells.

The kinase inhibitory region of SOCS3 attenuates reactive astrogliosis and astroglial scar in mice after traumatic brain injury

Traumatic brain injury (TBI) leads to reactive astrogliosis that impedes neural repair/regeneration. It has been proven that SOCS3 attenuates astrocyte activation by inhibiting the JAK2-STAT3 pathway. However, whether the kinase inhibitory region (KIR) of SOCS3 can be directly applied to mediate astrocyte activation after TBI is not clear. The present study aimed at investigating the inhibitory effect of KIR on reactive astrogliosis and its potential neuroprotection after TBI insult. For this purpose, A TBI model was developed by the free impact of heavy objects in adult mice. KIR was linked to the TAT peptide (TAT-KIR) to facilitate cell membrane penetration and intracranially injected into the cerebral cortex adjacent to the TBI lesion. Then reactive astrogliosis, activity of JAK2-STAT3 pathway, neuron loss, and function deficit were observed. Our results showed a decrease in neuron loss and an improvement in neural function. Meanwhile, Intracranial injection of TAT-KIR in TBI mice demonstrated a reduction of GFAP-positive astrocytes as well as C3/GFAP double-labeled A1 reactive astrocytes. Western blot analysis illustrated that the activity of the JAK2-STAT3 pathway was significantly inhibited by TAT-KIR. We conclude that exogenous treatment TAT-KIR, through suppression of JAK2-STAT3 activity, inhibits TBI -induced reactive astrogliosis induced, thereby alleviating the loss of neurons and relieving the neural function deficit. This investigation suggests that TAT-KIR could be a potential therapeutic strategy for enhancing neural regeneration following

Abstract 337: Antitumor effect of oncomagnetic therapy in glioblastoma

Abstract Background: Glioblastoma (GBM) is the most lethal primary malignant brain tumor with a median survival of 15-20 months. Concurrent temozolomide (TMZ) chemotherapy and radiation (XRT) remain the current standard of care (SOC) treatment for newly diagnosed GBM. The addition of tumor treating fields (TTFs) improves median survival from 16 to 20.9 months. This modest improvement underscores the urgent need to identify a new treatment modality as standalone therapy. We have reported recently that brief 2 - 6 h of daily brain stimulation with a new noninvasive, non-contact, magnetic stimulation device causes &amp;gt;30% reduction of contrast-enhanced tumor (CET) volume in an end-stage recurrent GBM patient after a ~30-day treatment. Purpose: The noninvasive Oncomagnetic device developed in our laboratory selectively kills glioblastoma (GBM) and other cancer cells while sparing normal developing neurons and astrocytes in vitro. The spinning oscillating magnetic field (sOMF) generated by this device disrupts electron transport in the mitochondrial respiratory chain leading to a marked increase in reactive oxygen species (ROS) and consequent cancer cell death. However, the downstream molecular mechanism of sOMF action is under investigation. We studied the cellular and molecular effects of sOMF treatment on GBM cell lines and investigated the response to Oncomagnetic monotherapy (OMT) in a syngeneic mouse model. We treated LN229 and U87 GBM cells with sOMF with and without a sub-therapeutic dose of TMZ in vitro. To study the anticancer effect of sOMF in vivo, we developed syngeneic GBM tumors in BALB/c mice by stereotactic intracranial injection of GL261WT cells. Tumor growth was monitored periodically by MRI scans. Results: Our results showed that sOMF significantly reduce cell proliferation and cell survival in vitro. We observed that sOMF induces DNA damage and arrests cells in G1 phase of cell cycle. OMT of tumor-bearing mice using a whole-body sOMF stimulation method caused a substantial retardation of tumor growth and reduction of the contrast-enhanced tumor volume in 9.4 T MRI scans. OMT also showed significant increase in overall survival in these mice. Conclusion: These experiments contribute to unraveling the mechanism of action underlying the response to OMT in GBM and provide a strong rationale for a standalone Oncomagnetic therapy for GBM. Citation Format: Arvind Pandey, Shashank Hambarde, David S. Baskin, Santosh A. Helekar. Antitumor effect of oncomagnetic therapy 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 337.

Abstract 1261: Mutant IDH-mediated suppression of the Th17 lineage in glioma: Biologic impact and therapeutic potential

Abstract Great excitement has been generated around the identification of tumor-specific hotspot mutations in isocitrate dehydrogenase I (IDH1) as they are the first potential therapeutic targets of low grade gliomas that have been identified in the last several decades. With a low number of viable therapeutic targets, mutations in IDH1 represent new hope in treating these devastating tumors that are invariably recurrent and confer a dismal prognosis in their most aggressive forms. In addition to their high frequency of mutation in gliomas, mutations in IDH1 are tumor-specific suggesting their effective targeting can reduce the collateral damage to the normal brain imparted by standard therapies. Furthermore, mutations in IDH1 lead to the overproduction of the oncometabolite D-2-hydroxyglutarate (D-2HG), which mediates many of mutant IDH1’s effects including blocks to normal differentiation, aberrant histone and DNA methylation profiles, perturbed metabolism, induction of an immunosuppressive microenvironment, among others. To gain an understanding of mutant IDH-mediated tumorigenesis, we generated genetically faithful mouse models of glioma that harbor the pathognomonic signature including the IDH1-R132H mutation and p53 deletion. This orthotopic intracranial injection model generates tumors that overproduce D-2HG, are histologically diffuse, and confer a prolonged survival compared to their wildtype counterparts. Additionally, these models display the proneural glioma subtype and are enriched for an oligodendrocyte precursor cell gene expression signature, both of which are typified by IDH mutations in human glioma. Exploring the impact of the IDH mutation on the tumor microenvironment, we identified several diminished immune cell subsets among the mutant IDH murine tumors, including plasma cells, NK cells, monocytes, and M2 macrophages, while a subtle increase among dendritic cells was observed as indicated through immune deconvolution of RNAseq data of murine brain tumors. Further analysis showed a reduced Th17 gene expression profile that was corroborated through in vitro studies showing a D-2HG-mediated suppression of IL17 producing Th17 cells and a perturbed metabolic activity skewing towards oxidative phosphorylation. Th17 cells have only relatively recently been identified and their biological impact appears to be cancer-context dependent with only a few studies being performed in gliomas. With the potential for Th17-mediated anti-tumor activity, as observed in cases of melanoma, the prospect for understanding this understudied lineage and exposing a new therapeutic avenue is exciting. As such, the biological significance of D-2HG-mediated Th17 suppression, the underlying mechanism, as well as the therapeutic potential and anti-tumor activity of Th17 restoration or IL17 overproduction are actively being investigated. Citation Format: Nathan M. Reynolds, Ashleigh J. Soedel, Ximena Bustamante-Marin, Emily Z. Miller, Paula K. Greer, David M. Ashley, Yiping He, Matthew S. Waitkus, Christopher J. Pirozzi. Mutant IDH-mediated suppression of the Th17 lineage in glioma: Biologic impact and therapeutic potential [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 1261.

Abstract 1647: In vivo efficacy and metabolism of a new synthetized dual EGFR-DNA targeting combi-molecule in a human stem cells-derived model of glioblastoma implanted intracranially in mice

Abstract Introduction. Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumours with an average survival rate of 12-18 months. The existing treatment (chemotherapy with temozolomide (TMZ)+radiotherapy) increases the median survival by 2-3 months. One protein, the epidermal growth factor receptor (EGFR) is expressed in 57.4% of GBM and half of them expresses its aggressive mutant, EGFRvIII, whose expression is associated with resistance to apoptosis, activation of DNA repair and ultimately resistance to chemotherapy. In the context of a strategy termed “combi-targeting”, we developed molecules designed to block EGFRvIII while being capable of inducing DNA damage. In vivo analysis suggests that the first prototype of such a class ZR2002 loses its DNA damaging function through metabolic oxidation. Recently, we surmised that a molecule designed to be metabolically stable may require a lower dose to be active in vivo. To test this hypothesis, we measured the cytotoxicity of the new combi-molecule, called "AF143", in a panel of glioblastoma cell lines and then we determined its efficacy and drug metabolism in an in vivo model of glioblastoma derived from human glioblastoma stem cells (GSC) with known resistance to TMZ. Material and Methods. Three glioblastoma cells line (U87-wt, U87-EGFR and U87-EGFRvIII) and the human GSC (1123IC7R) were used to determine IC50 of AF143 and ZR2002 with SRB or alamarBlue assay. Xenograft of tumors were obtained by intracranial injection of 1123IC7R-Luc GSC into the head of NSG female mice. One group of mice was treated with ZR2002 (N=8), the other with AF143 (N=7), the last with vehicle (N=8). Mice received 10 total doses: 2 cycles of 1 dose (100mg/kg) daily for 5 consecutive days followed by 2 days off, by oral gavage. The tumour burden was measured by bioluminescent imaging and drug metabolism by LC-MS. Body weights, mortality and toxicity were monitored. Survival curves and statistical analysis calculated. Results. In vitro growth inhibitory analysis showed that AF143 was 2-fold more potent than ZR2002 in U87-EGFR and 1123IC7R GSC and 3-fold more potent in U87-EGFRvIII. In vivo analysis of AF143 given alone showed significant antitumor activity (p&amp;lt;0.05) after 5 doses treatment, while ZR2002 induced an apparent tumour delay (p&amp;gt;0.05). Remarkably, AF143 and ZR2002 treatment significantly prolonged the overall survival of mice compared with the control group (p= 0.0004 for AF143 and p=0.04 for ZR2002). Analysis of drug metabolism revealed that in contrast to ZR2002, AF143 remained intact after 2h treatment, with moderate levels of N-acetyl and N6-dealkylated metabolites. Conclusion. The results in toto suggest that the intact structure of AF143 is stable to metabolism, can cross the blood brain barrier and induce antitumour activity in a EGFRvIII expressing glioblastoma stem cell model. Citation Format: Caterina Facchin, Ana Belen Fraga Timiraos, Brian Meehan, Nadia Babaa, Lata Adnani, Anne-Laure Larroque, Janusz Rak, Bertrand J Jean-Claude. In vivo efficacy and metabolism of a new synthetized dual EGFR-DNA targeting combi-molecule in a human stem cells-derived model of glioblastoma implanted intracranially in mice [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 1647.

466 Robust Model of Microglia Replacement With Circulation-Derived Microglia-Like Cells (CDMCs) in CNS Malignancies

INTRODUCTION: Tumor-associated microglia and macrophages (TAMs) represent a main cell type of brain malignancies and demonstrate complex interactions with cancerous cells and the tumor microenvironment. These interactions have important implications for the progression and treatment of malignant central nervous system (CNS) tumors. TAMs have therefore emerged as therapeutic targets, though their potential to date has been unfulfilled. METHODS: C57BL/6 mice were preconditioned for BMT with retroorbital busulfan injections (25 mg/kg), and native microglia were depleted via PLX5622 (1200 mg/kg, chow). Following transplantation, animals underwent intracranial stereotactic injection of syngeneic cell lines of either high-grade glioma (GL261) or breast cancer metastasis (E0771). Following animal sacrifice, we used single cell RNA sequencing of CD45-positive cells to profile the transcriptomic identity of glioma tumor-associated CDMCs (TA-CDMCs). RESULTS: Lineage tracing proved high intra- and peri-tumoral chimerism of BMT-graft derived cells among CNS myeloid cells in both models two weeks after tumor cell injection, demonstrating a robust ability to modify the tumor-immune microenvironment in animal models. TA-CDMCs showed a similarly activated morphology as naÏve TAMs under immunohistochemistry. Pathway enrichment analysis revealed an upregulation of immune response pathways in TA-CDMCs compared to regular TAMs, including the overexpression of genes relevant for the regulation of T cell activation, suggesting possible modification of the glioma immune-environment following CDMC repopulation. CONCLUSIONS: The present study offered a robust proof-of-concept that highly efficient TAM replacement could be achieved in orthotopic murine brain tumor models. Additionally, it appears that replaced CDMCs retain unique transcriptional patterns in the tumor-immune microenvironment, with the upregulation of several key immune response pathways. These efforts may represent a novel route of tumor-directed immunotherapy and offer exciting opportunities for ex-vivo CDMC manipulation to specifically target neoplastic cells.

457 CD39 Expression in the Glioblastoma Microenvironment does not Affect T Cell Exhaustion or Survival

INTRODUCTION: Glioblastoma (GBM), the most common malignant brain tumor, is notable for its dysfunctional T-cells. The initial optimism of Immune checkpoint inhibitors has been tempered as these agents have not been successful in treating GBM. CD39 is the first enzyme in the breakdown of ATP to adenosine and is highly upregulated in GBM. CD39 is also thought to contribute to T-cell exhaustion. However, inhibiting the CD39 ATP breakdown pathway did not improve survival in mice, even with PD-1 inhibition, leading to uncertainty about whether non-enzymatic functions of CD39 give it a role in T-cell exhaustion in GBM. METHODS: We performed intracranial injections of SB28 and GL261 GBM cell lines into CD39 global nulls (KO) and C57BL/6J (WT) mice and measured survival. Another cohort of mice were similarly injected and sacrificed after 2 weeks. These tumors were analyzed for T-cell exhaustion markers using flow cytometry. RESULTS: There was no survival benefit seen in either cell line when compared to wild type mice. The immune cell profile was not found to be statistically different between KO and WT mice. Next, expression of known exhaustion markers such as PD-1, TIM3, and LAG3 on tumor infiltrating lymphocytes (TILs) were analyzed. Expression of TIM3 and 2B4 were found to be increased in CD39KO TILs compared to WT (p &lt; 0.01). Additionally, numerous exhaustion markers such as LAG3, TIGIT, and CTLA4 were decreased on TILs compared to peripheral blood T-cells in tumor-bearing mice (p &lt; 0.01). CONCLUSIONS: These results suggest that CD39 lacks a direct impact on GBM outcomes but may affect the expression of other exhaustion markers. Further studies will continue to investigate the interaction between immune checkpoint molecules in GBM.

Toll-like receptors (TLR2 and TLR4) antagonist mitigates the onset of cerebral small vessel disease through PI3K/Akt/GSK3β pathway in stroke-prone renovascular hypertensive rats

ABSTRACT To examine the effect and mechanism of Toll-Like Receptors (TLR2, TLR4) antagonist in CSVD. The rat model of stroke-induced renovascular hypertension (RHRSP) was constructed. TLR2 and TLR4 antagonist was administrated by Intracranial injection. Morris water maze was used to observe the behavioral changes of rat models. HE staining, TUNEL staining and Evens Blue staining were performed to evaluate the permeability of the blood-brain barrier (BBB) and examine the CSVD occurrence and neuronal apoptosis. The inflammation and oxidative stress factors were detected by ELISA. Hypoxia-glucose-deficiency (OGD) ischemia model was constructed in cultured neurons. Western blot and ELISA were used to examine the related protein expression in TLR2/TLR4 signaling pathway and PI3K/Akt/GSK3β signaling pathway. The RHRSP rat model was successfully constructed, and the blood well and BBB permeability were altered. The RHRSP rats showed cogitative impairment and excessive immune response. After TLR2/TLR4 antagonist administration, the behavior of model rats were improved, cerebral white matter injury was reduced, and the expression of several key inflammatory factors including TLR4, TLR2, Myd88 and NF-kB were decreased, as well as the ICAM-1, VCAM-1, inflammation and oxidative stress factors. In vitro experiments showed that TLR4 and TLR2 antagonist increased the cell viability, inhibited the apoptosis, and decreased p-Akt and p-GSK3β expression. Moreover, the PI3K inhibitors resulted in decreased anti-apoptotic and anti-inflammatory effects of TLR4 and TLR2 antagonist. These results suggested that TLR4 and TLR2 antagonist achieved protective effect on the RHRSP through the PI3K/Akt/GSK3β pathway.

Translational Orthotopic Models of Glioblastoma Multiforme.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumorsdisplaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Cas9-mediated replacement of expanded CAG repeats in a pig model of Huntington's disease.

The monogenic nature of Huntington's disease (HD) and other neurodegenerative diseases caused by the expansion of glutamine-encoding CAG repeats makes them particularly amenable to gene therapy. Here we show the feasibility of replacing expanded CAG repeats in the mutant HTT allele with a normal CAG repeat in genetically engineered pigs mimicking the selective neurodegeneration seen in patients with HD. A single intracranial or intravenous injection of adeno-associated virus encoding for Cas9, a single-guide RNA targeting the HTT gene, and donor DNA containing the normal CAG repeat led to the depletion of mutant HTT in the animals and to substantial reductions in the dysregulated expression and neurotoxicity of mutant HTT and in neurological symptoms. Our findings support the further translational development of virally delivered Cas9-based gene therapies for the treatment of genetic neurodegenerative diseases.