Glial Research Articles

Biomarkers Unveiling the Interplay of Mind, Nervous System, and Immunity.

The field of psychoneuroimmunology has significantly expanded in the last few decades and so has our understanding of the bidirectional communications between the immune and central nervous systems (CNS). There is a preponderance of evidence supporting the fact that immunological pathways and neuroinflammation are involved in the pathophysiology of multiple neurological and mental health conditions. In this chapter, we have explored various neuroimmunological biomarkers involved in these pathways, responsible for developing and perpetuating different neuropsychiatric disorders. This chapter will examine inflammatory biomarkers and those associated with intestinal homeostasis, blood-brain barrier (BBB) permeability, glial cells, and neuronal injury. A range of tests has been developed to evaluate these markers, and we will also explore the existing methods currently employed for these techniques. Further studies of these inflammatory and neurological markers are needed to support their utility as biomarkers for diagnosis and prognosis and to inform treatment strategies for various neuropsychiatric disorders.

Targeting chaperone-mediated autophagy in neurodegenerative diseases: mechanisms and therapeutic potential.

The pathological hallmarks of various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease prominently feature the accumulation of misfolded proteins and neuroinflammation. Chaperone-mediated autophagy (CMA) has emerged as a distinct autophagic process that coordinates the lysosomal degradation of specific proteins bearing the pentapeptide motif Lys-Phe-Glu-Arg-Gln (KFERQ), a recognition target for the cytosolic chaperone HSC70. Beyond its role in protein quality control, recent research underscores the intimate interplay between CMA and immune regulation in neurodegeneration. In this review, we illuminate the molecular mechanisms and regulatory pathways governing CMA. We further discuss the potential roles of CMA in maintaining neuronal proteostasis and modulating neuroinflammation mediated by glial cells. Finally, we summarize the recent advancements in CMA modulators, emphasizing the significance of activating CMA for the therapeutic intervention in neurodegenerative diseases.

The Physiology of Enteric Glia.

Enteric glia are the partners of neurons in the enteric nervous system throughout the gastrointestinal tract. Roles fulfilled by enteric glia are diverse and contribute to maintaining intestinal homeostasis through interactions with neurons, immune cells, and the intestinal epithelium. Glial influences optimize physiological gut processes such as intestinal motility and epithelial barrier integrity through actions that regulate the microenvironment of the enteric nervous system, the activity of enteric neurons, intestinal epithelial functions, and immune response. Changes to glial phenotype in disease switch glial functions and contribute to intestinal inflammation, dysmotility, pain, neuroplasticity, and tumorigenesis. This review summarizes current concepts regarding the physiological roles of enteric glial cells and their potential contributions to gut disease. The discussion is focused on recent evidence that suggests important glial contributions to gastrointestinal health and pathophysiology.

Aged hippocampal single-cell atlas screening unveils disrupted neuroglial system in postoperative cognitive impairment.

Glia-neuron interaction is a crucial feature in aged hippocampus during the occurrence of postoperative cognitive impairment. However, the regulatory effects of microglia, astrocytes, and oligodendrocytes in this glia-neuron interaction, the potential mechanisms and gene targets are still to be elucidated. Here, single-cell RNA sequencing was performed to detect the perioperative genomic expression characteristics of neuroglial system in the hippocampus of aged mice, and to investigate the potential cross-cellular mechanisms and valuable treatment options for glia-neuron interaction-related cognitive impairment. We found that postoperative neurons and glia cells exhibited protein dysmetabolism and mitochondrial electron misrouting. Impaired autophagy and circadian rhythm worsened microglia activation/neuroinflammation, and exacerbated these metabolic alterations. Reactive microglia also aggravated astrocyte and oligodendrocyte cytotoxicity through the PGD2/DP and complement pathways, altering glutamate level and synaptic function via the "tripartite synapses" model, and affecting neuronal myelination. Ligand-receptor communication also indicated these synaptic and axonal dysfunctions via enhanced MDK and PTN pathways. Additionally, we found that anesthetic dexmedetomidine hold therapeutic potential within the disrupted neuroglial system. It enhanced neuronal metabolic rebalance (Atf3-related) and reduced neuroinflammation from a multicellular perspective, therefore improving postoperative cognitive impairment. Together, our study proposes an aged hippocampal cell atlas and provides insights into the role of disrupted glia-neuron cycle in postoperative cognitive impairment. Our findings also elucidate the therapeutic potential and mechanism of dexmedetomidine intervention.

Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells, which is involved in retinal ganglion cell apoptosis in glaucoma. Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma. In this study, we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation. Following this, we treated Müller glial cells in vitro and in vivo with the mGluR I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr9Asp overexpression. We found that both Kir4.1 and Kir4.1 Tyr9Asp overexpression inhibited activation of Müller glial cells. Subsequently, we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr9Asp in the eye, and observed similar results in Müller cells in vivo as those seen in vitro. Both Kir4.1 and Kir4.1 Tyr9Asp overexpression inhibited Müller cell activation, regulated the balance of Bax/Bcl-2, and reduced the mRNA and protein levels of pro-inflammatory factors, including interleukin-1β and tumor necrosis factor-α. Furthermore, we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr9Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia. In this co-culture system, we observed elevated adenosine triphosphate concentrations in activated Müller cells, increased levels of translocator protein (a marker of microglial activation), and elevated interleukin-1β mRNA and protein levels in microglia induced by activated Müller cells. These changes could be reversed by Kir4.1 and Kir4.1 Tyr9Asp overexpression in Müller cells. Kir4.1 overexpression, but not Kir4.1 Tyr9Asp overexpression, reduced the number of proliferative and migratory microglia induced by activated Müller cells. Collectively, these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma. Kir4.1 and Kir4.1 Tyr9Asp overexpression attenuated Müller cell activation, reduced ATP/P2X receptor-mediated interactions between glial cells, inhibited microglial activation, and decreased the synthesis and release of pro-inflammatory factors, consequently ameliorating retinal ganglion cell apoptosis in glaucoma.

Synapses and dendritic spines are eliminated in the primary visual cortex of mice subjected to chronic intraocular pressure elevation

Synaptic plasticity is essential for maintaining neuronal function in the central nervous system and serves as a critical indicator of the effects of neurodegenerative disease. Glaucoma directly impairs retinal ganglion cells and their axons, leading to axonal transport dysfuntion, subsequently causing secondary damage to anterior or posterior ends of the visual system. Accordingly, recent evidence indicates that glaucoma is a degenerative disease of the central nervous system that causes damage throughout the visual pathway. However, the effects of glaucoma on synaptic plasticity in the primary visual cortex remain unclear. In this study, we established a mouse model of unilateral chronic ocular hypertension by injecting magnetic microbeads into the anterior chamber of one eye. We found that, after 4 weeks of chronic ocular hypertension, the neuronal somas were smaller in the superior colliculus and lateral geniculate body regions of the brain contralateral to the affected eye. This was accompanied by glial cell activation and increased expression of inflammatory factors. After 8 weeks of ocular hypertension, we observed a reduction in the number of excitatory and inhibitory synapses, dendritic spines, and activation of glial cells in the primary visual cortex contralateral to the affected eye. These findings suggest that glaucoma not only directly damages the retina but also induces alterations in synapses and dendritic spines in the primary visual cortex, providing new insights into the pathogenesis of glaucoma.

Effect and mechanism of Tetramethylpyrazine in repair of sciatic nerve injury in rats.

Observing the effects of Tetramethylpyrazine (TMP) on the expression of Collagen IV and Laminin in neurovascular basement membrane and the apoptosis of vascular endothelial cells, and to study the mechanism of TMP in the treatment of sciatic nerve injury. Compared with the NS group, the TMP group had a significant increase in the sciatic nerve function index (P < 0.01).The miss times in TMP group was significantly lower than that in NS group (P < 0.01). The HE staining results of the TMP group showed irregular arrangement of some neuronal axons and Schwann cells, and more edema and rupture of cells. The proliferation of glial cells and inflammatory cells was significantly increased in TMP group. The results of immunohistochemistry showed that the expression of type IV collagen and laminin in the TMP group group was distributed around the blood vessels, vascular endothelial cells, basal membrane and glial cells after SNI. The expression of type IV collagen and laminin in TMP group increased significantly(P < 0.05). Immunofluorescence showed that compared with NS group, the apoptosis rate of TMP group was significantly decreased (P < 0.01). Flow cytometry results showed that compared with the NS group, the number of CECs in the TMP group was significantly decreased (P < 0.01). TMP can effectively improve the sciatic nerve functional index (SFI) of Sprague Dawley (SD) rats, enhance the proliferation of sciatic nerve vascular endothelial cells, reduce apoptosis, promote the expression of Collagen IV and Laminin in sciatic nerve microvascular basal membrane components, thereby promoting angiogenesis and improving lower limb function in rats.

Temporal dynamics of neurovascular unit changes following blood-brain barrier opening in the putamen of non-human primates.

Low-intensity focused ultrasound (LIFU) combined with intravenously circulating microbubbles has recently emerged as a novel approach for increasing delivery through the blood-brain barrier (BBB). This technique safely and transiently enables therapeutic agents to overcome the BBB, which typically poses a significant obstacle for treatment of brain disorders. However, the full impact of LIFU on the entire neurovascular unit (NVU), as well as the mechanisms and factors involved in restoring BBB integrity still require further elucidation. We conducted immunohistochemical analyses of the putamen in non-human primates to monitor changes over time [immediately post-treatment (3 h) and at 7- and 30-days post-BBB opening] in vascular, glial, and immune cells. Additionally, we examined the dynamic interactions among these elements and their role in the restorative process at the BBB level. A mild inflammatory response primarily involving microglia, astrocytes, and T- and B-lymphocytes was observed in the treated putamen acutely after BBB opening. These cells, recruited in response to the vascular changes, stimulate upregulation of PDGFRβ, a pericyte-specific marker, and VEGF-A, a pro-angiogenic factor. This was associated with vascular sprouting by 7 days post-BBB opening. Importantly, no notable long-term alterations were observed in the NVU 30 days post-BBB opening. These results offer further evidence regarding the efficacy and safety of LIFU in achieving BBB opening in the primate brain, indicating that nearly all changes in the NVU revert to baseline within 30 days post-treatment. This also suggests that angiogenesis may play an important role in restoring vascular integrity after BBB opening.

Drosophila glial system: an approach towards understanding molecular complexity of neurodegenerative diseases.

Glia is pivotal in regulating neuronal stem cell proliferation, functioning, and nervous system homeostasis, significantly influencing neuronal health and disorders. Dysfunction in glial activity is a key factor in the development and progression of brain pathology. However, a deeper understanding of the intricate nature of glial cells and their diverse role in neurological disorders is still required. To this end, we conducted data mining to retrieve literature from PubMed and Google Scholar using the keywords: glia, Drosophila, neurodegeneration, and mammals. The retrieved literature was manually screened and used to comprehensively understand and present the different glial types in Drosophila, i.e., perineurial, subperineurial, cortex, astrocyte-like and ensheathing glia, their relevance with mammalian counterparts, mainly microglia and astrocytes, and their potential to reveal complex neuron-glial molecular networks in managing neurodegenerative processes.

Pathogenic LRRK2 mutations cause loss of primary cilia and Neurturin in striatal parvalbumin interneurons.

Parkinson's disease-associated, activating mutations in the LRRK2 kinase block primary cilium formation in cell culture and in specific cell types in the brain. In the striatum that is important for movement control, about half of astrocytes and cholinergic interneurons, but not the predominant medium spiny neurons, lose their primary cilia. Here, we show that mouse and human striatal parvalbumin interneurons that are inhibitory regulators of movement also lose primary cilia. Without cilia, these neurons are not able to respond to Sonic hedgehog signals that normally induce the expression of Patched RNA, and their numbers decrease. In addition, in mouse, glial cell line-derived neurotrophic factor-related Neurturin RNA is significantly decreased. These experiments highlight the importance of parvalbumin neurons in cilium-dependent, neuroprotective signaling pathways and show that LRRK2 activation correlates with decreased Neurturin production, resulting in less neuroprotection for dopamine neurons.

Human Brain Ancestral Barcodes.

Dynamic CpG methylation "barcodes" were read from 15,000 to 21,000 single cells from three human male brains. To overcome sparse sequencing coverage, the barcode had ~31,000 rapidly fluctuating X-chromosome CpG sites (fCpGs), with at least 500 covered sites per cell and at least 30 common sites between cell pairs (average of ~48). Barcodes appear to start methylated and record mitotic ages because excitatory neurons and glial cells that emerge later in development were less methylated. Barcodes are different between most cells, with average pairwise differences (PWDs) of ~0.5 between cells. About 10 cell pairs per million were more closely related with PWDs < 0.05. Barcodes appear to record ancestry and reconstruct trees where more related cells had similar phenotypes, albeit some pairs had phenotypic differences. Inhibitory neurons showed more evidence of tangential migration than excitatory neurons, with related cells in different cortical regions. fCpG barcodes become polymorphic during development and can distinguish between thousands of human cells.

EXTH-29. EVALUATION OF THE ANTITUMOR EFFECT OF A CHEMOTHERAPY-LOADED DEVICE FOR THE TREATMENT OF PRIMARY BRAIN TUMORS

Abstract Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Tumor recurrence is therefore inevitable, and median survival is just over 14 months. Currently available treatments are inadequate and do not provide optimal tumor control. We believe that treatment localized directly at the tumor site will eliminate a greater number of infiltrating tumor cells while significantly reducing systemic toxicity. For this project, we are collaborating with a team specializing in bioengineering, who have produced a biomaterial enabling the gradual release of various chemotherapeutic agents. Our aim is to use this biomaterial to deliver chemotherapeutic agents to the tumor site through the surgical cavity. Cell-based assays were carried out using glial tumor cells in the presence of the biomaterial containing chemotherapeutics. A survival study was realized with the Fischer F98 rat model, which underwent partial tumor resection followed by injection of biomaterial containing chemotherapy. Histological studies of brains and resected tissues were also carried out to assess the effect of the biomaterial on the brain environment. In vitro assays showed a strong cytotoxic effect against glial tumor cells at 24h, 48h and 72h of biomaterial-drug incubation. The combination of two chemotherapeutic agents showed the best cytotoxic effect on glial tumor cells. We also performed a partial tumor resection and insertion of the chemotherapy-loaded biomaterial into the surgical cavity of the F98 glioma bearing rat. Similar to in vitro studies, the combination of two antineoplastic agents shows a significant improvement in animal survival. In conclusion, the in vitro and in vivo results of this study show that the combination of chemotherapy has the greatest antitumor effect. We hope that these exciting results will lead to the development of a new treatment for patients with glioblastoma and potentially other diseases where complete tumor resection is impossible.

The impact of adjuvant radiotherapy on overall survival in spinal low-grade gliomas: a propensity score-matched analysis.

Spinal low-grade gliomas (sLGGs) are a group of tumors that arise from glial cells in the spinal cord. Current evidence supporting the use of adjuvant radiotherapy for the management of sLGG is lacking. We hence aimed to compare overall survival rates in patients receiving surgery alone with those receiving surgery with adjuvant radiotherapy. The NCDB, a large, nationwide, US-based cancer registry was used. Relevant cases were identified using the following ICD-O-3 histological codes: 9382, 9384, 9400, 9410, 9411, 9420, 9421, 9424, 9425, and 9450, along with the ICD-O-3 topographical codes for spinal meninges (C70.1) and spinal cord (C72.0), excluding spinal ependymomas. Overall survival was the primary outcome. Propensity score matching 1:1 was used to balance the cohorts prior to Kaplan-Meier survival analysis. A total of 552 patients were included in the study, with 440 in the surgery alone group and 156 in the surgery with adjuvant radiotherapy group. Patients in the surgery with adjuvant radiotherapy group were significantly older (median age 40.0 vs. 24.0 years, p < 0.001), and exhibited higher proportions of WHO grade 2 tumors (p < 0.001). Adjuvant chemotherapy was more frequently administered in the surgery with adjuvant radiotherapy group (23% vs. 7%, p < 0.001). Overall, adjuvant radiotherapy was not associated with improved survival, with a significantly higher mortality in the radiotherapy group before propensity score matching (p < 0.0001). After matching, the difference in overall survival was no longer significant (p = 0.11). This study found no significant overall survival benefit associated with the use of adjuvant radiotherapy for spinal low-grade gliomas (sLGG). Although patients who received adjuvant radiotherapy initially demonstrated higher mortality rates, this difference was largely due to confounding factors such as more advanced disease in this group.

TMIC-80. HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC TUMOR MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Gliobastoma (GBM) is a highly aggressive solid tumor of the brain, characterized by a hypoxic tumor microevironment (TME) leading to histopathological features of necrosis, microvascular proliferation and deregulated hypervascularization. The heterogeneous nature of GBM results in a gradient of intra-tumoral hypoxia that causes molecular changes to specific cell populations within the bulk of the tumor. Our study is the first clinical study to utilize the exogenous oxygen-independent marker pimonidazole (PIMO) to identify the hypoxic TME using high-dimensional spatial and single-cell proteomics of specific cell populations within GBM. A cohort of 35 patients with primary GBM, recurrent GBM or IDH-mutant glioma were administered PIMO prior to surgical resection of tumor tissue for downstream bulk proteomic analysis of bulk tissue by serial immunohistochemistry and imaging mass cytometry (IMC) and single-cell analysis of dissociated tissue by Cytometry by time-of-flight (CyTOF). We utilized high-resolution imaging analyses to validate PIMO as a sensitive and stable marker for hypoxia against a panel of transiently expressed HIF-target genes and examine the inter- and intra-tumoral heterogeneity of hypoxia within each tumor subtype. A custom CyTOF marker panel was developed for further single-cell analyses of T-cell subsets and their functional states, myeloid cell subpopulations, natural killer cells, glial cells, and tumor cells in the hypoxic TME. We demonstrate the utility of PIMO to effectively study the hypoxic TME through spatial and single cell methods and further elucidate mechanisms of hypoxia-driven treatment resistance.

IMMU-42. UNRAVELING THE ROLE OF NLRX1 IN GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract •Gliomas are primary brain tumors that develop from glial cells within the central nervous system (CNS) and are among the deadliest human cancers. Glioblastoma (GBM) is the most aggressive glioma. Innate immune cells such as microglia and macrophages account for &amp;gt;50% of the cellular population within the GBM microenvironment. Innate immune cells express pattern recognition receptors (PRRs). NLRX1 is a PRR that regulates diverse signaling pathways and cellular processes including antiviral signaling, ROS production, apoptosis, autophagy, metabolic homeostasis, etc. Additionally, the tumor-suppressive and tumor-promoting functions of NLRX1 have been observed in many cancer types. For example, the tumor-suppressive role of NLRX1 has been observed in colitis-associated carcinogenesis, pancreatic cancer, and primary breast cancer. In contrast, the tumor-promoting role of NLRX1 was found in basal-like and metastatic breast carcinoma. Hence, the effect of NLRX1 on cancer may be context-dependent on cancer type or cell type aided by differences in the microenvironment. Further, how NLRX1 regulates GBM pathophysiology remains largely unexplored. This study aims to determine the expression pattern of NLRX1 in GBM cells and GBM-associated innate immune cells and investigate how NLRX1 expression regulates various cellular processes in GBM cell lines to regulate GBM pathophysiology. We report that NLRX1 is differentially expressed in GBM cell lines, GBM-associated innate immune cells, and glioma patient tissues. si-RNA-mediated silencing of Nlrx1 decreases the ability of the GBM cell line, LN-229 to proliferate and migrate. Further, Nlrx1-/- GBM cells show increased tunneling nanotube (TNT) formation capabilities. TNTs help in metabolic homeostasis maintenance by enabling the exchange of subcellular organelles such as mitochondria and endoplasmic reticulum. Nlrx1-/- GBM cells exhibit attenuated ability to form 3D spheroids. This research will aid the understanding of GBM pathophysiology, leading to novel diagnostic/prognostic markers and the discovery of signaling pathways that, in turn, may help create better GBM therapy approaches and improve overall survival.

EXTH-56. INVESTIGATING THE EFFECTS OF INDIRUBINS IN PEDIATRIC DIFFUSE HIGH GRADE GLIOMA MODELS

Abstract Diffuse midline glioma (DMG) is a highly invasive and fatal pediatric brain tumor that arises from glial cells within the pons of the brainstem. Despite focal radiation treatment, DMG continues to have a poor prognosis, with a median survival of less than 10 months. A significant challenge in treating DMG is the blood-brain barrier (BBB), which tightly restricts access to therapeutic agents, preventing drugs from reaching the brain at effective concentrations. Previous studies from the Lawler lab have shown that the indirubin derivative 6-bromoindirubin-3′-acetoxime (BIO-acetoxime/BIA) has anti-invasive properties and can enhance survival in glioblastoma xenograft models. We investigated the effects of BIA on pediatric glioma models using various assays. In an in vitro scratch migration assay, BIA significantly slowed cell migration at a concentration of 1µM in pediatric glioma cells over 72 hours. Additionally, BIA was shown to modulate tumor vasculature and improve drug delivery to tumors by targeting tight junctions in tumor-associated endothelium. BIA treatment increased dextran uptake into 3D BBB models and lowered endothelial cell permeability in vitro by reducing the expression of tight junction proteins, as shown by staining and a decrease in TEER values. Furthermore, BIA’s anti-angiogenic effects were demonstrated through staining, showing significant alterations in angiogenesis-related protein expression. To assess potential synergistic interactions, a drug screen was performed on a panel of pediatric glioma cell lines identifying several FDA-approved drugs that combine well with BIA. Drugs identified in the screen are currently under investigation. Our hypothesis is that BIA could be an effective therapeutic agent for DMG by targeting tumor invasion, angiogenesis, and improving drug potency and delivery through modulation of endothelial cell tight junctions. Future studies will focus on elucidating the underlying mechanisms and developing drug formulations for in vivo studies to assess the translational potential of this approach.

PATH-02. MESENCHYMAL-TYPE GENETIC MUTATIONS ARE LIKELY PRE-REQUISITE FOR GLIOBLASTOMA TO METASTASIZE OUTSIDE THE CENTRAL NERVOUS SYSTEM: AN ORIGINAL CASE SERIES AND SYSTEMATIC REVIEW OF THE LITERATURE

Abstract Glioblastoma multiforme (GBM) is locally aggressive, yet it metastasizes outside the central nervous system (CNS) in only 0.4% of cases. Little is known about what enables this subset of GBMs to survive outside the CNS, so we conducted a PRISMA-compliant systematic review of metastatic GBM wherein we reviewed 3579 search results and 1080 abstracts, ultimately analyzing data from 139 studies and 211 unique patients. Additionally, we analyzed four modern-day cases treated at our institution. In our patients, primary GBM lesions revealed consistent mutations in NF1, TERT, TP53, CDK4, and RB1/PTEN genes. Unique to the metastatic lesions were amplifications in integral cell-cycle regulating genes such as p53 and PDGFRA/KIT, as well as increased vimentin and Ki-67 expression. These data support the hypothesis that glial cells likely underwent mesenchymal-like mutations prior to extra-CNS involvement. How these cells reach the extra-axial environment is still under debate. In our systematic review, we found that metastases were discovered near previous surgical sites in at least 36.9% of cases, suggesting tumor seeding into the scalp during surgery may facilitate mesenchymal-type mutation in some cases. However, this is unlikely to be sole mechanism of spread as there are several reports of spread without surgery, and other hypothesized mechanisms include spontaneously circulating cells and glymphatics. Other sites of GBM metastasis included bone (47.9%), lung (25.6%), lymph nodes (25.1%), and liver (14.2%). On average, metastases were diagnosed 12.1 months after the most recent resection, and the mean survival from discovery was 5.7 months. In sum, there is strong evidence that GBMs acquire novel mutations to survive outside the CNS, are spread spontaneously or iatrogenically, and portend a poor prognosis once disseminated. Routine genetic and molecular characterization of both primary and metastatic GBMs and widespread reporting will help further characterize mechanisms of spread for this rare but deadly pathology.

EPCO-55. SPATIAL HETEROGENEITY IN THE TUMOR CORE AND INFILTRATING MARGIN OFIDHWT GLIOBLASTOMA

Abstract We integrated single-cell RNA-sequencing, Visium spatial transcriptomic, and Xenium in situ transcriptomic data to identify differences in cell composition between the tumor core and infiltrating edge in IDHWT glioblastoma (GBM). Using CellTrek, an algorithm that maps single cells to their most-likely locations in spatial transcriptomic data, we identified extensive spatial organization in GBM. Cell mapping revealed distinct regions enriched for tumor, immune, endothelial, neuronal, and glial cells. These were associated with the location of the tissue sample – edge regions were enriched for neurons and glia (as expected), while core regions were enriched for tumor cells and infiltrating immune cells, particularly macrophages. Within the core samples, the tumor and immune cells tended to occupy distinct spatial niches. Spatial clustering of mapped tumor cells across all samples revealed five spatially segregated tumor cell states, each associated with specific biological processes, including (1) histone methylation, (2) proliferation, (3) wound-healing, EMT, and antigen presentation, (4) hypoxia response, and (5) stem cell differentiation. These tumor cell states exhibited preferential colocalization with specific non-malignant cell types. For instance, antigen-presenting/wound-healing tumor cells tended to colocalize with diverse immune cells, while stem-like tumor cells tended to reside in neuron-rich regions at the tumor edge. Next, to identify ligand-receptor interactions governing this spatial organization, we analyzed known ligand-receptor pairs within all three data types. This revealed known and novel mediators of tumor cell interactions with vasculature, lymphoid cells, myeloid cells, neurons, and glia. While some interactions were shared across all tumor cell states (e.g. all tumor cell states interacted with myeloid cells via APP/CD74 interactions), others were specific to particular tumor cell states (e.g. wound-healing/antigen-presenting tumor cells interacted with macrophages via CSF1/CSF1R, while proliferating and stem-like tumor cells interacted with neurons through multiple known NRXN/NLGN interactions). These interactions suggest potential targets for clinical disruption of the tumor ecosystem.

TMOD-12. DEFINING THE ROLE OF THE DAAM2 R414W GERMLINE VARIANT IN FAMILIAL GLIOMA

Abstract Malignant glioma is the most common primary brain tumor in adults, leading to high morbidity and mortality due to its aggressive nature and limited efficacious treatment options. To improve this prognosis, research has focused on genetic factors that may contribute to glioma. While most glioma cases arise from sporadic somatic mutations, approximately 5-10% are classified as familial glioma, stemming from inherited genetic variations. Although several studies have explored mechanisms behind somatic mutations in glioma, less is known about hereditary variants that promote glioma susceptibility. We recently discovered five germline mutations in Daam2 (Dishevelled associated activator of morphogenesis 2) from familial glioma patients. To examine the role of these mutations in familial glioma, we overexpressed these mutations in patient-derived glioma stem-like cells, which were subsequently orthotopically xenografted into immunodeficient mice. We identified that one of these mutations, Daam2-R414W, promotes tumor cell proliferation both in vitro and in vivo. To further understand the role of the Daam2-R414W mutation in familial glioma, we generated a knock-in immunocompetent mouse model with the Daam2-R414W mutation. In these mutant mice, we found an increased number of proliferating glial progenitor cells in the subventricular zone compared to Daam2 wildtype mice, suggesting the potential of the Daam2-R414W mutation to influence glial progenitor expansion to stimulate glioma initiation. Furthermore, to establish a potential mechanism, we performed proteomic profiling of Daam2 wildtype and Daam2-R414W overexpression tumors and found enrichment of Git1, a GTPase-activating protein involved in cytoskeletal remodeling, suggesting that the Daam2-R414W variant may interact with Git1 to promote tumor invasion. Our findings thus far identify a hereditary genetic variant with the potential to increase the risk of glioma, shedding light on the repercussions of a human developmental gene variant and its potential to disrupt normal cellular machinery to promote malignancy and glioma development.

STEM-10. NEURAL PROGENITOR CELL MARKERS IN IDH MUTANT AND IDHWT GLIOMAS

Abstract INTRODUCTION Single-cell RNA sequencing (scRNA-seq) has helped to elucidate the cellular composition of cancer and its microenvironment. Recent scRNA-seq studies have highlighted the heterogeneity of glioblastoma (GBM). Moreover, single-cell GBM analyses have proposed resemblance of GBM cells to radial glia and outer radial glia, supporting the hypothesis that remnants of developmental tissue get reactivated in cancer. A recent study isolated neural progenitor cells (NPCs) from developing fetal human brain (gestational week 17–19) and classified NPCs based on their expression of THY1, CD24 and EGFR. Ventricular radial glia are THY1- CD24-EGFR+ whereas outer radial glia are THY1- CD24-EGFR-. Early neuron precursors are CD24+THY1-EGFR+ and glial progenitor cells (GPCs) are THY1+EGFR+. GPCs give rise to THY1+EGFR+PDGFRA+ pre-oligodendrocyte progenitor cells. The importance of EGFR in NPCs again highlights the resemblance to glioma. METHODS We aimed to apply the classification above to IDH mutant astrocytoma and oligodendroglioma as well as IDHwt glioblastoma samples. We used three publicly available datasets: Wang (paired 74 IDHwt primary and recurrent samples), Tirosh (6 primary oligodendroglioma samples) and Venteicher (10 primary IDH mutant astrocytoma). RESULTS In IDH mutant astrocytoma, 82.63% of cells express THY1+ (mostly EGFR+PDGFRA+) and 10.76% of cells are THY1-CD24-EGFR+. In oligodendroglioma, 75% of cells are THY1+ (mostly EGFR+PDGFRA+) and 12.07% are THY1-CD24-EGFR+. In IDHwt EGFR amplified primary GBM samples, 87.5% of cells are THY1-CD24-EGFR+. This percentage drops to 70.4% in the recurrent setting. THY1-CD24-EGFR- cells increase from 9.7% to 23.1% at recurrence. In IDHwt EGFRwt primary GBM samples, 48.6% of cells are THY1-CD24-EGFR+ and 44.15% are THY1-CD24-EGFR-. In the recurrent setting, 43.26% of cells are THY1-CD24-EGFR+ and 49.58% are THY1-CD24-EGFR-. CONCLUSION IDH mutant gliomas and IDHwt glioblastoma express different progenitor cell markers and may have a different cell of origin. THY1 is highly expressed in IDH mutant gliomas.