Neuronal-glioma interactions are increasingly recognized as critical in the development and progression of central nervous system tumors. Recent research highlights that gliomas can integrate into neural circuits through various mechanisms, including the synaptogenic factor thrombospondin-1 (TSP-1). This new mechanistic understanding of cancer neuroscience allows for novel insights into target discovery. Critically, therapies that modulate neuron-tumor interactions remain agnostic to other oncogenic changes within tumor cells yet may still target fundamental drivers of tumor growth. In line with these findings and controlling for critical confounding variables, we demonstrate a survival benefit associated with gabapentin (an antagonist of TSP-1) following surgical resection of newly diagnosed glioblastoma. This retrospective, multi-institutional cohort study included 1,072 patients, with a discovery cohort of 693 patients and an additional 379 patients from a separate site for external validation. Furthermore, our findings indicate that gabapentin administration is associated with reduced serum TSP-1 levels, suggesting its potential as a future biomarker.

The intricate relationship between neurons and cancer cells is critical for tumorigenesis and neuronal dysfunction, however underlying mechanisms and potential therapeutic opportunities are not well-established. Neuron-glioblastoma (GBM) interaction has been evaluated at the transcriptomic level; however, mRNA levels poorly represent the proteome. Selective, rapid, and highly precise translation of selected mRNAs is critical for synaptic transmission as well as cancer progression. However, there is a major gap in our understanding of how neuronal signaling regulates the cancer proteome, associated with progression and therapeutic resistance. We established a powerful neuron-human GBM PDX co-culture system, where neurons and GBM cells form synapses and drive neuronal hyperexcitability in vitro, as well as GBM aggressiveness, in vitro and in vivo. We demonstrate that neuronal signaling induced critical signaling pathways in GBM cells that converge on translation control signaling pathways upstream of translation control. Interestingly, neuronal signaling drove selective overexpression and activation of major cap-binding proteins eIF4E and the RNA helicase eIF4A1, the eIF4F translation initiation complex proteins critical in oncogenesis via regulating selective translation of a network of mRNAs. Most importantly, these changes preceded the increase in de novo protein synthesis and proliferation, suggesting that selective mRNA translation controlled by eIF4E and eIF4A1 represents early changes in neuronal signaling-driven GBM progression. We show that paracrine factors released in response to neuron-GBM communication contribute to proliferation and activate signaling that controls selective translation. Lastly, we show that neuronal-signaling-driven GBM aggression was independent of their genomic background. We are performing ribosome profiling and proteomics experiments, which will allow us to decipher the mechanisms of neuronal signaling-driven translational remodeling in GBM cells and dissect synaptic and paracrine signaling as upstream of translation regulation. These experiments will establish the role of translational remodeling control of GBM in response to neuronal signaling and promise to identify novel post-transcriptional vulnerabilities.

Diffuse gliomas infiltrate the cerebral cortex and profoundly alter brain function, yet the multiscale consequences of this invasion on human neural circuits remain poorly understood. Here, we integrate magnetoencephalography (MEG), electrocorticography, spatial transcriptomics, single-nucleus RNA sequencing, and high density single-unit Neuropixels recordings from WHO grade 2-4 glioma patients to interrogate the structural and functional integrity of infiltrated neocortex. We identify a reproducible gain of laminar dependent synaptic gene expression in glioma-infiltrated regions (n = 10 samples; 29,011 spots), strongly correlated with local tumor proliferation (n = 6 patients) despite greater tumor burden in deeper cortical layers (n = 19 patients). Single-nucleus profiling and deconvolution revealed subtype-specific vulnerability of neuronal subclasses accompanied by transcriptomic reprogramming. Single-unit analysis across cortical depth (n = 11 patients) highlighted a profound reduction in overall unit yield and spiking activity (Controls = 169.5 units vs Glioma-infiltrated cortex = 35 units, p-value < 0.05, Mann-Whitney Test) within glioma-infiltrated cortex, with selective loss of excitatory and inhibitory neurons. This cell-type-specific vulnerability varied by glioma subtype and cortical layer, with astrocytoma showing the greatest excitatory loss in deep layers. Power spectral analysis across depth revealed altered laminar oscillatory structure in infiltrated regions—characterized by increased delta (1–4 Hz) and depth-dependent decreases in beta (12–20 Hz) power—mirroring noninvasive MEG findings (n = 144 patients) and partially restored under propofol anesthesia. Cross-modal integration uncovered a core axis linking tumor infiltration, neuron loss, and circuit dysfunction across cortical layers. These findings define laminar signatures of glioma-induced cortical dysfunction, establish a foundational framework for mapping glioma-driven cortical collapse and identify therapeutic windows for circuit-level rescue in brain cancer.

IDH-mutant astrocytomas often progress from low-grade to higher-grade tumors, through incompletely defined mechanisms. The neuronal microenvironments through direct and paracrine-mediated signaling drive glioblastoma progression; therefore, we hypothesized its role in IDH-mutant astrocytoma evolution. In this study, we integrate sixteen patient-matched newly diagnosed and recurrent astrocytomas (CNS WHO grades 2–4), which were interrogated using spatial RNA sequencing, multiplexed immunofluorescence, and three-dimensional co-cultures comprising mouse cortical neurospheres (cNS) and patient-derived astrocytoma organoids (GliA; n = 17). Harmony-integrated UMAP of 10,189 spatial transcriptomes identified ten gene-expression programs across 8 IDH-mutant astrocytomas. CNS WHO grade 2 and grade 3 tumors shared overlapping transcriptomic profiles, whereas grade 4 tumors formed a distinct cluster. A 28-gene IDH-mutant glioma neural connectivity score, characterized by TSP1, NDRG2, and Cx43, correlated with overall survival. Immunofluorescent staining confirmed overexpression of TSP1, NDRG2, and Cx43 in cortex-infiltrative and dense tumor regions. Functional interrogation using novel in vitro co-culture model systems revealed a grade-specific increase in Ki67⁺ proliferating tumor cells throughout the tumor, not restricted to areas of glioma-neuron integration. This was quantified across 7 patient-matched GliA–cNS co-cultures (n = 172 regions) and GliA monocultures (n = 193 regions). Proliferation in GliA co-cultures decayed radially with increasing distance from the neuron–tumor interface, demonstrating that astrocytomas are most proliferative in close proximity to neurons. Furthermore, GliA-cNS co-culture demonstrated no increase in neuronal firing rates and synchrony, as quantified across 22 GliA–cNS co-cultures from 5 patients and 20 cNS monocultures. IDH-mutant astrocytomas display uniform, grade-dependent synaptic remodeling, revealing potential therapeutic targets and treatments for patients.

Neuronal activity and synchrony in both normal and pathological states are regulated by excitatory and inhibitory synaptic inputs, determined by ion channel permeability across the cell membrane. This study investigates neuron-glioma cross-talk in chloride flux and the role of chloride, the most abundant neuronal anion, in influencing neuronal excitability and glioblastoma (GBM) pathophysiology. We hypothesized that GBM’s aggressive proliferative and invasive phenotype is linked to chloride dysregulation and that inhibiting glioma chloride flux could reduce tumor proliferation and glioma-induced neuronal hyperexcitability. The direct effects of chloride flux on glioma growth and invasion were tested using optogenetic stimulation of chloride opsin-expressing glioma cells both in vitro and implanted in vivo in patient-derived xenograft (PDX) mice. To explore chloride cross-talk mechanisms between neurons and glioma cells, we performed single-nucleus RNA sequencing (sNuc-seq) of GBM-neuron co-cultures, analyzing 54,000 cells, and revealed the upregulation of genes involved in chloride homeostasis, including the voltage-gated ClC-3 chloride channel and the sodium-potassium-chloride cotransporter 1 (NKCC1). Mass spectrometry-based proteomic analysis of culture supernatant was performed to identify mechanistic targets and paracrine factors mediating chloride transfer. Light-activated chloride pumping into glioma cells significantly reduced tumor proliferation in vitro and in vivo, underscoring the link between intracellular chloride concentration and tumor growth. sNuc-seq also showed upregulation of NKCC1 and the ligand-gated chloride channel GABAA receptor in tumor-associated neurons co-cultured with NKCC1-overexpressing glioma cells, suggesting shared chloride signaling mechanisms in the tumor microenvironment. Proteomic profiling of conditioned media identified glioma-derived MK2 (MAPKAPK2) as a driver of neuronal NKCC1 upregulation and chloride dysregulation. Pretreatment of GBM cells with the MK2 inhibitor ralimetinib restored chloride balance and reduced neuronal hyperexcitability. Collectively, these findings reveal a paracrine mechanism of chloride transfer between glioma cells and neurons, highlighting chloride signaling as a novel therapeutic target to inhibit neuronal hyperexcitability and tumor proliferation in glioblastoma multiforme (GBM).

Diffuse gliomas remodel neuronal circuits with prognostic and therapeutic significance for patients. Electrophysiologic measures of cortical excitability hold promise for monitoring disease progression and evaluating therapeutic responses. The power law exponent (aperiodic slope) reflects the balance between excitatory and inhibitory activity within neuronal networks, a critical aspect of normal brain function often disrupted in neurological conditions. Despite its potential, the significance of the aperiodic slope in glioma-infiltrated tissue and its underlying cellular processes has not been fully investigated. Here, we integrate multimodal electrophysiological analysis with transcriptomic profiling to analyze the aperiodic slope in both normal and glioma-infiltrated cortex. We determine that glioma infiltration induces a flattening of the aperiodic slope, indicating a shift toward excitation dominance that varies according to tumor subtype and correlates with impairments in semantic naming. Single-nucleus RNA sequencing revealed that cortical regions with flat aperiodic slope exhibit transcriptional programs enriched in glutamatergic signaling, membrane depolarization, and excitatory synaptic transmission. The aperiodic slope responds to pharmacologically induced changes in cortical inhibition during propofol administration, a GABAA agonist. Our results establish the aperiodic slope as a robust biomarker of glioma-associated excitation-inhibition imbalance, with potential applications in tumor classification and treatment monitoring.

Neuronal activity-driven mechanisms influence glioblastoma cell proliferation and invasion, while glioblastoma remodels neuronal circuits. Although a subpopulation of malignant cells enhances neuronal connectivity, their impact on the immune system remains unclear. Here, we show that glioblastoma regions with enhanced neuronal connectivity exhibit regional immunosuppression, characterized by distinct immune cell compositions and the enrichment of anti-inflammatory tumor-associated macrophages (TAMs). In preclinical models, knockout of Thrombospondin-1 (TSP1/ Thbs1 ) in glioblastoma cells suppresses synaptogenesis and glutamatergic neuronal hyperexcitability. Furthermore, TSP1 knockout restores antigen presentation-related genes, promotes the infiltration of pro-inflammatory TAMs and CD8 + T-cells in the tumor, and alleviates TAM-mediated T-cell suppression. Pharmacological inhibition of glutamatergic signaling also shifts TAMs toward a less immunosuppressive state, prolongs survival in mice, and shows the potential to enhance the efficacy of immune cell-based therapy. These findings confirm that glioma-neuronal circuit remodeling is strongly linked with regional immunosuppression and suggest that targeting glioma-neuron-immune crosstalk could provide avenues for immunotherapy.

2062 Background: Diffuse gliomas disrupt neuronal dynamics, leading to excitation-inhibition (E/I) imbalance and associated functional impairments. The aperiodic component of the power spectral density (1/f slope) has emerged as a proxy for estimating E/I balance, offering a novel framework for understanding glioma-induced neural dysregulation. This study is the first to validate the relationship between 1/f slope and E/I dysregulation in glioma by integrating electrophysiological, genomic, and behavioral data. Methods: Resting-state intraoperative subdural electrocorticography (ECoG) data were recorded from 13 glioma patients. Power spectral analysis at a frequency of 70–150Hz (high-gamma) computed 1/f slopes, and electrodes were classified as glioma-infiltrated or normal-appearing based on preoperative MRI T2-FLAIR. Linear mixed-effects models assessed E/I balance across tissue and glioma subtypes. Single-nucleus RNA sequencing (snRNA-seq) was performed on 14 spatially annotated glioma tissue samples from regions classified as inhibitory or excitatory by 1/f slope. Behavioral analysis of language tasks examined functional correlates of E/I imbalance. Results: The cohort included 23.0% WHO grade 2 IDH-mutant oligodendrogliomas, 38.5% WHO grade 2-3 IDH-mutant astrocytoma, and 38.5% glioblastoma (GBM). Glioma-infiltrated electrodes (n=142) exhibited significantly lower 1/f slopes than normal-appearing electrodes (n=518;p < 0.0001), reflecting an excitation-dominant state. Subtype analysis revealed hierarchical E/I imbalance, with GBM showing the steepest reductions in 1/f slope compared to astrocytoma and oligodendroglioma (glioma-infiltrated: p<0.0001; normal-appearing: GBM vs. oligodendroglioma, p=0.012; GBM vs. astrocytoma, p=0.019). SnRNA-seq revealed elevated excitatory and reduced inhibitory signaling gene expression in glutamatergic and GABAergic neuronal populations across glioma-infiltrated (n=12; n=4 per subtype) and normal cortex (n=2) samples. Excitatory module scores were significantly higher in excitatory 1/f samples compared to inhibitory 1/f samples, validating the 1/f slope as a genomic correlate of E/I imbalance in human cortical tissue. Behavioral analysis of language tasks demonstrated error-related reductions in 1/f slope (e > i), emphasizing the functional impact of glioma-induced dysregulation. Conclusions: Diffuse gliomas are associated with a profound shift toward excitation dominance in both glioma-infiltrated and normal-appearing cortex. For the first time, the 1/f slope is validated as a robust measure of E/I imbalance through electrophysiological, genomic, and behavioral analyses. These findings position the 1/f slope as a physiologically relevant biomarker of glioma-induced neural dysregulation, offering significant potential to inform therapeutic strategies.

Neuronal activity profoundly influences glioblastoma (GBM) cell proliferation and invasion, while GBM actively remodels the surrounding neuronal circuitry. We recently demonstrated that intratumoral regions with heightened neuronal connectivity exhibit prominent regional immunosuppression. Our preclinical investigations revealed that tumor-secreted Thrombospondin-1 (TSP1) enhances glioma-neuronal circuit remodeling, contributing to an immunosuppressive tumor microenvironment (TME) characterized by increased anti-inflammatory tumor-associated macrophages (TAMs) and decreased CD8+ T cells. Notably, this immunosuppressive TME was alleviated either by eliminating tumor-derived TSP1 or by treatment with perampanel (PER), which inhibits glutamatergic excitatory signaling. However, the therapeutic implications of these findings remain to be fully elucidated. In this study, we conducted functional validation by isolating TAMs from mice bearing TSP1-WT or KO tumors and co-culturing them with carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells. Strikingly, the suppressive effect of TAMs on T cell proliferation was significantly alleviated in the TSP1-KO group (P = 0.03). Moreover, a similar trend was observed in TAMs from PER-treated tumor-bearing mice (P = 0.06). Building on this functional evidence, we explored combination therapeutic strategies. Specifically, we tested the combination of anti-EGFRvIII CAR-T cells and anti-PD1 antibody, with or without PER, in mice bearing EGFRvIII-expressing SB28 tumors. Complete tumor eradication occurred more frequently in the PER-treated group compared to controls (44% vs. 22%). Furthermore, in brain-infiltrating leukocytes isolated from five “cured” mice, persisting CAR-T cells were detected in all three PER-treated mice but only one of the two controls. Notably, a CD62L+CD44+ memory phenotype, associated with long-term tumor remission after treatment, was observed exclusively in the PER-treated group. In summary, our findings highlight the intricate crosstalk between glioma, neurons, and immune cells as a pivotal therapeutic vulnerability. Targeting neuronal activity may represent a promising strategy to modulate the immunosuppressive TME in GBM and enhance the efficacy of immunotherapeutic modalities, such as CAR-T cell therapy.

INTRODUCTION: A hallmark of the incurable nature of GBM is its highly migratory phenotype, making surgical resection only a method that prolonged the inevitable death of the patient. Neuron-glioma interactions have been shown to play a vital role in tumor cell migration, the level of functional connectivity in neuron-glioma networks and glioma-induced neuronal hyperexcitability. All of which have been shown to lead to worse prognosis. Preliminary from our lab shows that in tumor regions with high functional connectivity show excitatory GABAergic signaling mediated through NKCC1. We hypothesize that knocking down NKCC1 will lead to downregulated glioma cell migration, proliferation and network connectivity. METHODS: In neuron-glioma coculture, NKCC1 was knocked down through shRNA or the pharmacological inhibitor Bumetanide, and the effect on network activity and synchrony of neurons was captured using microelectrode array (MEA) and real-time calcium imaging. GBM cells with NKCC1 intact or NKCC1 knockdown were transplanted onto organotypic human and mouse cortical slices and performed live cell imaging to assess for tumor cell migration, depth of invasion, and presence of tumor microtubes. We performed immunostaining on both organotypic brain slices transplanted with GBM cells as well as neurons in co-culture with GBM cells to quantify proliferation and neuron-glioma synapses. RESULTS: GBM with NKCC1 knocked down demonstrated a decrease in network excitability and synchrony, tumor cell migration, depth of invasion, and network integration, but showed an upregulation in tumor microtube number and length. NKCC1 knockdown cells also showed decreased proliferation and number of neuron-glioma synapses. CONCLUSIONS: NKCC1-mediated excitatory GABAergic signaling is necessary for GBM network integration, migration, glioma proliferation, and Knocking down of NKCC1 has uncovered a possible therapeutic vulnerability in tumor cell migration.

Introduction Au sein des gliomes, l’interleukine 6 (IL6) exerce un rôle pro-tumoral, notamment en augmentant l’expression de PD-L1 (Programmed Cell Death Ligand 1) et en induisant ainsi une immunosuppression locale [1], [2]. Sur le versant neuronal, l’IL6 favorise la neurogenèse au cours de la vie intra-utérine [3] et participe au remodelage des circuits neuronaux chez les patients victimes d’une lésion traumatique cérébrale ou médullaire [4], [5]. Ainsi, l’objectif de ce travail était de déterminer si l’IL6 contribue au remodelage structural et fonctionnel des circuits neuronaux induits par les gliomes. Méthodes L’expression de l’IL6 et de GAP43, agent impliqué dans la formation des microtubes tumoraux, a été étudiée dans des zones de haute connectivité fonctionnelle et de basse connectivité fonctionnelle, identifiées par magnéto-électro-encéphalographie, dans un panel de 44 gliomes de différents grades. Des cellules tumorales issues de glioblastomes humains ont été cocultivées avec des neurones murins. L’index de prolifération, l’expression de marqueurs de connectivité et de marqueurs synaptiques ainsi que l’activité neuronale ont été quantifiés après traitement par Tocilizumab (TCZ), un inhibiteur du récepteur de l’IL6, ou par IL6 recombinante. Résultats L’expression d’IL6 et de GAP43, en ARN et en protéine, était significativement plus importante dans les régions de haute connectivité fonctionnelle que dans les régions de basse connectivité fonctionnelle, quel que soit le grade du gliome. Dans les cocultures neurone-glioblastome, l’index de prolifération, l’expression de GAP43 et le nombre de microtubes tumoraux par cellule étaient augmentés par l’exposition à l’IL6 recombinante et diminués par l’exposition au TCZ. La colocalisation de marqueurs pré-synaptiques et post-synaptiques et l’activité neuronale étaient augmentés par l’exposition à l’IL6 et diminués par l’exposition au TCZ. Conclusion L’IL6 augmente la prolifération tumorale et renforce les interactions entre les cellules tumorales et les neurones. Ces données suggèrent donc que les traitements ciblant l’IL6 pourraient représenter une nouvelle option thérapeutique chez des patients sélectionnés.

Oligodendrogliomas are initially slow-growing brain tumors that are prone to malignant transformation despite surgery and cytotoxic therapy. Understanding of oligodendroglioma evolution and new treatments for patients have been encumbered by a paucity of patient-matched newly diagnosed and recurrent tumor samples for multiplatform analyses, and by a lack of preclinical models for interrogation of therapeutic vulnerabilities that drive oligodendroglioma growth. Here we integrate spatial and functional analyses of tumor samples and patient-derived organoid co-cultures to show that synaptic connectivity is a hallmark of oligodendroglioma evolution and recurrence. We find that patient-matched recurrent oligodendrogliomas are enriched in synaptic gene expression programs irrespective of previous therapy or histologic grade. Analyses of spatial, single-cell, and clinical data reveal epigenetic misactivation of synaptic genes that are concentrated in regions of cortical infiltration and can be used to predict eventual oligodendroglioma recurrence. To translate these findings to patients, we show that local field potentials from tumor-infiltrated cortex at the time of resection and neuronal hyperexcitability and synchrony in patient-derived organoid co-cultures are associated with oligodendroglioma proliferation and recurrence. In preclinical models, we find that neurophysiologic drugs block oligodendroglioma growth and pathologic electrophysiology. These results elucidate mechanisms underlying oligodendroglioma evolution from an indolent tumor to a fatal disease and shed light on new biomarkers and new treatments for patients.

Therapeutic clinical trial enrollment does not match glioma incidence across demographics. Traditional statistical methods have identified independent predictors of trial enrollment; however, our understanding of the interactions between these factors remains limited. To test the interactive effects of demographic, socioeconomic, and oncologic variables on trial enrollment, we designed boosted neural networks (BNNs) for all glioma patients (n = 1042), women (n = 445, 42.7%), and minorities (n = 151, 14.5%) and externally validated these models [whole cohort, n = 230; women, n = 89 (38.7%); minority, n = 66 (28.7%)]. For the whole-cohort BNN, the most influential variables on enrollment were oncologic variables, including KPS [total effect (TE), 0.327], chemotherapy (TE, 0.326), tumor location (TE, 0.322), and seizures (TE, 0.239). The women-only BNN exhibited a similar trend. Conversely, for the minority-only BNN, socioeconomic variables [insurance status (TE, 0.213), occupation classification (TE, 0.204), and employment status (TE, 0.150)] were most influential. These results may help prioritize patient-specific initiatives to increase accrual.

Gliomas infiltrate eloquent cortex yet continue to participate in language processing, albeit with reduced information encoding. The molecular determinants of this dysfunction are unknown. The present work aims to define imaging and genomic predictors of aphasia in diffuse supratentorial gliomas. We studied 88 adults (median 59 y; 38 % female) with newly diagnosed WHO grade 2-4 gliomas who underwent targeted next-generation sequencing and Quick Aphasia Battery (QAB) testing (2017-2021). Support-vector regression lesion–symptom mapping localized voxels where tumor burden predicted QAB scores. A random-forest model integrating radiomics, molecular variants, and clinical variables ranked predictors of aphasia; multivariable linear regression validated key features. Single-nucleus RNA-seq of resected tumors clarified cellular expression patterns. Lesion–symptom mapping isolated a 61,745-voxel cluster in the left superior temporal gyrus whose tumor overlap strongly correlated with lower QAB scores. The random-forest model highlighted three radiographic metrics—T1-post-gadolinium, T2-FLAIR, and total lesion voxels within this cluster—and seven genomic alterations. The top molecular predictor, ARID2 mutation (3/88), was associated with markedly worse language performance (mean ± SD QAB 6.6 ± 1.4 vs 9.1 ± 1.3; p = 0.01). On multivariate analysis, ARID2 status (β = –1.42; 95 % CI –2.63 to –0.21; p = 0.02) and contrast-enhancing voxels in the cluster (β = –0.08 per 100-voxel increase; p < 0.001) independently predicted aphasia. Single-nucleus RNA-seq revealed ARID2 expression enriched in proliferating tumor cells, linking chromatin-remodeling dysregulation to cortical network dysfunction. An imaging-genomic signature comprising ARID2 mutation and contrast-enhancing burden in a dominant temporal language hub robustly predicts aphasia in diffuse glioma. These findings nominate ARID2 as a mechanistic driver of glioma-associated language impairment and provide actionable markers for precision surgical and adjuvant strategies to preserve cognition

Abstract Gliomas engage in bidirectional interactions with their microenvironment, wherein neuronal activity influences glioma proliferation and gliomas induce neuronal hyperexcitability. Although various models elucidate this complex interplay, a preference for a three-dimensional (3D) cell culture model has emerged to mimic intra-tumoral heterogeneity and microenvironmental interactions influencing glioma invasion, resistance, and recurrence. This study introduces an in vitro, self-assembled, scaffold-free, neuron-glioma spheroid fusion model to investigate the electrophysiological and functional interplay between glioma and its microenvironment. Primary mouse cortical neurons (MCN) were isolated from prenatal brain tissues and were cultured at 500,000 cells/spheroid on ultra-low attachment plates. Glioma organoids (WHO Grade 2-4) were developed from primary patient-derived tissue samples and were allowed to self-assemble for at least two weeks. After spontaneous spiking activity was detected in MCN spheroids, they were combined with glioma organoids in culture and allowed to fuse. A multielectrode array (MEA) was used to characterize the electrophysiological properties of the fusion model. Structural and functional characterizations were performed using immunofluorescence staining of proliferation, microglial, astrocytic, synaptic, and tumor markers. Additionally, the electrophysiological effects of the dual-IDH-mutant inhibitor, Vorasidenib, were assessed in WHO grade 2-4 IDH-mutant gliomas. Electrophysiological analysis of glioma-neuron co-cultures using MEA demonstrated a significantly increased network synchrony and firing rate in the fusion model when compared with the neuron-only condition, consistent with 2D models in the past. In correlation, the fusion model also demonstrated a significant increase in the Ki67 proliferation index across WHO grade 2-4 gliomas when compared to the glioma organoid-only condition. Vorasidenib treatment decreased neuronal firing rate and synchrony. Together, these results offer a high-throughput 3D model for recapitulating the tumor-brain microenvironment for both low and high-grade gliomas. Future studies will focus on the use of this model for drug screening and the exploration of malignant transformation in gliomas.

Neural-tumor interactions drive glioma growth as evidenced in preclinical models, but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients' survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma, primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like, astrocyte-like and oligodendrocyte-progenitor-like, alongside oligodendrocytes and excitatory neurons. In line with these findings, high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients, a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients' plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes.