This human iPSC-derived tumor–brain organoid platform provides a reliable and scalable system for studying complex tumor-neural interactions and exploring therapeutic approaches that aim to eliminate the tumor while preserving neural function.
Abstract
Background Glioblastoma (GBM) is characterized by neurological dysfunction caused by tumor cells that interact with and alter neuronal circuits. However, the specific neuronal populations and molecular mechanisms most susceptible to GBM invasion remain poorly understood. Methods We created a human tumor–brain organoid model by combining U87 glioblastoma cells with iPSC-derived neural organoids. This system enabled us to study tumor–neural interactions over an extended period under standard temozolomide (TMZ) treatment. We used single-cell transcriptomics to monitor cell-type-specific responses. Results Our model recapitulated the diffuse infiltration observed in patients, leading to extensive structural remodeling and a profound loss of neuronal and glial populations. Single-cell analysis revealed that TMZ suppressed proliferative and biosynthetic programs but enriched for stress-responsive, mesenchymal-like, and therapy-adapted tumor states. Notably, GABAergic neurons exhibited the greatest transcriptional vulnerability, with ∼36% (7,499 of 20,659) of genes differentially expressed. Invasion triggered endoplasmic reticulum stress and shut down metabolic, respiratory, synaptic, and ion-homeostatic pathways. Crucially, SLC12A5-expressing GABAergic neurons plummeted from 31% to 12%, accompanied by a sharp decline in KCC2 protein expression. While TMZ partially rescued neuronal metabolic and electron transport chain function, it failed to restore SLC12A5/KCC2 expression or inhibitory signaling. Conclusions GBM invasion leads to a continued imbalance of chloride in GABAergic networks, and this disruption remains even after undergoing tumor-targeted chemotherapy. This human iPSC-derived tumor–brain organoid platform provides a reliable and scalable system for studying complex tumor-neural interactions and exploring therapeutic approaches that aim to eliminate the tumor while preserving neural function.
Glioblastoma (GBM) is a highly aggressive malignant brain tumor accounting for 15% of all brain tumors and 50% of all gliomas. The exact cause of GBM is not fully understood but risk factors include age, genetic mutations, exposure to ionizing radiation, and certain genetic disorders. Symptoms of GBM include headaches, seizures, cognitive impairment, and weaknesses on one side of the body. Myeloid cells account for 30–50% of the tumor mass and are instrumental in shaping the complex tumor microenvironment (TME). Inflammation in the TME is an important driver of tumor growth and invasion; however, as the environment evolves, the immunosuppressive TME poses a significant hurdle as it hinders the immune-mediated killing of tumor cells. Our work utilizes neuroimmune organoids containing neurons, astrocytes, microglia, and vascular-like cells, to which we add patient-derived GBM cells and/or iPSC-derived macrophages to model the GBM TME. Model characterization was performed using single-cell RNA sequencing and supernatant proteomics to determine cell-specific changes during coculturing. Our findings are consistent with this 7-day coculture model recapitulating key aspects of GBM early tumor establishment and immune activation, with transcriptomic and secretome signatures suggestive of an emerging immune evasion phenotype.
Nina Y. Yuan, William D. Richards, Kailyn T. Parham et al.· Organoids· 0 citations
Glioblastoma (GBM) is the most aggressive primary brain tumour and is frequently accompanied by severe neurological symptoms, including epilepsy and cognitive impairment. Neurological symptoms often persist after surgical resection, indicating that GBM induces durable and self-sustaining changes in the surrounding neuronal networks. However, the mechanisms by which GBM reshapes network structure and function in the tumour periphery remain poorly understood. We present a compartmentalised in vitro platform enabling long-term coculture of iPSC-derived neurons and primary GBM cells to investigate these changes. Placed on high-density microelectrode arrays, the platform permits longitudinal electrophysiological recordings at single-neuron resolution. Using effective network inference, we find that GBM drives a reproducible structural progression: first toward a hyperconnected, hub-dominated architecture, then a collapse of community structure accompanied by a widespread neuron loss. This evolving structure shapes population dynamics, constraining features such as network burst rate and instantaneous synchrony. The reorganisation also carries computational consequences: signal propagation becomes progressively redundant and synergistic rather than unique. As a result, neurons lose the capacity to encode distinct input combinations independently, and the repertoire of accessible network states contracts. Together, these findings reframe GBM as a driver of neuronal network reorganisation rather than uniform hyperexcitability, and establish a compartmentalised, single-neuron-resolution platform for the longitudinal observation, dissection, and ultimately targeting of the network processes that underlie disease progression.
Giulia Amos, Luc Jordi, K. Ahuja et al.· bioRxiv· 0 citations
Introduction Gliomas integrate into neural circuits and heighten neuronal excitability, engaging in bidirectional communication whereby neuronal activity promotes tumor growth and proliferation. Aging reshapes the brain microenvironment through extracellular matrix changes, altered secretory factors, and immune dysfunction, creating conditions permissive to tumorigenesis and limiting immunotherapy efficacy in glioblastoma. However, its effect on neuronal excitability and signaling in glioblastoma remains poorly understood. Methods We developed a novel classification system for glioblastoma by leveraging three classes of DNA methylation-based aging biomarkers: chronological, biological, and mitotic clocks. This approach stratified tumors into accelerated and decelerated epigenetic aging subtypes, which we then characterized at the molecular, functional, and clinical levels using multimodal analyses. Guided by these profiles, we evaluated the in vitro effects of the FDA-approved agents levetiracetam and riluzole, alone and in combination with temozolomide, on U87MG and A172 cell lines. Specifically, we assessed changes in cell viability, apoptosis, and the expression of marker genes related to stemness, neuronal hyperexcitability, and immunosuppression. Results Tumors with decelerated epigenetic aging showed expression modules and CpG hypomethylation associated with neuronal activity and stemness, and carried significantly worse prognosis. Single-cell and spatial multi-omics analyses revealed enrichment for neurons and malignant neural stem-like cells in these tumors. They also displayed enhanced intercellular communication, driven predominantly by glutamate signaling across the malignant, neuronal, and immune compartments of the tumor microenvironment. In vitro pharmacological inhibition of glutamatergic signaling with levetiracetam and riluzole reduced cell viability, induced apoptosis, and suppressed expression of stemness, neuronal hyperexcitability, and immunosuppression markers. Both agents potentiated the cytotoxic and apoptotic effects of temozolomide, supporting glutamatergic inhibition as a strategy for improving chemosensitivity. Conclusion By establishing a framework for decoding glioblastoma heterogeneity through epigenetic aging, we identified the glutamatergic pathway as a clinically actionable vulnerability. Our findings suggest that combining anti-glutamatergic therapies with temozolomide exerts synergistic antitumor effects while mitigating adverse chemotherapy-induced phenotypes, such as increased stemness, neuronal hyperexcitability, and immunosuppression, thereby laying the groundwork for novel therapeutic strategies. Graphical Abstract
Abstract Brain metastases(BM) represent the most common intracranial tumors and are associated with a markedly worse prognosis compared with metastases at other sites. Metastatic lesions that colonize the brain adapt to its unique cellular and metabolic microenvironment. This adaptation drives the emergence of specialized tumor subpopulations with altered biological behavior. Understanding how metastatic tumors interact with and influence the surrounding neural tissue is therefore essential for developing strategies that preserve neurological function. The objective of this study was to investigate the interactions between BM and the surrounding brain parenchyma, with particular emphasis on the mechanisms underlying demyelination and neuronal impairment. Brain metastasis organoids(BMOs) were generated from surgically resected patient BM specimens and co-cultured with long-term viable human brain slices. Immunofluorescence staining was used to evaluate changes in myelination and cellular responses within the brain tissue. In parallel, transcriptomic profiling and tumor–brain parenchymal ligand–receptor interaction analyses were performed to identify dysregulated molecular signaling pathways with potential therapeutic relevance. BMOs successfully integrated with human brain slices and reproduced key characteristics of tumor behavior within the neural microenvironment. Myelin basic protein(MBP) immunostaining demonstrated significant demyelination in brain slices co-cultured with BMOs compared with control slices(P < 0.05). Transcriptomic analysis revealed clusters of differentially expressed genes, including marked downregulation of pathways associated with myelination and neurogenesis. Further investigation identified critical regulators of neuronal and glial function, including a downregulated gene network involved in myelin maintenance. Ligand–receptor interaction analysis revealed prominent dysregulation of NGF and TGF-β signaling. Pharmacologic targeting of these pathways in co-culture experiments significantly restored MBP signal intensity(P < 0.05). These findings demonstrate that BM actively disrupt myelin homeostasis and neural signaling within surrounding brain tissue. This ex-vivo platform provides a robust experimental system to study tumor–brain interactions and to evaluate therapeutic strategies aimed at limiting tumor-induced neurological damage.
Youssef M. Zohdy, Arman Jahangiri, Amelia Tong et al.· Neuro-Oncology Advances· 0 citations
Objective Glioblastoma (GBM) exhibits marked cellular plasticity, including partial acquisition of neuron-associated transcriptional features. We tested whether public GBM spatial and single-nucleus datasets reveal a tumor-neuronal program with spatial organization and distinction from canonical proneural-like identity. Methods We re-analyzed public multi-region GBM spatial transcriptomic data from Greenwald et al. and used healthy cortex data from Ravi et al. as a non-malignant reference. Tumor-neuronal (TN) classifications were defined by concurrent enrichment of malignancy-associated and neuronal-associated gene programs using permutation-derived empirical thresholds. Classification stability was assessed by bootstrap resampling, repeated threshold estimation, and Jaccard overlap. Greenwald-derived CNA/malignancy scores were used to evaluate tumor-associated signal in TN-classified spatial units. External validation was performed in an independent Wang malignant GBM-cell/single-nucleus dataset, including held-out TN-specific scoring after excluding genes used in the original scoring programs. Exploratory bulk-cohort analysis was performed in TCGA GBM. Results TN-classified spatial units were detected across tumor regions at higher frequency than healthy cortex and showed regional organization within the spatial discovery specimen. They expressed synaptic and vesicle-associated genes, including SYT1 and STMN2, while retaining malignant/glial-associated features such as GFAP, VIM, and CLU. Mature neuronal subtype markers were less enriched, supporting partial neuronal mimicry rather than lineage conversion. TN-associated genes formed a connected protein-interaction network centered on synaptic, neurite-associated, and cytoskeletal proteins. CNA/malignancy-score analysis showed tumor-associated signal in TN-classified spatial units. In the Wang malignant GBM-cell validation dataset, TN-classified cells/nuclei retained held-out TN-specific enrichment relative to non-TN and proneural-high non-TN malignant cells/nuclei. Conclusion Public spatial and single-nucleus GBM datasets identify a tumor-associated neuronal mimicry program that is spatially organized in a GBM discovery specimen and detectable in an independent malignant-cell validation dataset. This program is proneural-adjacent but not reducible to canonical proneural identity.
Claire Yuan, J. Chen· Frontiers in Oncology· 0 citations
Glioblastoma remains the most aggressive primary brain tumor in adults and is characterized by extensive invasiveness, high recurrence rates, and pronounced inter- and intratumoral heterogeneity. These features continue to limit the effectiveness of current therapies and highlight the need for experimental models that more faithfully capture the complexity of the disease. Conventional two-dimensional in vitro systems, while highly accessible and experimentally tractable, fail to reproduce the three-dimensional architecture, extracellular matrix organization, and dynamic tumor–microenvironment interactions that critically shape glioblastoma behavior. Although three-dimensional spheroid and organoid models partially address these limitations, they still incompletely reflect the structural and cellular complexity of native brain tissue. Organotypic brain tissue slice cultures have emerged as a powerful intermediate model that preserves native cytoarchitecture, cellular diversity, and key aspects of the tumor microenvironment. In this review, we provide an integrated overview of organotypic slice models derived from mouse, rat, and human tissue, with a focus on their application in studying glioblastoma invasion, tumor–microenvironment interactions, and therapeutic response. We discuss methodological approaches, advances in tissue preservation and bioengineering, and the integration of molecular and electrophysiological readouts. Particular emphasis is placed on human brain slice cultures, which retain patient-specific structural and cellular features and therefore offer a unique platform for investigating tumor heterogeneity and individualized treatment responses. At the same time, we address current limitations, including restricted viability, methodological variability, and the need for standardized experimental frameworks. By positioning organotypic brain slices as structurally preserved tumor ecosystems rather than simplified experimental systems, this review highlights their potential to bridge mechanistic discovery and translational application, and to contribute to the development of functionally informed precision oncology strategies in glioblastoma.
A. Bondar, Susanne Raulefs, Jette Wrana et al.· Frontiers in Oncology· 0 citations
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