Rotenone has potential chemical toxicity in the nervous system of both insects and mammals, but its deep molecular biological mechanisms have not been clarified. Here, the epigenetic regulatory mechanism underlying the toxicity of rotenone was studied using murine brain organoids (mBOs). Transmission electron microscopy indicated that rotenone destroyed mBOs'mitochondrial structure. RRBS-Seq showed that some promoter regions from the DLK1-DIO3 imprinted microRNA clusters were hypomethylated. But, rotenone stimulated hypermethylation significantly on the promoter DNA of miR-6991-3p. MiR-6991-3p in the rotenone-treated mBOs had the greatest decreased miRNA expression compared with the control. Meanwhile, luciferase report assay indicated that miR-6991-3p induced a decrease in luciferase activity via binding to specific sites on the 3'UTR of DEDD2 gene. To overexpression of miR-6991-3p attenuated mBO proliferated inhibition and cell death, accumulation for lipid peroxidation products significantly by rotenone inducing. Subsequently, results of cell staining and molecular biology experiment revealed that overexpression for miR-6991-3p significantly weakened expression levels of death-related genes (DEDD2, caspase-8, caspase-3, and caspase-1), but significantly elevated expression levels of cell proliferation-related genes (Ki67 and BCL2) in rotenone treated mBOs group. Here, we reveal a novel epigenetic mechanism of rotenone-induced neuronal death, in which rotenone induced promoter DNA hypermethylation of miR-6991-3p in the DLK1-DIO3 imprinted cluster. This caused miR-6991-3p transcriptional activity to be downregulated, which subsequently significantly increased the expression of its target gene, DEDD2, ultimately leading to neural organoid cell death.

Glioblastoma (GBM) is a malignant tumor of the nervous system, which is difficult to treat due to its strong invasiveness, rapid progression, and poor prognosis. To understand the complex biological behavior of glioblasts and the interaction between tumors and hosts, a new in vitro platform based on human cells is required, which can summarize the complex cellular structure and cell diversity of the human brain, as well as the biological behavior of GBM. Organoids are 3D self-organizing tissues, partially similar to source tissues, which can simulate the structure and physiological functions of organs or tissues in vitro. In this review, we underline the widespread application of different types of GBOs models in GBM pathogenesis, including cells derived, tumor tissues derived, and other co-culture models, as well as their application and shortcomings in the treatment of GBM.

Isocitrate dehydrogenase wild-type glioblastoma is an extremely aggressive and fatal primary brain tumour, characterised by extensive heterogeneity and diffuse infiltration of brain parenchyma. Despite multimodal treatment and diverse research efforts to develop novel therapies, there has been limited success in improving patient outcomes. Constructing physiologically relevant preclinical models is essential to optimising drug screening processes and identifying more effective treatments.

Traditional in-vitro models have provided critical insights into glioblastoma pathophysiology; however, they are limited in their ability to recapitulate the complex tumour microenvironment and its interactions with surrounding cells. In-vivo models offer a more physiologically relevant context, but often do not fully represent human pathology, are expensive, and time-consuming. These limitations have contributed to the low translational success of therapies from trials to clinic. Organoid and glioblastoma-on-a-chip technology represent significant advances in glioblastoma modelling and enable the replication of key features of the human tumour microenvironment, including its structural, mechanical, and biochemical properties. Organoids provide a 3D system that captures cellular heterogeneity and tumour architecture, while microfluidic chips offer dynamic systems capable of mimicking vascularisation and nutrient exchange. Together, these technologies hold tremendous potential for high throughput drug screening and personalised, precision medicine. This review explores the evolution of preclinical models in glioblastoma modelling and drug screening, emphasising the transition from traditional systems to more advanced organoid and microfluidic platforms. Furthermore, it aims to evaluate the advantages and limitations of both traditional and next-generation models, investigating their combined potential to address current challenges by integrating complementary aspects of specific models and techniques.

Cushing's disease (CD) is caused by pituitary tumors that overproduce adrenocorticotropic hormone (ACTH); however, effective medical treatments remain limited, significantly impairing patients' quality of life and prognosis. Despite extensive molecular analyses, the pathogenesis of CD remains unclear. Although previous molecular studies have relied heavily on rodent-derived cells and rodent transgenic models, significant species differences exist in the tumorigenesis of CD between humans and rodents. To date, an established human CD cell model is lacking, as human CD cells are limited in availability and sustainability over time. Additionally, the gene modifications used in transgenic models do not necessarily reflect the causative genes in CD. CD tumors exhibit wide phenotypic heterogeneity, which further complicates the development of an ideal genetic model. In this review, we provide an analysis of 11 genetic models used to study CD, outlining their historical development, strengths, and limitations. Additionally, we discuss the ongoing development of human induced pluripotent stem cell (iPSC)-derived pituitary organoids and further describe various models of pituitary organoids as an emerging novel approach to studying CD. By comparing all these models, we highlight the necessity of advancing genetic models to improve our understanding and treatment of CD.

Glioblastoma (GBM) progression and therapeutic resistance are significantly influenced by complex interactions between tumor cells and the brain microenvironment, particularly neurons. However, studying these interactions in physiologically relevant conditions has remained challenging due to limitations in existing model systems.

Here, we present hGliCS (human glioma-cortical spheroid), a novel fully human brain tumor model that overcomes key limitations of current approaches by combining patient-derived GBM cells with mature human cortical neurons derived from induced pluripotent stem cells.

We demonstrate that GBM cells in hGliCS develop three critical hallmark features observed in patients: (i) formation of tumor microtubes enabling intercellular communication, (ii) establishment of neuron-glioma synapses, and (iii) development of an interconnected network with coordinated calcium signaling. Single-cell RNA sequencing reveals that tumor cells in hGliCS exhibit cellular heterogeneity and transcriptional profiles remarkably similar to those observed in mouse xenografts, including activation of key oncogenic pathways and neuronal-like features. Notably, while GBM cells showed substantial transcriptional adaptation to the neural environment, neurons maintained their core identity with only subtle alterations in glutamate signaling and structural gene expression. We validate hGliCS as a drug screening platform by demonstrating resistance patterns to standard chemotherapy and radiation similar to clinical observations. Furthermore, we show the model's utility in testing standard and novel therapeutic compounds targeting cell proliferation and tumor-specific neurobiological features, respectively.

This physiologically relevant human model system provides new opportunities for studying GBM biology and tumor-neuron interactions in a controlled environment. By bridging the gap between simplified in vitro systems and complex in vivo models, hGliCS represents a promising platform for therapeutic development and personalized medicine approaches in GBM treatment.

Tumor organoids are important tools for cancer research, but current models have drawbacks that limit their applications for predicting response to therapy. Here, we developed a fast, efficient, and complex culture system (IPTO, individualized patient tumor organoid) that accurately recapitulates the cellular and molecular pathology of human brain tumors. Patient-derived tumor explants were cultured in induced pluripotent stem cell (iPSC)-derived cerebral organoids, thus enabling culture of a wide range of human tumors in the central nervous system (CNS), including adult, pediatric, and metastatic brain cancers. Histopathological, genomic, epigenomic, and single-cell RNA sequencing (scRNA-seq) analyses demonstrated that the IPTO model recapitulates cellular heterogeneity and molecular features of original tumors. Crucially, we showed that the IPTO model predicts patient-specific drug responses, including resistance mechanisms, in a prospective patient cohort. Collectively, the IPTO model represents a major breakthrough in preclinical modeling of human cancers, which provides a path toward personalized cancer therapy.

Glioblastoma (GBM) has a fatal prognosis because of its aggressive and invasive characteristics. Understanding the mechanism of invasion necessitates an elucidation of the relationship between tumor cells and the tumor microenvironment. However, there has been a scarcity of suitable models to investigate this. In this study, we established a glioblastoma-cerebral organoid assembloid (GCOA) model by co-culturing patient-derived GBM tumoroids and human cerebral organoids. Tumor cells from the tumoroids infiltrated the cerebral organoids, mimicking the invasive nature of the parental tumors. Using time-lapse imaging, various invasion patterns of cancer cells within cerebral organoids resembling a normal tissue milieu were monitored. Both single- and collective-cell invasion was captured in real-time. We also confirmed the formation of an intercellular tumor network and tumor-normal-cell interactions. Furthermore, the transcriptomic characterization of GCOAs revealed distinct features of invasive tumor cells. Overall, this study established the GCOA as a three-dimensional (3D) in vitro assembloid model to investigate invasion mechanisms and interactions between tumor cells and their microenvironment.

Glioblastoma (GBM) is an aggressive form of brain cancer that is highly resistant to therapy due to significant intra-tumoral heterogeneity. The lack of robust in vitro models to study early tumor progression has hindered the development of effective therapies. Here, this study develops engineered GBM organoids (eGBOs) harboring GBM subtype-specific oncogenic mutations to investigate the underlying transcriptional regulation of tumor progression. Single-cell and spatial transcriptomic analyses revealed that these mutations disrupt normal neurodevelopment gene regulatory networks resulting in changes in cellular composition and spatial organization. Upon xenotransplantation into immunodeficient mice, eGBOs form tumors that recapitulate the transcriptional and spatial landscape of human GBM samples. Integrative single-cell trajectory analysis of both eGBO-derived tumor cells and patient GBM samples reveal the dynamic gene expression changes in developmental cell states underlying tumor progression. This analysis of eGBOs provides an important validation of engineered cancer organoid models and demonstrates their utility as a model of GBM tumorigenesis for future preclinical development of therapeutics.

Human organoids are a widely used tool in cell biology to study homeostatic processes, disease, and development. The term organoids covers a plethora of model systems from different cellular origins that each have unique features and applications but bring their own challenges. This review discusses the basic principles underlying organoids generated from pluripotent stem cells (PSCs) as well as those derived from tissue stem cells (TSCs). We consider how well PSC- and TSC-organoids mimic the different intended organs in terms of cellular complexity, maturity, functionality, and the ongoing efforts to constitute predictive complex models of in vivo situations. We discuss the advantages and limitations associated with each system to answer different biological questions including in the field of cancer and developmental biology, and with respect to implementing emerging advanced technologies, such as (spatial) -omics analyses, CRISPR screens, and high-content imaging screens. We postulate how the two fields may move forward together, integrating advantages of one to the other.

Glioblastoma (GBM) is a relatively rare but highly aggressive form of brain cancer characterized by rapid growth, invasiveness, and resistance to standard therapies. Despite significant progress in understanding its molecular and cellular mechanisms, GBM remains one of the most challenging cancers to treat due to its high heterogeneity and complex tumor microenvironment. To address these obstacles, researchers have employed a range of models, including in vitro cell cultures and in vivo animal models, but these often fail to replicate the complexity of GBM. As a result, there has been a growing focus on refining these models by incorporating human-origin cells, along with advanced genetic techniques and stem cell-based bioengineering approaches. In this context, a variety of GBM models based on brain organoids were developed and confirmed to be clinically relevant and are contributing to the advancement of GBM research at the preclinical level. This review explores the preparation and use of brain organoid-based models to deepen our understanding of GBM biology and to explore novel therapeutic approaches. These innovative models hold significant promise for improving our ability to study this deadly cancer and for advancing the development of more effective treatments.

The generation of patient avatars is critically needed in neuro-oncology for treatment prediction and preclinical therapeutic development. Our objective was to develop a fast, reproducible, low-cost, and easy-to-use method of tumoroids generation and analysis, efficient for all types of brain tumors, primary and metastatic.

Tumoroids were generated from 89 patients: 81 primary tumors including 77 gliomas, and 8 brain metastases. Tumoroids morphology and cellular and molecular characteristics were compared with the ones of the parental tumor by using histology, methylome profiling, pTERT mutations, and multiplexed spatial immunofluorescences. Their cellular stability over time was validated by flow cytometry. Therapeutic sensitivity was evaluated and predictive factors of tumoroid generation were analyzed.

All the tumoroids analyzed had similar histological (n = 21) and molecular features (n = 7) to the parental tumor. The median generation time was 5 days. The success rate was 65 %: it was higher for high-grade gliomas and brain metastases versus IDH mutated low-grade gliomas. For high-grade gliomas, neither other clinical, neuro-imaging, histological nor molecular factors were predictive of tumoroid generation success. The cellular organization inside tumoroids analyzed by MACSima revealed territories dedicated to specific cell subtypes. Finally, we showed the correlation between tumoroid and patient treatment responses to radio-chemotherapy and their ability to respond to immunotherapy thanks to a dedicated and reproducible 3D analysis workflow.

Patient-derived tumoroid model that we developed offers a robust, user-friendly, low-cost, and reproducible preclinical model valuable for therapeutic development of all types of primary or metastatic brain tumors, allowing their integration into forthcoming early-phase clinical trials.

Tumor cells communicate with normal cells in various ways, typically leading to the exploitation of resources of the normal cells by tumor cells for their benefit. In this issue, Mangena and colleagues use three-dimensional organoid models to show the transfer of GFP and mRNA from malignant glioblastoma to nonmalignant cells in cerebral organoid models; this transfer is facilitated by extracellular vesicles and possibly tunneling nanotubes, demonstrating how nonmalignant cells in the tumor microenvironment can be exploited by neighboring malignant cells. See related article by Mangena et al., p. 299.

Glioblastoma (GBM) is characterized by heterogeneous malignant cells that are functionally integrated within the neuroglial microenvironment. In this study, we model this ecosystem by growing GBM into long-term cultured human cortical organoids that contain the major neuroglial cell types found in the cerebral cortex. Single-cell RNA sequencing analysis suggests that, compared with matched gliomasphere models, GBM cortical organoids more faithfully recapitulate the diversity and expression programs of malignant cell states found in patient tumors. Additionally, we observe widespread transfer of GBM transcripts and GFP to nonmalignant cells in the organoids. Mechanistically, this transfer involves extracellular vesicles and is biased toward defined GBM cell states and astroglia cell types. These results extend previous GBM organoid modeling efforts and suggest widespread intercellular transfer in the GBM neuroglial microenvironment. Significance: Models that recapitulate intercellular communications in GBM are limited. In this study, we leverage GBM cortical organoids to characterize widespread mRNA and GFP transfer from malignant to nonmalignant cells in the GBM neuroglial microenvironment. This transfer involves extracellular vesicles, may contribute to reprogramming the microenvironment, and may extend to other cancer types. See related commentary by Shakya et al., p. 261.

Brain metastasis stands as a leading contributor to mortality in lung cancer patients, yet the intricate mechanism underlying this phenomenon remains elusive. This underscores the need for robust preclinical models and effective treatment strategies. Emerging as viable in vitro models that closely replicate actual tumors, three-dimensional culture systems, particularly organoids derived from non-malignant cells or cancer organoids, have emerged as promising avenues. This review delves into the forefronts of fundamental research and clinical applications focused on lung cancer brain metastasis-derived organoids, highlighting current challenges and delineating prospects. These studies offer tremendous potential for clinical application despite being in nascent status.

The fight against Glioblastoma multiforme (GBM) is ongoing and the long-term outlook for GBM remains challenging due to low prognosis but every breakthrough brings us closer to improving patient outcomes. Significant hurdles in GBM are heterogeneity, fortified tumor location, and blood-brain barrier (BBB), hindering adequate drug concentrations within functioning brain regions, thus leading to low survival rates. The nasal passageway has become an appealing location to commence the course of cancer therapy. Utilization of the nose-to-brain (N2B) route for drug delivery takes a sidestep from the BBB to allow therapeutics to directly access the central nervous system (CNS) and enhance drug localization in the vicinity of the tumor. This comprehensive review provides insights into pertinent anatomy and cellular organization of the nasal cavity, present-day diagnostic tools, intracranial invasive therapies, and advancements in intranasal (IN) therapies in GBM models for better clinical outcomes. Also, this review highlights groundbreaking carriers and delivery techniques that could revolutionize GBM management such as biomimetics, image guiding-drug delivery, and photodynamic and photothermal therapies for GBM management.

Efforts to achieve precise and efficient tumor targeting of highly malignant brain tumors are constrained by the dearth of appropriate models to study the effects and potential side effects of radiation, chemotherapy, and immunotherapy on the most complex human organ, the brain. We established a cerebral organoid model of brain tumorigenesis in an autologous setting by overexpressing c-MYC as one of the most common oncogenes in brain tumors. GFP + /c-MYC high cells were isolated from tumor organoids and used in two different culture approaches: assembloids comprising of a normal cerebral organoid with a GFP + /c-MYC high tumor sphere and co-culture of cerebral organoid slices at air-liquid interface with GFP + /c-MYC high cells. GFP + /c-MYC high cells used in both approaches exhibited tumor-like properties, including overexpression of the c-MYC oncogene, high proliferative and invasive potential, and an immature phenotype as evidenced by increased expression of Ki-67, VIM, and CD133. Organoids and organoid slices served as suitable scaffolds for infiltrating tumor-like cells. Using our highly reproducible and powerful model system that allows long-term culture, we demonstrated that the migratory and infiltrative potential of tumor-like cells is shaped by the environment in which glia cells provide support to tumor-like cells.

Three-dimensional (3D) bioprinting holds immense promise for advancing stem cell research and developing novel therapeutic strategies in the field of neural tissue engineering and disease modeling. This paper critically analyzes recent breakthroughs in 3D bioprinting, specifically focusing on its application in these areas. We comprehensively explore the advantages and limitations of various 3D printing methods, the selection and formulation of bioink materials tailored for neural stem cells, and the incorporation of nanomaterials with dual functionality, enhancing the bioprinting process and promoting neurogenesis pathways. Furthermore, the paper reviews the diverse range of stem cells employed in neural bioprinting research, discussing their potential applications and associated challenges. We also introduce the emerging field of 4D bioprinting, highlighting current efforts to develop time-responsive constructs that improve the integration and functionality of bioprinted neural tissues. In short, this manuscript aims to provide a comprehensive understanding of this rapidly evolving field. It underscores the transformative potential of 3D and 4D bioprinting technologies in revolutionizing stem cell research and paving the way for novel therapeutic solutions for neurological disorders and injuries, ultimately contributing significantly to the advancement of regenerative medicine. STATEMENT OF SIGNIFICANCE: This comprehensive review critically examines the current bioprinting research landscape, highlighting efforts to overcome key limitations in printing technologies-improving cell viability post-printing, enhancing resolution, and optimizing cross-linking efficiencies. The continuous refinement of material compositions aims to control the spatiotemporal delivery of therapeutic agents, ensuring better integration of transplanted cells with host tissues. Specifically, the review focuses on groundbreaking advancements in neural tissue engineering. The development of next-generation bioinks, hydrogels, and scaffolds specifically designed for neural regeneration complexities holds the potential to revolutionize treatments for debilitating neural conditions, especially when nanotechnologies are being incorporated. This review offers the readers both a comprehensive analysis of current breakthroughs and an insightful perspective on the future trajectory of neural tissue engineering.

MicroRNAs are regulators of gene expression and their dysregulation can lead to various diseases. MicroRNA-135 (MiR-135) exhibits brain-specific expression, and performs various functions such as neuronal morphology, neural induction, and synaptic function in the human brain. Dysfunction of miR-135 has been reported in brain tumors, and neurodegenerative and neurodevelopmental disorders. Several reports show downregulation of miR-135 in glioblastoma, indicating its tumor suppressor role in the pathogenesis of brain tumors. In this review, by performing in silico analysis of molecular targets of miR-135, we reveal the significant pathways and processes modulated by miR-135. We summarize the biological significance, roles, and signaling pathways of miRNAs in general, with a focus on miR-135 in different neurological diseases including brain tumors, and neurodegenerative and neurodevelopmental disorders. We also discuss methods, limitations, and potential of glioblastoma organoids in recapitulating disease initiation and progression. We highlight the promising therapeutic potential of miRNAs as antitumor agents for aggressive human brain tumors including glioblastoma.

Glioblastoma (GBM) is a highly aggressive adult brain cancer, characterised by poor prognosis and a dismal five-year survival rate. Despite significant knowledge gains in tumour biology, meaningful advances in patient survival remain elusive. The field of neuro-oncology faces many disease obstacles, one being the paucity of faithful models to advance preclinical research and guide personalised medicine approaches. Recent technological developments have permitted the maintenance, expansion and cryopreservation of GBM explant organoid (GBO) tissue. GBOs represent a translational leap forward and are currently the state-of-the-art in 3D in vitro culture system, retaining brain cancer heterogeneity, and transiently maintaining the immune infiltrate and tumour microenvironment (TME). Here, we provide a review of existing brain cancer organoid technologies, in vivo xenograft approaches, evaluate in-detail the key advantages and limitations of this rapidly emerging technology, and consider solutions to overcome these difficulties. GBOs currently hold significant promise, with the potential to emerge as the key translational tool to synergise and enhance next-generation omics efforts and guide personalised medicine approaches for brain cancer patients into the future.

Recent advances in cancer biology and therapeutics have underscored the importance of preclinical models in understanding and treating cancer. Nevertheless, current models often fail to capture the complexity and patient-specific nature of human tumors, particularly gliomas. This review examines the strengths and weaknesses of such models, highlighting the need for a new generation of models. Emphasizing the critical role of the tumor microenvironment, tumor, and patient heterogeneity, we propose integrating our advanced understanding of glioma biology with innovative bioengineering and AI technologies to create more clinically relevant, patient-specific models. These innovations are essential for improving therapeutic development and patient outcomes.

Induced pluripotent stem cells (iPSCs) provide a novel approach to studying the pathophysiology of brain tumors and assessing various therapeutic techniques with greater precision. This study aims to systematically review the existing literature to critically analyze and synthesize current research findings. The objective is to evaluate the role of iPSCs in understanding brain tumors and in the development of innovative treatment strategies.

We systematically reviewed existing articles that utilized iPSC technology to assess either the pathophysiology of brain tumors or therapeutic techniques, following the standards of Preferred Reporting Items for Systematic review and Meta-Analysis guidelines. Key terms were comprehensively searched in electronic databases, including PubMed, EMBASE, and Scopus. Articles were screened based on specific inclusion and exclusion criteria. Ultimately, 22 relevant articles were chosen, and their data were extracted.

The summary of findings for each selected article was organized into two general categories: "Methods of Generating iPSCs" and "Applications of iPSCs." The methods of iPSC generation, including transfection and transduction, as well as the types of viral or non-viral vectors used, were extracted and reported for each study. Additionally, the main aims of the selected studies, whether modeling or therapeutic approaches, were gathered and reported in the results section.

iPSC technology is a novel vehicle that brings new solutions to overcome difficulties in brain tumor studies. In vivo and in vitro models generated from iPSCs provide suitable platforms to investigate the pathophysiology of brain tumors more precisely. Also, iPSCs have been utilized in various studies to examine how different antitumor agents may affect the target cells.

Gliomas are a group of primary brain tumors characterized by their aggressive nature and resistance to treatment. Infiltration of surrounding normal tissues limits surgical approaches, wide inter- and intratumor heterogeneity hinders the development of universal therapeutics, and the presence of the blood-brain barrier reduces the efficiency of their delivery. As a result, patients diagnosed with gliomas often face a poor prognosis and low survival rates. The spectrum of anti-glioma drugs used in clinical practice is quite narrow. Alkylating agents are often used as first-line therapy, but their effectiveness varies depending on the molecular subtypes of gliomas. This highlights the need for new, more effective therapeutic approaches. Standard drug-screening methods involve the use of two-dimensional cell cultures. However, these models cannot fully replicate the conditions present in real tumors, making it difficult to extrapolate the results to humans. We describe the advantages and disadvantages of existing glioma cell-based models designed to improve the situation and build future prospects to make drug discovery comprehensive and more effective for each patient according to personalized therapy paradigms.

Microglia represent the main resident immune cells of the brain. The interplay between microglia and other cells in the central nervous system, such as neurons or other glial cells, influences the function and ability of microglia to respond to various stimuli. These cellular communications, when disrupted, can affect the structure and function of the brain, and the initiation and progression of neurodegenerative diseases including Alzheimer's disease and Parkinson's disease, as well as the progression of other brain diseases like glioblastoma. Due to the difficult access to patient brain tissue and the differences reported in the murine models, the available models to study the role of microglia in disease progression are limited. Pluripotent stem cell technology has facilitated the generation of highly complex models, allowing the study of control and patient-derived microglia in vitro. Moreover, the ability to generate brain organoids that can mimic the 3D tissue environment and intercellular interactions in the brain provide powerful tools to study cellular pathways under homeostatic conditions and various disease pathologies. In this review, we summarise the most recent developments in modelling degenerative diseases and glioblastoma, with a focus on brain organoids with integrated microglia. We provide an overview of the most relevant research on intercellular interactions of microglia to evaluate their potential to study brain pathologies.

Pre-clinical animal models of human brain tumors have been invaluable tools for studying cancer pathogenesis and exploring novel treatment modalities. Such models recapitulate important aspects of the human disease such as the stem-progenitor-differentiated cell hierarchy. Although powerful, we argue that animal models are inherently limited in their ability to phenocopy certain important aspects of human brain tumor biology. We specifically highlight the inability of mouse models to generate certain forms aggressive pediatric medulloblastoma likely owing to cellular, anatomic, and genetic differences between the human and mouse brains. Additionally, we review some limitations of human brain tumor derived cell lines and outline why they are a sub-optimal system for purposes of pre-clinical modeling. Below, we present the case for human stem cell-based models of brain tumors, focusing mainly on glioblastoma and medulloblastoma. Drawing on several recently published studies, we review the exciting progress that has been made towards modeling human brain tumors using two-dimensional adherent stem cell cultures and three-dimensional organoids. We identify the important advances arrived at using these human stem cell-based models and suggest opportunities for future work in this direction. In this review article, we aim to highlight the utility and promises of human stem cell-based models of brain tumors as a complementary system to traditional transgenic animal and cell line systems.

Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus' (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus' oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors, enabling the translation of this approach to clinical trials, are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM, MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect, replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells, but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly, ZIKV rapidly replicated in GBM, MED, and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover, ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether, our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However, such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects, suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV.

Organoids are three-dimensional (3D) cultures, normally derived from stem cells, that replicate the complex structure and function of human tissues. They offer a physiologically relevant model to address important questions in cancer research. The generation of patient-derived organoids (PDOs) from various human cancers allows for deeper insights into tumor heterogeneity and spatial organization. Additionally, interrogating non-tumor stromal cells increases the relevance in studying the tumor microenvironment, thereby enhancing the relevance of PDOs in personalized medicine. PDOs mark a significant advancement in cancer research and patient care, signifying a shift toward more innovative and patient-centric approaches. This review covers aspects of PDO cultures to address the modeling of the tumor microenvironment, including extracellular matrices, air-liquid interface and microfluidic cultures, and organ-on-chip. Specifically, the role of PDOs as preclinical models in gene editing, molecular profiling, drug testing, and biomarker discovery and their potential for guiding personalized treatment in clinical practice are discussed.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging, we aimed to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question DIPG24 cells were cultured with pluripotent stem cell-derived cortical organoids. We created "mosaic" co-cultures enriched for tumour cell-neuronal cell interactions versus "assembloid" co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This suggested changes in cell interaction with the external environment reflected by decreased gene ontology terms associated with adhesion and extracellular matrix, and increased DNA synthesis and replication, in DIPG24 cells under either co-culture condition. By contrast, the mosaic co-culture was associated with neuron-specific brahma-associated factor (nBAF) complex signalling, a process associated with neuronal maturation. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially because of subventricular zone contact. Despite this, cross-talk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. In addition, GBM brain tumor-initiating cells (BTICs) increase expression of cathepsin B (CTSB) upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal that both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Last, we show LV-proximal CTSB up-regulation in patients, showing the relevance of this cross-talk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.

Glioblastoma (GBM) is an aggressive form of brain cancer that is highly resistant to therapy due to significant intra-tumoral heterogeneity. The lack of robust in vitro models to study early tumor progression has hindered the development of effective therapies. Here, we develop engineered GBM organoids (eGBOs) harboring GBM subtype-specific oncogenic mutations to investigate the underlying transcriptional regulation of tumor progression. Single-cell and spatial transcriptomic analyses revealed that these mutations disrupt normal neurodevelopment gene regulatory networks resulting in changes in cellular composition and spatial organization. Upon xenotransplantation into immunodeficient mice, eGBOs form tumors that recapitulate the transcriptional and spatial landscape of human GBM samples. Integrative single-cell trajectory analysis of both eGBO-derived tumor cells and patient GBM samples revealed the dynamic gene expression changes in developmental cell states underlying tumor progression. This analysis of eGBOs provides an important validation of engineered cancer organoid models and demonstrates their utility as a model of GBM tumorigenesis for future preclinical development of therapeutics.

Rare genetic diseases with neurodevelopmental disorders (NDDs) encompass several heterogeneous conditions (autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), specific learning disorder (SLD), among others). Currently, few treatments are available for these patients. The difficulty in accessing human brain samples and the discrepancies between human and animal models highlight the need for new research approaches. One promising approach is the use of the cerebral organoids. These 3D, self-organized structures, generated from induced pluripotent stem cells (iPSCs), enable the reproduction of the stages of human brain development, from the proliferation of neural stem cells to their differentiation into neurons, oligodentrocytes, and astrocytes. Cerebral organoids hold great promise in understanding brain development and in the search for treatments.

Since the emergence of the first cerebral organoid (CO) in 2013, advancements have transformed central nervous system (CNS) research. Initial efforts focused on studying the morphogenesis of COs and creating reproducible models. Numerous methodologies have been proposed, enabling the design of the brain organoid to represent specific regions and spinal cord structures. CNS organoids now facilitate the study of a wide range of CNS diseases, from infections to tumors, which were previously difficult to investigate. We summarize the major advancements in CNS organoids, concerning morphogenetic designs and disease models. We examine the development of fabrication procedures and how these advancements have enabled the generation of region-specific brain organoids and spinal cord models. We highlight the application of these organoids in studying various CNS diseases, demonstrating the versatility and potential of organoid models in advancing our understanding of complex conditions. We discuss the current challenges in the field, including issues related to reproducibility, scalability, and the accurate recapitulation of the in vivo environment. We provide an outlook on prospective studies and future directions. This review aims to provide a comprehensive overview of the state-of-the-art CNS organoid research, highlighting key developments, current challenges, and prospects in the field.

Glioblastoma poses a formidable challenge among primary brain tumors: its tumorigenic stem cells, capable of self-renewal, proliferation, and differentiation, contribute substantially to tumor initiation and therapy resistance. These glioblastoma stem cells (GSCs), resembling conventional stem and progenitor cells, adopt pathways critical for tissue development and repair, promoting uninterrupted tumor expansion. Long non-coding RNAs (lncRNAs), a substantial component of the human transcriptome, have garnered considerable interest for their pivotal roles in normal physiological processes and cancer pathogenesis. They display cell- or tissue-specific expression patterns, and extensive investigations have highlighted their impact on regulating GSC properties and cellular differentiation, thus offering promising avenues for therapeutic interventions. Consequently, lncRNAs, with their ability to exert regulatory control over tumor initiation and progression, have emerged as promising targets for innovative glioblastoma therapies. This review explores notable examples of GSC-associated lncRNAs and elucidates their functional roles in driving glioblastoma progression. Additionally, we delved deeper into utilizing a 3D in vitro model for investigating GSC biology and elucidated four primary methodologies for targeting lncRNAs as potential therapeutics in managing glioblastoma.

The adaptability of glioblastoma (GBM) cells, encouraged by complex interactions with the tumour microenvironment (TME), currently renders GBM an incurable cancer. Despite intensive research, with many clinical trials, GBM patients rely on standard treatments including surgery followed by radiation and chemotherapy, which have been observed to induce a more aggressive phenotype in recurrent tumours. This failure to improve treatments is undoubtedly a result of insufficient models which fail to incorporate components of the human brain TME. Research has increasingly uncovered mechanisms of tumour-TME interactions that correlate to worsened patient prognoses, including tumour-associated astrocyte mitochondrial transfer, neuronal circuit remodelling and immunosuppression. This tumour hijacked TME is highly implicated in driving therapy resistance, with further alterations within the TME and tumour resulting from therapy exposure inducing increased tumour growth and invasion. Recent developments improving organoid models, including aspects of the TME, are paving an exciting future for the research and drug development for GBM, with the hopes of improving patient survival growing closer. This review focuses on GBMs interactions with the TME and their effect on tumour pathology and treatment efficiency, with a look at challenges GBM models face in sufficiently recapitulating this complex and highly adaptive cancer.

A key challenge in cancer research is the meticulous development of models that faithfully emulates the intricacies of the patient scenario, with emphasis on preserving intra-tumoral heterogeneity and the dynamic milieu of the tumor microenvironment (TME). Organoids emerge as promising tool in new drug development, drug screening and precision medicine. Despite advances in the diagnoses and treatment of pediatric cancers, certain tumor subtypes persist in yielding unfavorable prognoses. Moreover, the prognosis for a significant portion of children experiencing disease relapse is dismal. To improve pediatric outcome many groups are focusing on the development of precision medicine approach. In this review, we summarize the current knowledge about using organoid system as model in preclinical and clinical solid-pediatric cancer. Since organoids retain the pivotal characteristics of primary parent tumors, they exert great potential in discovering novel tumor biomarkers, exploring drug-resistance mechanism and predicting tumor responses to chemotherapy, targeted therapy and immunotherapies. We also examine both the potential opportunities and existing challenges inherent organoids, hoping to point out the direction for future organoid development.

Cancers arise through acquisition of mutations in genes that regulate core biological processes like cell proliferation and cell death. Decades of cancer research have led to the identification of genes and mutations causally involved in disease development and evolution, yet defining their precise function across different cancer types and how they influence therapy responses has been challenging. Mouse models have helped define the in vivo function of cancer-associated alterations, and genome-editing approaches using CRISPR have dramatically accelerated the pace at which these models are developed and studied. Here, we highlight how CRISPR technologies have impacted the development and use of mouse models for cancer research and discuss the many ways in which these rapidly evolving platforms will continue to transform our understanding of this disease.

In cancer immunotherapy, chimeric antigen receptors (CARs) targeting specific antigens have become a powerful tool for cell-based therapy. CAR-natural killer (NK) cells offer selective anticancer lysis with reduced off-tumor toxicity compared to CAR-T cells, which is beneficial in the heterogeneous milieu of solid tumors. In the tumor microenvironment (TME) of glioblastoma (GBM), pericytes not only support tumor growth but also contribute to immune evasion, underscoring their potential as therapeutic targets in GBM treatment. Given this context, our study aimed to target the GBM TME, with a special focus on pericytes expressing CD19, to evaluate the potential effectiveness of CD19 CAR-iNK cells against GBM. We performed CD19 CAR transduction in induced pluripotent stem cell-derived NK (iNK) cells. To determine whether CD19 CAR targets the TME pericytes in GBM, we developed GBM-blood vessel assembloids (GBVA) by fusing GBM spheroids with blood vessel organoids. When co-cultured with GBVA, CD19 CAR-iNK cells migrated towards the pericytes surrounding the GBM. Using a microfluidic chip, we demonstrated CD19 CAR-iNK cells' targeted action and cytotoxic effects in a perfusion-like environment. GBVA xenografts recapitulated the TME including human CD19-positive pericytes, thereby enabling the application of an in vivo model for validating the efficacy of CD19 CAR-iNK cells against GBM. Compared to GBM spheroids, the presence of pericytes significantly enhanced CD19 CAR-iNK cell migration towards GBM and reduced proliferation. These results underline the efficacy of CD19 CAR-iNK cells in targeting pericytes within the GBM TME, suggesting their potential therapeutic value for GBM treatment.

The symbiotic interplay of organoid technology and advanced imaging strategies yields innovative breakthroughs in research and clinical applications. Organoids, intricate three-dimensional cell cultures derived from pluripotent or adult stem/progenitor cells, have emerged as potent tools for in vitro modeling, reflecting in vivo organs and advancing our grasp of tissue physiology and disease. Concurrently, advanced imaging technologies such as confocal, light-sheet, and two-photon microscopy ignite fresh explorations, uncovering rich organoid information. Combined with advanced imaging technologies and the power of artificial intelligence, organoids provide new insights that bridge experimental models and real-world clinical scenarios. This review explores exemplary research that embodies this technological synergy and how organoids reshape personalized medicine and therapeutics.