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Optimizing Tissue Harvesting Techniques for Establishing Patient-Derived Glioblastoma Organoids.
OBJECTIVE: Brain tumors display remarkable cellular and molecular diversity, significantly impacting the progression and outcomes of the disease. The utilization of tumor tissue acquired through surgical handheld devices for tumor characterization raises important questions regarding translational research. This study seeks to evaluate the integrity of tissue resected using a microdebrider (MD) in the context of establishing tumor organoids from glioblastomas (GBM). METHODS: Tumor samples were collected from patients with GBM using both tumor forceps (en bloc) and a MD. The time required to protocol completion and cell viability of paired samples was measured. H&E staining was performed to examine histologic morphology. RESULTS: Ten paired samples were obtained from GBM patients using tumor forceps and the MD. Samples collected with the MD demonstrated significantly shorter processing times compared to those obtained through en bloc resection, with overall means of 31.7 ± 2.4 mins and 38.8±3 mins, respectively (P < 0.001). Cell viability measured at the end of protocol completion was comparable between tissues obtained using both the MD and en bloc, with mean viabilities of 80.2 ± 12.4% and 79.1 ± 12.5%, respectively (P = 0.848). H&E examination of tissues revealed no significant differences in the cellular and histologic characteristics of paired samples obtained using both methods across GBM tumors, nor in the corresponding established organoids. CONCLUSIONS: Tumor tissues obtained using the MD and en bloc methods demonstrate a high success rate in establishing GBM organoids, with the MD offering the advantage of significantly reduced processing time. Both methods display comparable cell viability and maintain consistent histologic characteristics in the resected tissue and the corresponding organoids.
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Organoids and chimeras: the hopeful fusion transforming traumatic brain injury research.
Research in the field of traumatic brain injury has until now heavily relied on the use of animal models to identify potential therapeutic approaches. However, a long series of failed clinical trials has brought many scientists to question the translational reliability of pre-clinical results obtained in animals. The search for an alternative to conventional models that better replicate human pathology in traumatic brain injury is thus of the utmost importance for the field. Recently, orthotopic xenotransplantation of human brain organoids into living animal models has been achieved. This review summarizes the existing literature on this new method, focusing on its potential applications in preclinical research, both in the context of cell replacement therapy and disease modelling. Given the obvious advantages of this approach to study human pathologies in an in vivo context, we here critically review its current limitations while considering its possible applications in traumatic brain injury research.
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Cellular and inflammatory characteristics of patient‑derived glioblastoma organoids during in vitro cultivation.
Glioblastomas are characterized by extensive intra‑ and intertumoral heterogeneity, which drives their rapid invasive growth and frequent recurrence. As a result, prognosis remains poor, and developing effective treatments continues to be a major challenge. The survival and growth of glioblastomas are supported by a specific, protective tumor microenvironment (TME) that influences tumor morphology, growth rate, therapy resistance and recurrence. To better represent the complex TME with its immune cells and inflammatory mediators and the tumor heterogeneity, including glioma stem‑like cells (GSCs), realistic in vitro preclinical models are required. Patient‑derived glioblastoma organoids (GBOs), directly derived from tumor tissue obtained after surgical resection, were generated in the present study. Preserving the original tumor state in organoids, particularly regarding the TME and heterogeneity, is crucial. The present study evaluates the long‑term preservation of key tumor properties in cultivated GBOs, including glial structural proteins, GSCs, immune cells, endothelial cells, proliferation marker, and inflammatory mediators such as cytokines and chemokines. Expression levels of these components were measured at five defined time points in 10 independent GBO preparations, at both the mRNA and protein level, and statistical correlation analyses were performed. The findings of the present study indicate that the key components were generally maintained over a one‑month cultivation period. However, some markers showed reduced expression at later time points or recovered after an initial drop at early cultivation time points. Therefore, the optimal time window for employing patient‑derived organoids as an advanced preclinical model for glioblastoma research appears to be between 7‑14 days of cultivation.
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Development of Experimental Three-Dimensional Tumor Models to Study Glioblastoma Cancer Stem Cells and Tumor Microenvironment.
Glioblastoma (GBM) is the most common and dismal primary brain tumor. Unfortunately, despite multidisciplinary treatment, most patients will perish approximately 15 months after diagnosis. For this reason, there is an urgent need to improve our understanding of GBM tumor biology and develop novel therapies that can achieve better clinical outcomes. In this setting, three-dimensional tumor models have risen as more appropriate preclinical tools when compared to traditional cell cultures, given that two-dimensional (2D) cultures have failed to accurately recapitulate tumor biology and translate preclinical findings into patient benefits. Three-dimensional cultures using neurospheres, organoids, and organotypic better resemble original tumor genetic and epigenetic profiles, maintaining tumor microenvironment characteristics and mimicking cell-cell and cell-matrix interactions. This chapter summarizes our methods to generate well-characterized glioblastoma neurospheres, organoids, and organotypics.
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Transglutaminase 2 function in glioblastoma tumor efferocytosis.
Glioblastoma is an aggressive and incurable type of brain cancer. Regions of tissue necrosis are a distinctive pathological feature of this cancer. These arise through thrombosis of tumor vasculature, driven by tumor-derived pro-coagulation factors. In studies of transglutaminase 2 (TGM2), we observed that TGM2 mRNA expression in glioblastoma was primarily in a subset of tumor-infiltrating myeloid cells with hypoxia gene expression signatures. Analysis of xenograft and human glioblastoma samples by immunohistochemistry showed that macrophages in the vicinity of necrotic regions expressed very high levels of TGM2. These macrophages were engaged in the phagocytosis of apoptotic cells, a process known as efferocytosis. In cell culture, incubation of macrophages with apoptotic cells induced TGM2 expression in macrophages, and TGM2 inhibitors blocked efferocytosis. In patient-derived glioblastoma organoids cultured in 5% O2, a basal level of apoptosis was observed, and endogenous macrophages were observed in the process of clearing apoptotic cells. Clearance of apoptotic cells was reduced in organoids treated with a TGM2 inhibitor. Efferocytosis was absent or reduced in organoids grown in 20% O2. These data, together with previous work, define a model in which necrotic regions in glioblastoma induce hypoxia-driven apoptosis, which in turn promotes efferocytosis by macrophages. TGM2 is both a marker of efferocytosis and a target for efferocytosis inhibition in this process. Efferocytosis is a potent immunosuppressive mechanism, so this process provides an additional mechanism by which large glioblastoma tumors can evade immune responses.
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Bioethical implications of organ-on-a-chip on modernizing drug development.
Organ-on-chips are three-dimensional microdevices that emulate the structure, functionality, and behavior of specific tissues or organs using human cells. Combining organoids with microfabricated fluidic channels and microelectronics, these systems offer a promising platform for studying disease mechanisms, drug responses, and tissue performance. By replicating the in vivo microenvironment, these devices can recreate complex cell interactions in controlled conditions and facilitate research in various fields, including drug toxicity and efficacy studies, biochemical analysis, and disease pathogenesis. Integrating human induced pluripotent stem cells further enhances their applicability, thereby enabling patient-specific disease modeling for precision medicine. Although challenges like economy-of-scale, multichip integration, and regulatory compliance exist, advances in this modular technology show promise for lowering drug development costs, improving reproducibility, and reducing the reliance on animal testing. The ethical landscape surrounding organ-on-chip usage presents both benefits and concerns. While these chips offer an alternative to animal testing and potential cost savings, they raise ethical considerations related to community engagement, informed consent, and the need for standardized guidelines. Ensuring public acceptance and involvement in decision-making is vital to address misinformation and mistrust. Furthermore, personalized medicine models using patient-derived cells demand careful consideration of potential ethical dilemmas, such as modeling physiological functions of fetuses or brains and determining the extent of protection for these models. To achieve the full potential of organ-on-a-chip models, collaboration between scientists, ethicists, and regulators is essential to fulfil the promise of transforming drug development, advancing personalized medicine, and contributing to a more ethical and efficient biomedical research landscape.
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Comparison of the fatty acids of lipids of subcellular brain fractions.
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Superoxide dismutase isozymes in cerebral organoids from autism spectrum disorder patients.
Autism spectrum disorder is a pervasive neurodevelopmental disorder with a substantial contribution to the global disease burden. Despite intensive research efforts, the aetiopathogenesis remains unclear. The Janus-faced antioxidant enzymes superoxide dismutase 1-3 have been implicated in initiating oxidative stress and as such may constitute a potential therapeutic target. However, no measurement has been taken in human autistic brain samples. The aim of this study is to measure superoxide dismutase 1-3 in autistic cerebral organoids as an in vitro model of human foetal neurodevelopment. Whole brain organoids were created from induced pluripotent stem cells from healthy individuals (n = 5) and individuals suffering from autism (n = 4). Using Pierce bicinchoninic acid and enzyme-linked immunosorbent assays, the protein and superoxide dismutase 1, 2, and 3 concentrations were quantified in the cerebral organoids at days 22, 32, and 42. Measurements were normalized to the protein concentration. Results represented using medians and interquartile ranges. Using Wilcoxon matched-pairs signed-rank test, an abrupt rise in the superoxide dismutase concentration was observed at day 32 and onwards. Using Wilcoxon rank-sum test, no differences were observed between healthy (SOD1: 35.56 ng/mL ± 3.46; SOD2: 2435.80 ng/mL ± 1327.00; SOD3: 1854.88 ng/mL ± 867.94) and autistic (SOD1: 32.85 ng/mL ± 5.26; SOD2: 2717.80 ng/mL ± 1889.10; SOD3: 1690.18 ng/mL ± 615.49) organoids. Cerebral organoids recapitulate many aspects of human neurodevelopment, but the diffusion restriction may render efforts in modelling differences in oxidative stress futile due to the intrinsic hypoxia and central necrosis.
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Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids.
Due to an inability to ethically access developing human brain tissue as well as identify prospective cases, early-arising neurodevelopmental and cell-specific signatures of Schizophrenia (Scz) have remained unknown and thus undefined. To overcome these challenges, we utilized patient-derived induced pluripotent stem cells (iPSCs) to generate 3D cerebral organoids to model neuropathology of Scz during this critical period. We discovered that Scz organoids exhibited ventricular neuropathology resulting in altered progenitor survival and disrupted neurogenesis. This ultimately yielded fewer neurons within developing cortical fields of Scz organoids. Single-cell sequencing revealed that Scz progenitors were specifically depleted of neuronal programming factors leading to a remodeling of cell-lineages, altered differentiation trajectories, and distorted cortical cell-type diversity. While Scz organoids were similar in their macromolecular diversity to organoids generated from healthy controls (Ctrls), four GWAS factors (PTN, COMT, PLCL1, and PODXL) and peptide fragments belonging to the POU-domain transcription factor family (e.g., POU3F2/BRN2) were altered. This revealed that Scz organoids principally differed not in their proteomic diversity, but specifically in their total quantity of disease and neurodevelopmental factors at the molecular level. Single-cell sequencing subsequently identified cell-type specific alterations in neuronal programming factors as well as a developmental switch in neurotrophic growth factor expression, indicating that Scz neuropathology can be encoded on a cell-type-by-cell-type basis. Furthermore, single-cell sequencing also specifically replicated the depletion of BRN2 (POU3F2) and PTN in both Scz progenitors and neurons. Subsequently, in two mechanistic rescue experiments we identified that the transcription factor BRN2 and growth factor PTN operate as mechanistic substrates of neurogenesis and cellular survival, respectively, in Scz organoids. Collectively, our work suggests that multiple mechanisms of Scz exist in patient-derived organoids, and that these disparate mechanisms converge upon primordial brain developmental pathways such as neuronal differentiation, survival, and growth factor support, which may amalgamate to elevate intrinsic risk of Scz.
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Ultrastructure of oligodendrogliomas.
The study of 18 oligodendrogliomas shows a number of characteristics which indicate a common hypothelial stem cell. The main cytologic characteristics are: a) high number of mitochondria, frequently atypical; b) polygonal crystalline structures in the cytoplasm in most of the tumors studied, and c) cytoplasmic microtubuli. None of these characteristics can be considered as pathognomic for the oligodendrogliomas. However, rows of folding cell processes represent a fundamental structural principle of all oligodendrogliomas and since they have not been found in any other brain tumor and may be regarded as an inherent and specific characteristic of the oligodendroglial group.