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Flow cytometry protocol for cell death analysis in glioblastoma organoids: A technical note.
Tumor organoid models have emerged as a promising tool in cancer research. By preserving intra- and intertumoral heterogeneity and structural integrity they provide a physiologically relevant platform for drug-response studies. However, valid methodological approaches for cell death analyses applying flow cytometry, particularly in complex, large organoids, are lacking. Using glioblastoma organoids (GBOs), we developed a flow cytometry protocol to quantify cell death as an important readout in cancer research. Human GBOs were generated out of tumor material from six patients. Temozolomide (TMZ) and lomustine (CCNU) were used as cytotoxic agents commonly employed in glioblastoma therapy. After treatment for 144 and 288 hours, single cell suspensions from densely-packed GBOs were generated through a combined approach of enzymatic and mechanical dissociation. Cells were permeabilized with Triton X and subsequently stained with propidium iodide (PI). PI staining labels fragmented nuclear DNA, yielding a hypodiploid sub-G1 peak in flow cytometry that markes cell death. After treatment for 288 hours with physiologically-relevant concentrations of TMZ and CCNU cell death rates reached up to 63% in our GBO model. Across three GBO populations, the impact of CCNU at the given concentration was more pronounced compared to that observed with TMZ and the cell death rates of treatment for 288 hours surpassed that of the 144-hour treatment. Both biological and technical replicates showed low variability. Hoechst 33258 staining on the same samples confirmed trends in cell death rates obtained from PI-based analysis. We further validated the treatment-induced effect using a plate-based lactate dehydrogenase release assay and measurements of GBO diameter. Our single-stain flow-cytometry protocol scales to large, dense organoids and provides a practical balance of performance, hands-on time, cost, specificity, and throughput. This protocol could support development and evaluation of subtype-specific therapeutic strategies in translational cancer research.
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Multi-omics and spatial analysis of microgravity-grown glioblastoma organoids reveal superior modeling of advanced disease after long-term spaceflight.
Creating accurate organoid models remains a challenge, particularly for diseases like glioblastoma (GBM), an aggressive, treatment-resistant brain cancer with an immunosuppressive microenvironment. To overcome limitations of Earth-based models (sedimentation and disaggregation) and leverage the unique advantages of space (accelerated disease progression and immune dysregulation), this study utilizes a panel of GBM-myeloid organoids cultured for 40 d aboard the International Space Station. Spaceflight-grown GBM organoids have more uniform morphology compared to ground controls. We observed a context-dependent response to spaceflight, with the inclusion of macrophages and monocytes altering the bulk and spatial transcriptional response. While GBM-alone organoids have transcriptional markers of less aggressive disease in microgravity, organoids containing GBM cells + monocytes have increased expression of genes associated with disease progression and poor patient outcomes. Gene expression patterns are more spatially organized, with mesenchymal-related genes enriched in the core and inflammation-related genes enriched at the periphery, mimicking GBM tumor architecture. Microgravity-grown GBM+monocyte organoids exhibit increased secretion of proteins such as CXCL12 and LOX-1, associated with more aggressive disease. Infrared chemical imaging confirms spatial chemical gradients of DNA, protein, and lipid species in both space- and terrestrially-grown organoids. In summary, this work presents not only an advanced model of glioblastoma for basic and translational research but also demonstrates methods to acquire diverse data from organoids compatible with the unique constraints of in-space systems, helping establish a working model for orbital oncology.
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Attractor Landscape Analysis Reveals a Reversion Switch in the Transition of Colorectal Tumorigenesis.
A cell fate change such as tumorigenesis incurs critical transition. It remains a longstanding challenge whether the underlying mechanism can be unraveled and a molecular switch that can reverse such transition is found. Here a systems framework, REVERT, is presented with which can reconstruct the core molecular regulatory network model and a reversion switch based on single-cell transcriptome data over the transition process is identified. The usefulness of REVERT is demonstrated by applying it to single-cell transcriptome of patient-derived matched organoids of colon cancer and normal colon. REVERT is a generic framework that can be applied to investigate various cell fate transition phenomena.
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A Three-Dimensional Organoid Culture System Derived from Human Glioblastomas Recapitulates the Hypoxic Gradients and Cancer Stem Cell Heterogeneity of Tumors Found In Vivo.
Many cancers feature cellular hierarchies that are driven by tumor-initiating cancer stem cells (CSC) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2(+), OLIG2(+), and TLX(+) cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiotherapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displaying histologic features, including single-cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology. Cancer Res; 76(8); 2465-77. ©2016 AACR.
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Protective Effect of Human-Neural-Crest-Derived Nasal Turbinate Stem Cells against Amyloid-β Neurotoxicity through Inhibition of Osteopontin in a Human Cerebral Organoid Model of Alzheimer's Disease.
The aim of this study was to validate the use of human brain organoids (hBOs) to investigate the therapeutic potential and mechanism of human-neural-crest-derived nasal turbinate stem cells (hNTSCs) in models of Alzheimer's disease (AD). We generated hBOs from human induced pluripotent stem cells, investigated their characteristics according to neuronal markers and electrophysiological features, and then evaluated the protective effect of hNTSCs against amyloid-β peptide (Aβ1-42) neurotoxic activity in vitro in hBOs and in vivo in a mouse model of AD. Treatment of hBOs with Aβ1-42 induced neuronal cell death concomitant with decreased expression of neuronal markers, which was suppressed by hNTSCs cocultured under Aβ1-42 exposure. Cytokine array showed a significantly decreased level of osteopontin (OPN) in hBOs with hNTSC coculture compared with hBOs only in the presence of Aβ1-42. Silencing OPN via siRNA suppressed Aβ-induced neuronal cell death in cell culture. Notably, compared with PBS, hNTSC transplantation significantly enhanced performance on the Morris water maze, with reduced levels of OPN after transplantation in a mouse model of AD. These findings reveal that hBO models are useful to evaluate the therapeutic effect and mechanism of stem cells for application in treating AD.
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Imaging-Based Efficacy Evaluation of Cancer Immunotherapy in Engineered Tumor Platforms and Tumor Organoids.
Cancer immunotherapy is used to treat tumors by modulating the immune system. Although the anticancer efficacy of cancer immunotherapy has been evaluated prior to clinical trials, conventional in vivo animal and endpoint models inadequately replicate the intricate process of tumor elimination and reflect human-specific immune systems. Therefore, more sophisticated models that mimic the complex tumor-immune microenvironment must be employed to assess the effectiveness of immunotherapy. Additionally, using real-time imaging technology, a step-by-step evaluation can be applied, allowing for a more precise assessment of treatment efficacy. Here, an overview of the various imaging-based evaluation platforms recently developed for cancer immunotherapeutic applications is presented. Specifically, a fundamental technique is discussed for stably observing immune cell-based tumor cell killing using direct imaging, a microwell that reproduces a confined space for spatial observation, a droplet assay that facilitates cell-cell interactions, and a 3D microphysiological system that reconstructs the vascular environment. Furthermore, it is suggested that future evaluation platforms pursue more human-like immune systems.
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Reactive Oxygen Species Mediate Transcriptional Responses to Dopamine and Cocaine in Human Cerebral Organoids.
Dopamine signaling in the adult ventral forebrain regulates behavior, stress response, and memory formation and in neurodevelopment regulates neural differentiation and cell migration. Excessive dopamine levels, including those due to cocaine use in utero and in adults, could lead to long-term adverse consequences. The mechanisms underlying both homeostatic and pathological changes remain unclear, in part due to the diverse cellular responses elicited by dopamine and the reliance on animal models that exhibit species-specific differences in dopamine signaling. In this study, we use the human-derived ventral forebrain organoid model of Xiang-Tanaka and characterize their response to cocaine or dopamine. We explore dosing regimens of dopamine or cocaine to simulate acute or chronic exposure. We then use calcium imaging, cAMP imaging, and bulk RNA-sequencing to measure responses to cocaine or dopamine exposure. We observe an upregulation of inflammatory pathways in addition to indicators of oxidative stress following exposure. Using inhibitors of reactive oxygen species (ROS), we then show ROS to be necessary for multiple transcriptional responses of cocaine exposure. These results highlight novel response pathways and validate the potential of cerebral organoids as in vitro human models for studying complex biological processes in the brain.
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Mesoscale maladaptation in disease organoids.
In chronic diseases, multiple tissue components at a shared interface often deteriorate concurrently, and disease progression may depend on interactions among these failures rather than on any single defect. Multi-tissue organoid models can mimic disease-relevant pathology in vitro, but the field lacks a simple framework for comprehensive interrogation. Here, we propose mesoscale maladaptation as an operational concept for multi-tissue disease modelling, defined as a synergistic decline in interdependent functions that can occur between two or more tissue elements. To detect maladaptation, we introduce a stepwise workflow that identifies the failing tissue elements, defines directionality of potential interdependence between these elements and compares combined perturbations with single perturbations to evaluate synergistic decline. We apply this framework to intestinal neuromuscular, hepatic sinusoidal, blood-brain barrier and tumour-neural interfaces, but it can be extrapolated to other organs and systems. This framework will illuminate future directions for in vitro complex disease modelling by enriching biological insights to disentangle progressive pathology, shifting the focus from localised biological failures to concurrent multi-tissue failures that produce synergistic pathology.
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Establishing dorsal-ventral patterning in human neural tube organoids with synthetic organizers.
Precise dorsal-ventral (D-V) patterning of the neural tube (NT) is essential for the development and function of the central nervous system. However, existing models for studying NT D-V patterning and related human diseases remain inadequate. Here, we present organizers derived from pluripotent stem cell aggregate fusion ("ORDER"), a method that establishes opposing BMP and SHH gradients within neural ectodermal cell aggregates. Using this approach, we generated NT organoids with ordered D-V patterning from both zebrafish and human pluripotent stem cells (hPSCs). Single-cell transcriptomic analysis revealed that the synthetic human NT organoids (hNTOs) closely resemble the human embryonic spinal cord at Carnegie stage 12 (CS12) and exhibit greater similarity to human NT than to mouse models. Furthermore, using the hNTO model, we demonstrated the critical role of WNT signaling in regulating intermediate progenitors, modeled TCTN2-related D-V patterning defects, and identified a rescue strategy.
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Controllable Fabrication of Composite Core-Shell Capsules at a Macroscale as Organoid Biocarriers.
The cell encapsulation technology is promising for generation of functional carriers with well-tailored structures for efficient transplantation and immunoprotection of cells/tissues. Stem cell organoids are highly potential for recapitulating the intricate architectures and functionalities of native organs and also providing an unlimited cell source for cellular replacement therapy. However, it remains challenging for loading the organoids with hundreds of micrometers size by current existing cell carriers. Herein, a simple and facile coextrusion strategy is developed for controllable fabrication of Ca-alginate/poly(ethylene imine) (Alg/PEI) macrocapsules for efficient encapsulation and cultivation of organoids. Human-induced pluripotent stem cell (hiPSC)-derived islet organoids are encapsulated in the aqueous compartments of the capsules and immunoisolated by a semipermeable Alg/PEI shell. Via electrostatic interactions, a PEI polyelectrolyte can be incorporated in the shell for restricting its swelling, thus effectively improving the stability of the capsules. The Alg/PEI macrocapsules are featured with desirable selective permeability for immunoisolation of antibodies from reaching the loaded organoids. Meanwhile, they also exhibit excellent permeability for mass transfer due to their well-defined core-shell structure. As such, the encapsulated islet organoids contain islet-specific multicellular components, with high viability and sensitive glucose-stimulated insulin secretion function. The proposed approach provides a versatile encapsulation system for tissue engineering and regenerative medicine applications.