Tumor organoid-T-cell coculture systems.
Ontology highlight
ABSTRACT:
SUBMITTER: Cattaneo CM
PROVIDER: S-EPMC7610702 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture systems to evaluate the efficacy of immunotherapies on representative paediatric tumour models are lacking. Here, we describe a detailed procedure for the establishment of an ex vivo test coculture system of paediatric tumour organoids and immune cells that enables assessment of different immunotherapy approaches in paediatric tumour organoids. We provide a step-by-step protocol for an efficient generation of patient-derived diffuse intrinsic pontine glioma (DIPG) and neuroblastoma organoids stably expressing eGFP-ffLuc transgenes using defined serum-free medium. In contrast to the chromium-release assay, the new platform allows for visualization, monitoring and robust quantification of tumour organoid cell cytotoxicity using a non-radioactive assay in real-time. To evaluate the utility of this system for drug testing in the paediatric immuno-oncology field, we tested our in vitro assay using a clinically used immunotherapy strategy for children with high-risk neuroblastoma, dinutuximab (anti-GD2 monoclonal antibody), on GD2 proficient and deficient patient-derived neuroblastoma organoids. We demonstrated the feasibility and sensitivity of our ex vivo coculture system using human immune cells and paediatric tumour organoids as ex vivo tumour models. Our study provides a novel platform for personalized testing of potential anticancer immunotherapies for aggressive paediatric cancers such as neuroblastoma and DIPG.
| S-EPMC8466534 | biostudies-literature
Project description:Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within four to eight weeks, indicating the feasibility of personalized drug screening. Molecular, genetic and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to six months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.
| S-SCDT-EMM-2022-16001 | biostudies-other
Project description:Pancreatic ductal adenocarcinoma (PDA) is a clinically challenging disease with limited treatment options. Despite a small percentage of cases with defective mismatch DNA repair (dMMR), PDA is included in the most immune-resistant cancer types that are poorly responsive to immune checkpoint blockade (ICB) therapy. To facilitate drug discovery combating this immunosuppressive tumor type, a high-throughput drug screen platform is established with the newly developed T cell-incorporated pancreatic tumor organoid model. Tumor-specific T cells are included in the pancreatic tumor organoids by two-step cell packaging, fully recapitulating immune infiltration in the immunosuppressive tumor microenvironment (TME). The organoids are generated with key components in the original tumor, including epithelial, vascular endothelial, fibroblast and macrophage cells, and then packaged with T cells into their outside layer mimicking a physical barrier and enabling T cell infiltration and cytotoxicity studies. In the PDA organoid-based screen, epigenetic inhibitors ITF2357 and I-BET151 are identified, which in combination with anti-PD-1 based therapy show considerably greater anti-tumor effect. The combinatorial treatment turns the TME from immunosuppressive to immunoactive, up-regulates the MHC-I antigen processing and presentation, and enhances the effector T cell activity. The standardized PDA organoid model has shown great promise to accelerate drug discovery for the immunosuppressive cancer.
| S-EPMC10427404 | biostudies-literature
Project description:Cell fate progression of pluripotent progenitors is strictly regulated, resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study, we use human brain and retina organoid models and present single-cell profiling of H3K27ac, H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction, resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination.
| S-EPMC11239525 | biostudies-literature
Project description:Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.
| S-EPMC4500268 | biostudies-literature
Project description:In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.
| S-EPMC6656687 | biostudies-literature
Project description:Background & aimsPatient-derived organoid cancer models are generated from epithelial tumor cells and reflect tumor characteristics. However, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we developed a colorectal cancer organoid model that incorporates matched epithelial cells and stromal fibroblasts.MethodsPrimary fibroblasts and tumor cells were isolated from colorectal cancer specimens. Fibroblasts were characterized for their proteome, secretome, and gene expression signatures. Fibroblast/organoid co-cultures were analyzed by immunohistochemistry and compared with their tissue of origin, as well as on gene expression levels compared with standard organoid models. Bioinformatics deconvolution was used to calculate cellular proportions of cell subsets in organoids based on single-cell RNA sequencing data.ResultsNormal primary fibroblasts, isolated from tumor adjacent tissue, and cancer associated fibroblasts retained their molecular characteristics in vitro, including higher motility of cancer associated compared with normal fibroblasts. Importantly, both cancer-associated fibroblasts and normal fibroblasts supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. Organoids grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared with mono-cultures and closely resembled the in vivo tumor morphology. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by considerably deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. Thrombospondin-1 was identified as a critical factor for fibroblast invasiveness.ConclusionWe developed a physiological tumor/stroma model, which will be vital as a personalized tumor model to study disease mechanisms and therapy response in colorectal cancer.
| S-EPMC10141529 | biostudies-literature
Project description:The prevalence of tumor-colonizing bacteria along with advances in synthetic biology are leading to a new generation of living microbial cancer therapies. Because many bacterial systems can be engineered to recombinantly produce therapeutics within tumors, simple and high-throughput experimental platforms are needed to screen the large collections of bacteria candidates and characterize their interactions with cancer cells. Here, we describe a protocol to selectively grow bacteria within the core of tumor spheroids, allowing for their continuous and parallel profiling in physiologically relevant conditions. Specifically, tumor spheroids are incubated with bacteria in a 96-well low-adhesion plate followed by a series of washing steps and an antibiotic selection protocol to confine bacterial growth within the hypoxic and necrotic core of tumor spheroids. This bacteria spheroid coculture (BSCC) system is stable for over 2 weeks, does not require specialized equipment and is compatible with time-lapse microscopy, commercial staining assays and histology that uniquely enable analysis of growth kinetics, viability and spatial distribution of both cellular populations, respectively. We show that the procedure is applicable to multiple tumor cell types and bacterial species by varying protocol parameters and is validated by using animal models. The BSCC platform will allow the study of bacteria-tumor interactions in a continuous manner and facilitate the rapid development of engineered microbial therapies.
| S-EPMC10332800 | biostudies-literature
Project description:The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity, and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing. However, current workflows and methodologies are not suitable for simple artificial coculture systems where strain ratios are dynamic. Here, we established a workflow for coculture proteomics using an exemplar system containing two members, Azotobacter vinelandii and Synechococcus elongatus. Factors affecting the quantitative accuracy of coculture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated at the protein and cell level. We propose a new normalization method, named "LFQRatio", to reflect the relative contributions of two distinct cell types emerging from cell ratio changes during cocultivation. LFQRatio can be applied to real coculture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.
| S-EPMC10913063 | biostudies-literature
Project description:Precise reconstruction of heterotypic cell-cell interactions in vitro requires the coculture of different cell types in a highly controlled manner. In this article, we report a standing surface acoustic wave (SSAW)-based cell coculture platform. In our approach, different types of cells are patterned sequentially in the SSAW field to form an organized cell coculture. To validate our platform, we demonstrate a coculture of epithelial cancer cells and endothelial cells. Real-time monitoring of cell migration dynamics reveals increased cancer cell mobility when cancer cells are cocultured with endothelial cells. Our SSAW-based cell coculture platform has the advantages of contactless cell manipulation, high biocompatibility, high controllability, simplicity, and minimal interference of the cellular microenvironment. The SSAW technique demonstrated here can be a valuable analytical tool for various biological studies involving heterotypic cell-cell interactions.
| S-EPMC4188268 | biostudies-literature