Patient-Derived Tumor Organoid Rings for Histologic Characterization and High-Throughput Screening.
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ABSTRACT:
SUBMITTER: Nguyen HTL
PROVIDER: S-EPMC7546514 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundColorectal adenoma (CA), especially high-risk CA (HRCA), is a precancerous lesion with high prevalence and recurrence rate and accounts for about 90% incidence of sporadic colorectal cancer cases worldwide. Currently, recurrent CA can only be treated with repeated invasive polypectomies, while safe and promising pharmaceutical invention strategies are still missing due to the lack of reliable in vitro model for CA-related drug screening.MethodsWe have established a large-scale patient-derived high-risk colorectal adenoma organoid (HRCA-PDO) biobank containing 37 PDO lines derived from 33 patients and then conducted a series of high-throughput and high-content HRCA drug screening.ResultsWe established the primary culture system with the non-WNT3a medium which highly improved the purity while maintained the viability of HRCA-PDOs. We also proved that the HRCA-PDOs replicated the histological features, cellular diversity, genetic mutations, and molecular characteristics of the primary adenomas. Especially, we identified the dysregulated stem genes including LGR5, c-Myc, and OLFM4 as the markers of adenoma, which are well preserved in HRCA-PDOs. Based on the HRCA-PDO biobank, a customized 139 compound library was applied for drug screening. Four drugs including metformin, BMS754807, panobinostat and AT9283 were screened out as potential hits with generally consistent inhibitory efficacy on HRCA-PDOs. As a representative, metformin was discovered to hinder HRCA-PDO growth in vitro and in vivo by restricting the stemness maintenance.ConclusionsThis study established a promising HRCA-PDO biobank and conducted the first high-throughput and high-content HRCA drug screening in order to shed light on the prevention of colorectal cancer.
| S-EPMC10478412 | biostudies-literature
Project description:The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.
| S-EPMC5030729 | biostudies-literature
Project description:Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine.
| S-EPMC6013403 | biostudies-literature
Project description:Mammary tumor organoids have become a promising in vitro model for drug screening and personalized medicine. However, the dependency on the basement membrane extract (BME) as the growth matrices limits their comprehensive application. In this work, mouse mammary tumor organoids are established by encapsulating tumor pieces in non-adhesive alginate. High-throughput generation of organoids in alginate microbeads is achieved utilizing microfluidic droplet technology. Tumor pieces within the alginate microbeads developed both luminal- and solid-like structures and displayed a high similarity to the original fresh tumor in cellular phenotypes and lineages. The mechanical forces of the luminal organoids in the alginate capsules are analyzed with the theory of the thick-wall pressure vessel (TWPV) model. The luminal pressure of the organoids increase with the lumen growth and can reach 2 kPa after two weeks' culture. Finally, the mammary tumor organoids are treated with doxorubicin and latrunculin A to evaluate their application as a drug screening platform. It is found that the drug response is related to the luminal size and pressures of organoids. This high-throughput culture for mammary tumor organoids may present a promising tool for preclinical drug target validation and personalized medicine.
| S-EPMC8564453 | biostudies-literature
Project description:Organoids are in vitro self-assembling, organ-like, three-dimensional cellular structures that stably retain key characteristics of the respective organs. Organoids can be generated from healthy or pathological tissues derived from patients. Cancer organoid culture platforms have several advantages, including conservation of the cellular composition that captures the heterogeneity and pharmacotypic signatures of the parental tumor. This platform has provided new opportunities to fill the gap between cancer research and clinical outcomes. Clinical trials have been performed using patient-derived organoids (PDO) as a tool for personalized medical decisions to predict patients' responses to therapeutic regimens and potentially improve treatment outcomes. Living organoid biobanks encompassing several cancer types have been established, providing a representative collection of well-characterized models that will facilitate drug development. In this review, we highlight recent developments in the generation of organoid cultures and PDO biobanks, in preclinical drug discovery, and methods to design a functional organoid-on-a-chip combined with microfluidic. In addition, we discuss the advantages as well as limitations of human organoids in patient-specific therapy and highlight possible future directions.
| S-EPMC8755639 | biostudies-literature
Project description:Identifying drugs for the treatment of glioblastoma (GBM), a rapidly fatal disease, has been challenging. Most screening efforts have been conducted with immortalized cell lines grown with fetal bovine serum, which have little relevance to the genomic features found in GBM patients. Patient-derived neurosphere cultures, while being more physiologically relevant, are difficult to screen and therefore are only used to test a few drug candidates after initial screening efforts. Laminin has been used to generate two-dimensional cell lines from patient tumors, preserving the genomic signature and alleviating some screening hurdles. We present here the first side-by-side comparison of inhibitor sensitivity of laminin and neurosphere-grown patient-derived GBM cell lines and show that both of these culture methods result in the same pattern of inhibitor sensitivity. We used these screening methods to evaluate the dependencies of seven patient-derived cell models: three grown on laminin and four grown as neurospheres, against 56 agents in 17-point dose-response curves in 384-well format in triplicate. This allowed us to establish differential sensitivity of chemotherapeutic agents across the seven patient-derived models. We found that MEK inhibition caused patient-sample-specific growth inhibition and that bortezomib, an FDA-approved proteasome inhibitor, was potently lethal in all patient-derived models. Furthermore, the screening results led us to test the combination of the Bcl-2 inhibitor ABT-263, and the mTOR inhibitor AZD-8055, which we found to be synergistic in a subset of patient-derived GBM models. Thus, we have identified new candidate therapeutics and developed a high-throughput screening system using patient-derived GBM samples.
| S-EPMC4538440 | biostudies-literature
Project description:Three-quarters of compounds that enter clinical trials fail to make it to market due to safety or efficacy concerns. This statistic strongly suggests a need for better screening methods that result in improved translatability of compounds during the preclinical testing period. Patient-derived organoids have been touted as a promising 3D preclinical model system to impact the drug discovery pipeline, particularly in oncology. However, assessing drug efficacy in such models poses its own set of challenges, and traditional cell viability readouts fail to leverage some of the advantages that the organoid systems provide. Consequently, phenotypically evaluating complex 3D cell culture models remains difficult due to intra- and inter-patient organoid size differences, cellular heterogeneities, and temporal response dynamics. Here, we present an image-based high-content assay that provides object level information on 3D patient-derived tumor organoids without the need for vital dyes. Leveraging computer vision, we segment and define organoids as independent regions of interest and obtain morphometric and textural information per organoid. By acquiring brightfield images at different timepoints in a robust, non-destructive manner, we can track the dynamic response of individual organoids to various drugs. Furthermore, to simplify the analysis of the resulting large, complex data files, we developed a web-based data visualization tool, the Organoizer, that is available for public use. Our work demonstrates the feasibility and utility of using imaging, computer vision and machine learning to determine the vital status of individual patient-derived organoids without relying upon vital dyes, thus taking advantage of the characteristics offered by this preclinical model system.
| S-EPMC8724556 | biostudies-literature
Project description:Tumor organoids maintain cell-cell interactions, heterogeneity, microenvironment, and drug response of the sample they originate from. Thus, there is increasing interest in developing tumor organoid models for drug development and personalized medicine applications. Although organoids are in principle amenable to high-throughput screenings, progress has been hampered by technical constraints and extensive manipulations required by current methods. Here we introduce a miniaturized method that uses a simplified geometry by seeding cells around the rim of the wells (mini-rings). This allows high-throughput screenings in a format compatible with automation as shown using four patient-derived tumor organoids established from two ovarian and one peritoneal high-grade serous carcinomas and one carcinosarcoma of the ovary. Using our automated screening platform, we identified personalized responses by measuring viability, number, and size of organoids after exposure to 240 kinase inhibitors. Results are available within a week from surgery, a timeline compatible with therapeutic decision-making.
| S-EPMC6389967 | biostudies-literature
Project description:A total of 3 independently derived organoids were compared to patient tumor samples and patient normal samples to determine if the tumor-derived organoids represent the genomic landscape of the tumor from which they were derived.
2023-01-02 | E-MTAB-10353 | biostudies-arrayexpress
Project description:Cancer drug screening in patient-derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix-dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy-based assay to resolve drug-induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug-induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.
| S-EPMC5731348 | biostudies-literature