Currently, toxicology studies require a large number of animal testing to identify better and safer drug candidates. In an attempt to reduce the use of large animals in pharmaceutical industry, there is a need to bridge the gap between the conventional in vitro cell-based assay and the non-human animal studies. Recently, three- dimensional models are gaining momentum and replacing conventional 2D cell culture assay. 3D organoids derived from animals can serve the purpose of drug testing recapitulating the drug effect in the whole animal. Organoids derived from dog, with many shared disease pathophysiology, is advantageous since multiple biopsy samples can be operated without sacrificing the animal, allowing longitudinal observation of the same animal under the disease pathology. Dog spontaneously develops cancer with similar clinical pathology appearance, therapeutic responses, and acquired resistance to treatments, similar to that in human. Growing and investigating 3D organoids in large quantities from fresh and frozen biopsy samples are pivotal roles in implementing the large-scale screening approach using the organoid-based approach. In order to image a large number of organoids for high-throughput phenotypic screens, we will pursue our goals in two specific aims: 1) we will develop the microfluidic device and characterize the culturing, and 2) perform dose-dependent toxicity test by treating the enteroids and coloinds with doxorubicin, an intravenous (IV) chemotherapy agent. In Aim 1.1, we will design a new microfluidic platform to immobilize and image ten organoids from eight different populations using a confocal microscope. The specific geometry of the chip will immobilize the organoids completely in an optically favorable orientation while eliminating motion blur during stage motion to move the samples. In Aim 1.2 we will characterize the growth and physiological states of the two organoids (enterdoids and colonoids) using multiple passages from both fresh and frozen tissue samples. On the successful characterization of the cultures and the imaging method, the organoids will be evaluated for dose toxicity in Aim 2.1 by treating with Doxorubicine, which causes gastrointestinal toxicity (mucositis) in patients, using multiple staining. The dose responses will be measured and analyzed using the MCP-Mod statistical- based method and compared to toxicity effects measure on human organoids, in Aim 2.2, to demonstrate the clinical relevance and the predictive nature of our approach. A streamlined protocol and chip fabrication pipeline for large-scale production will follow the development of a fully automated large-scale organoid screening platform. In Phase II, we will expand the chip to 96- and 384- well format for high-throughput and develop image analysis algorithm to handle a large volume of images generated from such phenotypic screens. Leveraging our unique expertise, Newormics will be able to sale OrganoidChipsTM to the consumer market and simultaneously have the organoid biobank to provide services with the large collection of canine-based organotypic models from multiple genetic backgrounds.
Public Health Relevance Statement: Project narrative: The lengthy and escalating price of the drug development process suffers from nearly 90% failure rates, a large portion of which is contributed to toxicity and efficacy of new drug molecules. Currently with 7,000 drugs in development, faster and better models are needed to screen these compounds to improve the translation and meet the patientsâ need, especially for chronic and life-threatening disorders. Organotypic models derived from large animals such as dogs recapitulates the human disease pathology better and can be screened in large quantities to identify most effective drug with lowest toxicology effect prior to the first-in-human test.
Project Terms: 3-Dimensional; Algorithmic Analysis; Animal Model; Animal Organ; Animal Testing; animal tissue; Animals; Appearance; Automation; base; biobank; Biological; Biological Assay; Biological Markers; Biological Models; Biopsy; Biopsy Specimen; Canis familiaris; Cell Culture Techniques; Cell surface; Cells; Chemicals; chemotherapy; Chronic; Clinical Pathology; Clinical Pharmacology; clinically relevant; Collection; Colon; cost; Data; Data Collection; design; Development; Disease; Dose; Doxorubicin; drug candidate; drug development; Drug Industry; Drug Screening; drug testing; Drug toxicity; Duodenum; Endoscopic Biopsy; Engineering; Epithelial Cells; experience; Expression Profiling; Failure; first-in-human; follow-up; Freezing; Functional disorder; gastrointestinal; Gene Expression; Genetic; Geometry; Goals; Growth; high resolution imaging; high throughput screening; Histologic; Human; human disease; Image; Image Analysis; imaging modality; imaging platform; imaging system; Immobilization; Immunity; Immunohistochemistry; improved; In Situ Hybridization; In Vitro; in vivo; Intestines; Intravenous; Iowa; large scale production; Life; Liquid substance; Malignant Neoplasms; Measures; Methods; Microfluidic Microchips; Microfluidics; Microscope; Modeling; Molecular; Motion; Mucositis; Mus; Nature; next generation; novel therapeutics; Optics; Organoids; Pathology; Patients; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Population; pre-clinical; preclinical safety; preclinical study; preclinical toxicity; Price; Procedures; Process; Proteins; Protocols documentation; repository; Resistance; response; RNA; Robotics; Role; safety study; Sales; Sampling; Science; screening; Services; Shapes; Site; Stains; Statistical Models; Study models; System; Testing; three-dimensional modeling; Tissue Sample; Tissues; Toxic effect; Toxicity Tests; Toxicology; Translations; treatment response; tumor microenvironment; Universities; Wor
