Assay establishment and validation of a high-throughput organoid-based drug screening platform.

BACKGROUND: Organoids are three-dimensional structures that closely recapitulate tissue architecture and cellular composition, thereby holding great promise for organoid-based drug screening. Although growing in three-dimensional provides the possibility for organoids to recapitulate main features of corresponding tissues, it makes it incommodious for imaging organoids in two-dimensional and identifying surviving organoids from surrounding dead cells after organoids being treated by irradiation or chemotherapy. Therefore, significant work remains to establish high-quality controls to standardize organoid analyses and make organoid models more reproducible. METHODS: In this study, the Z-stack imaging technique was used for the imaging of three-dimensional organoids to gather all the organoids' maximum cross sections in one imaging. The combination of live cell staining fluorescent dye Calcein-AM and ImageJ assessment was used to analyze the survival of organoids treated by irradiation or chemotherapy. RESULTS: We have established a novel quantitative high-throughput imaging assay that harnesses the scalability of organoid cultures. Using this assay, we can capture organoid growth over time, measure multiple whole-well organoid readouts, and show the different responses to drug treatments. CONCLUSIONS: In summary, combining the Z-stack imaging technique and fluorescent labeling methods, we established an assay for the imaging and analysis of three-dimensional organoids. Our data demonstrated the feasibility of using organoid-based platforms for high-throughput drug screening assays.

SIRT1 inhibitors mitigate radiation-induced GI syndrome by enhancing intestinal-stem-cell survival.

High-dose radiation exposure induces gastrointestinal (GI) stem cell death, resulting in denudation of the intestinal mucosa and lethality from GI syndrome, for which there is currently no effective therapy. Studying an intestinal organoid-based functional model, we found that Sirtuin1(SIRT1) inhibition through genetic knockout or pharmacologic inhibition significantly improved mouse and human intestinal organoid survival after irradiation. Remarkably, mice administered with two doseages of SIRT1 inhibitors at 24 and 96 h after lethal irradiation promoted Lgr5+ intestinal stem cell and crypt recovery, with improved mouse survival (88.89% of mice in the treated group vs. 0% of mice in the control group). Moreover, our data revealed that SIRT1 inhibition increased p53 acetylation, resulting in the stabilization of p53 and likely contributing to the survival of intestinal epithelial cells post-radiation. These results demonstrate that SIRT1 inhibitors are effective clinical countermeasures to mitigate GI toxicity from potentially lethal radiation exposure.