Whole-process 3D ECM-encapsulated organoid-based automated high-throughput screening platform accelerates drug discovery for rare diseases

Organoid-based high-throughput screening (HTS) is revolutionizing pharmaceutical development. However, the complexity of handling extracellular matrix (ECM) components with traditional HTS devices leads to the use of suspension cultures for organoids during HTS, which alters their transcriptomic landscape and drug responses. Although automated generation techniques for 3D ECM-encapsulated organoids have been established, limitations in operational simplicity and time efficiency remain barriers to achieving high throughput. Here, we develop a whole-process 3D ECM-encapsulated organoid-based automated HTS (wp3D-OAHTS) platform, which achieves superior throughput compared to existing reported systems for 3D organoid drug screening. Utilizing this automated platform, we generated more than 10,000 homogeneous 3D organoid domes of neuroendocrine cervical cancer (NECC) and evaluated their drug responses to 2802 compounds in 13 days. This highly efficient and reproducible approach finally enabled the identification of 5 top hits that significantly inhibited NECC organoids <i>in vitro</i> with half-maximal inhibitory concentration (IC₅₀) of lower than 10 nM. The representative candidate, Quisinostat 2HCl, demonstrated significantly stronger anti-tumor efficacy than clinically used agents <i>in vivo</i>. This platform significantly improves the rapidity and efficiency of 3D ECM-encapsulated organoid drug screening and facilitates new drug discovery for rare diseases.