Abstract
We introduce a protocol for generating 3D organoids from circulating tumor cells (CTCs), enabling longitudinal functional and molecular analyses in pancreatic cancer patients, including those with unresectable disease, which constitutes the majority of cases. We outline the process for isolating and characterizing CTCs from the blood of pancreatic cancer patients and provide detailed instructions for initiating, passaging, and phenotyping CTC-derived organoids. Additionally, we describe techniques for utilizing these organoids in drug screening with a focus on stemness-related pathways. For complete details on the use and execution of this protocol, please refer to Tang et al.1.