Abstract
Patient-derived tumor organoids are three-dimensionally cultured cancer cells that enable a suitable platform for studying heterogeneity and plasticity of cancer. We present a protocol for tracking the growth fate of single cells and isolating slow-growing cells in human colorectal cancer organoids. We describe steps for organoid preparation and culturing using the cancer-tissue-originating spheroid method, maintaining cell-cell contact throughout. We then detail a single-cell-derived spheroid-forming and growth assay, confirming single-cell plating, monitoring growth over time, and isolating slow-growing cells. For complete details on the use and execution of this protocol, please refer to Coppo et al.1.