Functional single-cell analysis of the stem cell plasticity associated with drug-tolerant persister cells in colorectal cancer

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Using patient-derived colorectal cancer (CRC) organoids, we previously generated an analysis platform consisting of slow-growing CRC cells isolated from different human samples to represent the tumor heterogeneity. In this study, we tracked the fate of each cell through a clonogenic growth assay and found that the CRC cells showed a wide range of growth ability. Further rounds of the clonogenic growth assay revealed that the spheroid forming cells in CRC organoids consisted of distinct subpopulations; the cells generating large spheroids (L-cells) and the cells generating small spheroids (S-cells). The cells derived from the small spheroids gave rise to only small spheroids, consisting of slow-growing cells (S-pattern). While the cells derived from the large spheroids gave rise to both small and large spheroids, showing a dual-growing phenotype (D-pattern). Although the S-pattern spheroids never gave rise to large spheroids once isolated, transition to the D-pattern occurred by various extrinsic triggers, in which Musashi-1 (MSI1) played a key role. We revealed that the suppression of MSI1 in large spheroids, by using the CRISPR/Cas9 system, induced a transition from the D- to the S-pattern. We also found that the S-pattern spheroids were resistant to chemotherapy and transited to the D-pattern upon drug treatment. In conclusion, the isolated S-cells could be a novel platform for investigating drug-tolerant persister cells (DTPs) and developing the DTP targeting treatment. As the transition is linked to the drug resistance, it can be a therapeutic target.