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Colorectal cancer results from genetic aberrations which accumulate over a long period of time, with malignant and metastatic properties acquired at a relatively late stage. A subpopulation of CD26+ colorectal cancer stem cells are known to be implicated in metastasis. We quantified CD26+ cancer cells in 11 primary tumor samples by flow cytometry, and showed that tumors having confirmed or suspected metastases harbored a relatively high CD26+ level in these samples. We hypothesized that this subpopulation of cancer stem cells arises in the late stage of carcinogenesis from the bulk of tumor daughter cells which are CD26-. The manipulation of PIK3CA and TP53 , two genes commonly deregulated in the late stage, had an effect on the maintenance of the CD26+ cell population. When CD26- tumor daughter cells were sorted and cultured, the emergence of tumor spheres containing CD26+ cells occurred. These findings shed light to the origin of colorectal cancer stem cells with metastatic properties, which has an implication on conventional treatments by surgery or adjuvant chemotherapy for tumor debulking., Competing Interests: The authors declare no conflict of interest.