Although our hypothesis is that Akt expression and its signaling activity are increased during the development and metastasis/recurrence of colorectal cancer, total Akt levels were not changed in more tumorigenic spheroid-forming CSCs due to the following limitation of our in vitro culture model of colorectal CSCs: our in vitro models of colorectal CSCs are 3D-sphere-forming culture system or sorted cell culture system with putative CSC-marker (CD44 and CD133) of same colon cancer cell line (HCT116), whose expression levels of total Akt were not altered during 3D culture or cell sorting. This evidence concerns the gene AKT1 and malignant colon neoplasm.