Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation
is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro
method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated
cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In
this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated
cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon
spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette
(ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore,
cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in
vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient
(NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs.
Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization
from the Caco-2 colorectal cancer cell line.