Data from Induction of the Stem-like Cell Regulator CD44 by Rho Kinase Inhibition Contributes to the Maintenance of Colon Cancer–Initiating Cells

The difficulty in expanding cancer-initiating cells in vitro is one of major obstacles for their biochemical characterization. We found that Rho kinase (ROCK) inhibitors as well as blebbistatin, a myosin II inhibitor, greatly facilitated the establishment of spheroids from primary colon cancer. The spheroid cells expressed cancer stem cell markers, showed the ability to differentiate, and induced tumors in mice. The spheroids were composed of cells that express various levels of CD44, whereas CD44^high cells were associated with increased sphere-forming ability, expression of the activating form of β-catenin, and elevated levels of glycolytic genes, CD44^−/low cells showed increased levels of differentiation markers and apoptotic cells. The spheroid cells expressed variant forms of CD44 including v6, and the induction of the variants was associated with the activating phosphorylation of c-Met. As expected from the predicted hierarchy, CD44^high cells differentiated into CD44^−/low cells. Unexpectedly, a fraction of CD44^−/low cells generated CD44^high cells, and the ROCK inhibitor or blebbistatin primed the transition by inducing CD44 expression. We propose that the transition from CD44^−/low to CD44^high state helps to maintain a CD44^high fraction and the tumorigenic diversity in colon cancer. Cancer Res; 72(19); 5101–10. ©2012 AACR.