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

Abstract

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 b-catenin, and elevated levels of glycolytic genes, CD44 À/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 cMet. As expected from the predicted hierarchy, CD44 high cells differentiated into CD44 À/low cells. Unexpectedly, a fraction of CD44 À/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 À/low to CD44 high state helps to maintain a CD44 high fraction and the tumorigenic diversity in colon cancer.

Establishment and Characterization of an In Vitro Model of Ovarian Cancer Stem-like Cells with an Enhanced Proliferative Capacity

“…For serial passage, spheroid cells were dissociated with Accumax (Innovative Cell Technologies) once every 10 days. Xenograft establishment after transplantation of spheroid cells was performed as previously described (21).…”

“…Western blot analyses were performed as previously described (21). Antibodies against SOX2, Nanog, CD44, CK7, ALDH1A1, actin, and CD133 were purchased from Cell Signaling Technology, Abcam, Thermo Scientific, Dako, Santa-Cruz Biotechnology, Sigma-Aldrich, and Miltenyi, respectively.…”

“…Immunostaining of spheroid cells, mouse xenograft, or primary tumors was performed as previously described (21). For immunostaining of clinical specimen, SOC samples were stained with anti-ALDH1A1 (ab52492, Abcam), and the extent of the staining was visually evaluated on a scale of 0 (no staining) to 3 (strong staining).…”

“…Previously, we demonstrated that the inhibition of Rho kinase (ROCK) markedly improved the maintenance of a spheroid culture of colon CSCs in vitro (21). The improved efficiency of the spheroid formation by ROCK inhibition was associated with the sustained expression of CD44 variants (CD44v), which are known to be among the established CSC markers of various types of cancer (21).…”

“…The improved efficiency of the spheroid formation by ROCK inhibition was associated with the sustained expression of CD44 variants (CD44v), which are known to be among the established CSC markers of various types of cancer (21).…”