blister and Colonic-Neoplasms

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.

Topics: Amides; Animals; Apoptosis; Blotting, Western; Cell Proliferation; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression Profiling; Hep G2 Cells; Heterocyclic Compounds, 4 or More Rings; HT29 Cells; Humans; Hyaluronan Receptors; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasms, Experimental; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; rho-Associated Kinases; RNA Interference; Spheroids, Cellular; Transplantation, Heterologous; Tumor Cells, Cultured

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.

Topics: Actins; Biomechanical Phenomena; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Humans; Mechanical Phenomena; Neoplasm Metastasis; Phenotype