Mechanical Force Affects Expression of an In Vitro Metastasis-Like Phenotype in HCT-8 Cells

Abstract

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.

A dual phenotype of MDA-MB-468 cancer cells reveals mutual regulation of tensin3 and adhesion plasticity

“…These suspension subcultures were acquired by enriching non-adherent single cells that spontaneously dissociated from MDA-MB-468 monolayer cultures. In a similar, approach Tang et al generated an in vitro metastasis model consisting of metastasis-like dissociated round cells (R-cells) and adherent epithelial cells (E-cells) from HCT8 colon adenocarcinoma cells (Tang et al, 2010(Tang et al, , 2014. In HCT8 colon cancer, R-cells occurred at very low frequency but the R-cell:E-cell ratio could be increased by appropriate physical properties of the culture substrate.…”

Physical Biology in Cancer. 4. Physical cues guide tumor cell adhesion and migration

“…Importantly, numerous cell types, including fibroblasts (49,108,127), ECs (13,15,16,31,64,80,115,126,128,131,152,154), epithelial cells (30,108), cardiomyocytes (8,34,68), dorsal root ganglia neurons (4,146), neutrophils (103,129), and stems cells (35), are sensitive to matrix mechanical properties. Intriguingly, tumor cell migration and adhesion can also be driven by matrix mechanical properties on 2D substrates (75,79,139), which may induce metastasis (133), while some types of cancer cells may lose mechanosensitivity upon transition to a metastatic phenotype (134).…”

Biomechanical Forces Shape the Tumor Microenvironment

“…56 Substrate stiffness also affects the phenotype of human colon carcinoma cells, where adhesion to a 2D surface of physiological elasticity (21-47 kPa) induces a metastatic phenotype. 98 Cells from neuroblastoma, a pediatric neuroendocrine tumor, are highly sensitive to the biomechanical properties of their microenvironment and exhibit changes in differentiation behavior. 60,63 One of the ways that all cells respond to the stiffness of their substrate or 3D surroundings is through alterations in the cell cytoskeleton and intracellular stiffness.…”