Changes in cellular mechanical properties during onset or progression of colorectal cancer

Table 1 List of experimental studies investigating the effect of external pressure on colon cancer cells proliferation

Ref.	Applied load	Pressure loading system and experimental conditions	Results
Hirokawa et al[44], 1997	40-120 mmHg (5-16 kPa)	The pressure-loading apparatus consists of a flask of which cap was pierced and connected to a tubing by which compressed He gas was introduced to raise internal pressure.	Pressurization from 40 to 120 mmHg for 48 h significantly increased cell (IEC18) number with peak proliferation at 80 mmHg. Pressure-induced DNA synthesis was further enhanced by the addition of interleukin-2, suggesting the regulation of intestinal epithelial growth by pressure could be dependent on cytokines.
Hirokawa et al[43], 2001			Applied pressure for 48 h induced proliferation of IEC18 cell, with a significantly peak at 80 mmHg. The pattern of F-actin distribution was not significantly altered. The pressure-induced increase in phosphorylation of Elk-1 fusion protein corresponding to the activation of MAPK.
Basson et al[63], 2000	15 mmHg (2 kPa)	Cell plates was positioned in an airtight acrylic box, in which pressurized gas was introduced by a tubing to increase pressure.	Increasing ambient pressure stimulated the adhesion of human Caco-2, SW1116, SW620, and HT-29 cells to Matrigel, type I collagen, laminin, and fibronectin.
Whitehead et al[6], 2008	0.8 kPa	A controlled mechanical strain was applied on short segments of colon explants obtained from normal and APC1638N/+ mice. Tissues were placed into a mechanical deformation box and compressed in the z-direction of approximately half of their relaxed thickness for 20 min.	APC1638N/+ mice showed the expression of the two oncogenes Myc and Twist1, not observed in wild-type colon explants. Myc and Twist1 activation was found to be correlated with an increased presence of nuclear β-catenin . Almost no nuclear β-catenin was detected in the wild-type colon epithelium.
			The mechanical stimulation of APC1638N/+ tissue leads to the phosphorylation of β-catenin at tyrosine 654, the site of interaction with E-cadherin, affecting cell adhesions properties.
Fernández-Sánchez et al[5], 2015	1.2 kPa	A controlled pressure was applied in vivo in APC1638N/+ and control mice by subcutaneously inserting a magnet close to the mouse colon. The magnet generates a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts.	The magnetically induced load led to a rapid Ret activation and the phosphorylation of β-catenin on Tyr654, impairing its interaction with E-cadherin.
			β-catenin nuclear translocation was observed after 15 days with a consequent increased expression of β-catenin-target genes at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci.
			Such malignant behavior was induced in, both, APC1638N/+ and control mice, irrespective of the presence of prior genetic abnormalities.
Avvisato et al[27], 2007	1.5 kPa	Cells were plated on 38 mm × 76 mm slides and subjected to a laminar shear stress in a rectangular flow channel for 12 h.	β-catenin signalling of SW480 cells decreased to 22% of control values. The β-catenin signalling were measured for 0-24 h during shear stress exposure, it decreased significantly following 12 h of flow, reaching a minimum after 24 h.

Table 2 List of cell properties that depends on substrate stiffness

Substrate-related mechanical properties	Substrate stiffness	Substrate type and composition	Outcomes	Related-biochemical and genetic pathway	Ref.
E-to-R transition	1 kPa	Laminin	The E to R transition is not observed.	Not applicable.	Tang et al[24], 2015
		or fibronectin coated PA gel
	21 kPa	Laminin coated PA gel	Approximately 70%-90% of E cells start transiting to R cells after culturing for 7 d. Transition takes approximately 5-10 h.	E-Cadherin decreases in dissociated
				R cell by a factor 4.73 ± 1.4.
		Fibronectin coated PA gel	Approximately 70%-90% of E cells start transiting to R cells after culturing for 15 d. Transition takes approximately 5-10 h.	Replanted cells retain their dissociated phenotype irrespective of the substrate stiffness.
	3.6 GPa	Fibronectin coated PA gel	Not observed.	Not applicable.
	20 kPa	E-cadherin coated PA gel	E cells transit to R cells in 6 h.	Vinculin in mainly located at the cell-cell junction.
		Fibronectin coated PA gel	E cells transit to R cells in 6 h.	Vinculin in mainly located at the cell- substrate junction.	Ali et al[45], 2014
	~70 GPa	E-cadherin coated stiff glass substrates	Transition is not observed.	Not applicable.
	Extremely stiff1	Plastic/glass stiff substrate	Occasionally E cells transit to R cell (1 cell over 2 × 105).	R cells are deficient in αE-catenin (protein linking the cell-cell adhesion molecule E-cadherin to the action cytoskeleton).	Vermeulen et al[46-48], 1995, 1998, 1999
Cell colony sizes	1-20 kPa	Gradient stiffness fibronectin coated PA gel	E type: colony size positively correlated with substrate stiffness	Not applicable.	Tang et al[25], 2012
			R type: colony size (smaller than E-colony size) positively correlated with substrate stiffness.
	Soft1	Agar gel	Equal numbers of E and R cells were plated and examined after 10 d, 75% of the E cells plated formed colonies while R cells formed no colonies.		Rosenthal et al[72], 1977
Adhesion	1-20 kPa	Gradient stiffness fibronectin coated PA gel	E type: cells show a strong cell-cell adhesion and cell-substrate adhesion evaluated through the measurement of the cell-substrate contact area (188.1 ± 80.7 μm2) by confocal microscopy. Moreover, a strong aspecific adhesion of ~250 nN is detected trough a novel MEMS system.	Reduced E-cadherin expression on R cells.	Tang et al[25], 2012
			R type: cells show a weak cell-cell adhesion on very soft substrate (1 kPa). No cell-cell contact is observed on stiffer substrate (5-10-15-20 kPa). A weak cell/substrate adhesion is demonstrated through the measurement of the cell/surface contact area (49.5 ± 20.9 μm2).
			A week aspecific adhesion of ~2.5 nN is measured through a MEMS system.

¹Young’s modulus not provide.