Table 2.

Effect of matrix stiffness on CSCs population and characteristics

Cancer	Cell type	Material of scaffold	Stiffness	CSC markers/stemness-related genes and genes affecting CSCs	Methods of investigation	Results	Ref.
Breast	4T1, MCF-7	polyethylene glycol diacrylate hydrogels (PEGDA)	2.5, 5.3, 26.1, and 47.1 kPa	ABCG2, CD44	qRT-PCR	5,3 kPa was the optimal stiffness for higher tumorsphere size and CSCS marker expression	[110]
	MCF-7, MDA-MB-231	PEGDA	2–70 kPa	CD44	qRT‐PCR, Flow-cytometry	5 kPa is the optimum matrix stiffness for BCSC growth and CSC marker expression	[109]
	MDA-MB-231	Polycaprolactone (PCL)	3D: 7 kPa and 2D	CD44, Nanog, Sox2, Oct4	q-PCR	7 kPa-cultured cells express higher levels of the stemness marker CD44	[107]
	MDA-MB-231	GelMA/collagen	2 and 12 kPa	MENA	qRT-PCR	Stiff matrix-grown cells express more MENA, a breast cancer metastasis-associated invadopodia protein. MENA overexpression increases the cancer stem cell-like phenotype	[111]
	MDA-MB-231, 4T1	Polyacrylamide (PA)/collagen	130, 4020 Pa	CD44, Nanog, CD49f, ALDH	qRT-PCR, Immunofluorescence	25% of cells in the stiff scaffold and 4% in the soft scaffold expressed CD44	[19]
Lung	A549	PA crosslinked with bisacrylamide	0.2, 2, and 25 kPa	c-Met, EGFR, snail	RT-PCR, Western blot	With increasing matrix stiffness, EGFR and c-Met expression increased, causing tumor cell proliferation and resistance to EGFR and c-Met inhibitors	[124]
	A549, NCI-H1395, NCI-H1650, and PC9	2D 3D: (very stiff: ABS, HIPS, PLA) and stiffness comparable to lung cancer (GelMA-PEGDA)	> 90 M pa < 0.01Mpa	several stem markers such as ALDH1A1, NANOG, CD44, …	qRT-PCR	More CSC markers were expressed by cells cultured on scaffolds than in 2D culture. Less CSC markers were expressed by cells seeded on ABS, HIPS, and PLA scaffolds than on GelMA-PEG scaffolds	[126]
	H1299	Matrigel coated with collagen	0.5, 4 and 25 kPa	CD133, NANOG	Immunoblotting, IHC, RT-PCR	H1299 cells cultured in a 4 kPa scaffold showed higher CD133 and NANOG expression than cells cultured in a 0.5 or 25 kPa scaffold	[20]
Liver	MHCC97H, Hep3B, and HepG2, Huh7	Matrigel	1 to 40 kPa	EpCAM, CK7, CK19	qRT-PCR	Higher expression in cells cultured in medium stiffness	[144]
	Huh7, HepG2	PA coated with collagen	1–12 kPa	OCT4, NANOG, c-kit, CD44, CXCR4, CD133	qRT-PCR	CSCs' marker expression was higher in cells cultured on a softer substrate	[150]
	Huh7, Hep3B	PA coated with collagen	6, 10, and 16 kPa	EpCAM, CD133, Nanog, SOX2	qRT-PCR, Flow cytometry	In the stiffer matrix, cells express more stemness-related genes, including SOX2 and Nanog, have high self-renewal capacity, and are CD133( +)/EpCAM	[143]
	Hep3B, Huh7	PA coated with collagen	1, 6 and 12 kPa	EpCAM, CD133, ALDH-1, CXCR4 and EMT Marker	qRT-PCR, Western blot, IHC	CXCR4, N-cadherin, and vimentin are upregulated in cells with a stiffer matrix, while E-cadherin is downregulated. The levels of EpCAM, CD133, and ALDH-1 were higher in stiff gels than in soft gels	[151]
	MHCC-97H	PA	0.4 to 25.6 kPa	miR-3682-3p	qRT-PCR	Stiffness of 25.6 kPa can enhance miR-3682-3p. Upregulation of MiR-3682-3p increased HCC spheroid formation, side population cell fractions, and CSC marker expression	[147]
	SMMC-7721, HepG2	PA	12, 16 kPa	CD133	Flow cytometry	Increased colony number and CD133 expression in stiff scaffolding	[135]
PDAC	PDAC organoids	HELP (Hyaluronan and elastin-like protein) Low, or HELP High matrices	279, 1253, and 3040 pa,	CD44, ABCG2, and CD24	Flow cytometry	The levels of CD44, ABCG2, and CD24 were higher in HELP High matrices. Moreover, significantly more side populations (SP) were seen in organoids grown in a high matrix (3.74%) than in a low matrix (0.74%)	[171]
Colorectal cancer	HCT-8	polystyrene	3.6 GPa	ALDH3A1, TNS4, CLDN2, and AKR1B10	qRT-PCR, Immunofluorescent	TNS4, CLDN2, AKR1B10, and ALDH1A1 are upregulated on scaffolds at 20 kPa	[174]
		PA	1–20 kPa
	CCD18 (colon fibroblast cells)	fibronectin-functionalized PA	2, 10, 40, 95, and 120 kPA	activin A	ELISA	At 40 kPa, the level of Activin A in the conditioned medium of CCD18 cells reached a plateau. Activin A promoted invasive ALDHhi CSC-like phenotypes, cancer cell plasticity, and metastatic potential	[190]
	HCT‐116	collagen‐coated PA	2 to 20 kPa	CD133, ALDH‐1, Lgr‐5	qRT‐PCR, Western blots	HCT116 cells on stiff scaffold displayed higher expression of CD133, ALDH1, and Lgr5, as well as YAP nucleation	[186]
	HCT-116	PEGDA	2–70 kPa	CD44	qRT‐PCR, Flow-cytometry	25 kPa is the optimal matrix stiffness for HCT116 tumor stem cell growth and marker expression	[109]