Table 2 Effect of matrix stiffness on CSCs population and characteristics

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]