br the cells feel stiffness however tand value might
the cells feel stiffness; however, tand value might be more favorable to understand the basic of celleECM interactions and mechano-transduction. For this reason, tand value has robust correlation to the morphological parameters of cytoplasm roundness (deform-ability) and cellular spreading factor (AN/AC) in MDA-MB-231 cells (Fig. 8(a) and (b)) in hypoxia.
The response of MCF-7 cells does not follow the same trend, suggesting colonization inhibits the deformation of cells, as mentioned previously. The damping coefficient might not be responsible for the cellular morphologies, although the upregula-tion of deformability (Fig. 8(d)) and nuclear elongation factor
(Fig. 8(f)) is undergoing in less invasive MCF-7 cells of the substrates.
3.5. AC gel substrates induced EMT
The cellular morphologies were strongly influenced by the substrate stiffness for more invasive MDA-MB-231 cells. Then, the gene expression changes were investigated to understand the role of the ECM for the malignant phonotype. In this study, a typical mesenchymal marker of vimentin [32e36] was chosen because EMT is a critical phenomenon that induces cancer metastasis
Fig. 6. Relationships between cellular migration speed (S) and morphological parameters of (a and d) cytoplasm roundness, (b and e) AN/AC, and (c and f) nuclear elongation factor for (aec) MDA-MB-231 and (def) MCF-7 at day 3 cultured under hypoxic condition on four different substrates. AC, cytoplasm area; AC, acrylamideeN-acryloyl-6-aminocaproic Hygromycin B copolymer; AN, nuclear area; TCP-coat, tissue culture plate coated with type I collagen.
Fig. 7. Relationships between (a and d) cellular migration speed (S), (b and e) persistent time (P), and (c and f) cellular diffusivity (D) against damping coefficient (tand) and/or relative storage modulus (G0/r) for MDA-MB-231 cells at day 3 cultured on four different substrates in hypoxia. AC, acrylamideeN-acryloyl-6-aminocaproic acid copolymer; TCP-coat, tissue culture plate coated with type I collagen.
Fig. 8. Relationships between morphological parameters and damping coefficient (tand) for (aec) MDA-MB-231 and (def) MCF-7 cells at day 3 cultured on four different substrates in hypoxia. AC, acrylamideeN-acryloyl-6-aminocaproic acid copolymer; TCP-coat, tissue culture plate coated with type I collagen.
[28e31,37e42]. To know details of EMT and metastasis, TGF-b [32,43,44], SNAI2 [32,45,46], and ZEB1 [43,45e48] were added to analysis. TGF-b is known to induce EMT. SNAI2 and ZEB1 are potent repressor of epithelial cell marker E-cadherin gene expression. Cancer cells respond to the hypoxic microenvironment through the activity of HIF-1a [49e51] that behaves as a promoter of EMT. In each of the substrate conditions tested in this study, the effect on EMT is not beneficial for rather less invasive and epithelial-type (MCF-7) cells. Fig. 9 shows the gene expression of HIF-1a, TGF-b, SNAI2, ZEB1, and vimentin for MDA-MB-231 cells cultured on different substrates in hypoxia at day 3.
For comparison, the expression levels at each substrate were normalized to the data of AC-soft. The HIF-1a level under hypoxic condition is markedly increased in MDA-MB-231 cells incubated on AC-stiff and the TCP-coat in comparison with those incubated on AC-soft and AC-mid (Fig. 9(a)). The cells incubated on the TCP-coat exhibit significant increase in TGF-b (Fig. 9(b)) and SNAI2
expression (Fig. 9(c)), indicating the cells are undergoing EMT in-duction because of the EMT-promoting gene [32,43]. As expected, the transcription factor (SNAI2) associated with EMT is activated by the decrease in tand and/or the increase in deformability (cyto-plasm roundness) of cells on the substrates, leading to robust cor-relation (R2 ~ 0.99) (Fig. S7: Supplementary data).
The MDA-MB-231 cells incubated on the TCP-coat in hypoxia express a significant level of repression in vimentin in comparison with those on AC gel substrates (Fig. 9(e)). The induction of EMT is believed to promote tumor cell motility with acquisition of the mesenchymal phenotype (vimentin expression). Our finding shows no correlation between induction and acquisition of the EMT. In this study, the AC gel substrates are more favorable to enhance vimentin expression for MDA-MB-231 cells, that is, vimentin expression has no effect on cellular migration speed (S). Similar results were obtained when MDA-MB-231 cells were treated under normoxic condition (Fig. S8: Supplementary data).
Fig. 9. Effect of the substrates on gene expression of (a) HIF-1a, (b) TGF-b, (c) SNAI2, (d) ZEB1, and (e) vimentin for MDA-MB-231 cells after 3 days of culture in hypoxia. Expression levels at each substrate were normalized to the data of AC-soft. (**p < 0.01). AC, acrylamideeN-acryloyl-6-aminocaproic acid copolymer; TCP-coat, tissue culture plate coated with type I collagen.