br Linkage to EMT in cellular morphologies br The
3.4. Linkage to EMT in cellular morphologies
The cellular morphological parameters are chosen to clarify the relationship between the morphology induced by physical prop-erties of substrates and gene expression of typical EMT markers (Figs. 6, 7, and 8).
Relative TGFB (Fig. 6), HIF1A (Fig. 7), and vimentin (Fig. 8) expressions are plotted as a function of morphological parame-ters. For MDA-MB-231 Erteberel under both oxygen concentration conditions, linear relations between four cellular morphologies (cytoplasm roundness, cytoplasm elongation factor, nuclear elongation factor, and nuclear area to cytoplasm area ratio [AN/ AC]) and TGFB and HIF1A expression are found on PLLA substrate with different stiffness, indicating that the elongation of cells induces the expression of both genes. In addition, the significant increasing of slope in the linear relation under hypoxia, this
evidence suggests that hypoxia upgrades EMT in MDA-MB-231 cells on PLLA substrate.
A significant level of repression in vimentin expression with increasing of area ratio (AN/AC) is observed (Fig. S7) in both cancer cells under both oxygen concentration conditions. The slope under hypoxic condition reflects the upregulation of EMT phenomenon. Interestingly, the cells on the PCL substrates can downregulate vimentin expression compared with the cells on the PLLA sub-strates. Schrader et al. demonstrated that the vimentin expression was upregulated with increasing stiffness of the substrate . In this study, the PLLA substrate is more favorable to enhance vimentin expression for both cancer cells because of the synergetic effect of fiber alignment and stiffness.
Relative CDH1 expression is plotted as a function of four morphological parameters (Fig. S7: Supplementary data). For MDA-MB-231 cells on the PLLA substrate, the expression has no relation to the morphological parameters or the elongation of cells. This behavior is consistent with the results of MCF-7 cells incubated on PLLA substrate, where the elongation of cells inhibits the re-pressions of CDH1 (Fig. S8: Supplementary data). In addition, the expression of CDH2 (N-cadherin) in MDA-MB-231 cells on PLLA substrate at day 3 exhibits similar relation in the elongation of cells (Fig. S9: Supplementary data). Both cadherin expressions in cancer cells may be not responsible for the cellular morphologies although the upregulation of CDH2 and downregulation of CDH1 are un-dergoing in both cancer cells of the PLLA substrate.
In each of the substrate conditions tested in this study, the topographical effect on EMT is much more beneficial for mesen-chymal-type (MDA-MB-231) cells than the epithelial-type (MCF-7) cells, in which a significant changes of SNAI2 and ZEB1 are evident for MDA-MB-231 cells (Fig. S10: Supplementary data). The
Fig. 8. Relationships between gene expression of vimentin and morphological parameter (ratio of AN and AC) for MDA-MB-231 (a, b) and MCF-7 (a’, b’) cells cultured on six different substrates under normoxic and hypoxic conditions at day 7.
Fig. 9. Representative trajectory of MDA-MB-231 cells cultured on six different substrates under normoxic (a) and hypoxic (b) conditions over 12 h at day 7. Arrows indicate the aligned fiber direction of the substrate. PLLA, poly(L-lactic acid); PCL, poly(ε-caprolactone).
transcription factors associated with EMT (SNAI2 and ZEB1) are activated by both hypoxic condition and elongation of cells. We also found that the elongation of cells promotes the upregulation of the transcription factors in MDA-MB-231 cells.
3.5. Effect of substrate properties on motility of cells
The representative recorded trajectories of both breast cancer cells cultured on different substrates and under both oxygen con-centration conditions over 12 h at day 7 point are shown in Fig. 9 (MDA-MB-231) and Fig. S11 (Supplementary data) (MCF-7). The cells on A-PLLA and A-PCL substrates generally migrated along the underlying fiber structures, while the cells on the random and flat surface substrates migrated in random directions.
As reported in our previous article , for MDA-MB-231 cells, their migrations were restricted on R-PLLA and R-PCL compared with F-PLLA and F-PCL substrates. As observed in short displace-ment traveled with isotropic nature, the fibers might act as an obstacle presumably due to the unevenness fiber structure with a certain height (fiber diameter ~ 1e2 mm) than flat substrate when the cells migrate across the fibers. These finding are in accordance with both alignment and migration of cells with a guide [65e67]. As expected, increasing in the distance traveled along the fiber direction is seen in MDA-MB-231 cells cultured on both A-PLLA and A-PCL substrates. This behavior leads cells to move farther away, regardless the decrease in displacement for aligned fibers sub-strates compared with the flat substrate (Fig. 9).