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  • br Another interesting feature is


    Another interesting feature is that MDA-MB-231 1035072-16-2 cultured on PLLA substrates have some adverse effect on migration with a significant decrease in displacement than that on PCL substrates. This trend is supported by the stiff substrate that decreases cellular migration [68,69]. Interestingly, the same trend is observed under both oxygen concentration conditions.
    MCF-7 cells follow the similar trend of cellular migration on the substrates under both oxygen concentration conditions (Fig. S11: Supplementary data). MCF-7 cells are less affected by the aligned
    fibers substrates showing less guided along fiber direction. This observation is in accordance with the developed cellular mor-phologies with less nucleus elongation by aligned fibers substrates (see Fig. 4(a), (a’), (b), and (b’)).
    3.5.2. Cellular migration analysis by persistent random walk (PRW) model
    To discuss the diffusion of the seeded cells on the substrates (Fig. S12: Supplementary data) the calculated MSD values and characteristic parameters, cellular migration speed (S), persistent time (P), and cellular diffusivity (D) given by Equation S5 (Supple-mentary data) are presented in Fig. 10. MDA-MB-231 cells cultured on A-PLLA substrates exhibit the highest value of S (0.98 mm/min) at day 7, while decreasing in S under hypoxic condition (0.16 mm/min) was observed on the aligned substrate. However, the P value of MDA-MB-231 cells under hypoxia changes obviously higher (15-fold in comparison with normoxia) on A-PLLA substrate. The D value is the balance between S and P. Overall, D is significantly downregulated under hypoxic condition, and the cellular motility is restricted in comparison with that of under normoxic condition.
    For MCF-7 cells under hypoxia, the cellular motility is not restricted presumably due to the multicellular aggregation (Fig. 2). In this regard, MCF-7 cells are rather less-invasive cancer cells. D is elevated under hypoxic condition due to the upregulation of vimentin expression (Fig. S6(a’)).
    D is significantly upregulated with increasing vimentin expression for both cancer cells (Fig. 11). The D value is important to understand how quick the cancer cells can invade to vasculature or lymph node. MDA-MB-231 cells cultured on both PLLA and PCL fibers substrates lead to a significant change in D between hypoxia and normoxia. In addition, PCL substrate exhibits large D value (fourfold for normoxia and twofold for hypoxia) at same extent of expression in vimentin compared with that of PLLA substrate because the stiff substrate decreases cellular migration [68,69]. In
    general, metastatic cells can be distinguished from non-invasive cancer cells by reduced cytoskeletal stiffness (ratio of AN and AC: Fig. 8) and increased deformability. As seen in Fig. S13
    Fig. 10. Summary of the cellular migration parameters calculated from MSD: cellular migration speed (S) ((a) and (a’)), persistent time (P) ((b) and (b’)), and cellular diffusivity (D) ((c) and (c’)) at day 7 cultured on six different substrates. PLLA, poly(L-lactic acid); PCL, poly(ε-caprolactone).
    Fig. 11. Relationships between gene expression of vimentin and cellular diffusivity (D) for MDA-MB-231 ((a) and (b)) and MCF-7 ((a’) and (b’)) cells at day 7 cultured under normoxic and hypoxic conditions on six different substrates. GAPDH, glyceraldehyde-3-phosphatase dehydrogenase.
    (Supplementary data), the D values are upregulated with increasing deformability (ratio of AN and AC) under both oxygen concentration conditions although such relationships in MDA-MB-231 cells incubated on PLLA substrates do not lead to robust correlation.
    For MCF-7 cells under hypoxia, same trend is obtained (Fig. 11(b’)). However, the vimentin expression has no effect on the D value in MCF-7 cells on substrates under normoxic control (Fig. 11(a’)).
    4. Conclusions
    For MDA-MB-231 cells under both oxygen concentration con-ditions, linear relations between four cellular morphologies 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 sug-gested that hypoxia upgrades EMT in MDA-MB-231 cells on PLLA substrate.
    A significant level of vimentin expression with increasing of the elongation of cells was observed in both cancer cells under both oxygen concentration conditions. The hypoxic condition reflected the upregulation of EMT phenomenon. The cells on the PCL sub-strates can downregulate vimentin expression compared with on the PLLA substrates. The PLLA substrate was more favorable to enhance vimentin expression for both cancer cells because of the synergetic effect of fiber alignment and stiffness.