br where x and y are position of
where x and y are position of cells, t is time, t is the time lag be-
factor) are summarized in Fig. 3.
tween positions of cells, n is the number of tracked cells, < … >
indicates time average, and […] indicates cell average. Calculated cells are elongated along an arbitrary direction without the multi-
MSD was assessed by applying the persistent random walk (PRW) cellular aggregate (colonization). In contrast, for the cells incubated
model reported by several researchers [22e24]. Two-dimensional on AC-soft or AC-mid substrates (Fig. 2(a) and (b)), the cells are not
MSD for the PRW model can be expressed by equation (4).
elongated, but the cells are slightly deformed when they are incu-
bated on the AC-stiff substrate (Fig. 2(c)). The MCF-7 cells incubated
(4) on AC gel substrates are more aggregated in comparison with MDA-
similar trend is observed in Fig. 2(h) (incubated on the TCP-coat).
Fig. 1. Damping coefficient (tand) and relative storage modulus (G0/r) map for AC gels and various conventional materials at 25 C. The solid line represents a viscoelastic figure of merit (VFOM) (G0/r)x(tand)0.5 ¼ 105 m2s 2, for steel, aluminum, poly(methyl methacrylate) (PMMA), TCP (PS), and natural rubber . The biomedical properties of a tissue in terms of stiffness (elastic modulus) are shown on the x-axis . AC, acrylamideeN-acryloyl-6-aminocaproic AUY 922 copolymer; PS, polystyrene; TCP, tissue culture plate.
Fig. 2. Morphological comparison of (aed) MDA-MB-231 and (eeh) MCF-7 cultured on different substrates (AC gels and TCP-coat) under hypoxic condition for 3 days. Immu-nofluorescence of breast cancer cells was imaged with Hoechst 33342 (blue) and phalloidin (green). Scale bar: 100 mm. AC, acrylamideeN-acryloyl-6-aminocaproic acid copolymer; TCP-coat, tissue culture plate coated with type I collagen.
Both cells cultured on stiff substrates (TCP-coat, TCP, and AC-stiff) produce spread and less circular morphologies than those on the other substrates (Fig. 3(a)). The nuclear areaetoecytoplasm area ratio (AN/AC) (Fig. 3(b)) and the nuclear elongation factor (Fig. 3(c)) represent the extent of stresses transmitted to nucleus. With this in mind, MDA-MB-231 cells cultured under hypoxic condition on stiff substrates (TCP-coat and TCP) exhibit more spread and elongated morphologies because of much lower values in AN/AC and higher values in the nuclear elongation factor in comparison with those of MCF-7 cells. This phenomenon was similar to the results reported by several researchers [13,16,27]. The cells could spread and adhere to the stiff substrate rather than the soft substrate.
The response against AC gel substrates, the TCP-coat, and the TCP for incubation of MCF-7 cells does not exactly follow the same trend of the feature of MDA-MB-231 cells. This is presumably due to the colonization, which leads to an enhanced cellecell contact via
E-cadherin, following the less traction force (contractility) gener-ation and transmitted stresses to nucleus occurring .
The similar trend is observed in normoxic condition (20% O2) (Fig. S3(a)-(c): Supplementary data). As seen in Fig. 3 and Fig. S3 (Supplementary data), the hypoxic treatment had no remarkable effect on morphologies of both cells. This is supported through detailed cellular migration analysis under both normoxic and hypoxic conditions at day 3.
3.3. Effect of substrate properties on motility of cells
The representative recorded trajectories of both breast cancer cells cultured on different substrates of AC gels and the TCP-coat under hypoxic condition over 16 h at day 3 point are shown in Fig. 4. For both cells on AC-soft and AC-mid substrates, their
Fig. 3. Quantification of the cellular morphologies of (a) cytoplasm roundness, (b) AN/ AC ratio, and (c) nuclear elongation factor as box plots for MDA-MB-231 and MCF-7 cells cultured on different substrates of AC gels and TCP-coat under hypoxic condi-tion at day 3. AC, acrylamideeN-acryloyl-6-aminocaproic acid copolymer; TCP-coat, tissue culture plate coated with type I collagen.
migrations are restricted compared with those on the AC-stiff substrate, TCP-coat, and TCP, while the cells migrated in random directions. As expected, increase in stiffness of the substrates leads cells to move generally (Fig. 4(c)) and farther away with higher motility (Fig. 4(d)), regardless of any direction.
Another interesting feature is that both cells cultured on the TCP have some adverse effect on migration with a significant decrease in displacement compared with that on the TCP-coat. This trend is supported by the substrate surface that is coated with type I collagen. Interestingly, the same trend is observed under normoxic condition (Fig. S4: Supplementary data).