Viscoelastic Liquid Matrix with Faster Bulk Relaxation Time Reinforces the Cell Cycle Arrest Induction of the Breast Cancer Cells via Oxidative Stress

Abstract

The reactivating of disseminated dormant breast cancer cells in a soft viscoelastic matrix is mostly correlated with metastasis. Metastasis occurs due to rapid stress relaxation owing to matrix remodeling. Here, we demonstrate the possibility of promoting the permanent cell cycle arrest of breast cancer cells on a viscoelastic liquid substrate. By controlling the molecular weight of the hydrophobic molten polymer, poly(ε-caprolactone-co-D,L-lactide) within 35-63 g/mol, this study highlights that MCF7 cells can sense a 1000 times narrower relaxation time range (80-290 ms) compared to other studies by using a crosslinked hydrogel system. We propose that the rapid bulk relaxation response of the substrate promotes more reactive oxygen species generation in the formed semi-3D multicellular aggregates of breast cancer cells. Our finding sheds light on the potential role of bulk stress relaxation in a viscous-dominant viscoelastic matrix in controlling the cell cycle arrest depth of breast cancer cells.

Keywords: breast cancer; material-induced senescence; multicellular aggregates; oxidative stress; stress relaxation.

Figure 1

(a) Bulk loss factor of the copolymer substrates obtained by rheometer at 1 Hz angular frequency and 37 °C, (b) bulk stress relaxation of the copolymer substrates obtained by rheometer at 10% strain concentration (normalized to their each maximum individual stress), (c) bulk relaxation time of the copolymer substrate obtained by fitting the stress relaxation curve to the standard linear solid model, (d) the contact angle of the copolymer substrates, (e) the quantified amount of adsorbed fibronectin on each substrate, and (f) the schematic illustration of the fibronectin (green)-coated copolymer substrate used as a cell culture substrate. (Statistical analyses: Student’s t-test n.s. p > 0.05; # is the comparison between samples p < 0.05; * is the comparison between different treatment p < 0.05).

Figure 2

(a) The brightfield image (x–y) of the morphology of the multicellular aggregates of MCF-7 on day 5, (b) The actin (red) stained and z-axis view of DAPI (blue) stained- cells on day 5, (c) the number of generated multicellular aggregates on day 5, (d) the average diameter of the multicellular aggregates on day 5, and (e) the diameter distribution frequency of the multicellular aggregates on day 5. (Statistical analyses: Student’s t-test n.s. p > 0.05; # p < 0.05).

Figure 3

(a) The immunofluorescence-stained images of the cells on day 5 for paxillin, (b) the quantified expression of paxillin on day 5 (normalized to the paxillin expression in control substrate, polystyrene), and (c) the expression of chromogenic senescence marker (SA-β-Gal) with various condition (untreated, or treated with a ROCK inhibitor, Y27632). (Statistical analyses: Student’s t-test n.s. p > 0.05).

Figure 4

(a) The metabolic activity (NAD+ → NADH) of the cells on day 5, (b) the proliferation ability of the cells on day 5, (c) the expression of apoptosis & necrosis marker of the cells on day 5 on the substrates with various bulk relaxation time (from left to right: 80, 210, 290, and 10⁸ ms), (d) the percentage of cell death marker-LDH on day 5, and (e) the cell cycle distribution on substrates with various bulk relaxation time on day 5. (Statistical analyses: Student’s t-test ns p > 0.05; # is the comparison between each time point p < 0.05; * is the comparison between samples at the same time point p < 0.05).

Figure 5

(a) The immunofluorescence staining images of the phosphorylated p38/MAPK on day 5, (b) the quantified expression of phosphorylated p38/MAPK based on its immunofluorescence images, (c) the expression of chromogenic senescence marker (SA-β-Gal) of untreated and ROS inhibitor (MitoQ) treated cells after day 5 of cell culture with scale bar= 100 µm, (d) the quantified SA-β-Gal before and after ROS inhibition with MitoQ, and (e) the western blot image of the HIF1A protein at day 5. (Statistical analyses: Student’s t-test ns p > 0.05; # is the comparison between drug-treated and untreated p < 0.05; * is the comparison between different samples p < 0.05).

Figure 6

The western blot images of (a) Ki67, (b) p27/Kip1, (c) p21, and (d) p53 on day 5, and their quantified expression normalized to that of GAPDH, (e) the bulk ${\tau }_{1/2}$ of the copolymer substrates-dependent Ki67 expression level & DAPI staining images of the re-plated cells on the polystyrene dish after 2 days of cell culture, and (f) the metabolic activity of the cells after 2 days of cell culture on the polystyrene dish before and after supplementation with CM. (Statistical analyses: Student’s t-test ns p > 0.05; # is the comparison between untreated and CM-treated p < 0.05; * is the comparison between samples p < 0.05 [in (a–d), * refers to the comparison between each sample to 10⁸ ms substrate).