Substrate stiffness coupling TGF-β1 modulates migration and traction

blebbistatin decreased TGF-β1-enhanced traction force, but increased TGF-β1- .... traction force in the leading-edge of cell migration, and then dep...
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Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Substrate Stiffness Coupling TGF-β1 Modulates Migration and Traction Force of MDA-MB-231 Human Breast Cancer Cells in Vitro Feng Lin,† Haihui Zhang,‡ Jianyong Huang,† and Chunyang Xiong*,†,‡ †

Department of Mechanics and Engineering Science, College of Engineering, and ‡Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China S Supporting Information *

ABSTRACT: Cancer cell migration is the hallmark of tumor metastasis; however, the mechanisms of cancer cell migration have not been fully understood. Considering the fact that biophysical and biochemical properties of the tumor microenvironment are altered during tumor progression, it is instinctive to think about whether the changed microenvironment can regulate cancer cell migration. Herein, we cultured human breast cancer cells (MDA-MB-231) on polyacrylamide gel substrates with different stiffnesses (1, 5, 10, and 20 kPa) with and without transforming growth factor-β1 (TGF-β1, 2 ng/mL) treatment to evaluate the effects of the altered tumor microenvironment on cancer cell migration in addition to the response of traction force generation and cytoskeleton remodeling. The results demonstrated that MDA-MB-231 migration increased with increasing substrate stiffness and was further enhanced with TGF-β1 addition. Traction forces and cytoskeleton remodeling were also found to be enhanced in response to TGF-β1 treatment. Furthermore, inhibiting myosin IIA-mediated contraction by blebbistatin decreased TGF-β1-enhanced traction force but increased TGF-β1-enhanced migration. These results imply that both biophysical (like stiffness) and biochemical (like TGF-β1) factors could orthogonally regulate cancer cell migration. KEYWORDS: substrate stiffness, transforming growth factor, cell migration, traction force, breast cancer cells

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matrix stiffness may facilitate cancer cell adhesion, stress fiber formation, and cell motility. Considering these important roles of matrix stiffness on cell functions, it is crucial to reveal the related mechanisms of the ECM stiffness-regulated cell migration. Besides the biophysical cues, the biochemical cues like some growth factors from the tumor microenvironment can also regulate cell migration. In the present study, a growth factor of interest, transforming growth factor-β (TGF-β), was also investigated for its role in regulating cancer cell migration. TGF-β is a multifunction cytokine that is essential for many physiological and pathological processes, including immune function, organ fibrosis, and cancer progression.10 However, because of its pleiotropic function, the precise role of TGF-β in some physiological and pathological phenomena is not well understood. For instance, TGF-β acts as a tumor suppressor in early tumor progression and switches its role as a tumor and metastasis promoter in later tumor progression.11−13 Although the function of TGF-β switching from tumor suppressor to promoter during cancer progression is well documented, the detailed mechanisms of this switch are still currently unclear.

INTRODUCTION Cell migration plays a central role in various physiological and pathological phenomena, including wound healing, embryo development, and transmigration of tumor cells during cancer metastasis.1−3 Simultaneously, it was also previously reported that cancer cell migration can be regulated by both external cues from the cancer microenvironment, such as the biochemical and biophysical properties, and intrinsic motivation of cancer cells themselves, such as cancer cell contraction.4,5 Although cancer cell migration has been widely studied because inhibiting the migration of cancer cells can prevent cancer progression, cancer is often not completely curable at present because of the lack of a full understanding of the mechanism(s) of cancer cell migration. It is published that biophysical cues from the extracellular microenvironment, especially the extracellular matrix (ECM) stiffness, can regulate various cell functions, including cell proliferation, cell apoptosis, stem cell differentiation, and cell migration.6−8 Cell proliferation is increased and apoptosis is decreased when the cells are cultured on a stiffer substrate,6,9 and the differentiation of mesenchymal stem cells is also directed by matrix stiffness for neural lineage differentiation on a softer matrix and myotube differentiation on a stiffer matrix.7 The importance of ECM stiffness in cell functions is apparent in regulating cancer cell metastasis because the increased cancer © XXXX American Chemical Society

Received: October 31, 2017 Accepted: February 27, 2018 Published: February 27, 2018 A

DOI: 10.1021/acsbiomaterials.7b00835 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

compare the wound healing potential of MDA-MB-231 cultured on PA gel with different stiffnesses either with or without TGF-β1 (2 ng/ mL) treatment through an in vitro wounding healing model (CytoSelect 24-well wound healing assay with 0.9 mm gap for insert, BIOCAT GmbH, Heidelberg, Germany) according to previous studies.23,24 In brief, MDA-MB-231 was digested at a density of 2 × 105 cells/mL, and then each 250 μL of cell suspension was added to each side of the inset, so that, finally, 1 × 105 cells/well were seeded. After ∼12 h when the cells formed a monolayer around the inset, the insets were removed from the wells, and cells were changed to DMEM either with or without TGF-β1 (2 ng/mL) and then imaged at 0, 12, and 24 h using an Olympus IX71 (Olympus, Japan). For the migration capacity of MDA-MB-231 to be analyzed in response to the microenvironment, the wound closure was measured and defined by the ratio between the distances of each time point to that of 0 h. Single Cell Migration. For further testing the effect of biophysical and biochemical cues from the cancer cell microenvironment on cell migration, cell migration speed, mean square displacement (MSD), and migration persistence were measured. In brief, MDA-MB-231 cells were seeded into 35 mm dishes attached by PA gel with different stiffnesses at a density of ∼1 × 104 cells/well. After the cells completed adhesion and spreading, they were changed to DMEM either with or without TGF-β1 (2 ng/mL), incubated in Nikon microscopy with a live-cell station, and imaged for cell positions per 5 min for a duration of 2 h. From every image in the time-lapse video, the cell center (X,Y coordinates) of each cell was extracted using volocity software (PerkinElmer, Massachusetts, USA). The migration velocity of the cell, V, was defined as the ratio between displacement (D) and time (T) and calculated by the formula as follows according to a previous study25

One possibility of this switch is that the changed tumor microenvironmental matrix could affect the TGF-β signaling pathway14 and guide the cell function in response to TGF-β.15 Simultaneously, considering the fact that a number of cell functions that are mediated by TGF-β can also be regulated by matrix stiffness as mentioned above, we hypothesized that matrix stiffness can couple with TGF-β to regulate cancer cell migration. As we know, cell migration involves the sensing of biophysical and biochemical stimulations from its microenvironment, remodeling the cellular cytoskeleton to generate cell traction force at the leading-edge of cell migration and then depolymerizing F-actin through actomyosin contractility at the trailing-edge of cell migration,16,17 which can be mediated by some mechanotransduction pathways like binding of ECM ligands through integrin to generate cell tension, developing focal adhesions, and activating force-related kinases to interact with small GTPases (i.e., Rho, Rac).17−19 Together, these facilitate the changes in cytoskeletal organization and actinmyosin contractility to enhance cell migration.20,21 To this end, we investigated whether biophysical (stiffness) and biochemical (TGF-β1) factors can orthogonally affect breast cancer cell migration. We selected MDA-MB-231, one of the highly metastatic potential human breast cancer cell lines with extensive studies, to perform experiments. The experimental results demonstrate that MDA-MB-231 migration was increased with increasing substrate stiffness and was further enhanced with the addition of TGF-β1. Traction forces and cytoskeleton remodeling were also found to be enhanced in response to TGF-β1 treatment. Furthermore, inhibiting myosin IIA-mediated contraction by blebbistatin decreased TGF-β1enhanced traction force but increased TGF-β1-enhanced migration. These results imply that both biophysical (like stiffness) and biochemical (like TGF-β1) factors could orthogonally regulate cancer cell migration, which may help to determine the underlying mechanisms of cancer cell migration.

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V=

∑ (Xi − Xi − 1)2 + (Yi − Yi − 1)2 D = T T

(1)

where Xi and Yi are the coordinates of the cell center at the ith time point and T is the total time of the migration duration. The MSD, ⟨d2(t)⟩, was defined by the formula as follows according to a previous study25 ⟨d 2(t )⟩ = MSD(nΔt ) =

1 N−n

N−n

∑ ⎡⎣(Xi + n − Xi)2 + (Yi + n − Yi)2 ⎤⎦ i=1

(2)

MATERIALS AND METHODS

where Δt corresponds to the time interval between two frames and n and N denote the number of time steps and frames, respectively. Assessment of Cell Proliferation. For examining whether the observed wound healing behavior could be caused by cell proliferation, the proliferation of MDA-MB-231 cultured on PA gel with different stiffnesses either with or without TGF-β1 (2 ng/mL) treatment was tested by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In brief, 15 mm cover glasses attached by PA gel with different stiffnesses were placed into a 24-well plate, and MDA-MB231 was seeded at a density of 2 × 104 cells/well. Then, the cells were cultured with DMEM either with or without TGF-β1 for 12 and 24 h in the cell incubator. Subsequently, the cells were added with MTT solution (5 mg/mL in PBS) and incubated for 4 h at 37 °C. Finally, the purple formazan was dissolved with dimethyl sulfoxide (DMSO), and measurements of absorbance at 490 nm were carried out. Measurement of Cell Traction Force. Cell traction force was measured by traction force microscopy (TFM) as published before.26,27 In brief, MDA-MB-231 was seeded on the PA gel (1 kPa) with fluorescence beads at a density of ∼10,000 cells/well; after the cells completed adhesion and spreading, they were incubated with DMEM either with or without TGF-β1 (2 ng/mL) for 12 h. Then, single cells were imaged in phase contrast, and fluorescence images of the fluorescence beads before and after cell detachment were also recorded. The cell traction force was computed from the displacement field of the fluorescence beads before and after cell detachment, and then strain energy was calculated by the formula28

Polyacrylamide Gel Preparation. For the microenvironment of the cancer cells to be made with different stiffnesses, polyacrylamide (PA) gel substrates with different stiffnesses were prepared according to previous studies.12,22 Briefly, the glass bottom of the 35 mm dishes and cover glasses with a diameter of 15 mm were pretreated with bound-silane to ensure PA gel attachment. Then, the gel solution with different ratios of 40% acrylamide and 2% bis-acrylamide was used to generate gels with various Young’s moduli (% acrylamide; % bisacrylamide, 1 kPa (3; 0.1), 5 kPa (5; 0.15), 10 kPa (5; 0.3), and 20 kPa (8; 0.264)). After gel polymerization, the gel substrates were activated by sulfo-SANPAH (Pierce, Rockford, USA) and then functionalized with 20 μg/mL of collagen solution (type I, Sigma) overnight at 4 °C. Then, the substrates were hydrated with Dulbecco’s modified Eagle’s medium (DMEM) and incubated at 37 °C and 5% CO2 for at least 24 h before use. For the cell traction force test in particular, red fluorescence beads (diameter of 0.2 μm) were added to the gel solution, and gel substrates were coated with 200 μg/mL of type I collagen. Cell Culture. The MDA-MB-231 cell line was purchased from ATCC and cultured in an incubator at 37 °C and 5% CO2 with DMEM supplied with 10% (v/v) fetal bovine serum (FBS), 100 μg/ mL of streptomycin, and 100 U/mL penicillin. Cells were subcultured when they grew to 80 or 90% confluence. Wound Healing. For the migration of MDA-MB-231 to be analyzed in response to biophysical and biochemical stimulations from the cell microenvironment, a wound healing assay was used to B

DOI: 10.1021/acsbiomaterials.7b00835 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering U = (1/2)

∫ T⃗( r )⃗ ·u⃗( r ⃗) dx dy

(3)

where T⃗ denoted the local cellular traction stress (Pa), and u⃗ is the displacement field of substrates. Assay of Cell Cytoskeleton Structure. For the cytoskeleton structure to be evaluated, MDA-MB-231 cells were labeled simultaneously with fluorescent probes for F-actin and vinculin similar to previous research.29 In brief, MDA-MB-231 cells were cultured on PA gel with different stiffnesses either with or without TGF-β1 (2 ng/ mL) treatment for 12 h and fixed with 4% formaldehyde for 30 min at room temperature, and then the fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. After being washed with PBS, the cells were blocked with 5% BSA in PBS for 30 min at room temperature. F-actin was labeled by rhodamine-phalloidin (5 μg/mL; cytoskeleton) for 30 min at room temperature. For labeling vinculin, the cells were treated with a monoclonal antivinculin antibody (Abcam, Cambridge, UK, and 1:100 dilution in 1% BSA) overnight at 4 °C and further incubated with a secondary antibody, FITC-labeled goat anti-mouse IgG (ProteinTech, Chicago, IL, USA, and 1:50 dilution in 1% BSA), for 2 h at room temperature. Then, the stained cells were viewed and imaged by laser confocal microscope (PerkinElmer, Massachusetts, USA). Modulation of Cell Contraction. For assessing the role of internally generated cell contractility in the MDA-MB-231 migration function in response to biophysical and biochemical cues from the microenvironment, blebbistatin was used to inhibit the function of nonmuscle myosin IIA, which is one of the components of the cytoskeleton, to regulate cell contraction.30−32 For the testing of cell migration, traction force, and cytoskeleton structure, MDA-MB-231 cells cultured on the softest (1 kPa) and stiffest (20 kPa) gels were pretreated with blebbistatin (25 μM) for 30 min and then incubated on DMEM with TGF-β1 (2 ng/mL). Statistical Analysis. All of the values are shown as mean ± standard deviation (SD). Statistical analysis for multiple comparisons was performed with one-way analysis of variance (ANOVA) followed by Tukey test. A level of p < 0.05 was accepted as statistically significant.

Figure 1. Wound healing of MDA-MB-231 cultured on PA gel with different stiffnesses either with or without TGF-β1 treatment. (A) Representative optical images of wound healing of MDA-MB-231 treated without (left panel) and with TGF-β1 (right panel). The panels from upper to lower exhibited cells cultured on PA gel substrates with stiffnesses of 1, 5, 10, and 20 kPa, respectively, and from left to right displayed the beginning of the assay (0 h, left) and wound closure after 12 h (middle) and 24 h (right). It showed that the gap of the wound decreased with increasing substrate stiffness at each time point and further closed with the addition of TGF-β1. Scale bar, 100 μm. (B) Quantitative wound healing rate of MDA-MB-231 corresponding to images as shown in (A). From left to right is the rate of wound closure of cells cultured on 1, 5, 10, and 20 kPa substrates either with or without TGF-β1 treatment. Data are shown as mean ± SD (n = 5, ***p < 0.001).

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RESULTS Substrate Stiffness Coupled to TGF-β1 Increases Wound Healing. For the effect of substrate stiffness and TGF-β1 on MDA-MB-231 migration to be tested, wound healing assays were performed to investigate the migration behavior of monolayer MDA-MB-231. Figure 1A shows representative images of MDA-MB-231 cultured on PA gels with different stiffnesses either with or without TGF-β1 treatment. It can be clearly seen from the images that there was a gradual closure of the wound as cells migrated into the scratch over time (0, 12, and 24 h from left to right panels, respectively), and the wound was closed faster with increased substrate stiffness (1, 5, 10, and 20 kPa from upper to lower panels, respectively). Moreover, the addition of TGF-β1 significantly enhanced the migration as cells migrated faster into the wound in comparison to control (cells not exposed to TGF-β1). Figure 1B shows the quantitative wound healing rate of MDA-MB-231 corresponding to images in Figure 1A. It can be seen that cell migration was significantly increased with increasing substrate stiffness (p < 0.001), and TGF-β1 significantly further enhanced the migration (p < 0.001), demonstrating that substrate stiffness can couple with TGF-β1 to strengthen MDA-MB-231 migration. We also analyzed the wound healing rate between the first and second 12 h of MDAMB-231 cultured on increasing stiffness substrate either with or without TGF-β1 treatment (Figure S1). We found that there was no difference in the wound healing rate between the first 12 and second 12 h if the cells were just cultured on PA gel with

increasing stiffness (Figure S1A), whereas the cells treated with TGF-β1 had a wound healing rate for the second 12 h that was significantly lower than that of the first 12 h in all cases of substrate stiffness (Figure S1B). Substrate Stiffness Coupled to TGF-β1 Increases Single Cell Migration. For characterizing single MDA-MB231 cell migration behavior in response to substrate stiffness and TGF-β1, cell motility of individual cells was recorded (5 min interval) using time-lapse video, and the mean migration speed over a 2 h period was computed for each cell cultured on PA gel with different stiffnesses either with or without TGF-β1 treatment. As shown in Figure 2, average cell migration speed (20 cells per sample) was significantly increased from 0.007 μm/s for the 1 kPa gel to 0.013 μm/s for the 20 kPa gel (p < 0.001). After exposure to TGF-β1, migration speed of MDAMB-231 was significantly increased in all cases of substrate stiffness compared with no TGF-β1 treatment, ranging from 0.008 μm/s for the 1 kPa gel to 0.016 μm/s for the 20 kPa gel (p < 0.001). Substrate Stiffness Coupled to TGF-β1 Changes the Mean Square Displacement and Migration Persistence. For the cell migration behavior to be further determined, the trajectories of migrating MDA-MB-231 were also analyzed to obtain the mean square displacement (MSD) as a function of time interval (Δt) as described in the Materials and Methods. C

DOI: 10.1021/acsbiomaterials.7b00835 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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without 2 ng/mL of TGF-β1 together with measures of cell migration speed, mean square displacement (MSD), and migration persistence (β index). The results showed that the migration of MCF7 on stiffer substrate (60 kPa), including migration speed, MSD, and migration persistence (β index), was stronger than that of MCF7 cultured on a softer substrate (5 kPa), and the migration was also further enhanced after addition of TGF-β1 (Figure S2A−C). Substrate Stiffness and TGF-β1 Did Not Affect Cell Proliferation. It is reported that both cell migration and proliferation abilities will contribute to the wound healing behavior. Thus, to confirm what is the main contribution to the different wound healing behaviors found for MDA-MB-231 under the indicated culture conditions, we also need to measure the proliferation of MDA-MB-231 under the indicated culture conditions besides testing for the single cell migration behavior. MTT assays were performed to measure whether MDA-MB231 wound healing behavior could be caused by cell proliferation. The absorbance of purple formazan, which is produced by newborn cells during the MTT assay, at 490 nm reflects the ability of cell proliferation. As shown in Figure 4, OD absorbance of MDA-MB-231 under the indicated culture conditions was not significantly different in most of the cases. For the OD absorbance of the 12 h case, the mean value of OD absorbance was similar among each condition, and OD absorbance difference just emerged in the comparison between 5 kPa vs 5 kPa plus with TGF-β1. Similarly, for the OD absorbance of the 24 h case, the mean value of OD absorbance was also similar among each condition, and the OD absorbance difference just emerged in the comparison between 20 kPa vs 20 kPa plus with TGF-β1. These together indicated that cell proliferation may not be significantly affected by the substrate stiffnesses, which were used in the present study, and the addition of low dose TGF-β1 (2 ng/mL), suggesting that MDA-MB-231 wound healing behavior could not be caused by cell proliferation in the present study. TGF-β1 Increased Cell Traction Force. For investigating whether the different migration behaviors of MDA-MB-231 regulated by the biophysical and biochemical cues from its microenvironment could be ascribed to its altered ability in cell contraction, traction force generation of MDA-MB-231 that was cultured on PA gel (1 kPa) and treated either with or

Figure 2. Migration speed of MDA-MB-231 cultured on PA gel with different stiffnesses either with or without TGF-β1 treatment. From left to right is the migration speed of cells cultured on gel substrates with stiffnesses of 1, 5, 10, and 20 kPa and together without (solid bar) and with TGF-β1 (open bar) treatment, respectively (n = 20, ***p < 0.001).

As shown in Figure 3A, MSD of MDA-MB-231 was computed during 2 h of cell migration. Similar to the previous research,30,33 one can also see from our results that MSD increased with time according to a power-law relationship in all cases of the substrate stiffness either with or without TGF-β1 treatment; MSD = D(t/Δt)β, where Δt is the time interval (5 min) and the exponent β is a measure of persistence. Importantly, MSD of MDA-MD-231 was also increased with increasing substrate stiffness and was further enhanced after TGF-β1 addition. Figure 3B exhibits the quantitative value of exponent β, which was computed from the slope of the powerlaw relationship. The data showed that the migration of cell cultured on PA gel with higher stiffness and treated with TGFβ1 was more persistent, as reflected by the higher value of β (p < 0.05, p < 0.01). These results suggested that substrate stiffness and TGF-β1 can jointly increase the speed and persistence of MDA-MB-231 migration. Similar trends were also found on the effects of substrate stiffness and TGF-β1 on the migration of epithelial-like breast cancer cells (MCF7). Briefly, we cultured MCF7 on a softer substrate (5 kPa) and a stiffer substrate (60 kPa) and treated the cells either with or

Figure 3. Mean square displacement (MSD) and migration persistence of MDA-MB-231 cultured on PA gel with different stiffnesses either with or without TGF-β1 treatment. (A) MSD of MDA-MB-231 was increased in ascending order corresponding to the substrate stiffness of 1 (squares), 5 (circles), 10 (triangles), and 20 kPa (diamonds), respectively, and further enhanced after addition of TGF-β1 (open dots). (B) Calculated slope for the power-law exponent β. From left to right is the migration persistence of cancer cells cultured on 1, 5, 10, and 20 kPa gel substrates and treated without (solid bar) and with TGF-β1 (open bar), respectively (n = 20, *p < 0.05, **p < 0.01). D

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generation of MCF7 (Figure S2D). We also measured the traction force generation of MDA-MB-231 cultured on PA gel with a stiffness of 5 kPa. We found that strain energy calculated from the cells cultured on 5 kPa gel with TGF-β1 treatment was also significantly enhanced compared to that of just the 5 kPa gel (Figure S3), suggesting that TGF-β1 indeed increased traction force generation of MDA-MB-231. Substrate Stiffness Coupled to TGF-β1 Remodels the Cell Cytoskeleton. Immunofluorescence assay was used to investigate the effect of substrate stiffness and TGF-β1 on remodeling of the MDA-MB-231 cytoskeleton structure. Figure 6 displays representative images of F-actin (red) and vinculin

Figure 4. Proliferation of MDA-MB-231 cultured on PA gel with different stiffnesses either with or without TGF-β1 treatment. From left to right is the OD absorbance of cancer cells cultured on 1, 5, 10, and 20 kPa substrates and treated either without (solid bars) or with TGF-β1 treatment (hashed bars). Substrate stiffness and TGF-β1 had no effect on MDA-MB-231 proliferation (n = 5, *p < 0.05).

without TGF-β1 (2 ng/mL) for 12 h was determined by TFM. Figure 5A exhibits the computed field of cell traction force of single MDA-MB-231 cells cultured on PA gel substrate with or without TGF-β1 treatment. Strain energy (Figure 5B) of each MDA-MB-231 was quantified from the traction force field. It can be seen from the results that cell strain energy was significantly increased after TGF-β1 treatment as compared to control (p < 0.01). A similar trend was also found on MCF7 that TGF-β1 significantly enhanced the traction force

Figure 6. Cytoskeleton structure of MDA-MB-231 cultured on PA gel with different stiffnesses either with or without TGF-β1 treatment. Representative images of MDA-MB-231 cytoskeleton that was characterized by fluorescently labeled F-actin (red panel) and vinculin (green panel) and visualized by confocal microscopy. The left and right column panels represented MDA-MB-231 cells treated without and with TGF-β1 treatment, respectively, and the panels from upper to lower showed the cells cultured on gel substrates with stiffnesses of 1, 5, 10, and 20 kPa, respectively. Substrate stiffness and TGF-β1 significantly remodeled cell cytoskeleton structure as shown with more stress fibers and matured focal adhesions. Scale bar, 50 μm.

(green) labeled by fluorescence probes. Images from upper to lower represented the cytoskeleton structure of MDA-MB-231 cultured on 1, 5, 10, and 20 kPa PA gel substrates, respectively. It can be seen from the images that both F-actin and vinculin exhibited different structures and distributions on substrates with different stiffnesses. F-actin structure was dispersedly distributed when cells were cultured on the softest gel (1 kPa) and then became stronger and showed stress fibers with increasing stiffness (5, 10, and 20 kPa). Simultaneously, vinculin had a similar response trend with F-actin, for vinculin was a blur and not fully matured when cells were cultured on the softest gel (1 kPa) and then became typically matured focal adhesions (FAs) with increasing stiffness (5, 10, and 20 kPa). Addition of TGF-β1 further significantly enhanced the cytoskeleton structure as cells exhibited stress fibers and matured FA even cultured on the softest gel (1 kPa), suggesting that substrate stiffness and TGF-β1 can jointly remodel the cell cytoskeleton. We also quantitatively analyzed the area (Figure S4A), shape index (Figure S4B), and length (Figure S4C) of focal adhesion (FA) of MDA-MB-231 under the indicated culture conditions to further investigate the effects of substrate stiffness and TGF-β1 on FA formation. The outcome revealed

Figure 5. Traction force generated by MDA-MB-231 that either were or were not treated with TGF-β1. (A) Representative images of traction force field generated by control group (without TGF-β1 treatment, left panel) and experimental group (with TGF-β1 treatment, right panel), which was tested by Fourier transform traction microscopy. (B) Strain energy computed from the traction force map as shown in (A). TGF-β1 significantly enhanced cell traction as shown by higher strain energy (n = 20, **p < 0.01). E

DOI: 10.1021/acsbiomaterials.7b00835 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 7. The effect of myosin IIA-mediated contraction on substrate stiffness and TGF-β1-enhanced MDA-MB-231 migration. (A−D) Depletion of myosin IIA-mediated contraction affected substrate stiffness and TGF-β1-enhanced cell migration, including increasing wound healing (A), migration speed (B), MSD (C), and decreasing migration persistence (D). (E) Myosin IIA depletion impaired cell traction force, which was increased by TGF-β1 (*p < 0.05, **p < 0.01, ***p < 0.001).

that the area, shape index, and length of FA was remarkably different. FA was smaller, rounder, and shorter on the softer gel as compared to that on stiffer gels, whereas FA became larger and longer after addition of TGF-β1. Cell Contraction Regulated Substrate Stiffness- and TGF-β1-Enhanced Cell Migration. For the role of cell contraction in regulating tumor microenvironment-enhanced cancer cell migration to be further assessed, cell contraction was preinhibited through blocking myosin IIA by its special ATPase inhibitor (blebbistatin, 25 μM); then, MDA-MB-231 cultured on the softest (1 kPa) and stiffest (20 kPa) PA gel substrates together with TGF-β1 treatment was tested for migration behavior, cell traction force, and cytoskeleton structure as shown in Figure 7. It can clearly be seen from the results that both MDA-MB-231 wound healing (Figure 7A) and migration speed (Figure 7B) were significantly increased with blebbistatin pretreatment (p < 0.05, p < 0.001). Interestingly, although the migration velocity (Figure 7B) and MSD (Figure 7C) were increased in myosin IIA disrupted cells, the overall directional persistence defined by exponent β (Figure 7D) was decreased (p < 0.01, p < 0.001). Blebbistatin treatment also significantly decreased traction force generation of MDA-MB-231 (Figure 7E, p < 0.01). Simultaneously, F-actin staining results also revealed dramatic disruption of F-actin stress fiber structure in the cells pretreated with blebbistatin (Figure 8), and the cells displayed larger lamellipodia. These results suggested that myosin IIA-mediated contraction of MDA-MB-231 can regulate cell migration behavior probably through remodeling of the cytoskeleton structure.

Figure 8. The effect of myosin IIA-mediated contraction on substrate stiffness and TGF-β1-enhanced cytoskeleton remodeling. Representative fluorescence images show that inhibiting myosin IIA impaired cytoskeleton structure, which was remodeled by substrate stiffness and TGF-β1. Scale bar, 50 μm.

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DISCUSSION The primary findings of this study were that substrate stiffness can couple with TGF-β1 to increase migration of MDA-MB231 probably through remodeling of the cytoskeleton structure to alter cell traction force generation. MDA-MB-231 migration characterized by wound healing, cell migration speed, MSD, and migration persistence (β index) was increased with the matrix stiffness increase and further enhanced with the addition of TGF-β1. Notably, cell traction force generation and cytoskeleton structure remodeling were also found to be enhanced in response to TGF-β1; moreover, inhibiting myosin IIA-mediated contraction by blebbistatin decreased TGF-β1F

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ACS Biomaterials Science & Engineering

motion and β with values smaller than 1 or larger than 1 corresponding to sub- or superdiffusive motion, respectively.25,43 According to this, one that emerged from our experimental results was that MDA-MB-231 cultured on each substrate with different stiffnesses and treated either with or without TGF-β1 exhibited superdiffusive motion manner, and the superdiffusive trend was positive with the increase of substrate stiffness and the addition of TGF-β1 as the exponent β becoming higher. In the present research, cell traction force and cytoskeleton remodeling were also found to be enhanced in response to TGF-β1 treatment. Cell traction force is the force that an adherent cell exerts on the underlying substrate. It can help cells sense the biophysical and biochemical properties of their underlying substrate and characterize cell migration capacity.44 Our experimental results demonstrated that cell traction force (referred as strain energy in the present study), which was generated by MDA-MB-231 in response to TGF-β1 treatment, was significantly enhanced when compared to that of the control (without TGF-β1 treatment). The increase in cell traction force generated by cancer cells with TGF-β1 treatment could be ascribed to various factors not limited to strengthening of the cytoskeletal structure45 and transmitting force to the substrate via focal adhesion.46 Our results exhibited that the cytoskeleton structure of MDA-MB-231 (as shown in Figure 6) was significantly remodeled in response to the increase in substrate stiffness and was further enhanced with the addition of TGF-β1. Together, these would increase the generation of cell traction force and then increase cell migration. In the other aspect, cell traction force is also known as being generated through the interaction of actin filaments with myosin-II (a cellular motor protein). The actomyosin cytoskeleton carries the contractile prestress that is necessary for controlling cell morphology, providing mechanical properties, and regulating cell functions.31,47 Blocking myosin IIAmediated contraction with the myosin IIA inhibitor blebbistatin significantly impaired the ability of cell traction force generation triggered by TGF-β1 but increased wound healing behavior, migration speed, and MSD of MDA-MB-231 cultured on both the softest (1 kPa) and stiffest (20 kPa) substrates together with TGF-β1 treatment, and decreased directional persistence (as the β value became smaller) of MDA-MB-231 migration, indicating that MDA-MB-231 tended to migrate in a random motion fashion. The increase in wound healing and migration speed of MDA-MB-231 with blebbistatin treatment was somewhat weird, but not completely surprising, because it is well-known that migration behavior could be regulated by cell contraction and that both positive and negative correlations between increasing metastatic and migration potential and producing traction force had been reported.48−50 The migration speed that positively and/or negatively correlates with cell traction force could be ascribed to various factors but not limited to traction force generation, adhesion strength, type of substratum (extracellular matrix ligands and other cells), external migration cues, topography of extracellular matrix, cellular polarity machinery, receptor signaling, integrin trafficking, integrin coreceptors, actin turnover, polarized force generated, and control of lamellipodia protrusion.51,52 The migration of blebbistatin-treated cells was faster than that of control and TGF-β1-only treated cells, which was similar to previous reports and may also be attributed to inhibiting the ability of myosin IIA to periodically interrupt actin polymerization and hinder protrusions at the leading edge,32,53,54

enhanced traction force but increased TGF-β1-enhanced cell migration. These results imply that both biophysical (like stiffness) and biochemical (like TGF-β1) factors could orthogonally regulate cancer cell migration, which may help to determine underlying mechanisms of cancer cell migration. It is reported that tissue biophysical properties can be altered during cancer progression; in particular, tissue stiffness can be increased due to the deposition and cross-linking of ECM proteins and the stiffening of tissue cells.34 In breast cancer, the healthy mammary gland is compliant with stiffness of ∼200 Pa, whereas the lesion tissue like breast is over an order of magnitude stiffer with the stiffness of several kilopascals and even tens of kilopascals.35 Simultaneously, the widely used elastic substrate to study the effect of matrix stiffness on cell behavior and function is based on varying the curing ratio of PA gel, whose stiffness ranges from tens of pascals to hundreds of kilopascals, which covers the stiffness range of healthy and diseased breast tissue. Considering the fact that MDA-MB-231 is a kind of breast cancer cell line, in the present study, the PA gels with stiffnesses of 1, 5, 10, and 20 kPa, which were more like the stiffness of lesion tissue of breast, were prepared to investigate the role of substrate stiffness in inducing MDA-MB231 migration. The finding that the migration of MDA-MB-231 was increased in response to increasing substrate stiffness is consistent with the previous reports that matrix rigidity can increase migration of various cancer cells, including pancreatic cancer cells, colorectal cancer cells, breast cancer cells, and so forth,35−38 which may be attributed to the fact that the increased tumor microenvironment stiffness is essential for maintaining the biophysical phenotype of the tumor, altering cell morphology, upregulating focal adhesion, enhancing cell contractility, and increasing invasion. It is also reported that not only the biophysical factors but also the biochemical factors of the tumor microenvironment can regulate tumor progress and tumor cell metastasis. In the present study, a biochemical factor in the tumor microenvironment, TGF-β1, was observed for its role in affecting MDA-MB231 migration. It was found that TGF-β1 enhanced the migration of MDA-MB-231 in all cases of substrates stiffness. TGF-β1 is a multifunctional cytokine with pleiotropic cellular functions, including regulating cell fate and inducing angiogenesis, epithelial to mesenchymal transition (EMT), and cancer cell migration.8,10,12,39,40 TGF-β1 can regulate cancer cell migration through various pathways such as the TGFSMAD signaling pathway and non-SMAD-related signaling pathway according to previous research.41 For instance, TGFβ1 can bind to type I and II serine/threonine kinase receptors (TβRI/II), induce the phosphorylation of Smad 2 and Smad 3, and then regulate the expression of target genes to further trigger motility and invasiveness of aggressive human prostate cancer cells;39 on the other hand, TGF-β1 can upregulate the expression of cyclin D1 and reorganize actin to mediate cell migration in MDA-MB-231.42 In the present study, the experimental results that TGF-β1 coupled with substrate stiffness enhance MDA-MB-231 migration could also be ascribed to these mechanisms, but it is beyond the focus of this research. In the current work, we observed that the migration of MDAMB-231 cultured on stiffer substrates and treated with TGF-β1 was more persistent as reflected by their higher β value (as shown in Figure 3B). It is well-known that the exponent β reflects cell migration strategy for a power law exponent β with the value of 1 reflecting the behavior of random Brownian G

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ACS Biomaterials Science & Engineering because our data also showed that F-actin stress fiber structure was dramatically remodeled and disrupted after blebbistatin treatment, and the cells with inhibited myosin IIA function displayed larger lamellipodia. Together, these will regulate actin turnover and control lamellipodial protrusion, which will cause increased cell migration.

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CONCLUSIONS In conclusion, our findings suggest through in vitro evidence that MDA-MB-231 traction force and cytoskeleton structure were involved in substrate stiffness and TGF-β1-enhanced MDA-MB-231 migration. Our data demonstrate that cell traction force and cytoskeleton remodeling was enhanced in response to TGF-β1 treatment to strengthen the migration of MDA-MB-231 and that blocking myosin IIA-mediated contraction using blebbistatin decreased TGF-β1-enhanced traction force but further increased MDA-MB-231 migration enhanced by substrate stiffness and TGF-β1. These results together suggest that both biophysical (like stiffness) and biochemical (like TGF-β1) factors could orthogonally regulate cancer cell migration, possibly revealing potential biomechanical mechanisms of the effect of substrate stiffness and TGF-β1 on contributing to breast cancer cell migration, which thus may help find novel clinical therapeutics to target the tumor microenvironment to treat tumors.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00835. Would healing rates, migration behaviors, traction forces, and the effects of stubstrate stiffness and TGF-β1 on FA structure (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Feng Lin: 0000-0003-4540-9208 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the support of the National Natural Science Foundation of China (NSFC) under Grants 11472013, 11772006, and 11772004.

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REFERENCES

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DOI: 10.1021/acsbiomaterials.7b00835 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX