Cell culture and preparation
All cell lines described below were purchased and used for the study. MDA-MB-231 cells (ATCC HTB-26), an invasive human breast adenocarcinoma cell line, were cultured in Dulbecco’s modified Eagle medium (DMEM) with 2 mM L-glutamine (# 11965–092, Thermo Fisher, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, # 35–010-CV, Thermo Fisher), 100 units/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL fungizone, and 5 μg/mL gentamicin (# 15750–060, Invitrogen, Carlsbad, CA) at 5% CO2 and 37 °C. 4 T1 cells (ATCC CRL-2539, a mouse breast cancer cell line) purchased from BeNa Culture Collection Corporation (# BNCC273810, Beijing, China) were cultured in RPMI-1640 (Gibco-Invitrogen, Carlsbad, CA) supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% FBS. For matrix degradation and invadopodia experiments, cells were incubated in invadopodia medium containing DMEM supplement with 5% Nu-Serum (# 355104, Corning, NY), 10% FBS, and 20 ng/mL EGF.
For labeling actin in live cells, stable cell lines expressing Lifeact-RFP were generated via lentiviral transfection. The lentiviral transfer plasmids pLVX-puro-GFP-Lifeact and pLVX-puro-RFP-Lifeact were cloned from RFP-Lifeact plasmid obtained from Dr. Gaudenz Danuser (UT-Southwestern). Briefly, lentiviruses were produced by transfecting human embryonic kidney 293 T cells (ATCC CRL-3216) with psPAX2 and pMD2.G (Addgene) and pLVX-puro-GFP-Lifeact viral vectors. Conditioned medium containing viruses were collected after 5 days and then used immediately to infect cells or stored at − 80 °C. Transduced target cells were selected with puromycin for 72 h.
For optical imaging of dynamic calcium signaling and caveolae localization in live cells, cell lines transiently expressing G-GECO (a green fluorescent genetically encoded calcium indicator) and caveolin-1 (Cav-1)-EGFP respectively were generated via plasmid transfection. The plasmids expressing G-GECO were a generous gift from Takanari Inoue (Johns Hopkins University) [60], and those expressing Cav-1-EGFP were from Ari Helenius (ETH Zurich). Briefly, cells were transfected with Lipofectamine-2000 (# 11668–019, Life Technologies, Carlsbad, CA). For 35 mm glass-bottom dishes, 6 μg plasmid DNA in OptiMEM transfection medium (# 31985062, Gibco, Waltham, MA) was used for each transfection. After 24 h at 37 °C, the transfection medium was replaced with a complete medium, and cells were processed 24–48 h later.
Drug treatments
For experiments involving inhibitors, cells were exposed to the inhibitor for 0.5 h, unless stated otherwise, in the presence or absence of compression. For inhibiting the function of mechanically sensitive ion channels, cells were treated with gadolinium chloride (Gd3+, 5 μM, # 203289, Sigma) or GsMTx4 (5 μM, #ab141871; Abcam, Cambridge, MA). To remove calcium ions from the DMEM, EGTA (2 mM, # E3889; Sigma) was added to the medium. To disrupt caveolae in the membrane, cells were treated with 5 mM of methyl-β-cyclodextrin (MβCD, # SLBP3372V, Sigma). To evaluate the impact of HIF-1a, cells were treated with inhibitor CAY10585 (10 μM, # ab144422, Abcam). For inhibiting the activity of Src, cells were treated with PP2 (10 μM, Calbiotech, Spring Valley, CA). For inhibiting the activity of MMP, cells were treated with GM-6001 (a broad-spectrum MMP inhibitor, 15 μM, #CC1000; Sigma).
Antibodies for immunofluorescence and Western blot
Antibodies used in immunofluorescence and Western blot include: anti-Tks5 polyclonal antibody (# 09–403-MI) and anti-GAPDH mouse monoclonal antibody (# CB1001) purchased from EMD Millipore (Billerica, MA); anti-Src rabbit antibody (# 2108), anti-p-Src (Y416) rabbit antibody (# 2101), anti-p44/42 MAPK (ERK1/2) mouse monoclonal antibody (# 4696), and anti-p-ERK1/2 (Thr202Tyr204) rabbit monoclonal antibody (# 4370) obtained from Cell Signaling Technology (Danvers, MA), respectively; anti-cortactin rabbit monoclonal antibody (# Ab81208) purchased from Abcam; anti-Piezo1 rabbit polyclonal antibody (# PA5–72974) and anti-Cav-1 rabbit polyclonal antibody (# PA1–064) obtained from Thermo Fisher.
In vitro compression device
To investigate the effect of compression on cell behaviors, we used a previously described setup [9, 61]. Briefly, cells were grown either in a 35 mm culture dish with a glass bottom (# 12–565-90, Thermo Fisher, Waltham, MA) that was coated with/without gelatin, or in a transwell chamber with a permeable membrane of 8-μm pores that were coated with Matrigel. Then the cells were covered with a 1% soft agarose disk layer, and subsequently, a piston of specific weight was placed on top of the agarose disk to apply given compression to the cells underneath indirectly. The cross-sectional area of the piston (24 mm diameter) was 4.52 cm2 but its weight was variable at 9.22 g, 18.45 g, and 27.67 g, corresponding to a stress of 200 Pa, 400 Pa, and 600 Pa, respectively, on the cells. Cells prepared as such but not subjected to piston weight were used as control (Ctr). It needs to note that even cells in the control groups were also exposed to 1% agarose, a constant atmosphere pressure, and culture medium.
RNA interference
To silence the expression of Piezo1 and Cav-1, Negative Control Medium GC Duplex #2 and siRNA interference for Piezo1 (# AM16708, Assay ID:138387, Thermo Fisher) and Cav-1 (# AM16708, Assay ID: 10297, Thermo Fisher) were used. Briefly, cells were seeded in 6-well plates at 1 × 106 cells/well for 24 h before transfection. At 90% confluence, the cells were transfected with 30 nmol/L siRNA using Lipofectamine RNAi MAX (# 13778, Invitrogen) in OptiMEM according to the manufacturer’s instructions. Transfection mixes were applied to the cells for 24 h, subsequently removed and replaced with 2 mL of growth media. The cells were cultured for 48 h before use in experiments. The protein expression levels of Piezo1 and Cav-1 were ascertained by Western blot.
In vitro transwell invasion assay
To assay the effect of compression on cell invasion, standard transwell invasion assay adapted from Bravo-Cordero [62, 63] was performed using 6-well Transwell chambers that were separated as upper and lower chambers by filter membrane with 8 μm pores (# 07–200-169, Corning). For the assay, the transwell filter membrane was coated with 300 μl Matrigel (12 mg/mL, # E1270, Sigma, Burlington, MA) diluted in serum-free DMEM (2 mg/mL final concentration), followed by incubation for 1 h at 37 °C. MDA-MB-231 cells in serum-free medium (5 × 105 cells/well) were placed in the upper chamber, while the lower chamber was filled with 2 mL complete medium. Cells were allowed to grow for 6 h and then compressed for 18 h before being fixed with 4% paraformaldehyde (# 30525–89-4, Electron Microscopy Sciences, Hatfield, PA). The non-invasive cells on the upper chamber were removed with cotton swabs, and the invaded cells in the lower chamber were stained with 0.1% crystal violet (# C6158; Sigma) for 10 min at room temperature, before being examined and imaged by light microscopy at 10X magnification (Olympus BX60; Olympus Corporation, Tokyo, Japan). Then the number of stained cells was counted using ImageJ software (National Institute of Health, Bethesda, MD) and the enhancement of cellular invasion induced by compression was quantified as a percentage (%) of the number of compressed cells over that of the non-compressed cells that had invaded through the filter membrane, i.e. [# of cells in the lower chamber in the presence of a specific weight (experiment group)]/[# of cells in the lower chamber in the absence of a specific weight (control group)]. Results are based on the analysis of 10 random fields per transwell in each condition and each experiment was repeated three times.
Live fluorescence microscopy
To observe the dynamics of actin, Cav-1, and calcium signaling, live cells expressing Lifeact-RFP, Cav-1-EGFP, and G-GECO were imaged with a spinning disk confocal microscope with a 60X or 100X oil immersion objective (Olympus IX73 with Yokogawa CSU-X1). For live fluorescence microscopy, cells were seeded in a 35 mm glass-bottom dish that was placed in an environmental chamber mounted on the microscope to maintain constant 37 °C, 5% CO2, and humidity. Cav-1-EGFP was observed at the excitation wavelength of 488 nm. For dynamic tracking of actin in live cells, the cells were consecutively imaged for up to 60 min, and the images were processed using ImageJ. Cells were observed from both top-down and side view for spatial localization of actin, and caveolae by 3D reconstruction of images in Z-stacks (0.4 μm increments).
Cell height and nuclear area assay
Cell height and nuclear area can be used to indicate the effect of compression on cells. MDA-MB-231 cells transduced with Lifeact-RFP were plated in glass-bottom dishes at a density of 2 × 105 cells/mL and cultured for 24 h at 37 °C and 5% CO2. At 24 h, the cells were incubated with Hoechst 33342 in PBS (1: 2000) for 20 min. 1% agarose disks were UV-treated, incubated in media for 1 h at 37 °C, and then placed on top of the cells. Weights were applied to achieve 200 Pa, 400 Pa, and 600 Pa. For the condition of a control group, an agarose disk was applied without any weight. The agarose disks allow nutrient diffusion and sit in between the weight and the cells. Fluorescence live-cell imaging was performed using a spinning disk confocal microscope. Hoechst and Lifeact-RFP were excited at wavelengths of 405 nm and 561 nm, respectively. Image stacks were taken at 30 min intervals for 2 h. Cell height and nuclear area were quantified by the side-view profiles of Lifeact-RFP images and the top-view profiles of Hoechst 33342 images, respectively, using ImageJ.
Cell proliferation assay
MDA-MB-231 cells were plated in transwell cell culture inserts at a density of 2 × 105 cells/mL and cultured for 24 h at 37 °C and 5% CO2. The cells were then transfected with scramble siRNA or Piezo1 siRNA using Lipofectamine 3000 and cultured for another 24 h. Pre-incubated agarose disks were placed on top of the cells, and weights were applied on top of the agarose disks. After 24 h, the weights and the agarose disks were removed. The media was collected in labeled centrifuge tubes. The cells were detached using 0.05% trypsin, transferred to the corresponding tubes, and spun down at 1000×g for 5 min. The cells were then resuspended in 1 mL of fresh media. 50 μL of cell suspension, 55 μL of DMEM, and 5 μL of WST-8 solution were added to each well in a 96-well plate, mixed gently on an orbital shaker, and incubated for 2 h at 37 °C and 5% CO2. The absorbance of the samples at a wavelength of 450 nm was measured using a plate reader.
Cell migration assay
To assay the effect of compression on cell migration, standard wound healing assays were performed using 6-well Transwell chambers that were separated as upper and lower chambers by a filter membrane with 0.4 μm pores (# 07–200-148, Corning). For the assay, MDA-MB-231 cells (1 × 106 cells/well) were placed in the upper chamber, while the lower chamber was filled with 2 mL complete medium. Cells were allowed to grow for 24 h to achieve a confluent monolayer. An experimental wound was made using a sterile micropipette tip, then the cells were washed 3 times with sterile PBS and compressed for 24 h. Wound areas were observed and recorded at 24 h by using a Nikon TiE Perfect Focus System microscope equipped with an 10X objective, an sCMOS camera (Flash 4.0, Hamamatsu Photonics, Japan), and a laser launch controlled by an acousto-optical tunable filter (AOTF). The experimental wound area was quantified manually using “Area measurement” in ImageJ software and normalized to the wound area at the start of the experiment, and the ratio of cell migration was defined by the ratio of the wound healing area of compression-treated groups to that of control groups. Results are based on the analysis of 3 random fields per transwell in each condition and each experiment was repeated three times.
Evaluation of invadopodia formation and ECM degradation
To determine whether compression enhances cells’ ability to degrade ECM, we examined cells cultured on gelatin substrate for their tendency to form invadopodia and associated gelatin degradation, according to a protocol adapted from Artym et al. [64]. Briefly, glass-bottom dishes were treated with 20% nitric acid for 1 h, washed with H2O for 4 times, then incubated with 50 μg/mL poly-L-lysine (# P8920, Sigma) in phosphate buffer solution (PBS) for 15 min and washed with PBS, then further incubated with 0.5% glutaraldehyde in PBS on ice for 15 min followed by thorough washes with PBS. Subsequently, the dishes were coated with 1 mL of gelatin in PBS (1:9 of 0.1% fluorescein isothiocyanate (FITC)-gelatin (# G13186, Invitrogen): 2% porcine gelatin), then washed in PBS, incubated with 5 mg/mL sodium borohydride (NaBH4) for 3 min, rinsed in PBS, and then incubated in 10% FBS/DMEM at 37° for 2 h. Afterward, MDA-MB-231 cells were seeded in each dish at 5 × 105 cells per well and incubated for 8 h, and then subjected to compression of either 200 Pa, 400 Pa, or 600 Pa, respectively, for 8 h as aforementioned.
Upon completion of compression, the cells were imaged with live fluorescence microscopy (60X) and the microscopic images were analyzed by using ImageJ to assess the formation of invadopodia and the degradation of gelatin matrix. Invadopodia were defined as F-actin-positive puncta protruding from the cells into the gelatin matrix underneath the cell in our experiments [65]. For each independent experiment that was performed in triplicates, the number of invadopodia per cell was quantified with cells imaged randomly in > 15 microscope view fields, representing a total of ~ 100 cells per experimental condition. At the same time, degradation of the gelatin matrix was quantified as the percentage of the degraded area (dark spots comprised of dense degraded protein products) in the whole area underneath each cell.
Intracellular Ca2+ measurement
To evaluate the intracellular calcium concentration ([Ca2+]), we used cells labeled with Fluo-4/AM (# F14201, Thermo Fisher) or transiently expressed with calcium-sensitive reporter G-GECO [66] and then evaluated the intensity of intracellular calcium signaling. For the Fluo-4/AM system, cells were incubated with Fluo-4/AM for 1 h at room temperature (25 ± 2 °C) followed by a 0.5 h wash at 37 °C. For G-GECO systems, cells transfected with G-GECO for 48 h were plated into a glass-bottom dish, which was further incubated for 24 h. Subsequently, the cells were imaged with the spinning disk confocal microscope (60X objective), with fluorescence excitation and emission at 488 nm and 533 nm, respectively. For each experimental group, twenty cells were randomly selected and the fluorescence intensity per cell was quantified using ImageJ.
Western blot
Western blot assay was used to examine the protein expression and/or activity of Piezo1, Cav-1, Src, and ERK in MDA-MB-231 cells after exposure to control groups or mechanical compression conditions for 4 h. Cells grown on glass-bottom dishes under described assay conditions were lysed using RIPA buffer (# R0278, Sigma) with an added cocktail of protease and phosphatase inhibitors (MS-SAFE, Sigma). The protein concentration of cell lysates was determined using the Protein Assay Reagent (#23227, Thermo Fisher). Cell lysis buffer was combined in 4× SDS sample buffer and 2-mercaptoethanol and incubated at 95 °C for 5 min. After loading an equal amount of protein per lane, SDS-PAGE was performed. The proteins were transferred onto 0.22 μm nitrocellulose membranes (# 66485, Pall Life Sciences) using Pierce G2 Fast Blotter (Thermo Fisher). Following the transfer, the membranes were cut before probing with antibodies to save antibodies. Membranes were first blocked using 5% nonfat milk in 1x TBST (Tris-buffered saline and 0.1% of Tween-20) for 1 h at RT with gentle agitation and incubated with the primary antibodies overnight at 4 °C under mild shaking condition. After washing three times with 1x TBST, membranes were incubated with goat anti-rabbit secondary antibody (DyLight 800, # SA5–10036, Thermo Fisher) or goat anti-mouse secondary antibody (DyLight 680, # 35518, Thermo Fisher) at RT for 1 h. Signals of immunoblots were detected using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). Images were cropped to only show the molecular weight regions that are informative for our proteins on interest and were grouped into panels for clearer presentation and easier comprehension. For quantification, the intensity of the gel band was calculated after subtracting the background. The relative protein expression was expressed as a ratio of the band intensity to that of the control group after both of them were normalized to that of GAPDH.
Immunofluorescence and colocalization analysis
Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% TritonX-100 for 10 min at room temperature. Non-specific sites were blocked using 5% non-fat milk in PBS for 1 h at room temperature. Cells were then incubated in 5% non-fat milk in PBS containing primary antibodies at 1:100 dilution for 1 h at room temperature. After washing with PBS, cells were incubated with Alexa Fluor 594 or 640 conjugated secondary antibody for 60 min at room temperature. Cells were visualized using the spinning disk confocal microscope with a 60X oil immersion objective. For F-actin staining, cells were incubated with 1:100 rhodamine-phalloidin (# PHDR1, Cytoskeleton Inc.) for 60 min at room temperature.
Colocalization of Piezo1 and Cav-1 was analyzed using Fiji software [67] containing a procedure for colocalization analysis, designated as Coloc2, which is based on pixel-intensity-correlation measurements. Pearson coefficient and 2D intensity histograms were recorded to quantify the degree of the colocalization between Piezo1 and Cav-1.
Statistical analysis
Statistical analysis was done using one-way analysis of variance (ANOVA), followed by post hoc student’s t test for multiple comparisons. Statistical significance set to *p < 0.05 and **p < 0.01. All experiments were repeated at least three times and the data expressed as means ± s.e.m. (standard error of the mean).