The authors thank Tom Kodger, Michael Landry, and Christopher J. Barrett for assistance with bead synthesis. A.J.E. acknowledges Natural Sciences and Engineering Research Council grants RGPIN/05843-2014 and EQPEQ/472339-2015, Canadian Institutes of Health Research grant no. 143327, Canadian Cancer Society grant no. 703930, and Canadian Foundation for Innovation Project #32749. R. Krishnan acknowledges National Institutes of Health grant no. R21HL123522 and R01HL136209. H.Y. was supported by Fonds de recherche Sant&#233; Qu&#233;bec, and Fonds de recherche Nature et Technologies Qu&#233;bec. The authors thank Johanan Idicula for assistance with the video and manuscript and Zixin He for assistance in preparing the video.

NOTE: The following protocol will guide researchers in fabricating and using the multi-well TFM dish shown in Figure 1.
1. Preparation of PDMS silicone substrates
<ol>
	<li>Preparation of PDMS silicone rubber mixture based on a composite mixture of two commercially available kits.
	<ol>
		<li>Add Part A and Part B of PDMS kit (e.g., GEL-8100, see the Table of Materials) in a 1:1 weight ratio into the 50 mL tube. 
		NOTE: The mixture is mixed on a rotator at a speed slow enough for the mixture to flow back and forth during revolution to ensure complete mixing.</li>
		<li>Add the required amount of curing agent for the desired modulus of the substrate. 
		NOTE: The amount of curing agent to be added to the mixture depends on the desired modulus of the substrate and may typically range from 0.1% to 1.8%. Refer to Table 1 and Figure 3 for a guide to specific crosslinker concentrations and resulting moduli.</li>
		<li>Mix the formulation on the rotator for 30-45 min; ensure the rotation is slow enough for thorough mixing.</li>
	</ol>
	</li>
	<li>Bottom layer: coating PDMS substrates on the glass slide
	<ol>
		<li>Place the custom-built chuck illustrated in Figure 2 on the spin-coater. Clean the glass with ethanol or isopropanol, and dry with a lint-free wipe. Place the glass slide in the chuck and turn on the vacuum to hold the slide in place.</li>
		<li>Apply uncured PDMS approximately 1 cm from the edges and work in towards the center. Apply enough (3-4 mL) PDMS to ensure the whole surface will be covered. 
		NOTE: To ensure that the PDMS is evenly spread on the surface of the glass, a pipette tip may be helpful to spread the PDMS mixture from the center to the edges.</li>
		<li>Spin the glass with the PDMS mixture on a spin-coater with the following protocol.
		<ol>
			<li>To spread the uncured PDMS on the slide, accelerate at 50 rpm/s from 0 to 200 rpm; hold at 200 rpm for 1 min.</li>
			<li>To achieve a 100 &#181;m thick PDMS layer, accelerate at 50 rpm/s to 300 rpm and hold for 1 min at 300 rpm. Different desired thicknesses other than 100 &#181;m will require specific speed values.</li>
			<li>To remove, decelerate at 50 rpm/s to 0 rpm. Disable vacuum and remove the coated slide, taking care not to touch the coated surface. 
			NOTE: It is important to include the acceleration and deceleration steps to ensure a smooth continuous surface. 
			CAUTION: To ensure that the sample does not fly off the chuck, the custom-made holder should be used to hold the slide, and not rely simply on vacuum and the existing flat chuck. The details and specifications of this holder are given in Figure 2.</li>
		</ol>
		</li>
		<li>Place the spin-coated sample in the oven at the manufacturer recommended temperature (100 &#176;C) for 2 h. 
		NOTE: The surface of the oven where the sample is placed should be solid (i.e., not a wire rack) and level surface to ensure the uniform heating and thickness of the sample. A ceramic or steel plate makes an ideal surface.</li>
	</ol>
	</li>
	<li>Top bead layer
	<ol>
		<li>Add bead solution in the appropriate ratio to the remaining PDMS mixture. 
		NOTE: This ratio depends on the concentration of the stock bead solution and the desired bead density on the sample. Typical final values are 9.2 x 1011 beads/mL and 0.05-0.2 beads/&#181;m2, and an excess of beads is generally preferable to an inadequate amount.</li>
		<li>Mix the bead solution with the uncured PDMS. This may be accomplished by placing the tube on a rotator for approximately 30 min, vortexing for 1-2 min, or sonication for 30 min. These methods may be combined. 
		NOTE: In our application, we find that sonication is effective in breaking bead aggregates, and rotation is effective in mixing. Synthesized fiduciary beads may aggregate in storage. Prior to use, they may be resuspended in hexane and sonicated. If there are significant large aggregates, one can filter the bead suspension through a 5 &#181;m syringe filter. This filtration step is optional; it helps to coat the sample with monodispersed beads, but a significant fraction of beads may be lost in the filter.</li>
		<li>Take out the slide from the oven, allow to cool to room temperature, and place it on the spin-coater.</li>
		<li>Add 3-4 mL of the bead and uncured PDMS mixture onto the surface of the coated sample. 
		NOTE: The mixture with beads added is less viscous due to the hexane. Make sure not to touch the surface of the substrate as it may damage the already-coated PDMS substrate. Additionally, the bead mixture may initially not wet the surface; take care that the mixture does not immediately flow off the cured PDMS surface.</li>
		<li>Spin the sample with the following protocol.
		<ol>
			<li>To spread the bead and uncured PDMS mixture, accelerate at 100 rpm/s from 0 to 500 rpm; hold at 500 rpm for 1 min.</li>
			<li>To achieve a thin layer of bead-embedded PDMS (~1 &#181;m), accelerate at 200 rpm/s from 500 to 5000 rpm; hold at 5000 rpm for 10 s.</li>
			<li>To remove, decelerate at 100 rpm/s to 0 rpm. Disable vacuum and remove coated slide, taking care not to touch the coated surface.</li>
		</ol>
		</li>
		<li>Place the spin-coated sample in the oven at 100 &#176;C for 1 h. 
		NOTE: Temperatures above 100 &#176;C or durations longer than 1 h can reduce the bead fluorescence. Make sure that the oven temperature is set to 100 &#176;C and not higher.</li>
		<li>The protocol can be paused here. To store the sample, cover the surface to avoid dust and light exposure. Make sure that nothing touches the surface. The sample is shelf-stable at room temperature indefinitely.</li>
	</ol>
	</li>
	<li>Assembling the plate
	<ol>
		<li>Add Part A (base) and Part B (curing agent, e.g., Sylgard) of a PDMS elastomer kit in a 10:1 weight ratio into the 50 mL tube.</li>
		<li>Mix the mixture on the rotator for 30-45 min. 
		NOTE: For one plate, mix 5 mL of base with 0.5 mL of curing agent. Up to 1 mL of hexane can be added to reduce the viscosity of the mixture.</li>
		<li>Apply the mixture to the bottom of the divider and spread the mixture. 
		NOTE: The divider should be placed upside-down.</li>
		<li>Lay the substrate onto the divider upside-down.</li>
		<li>Place the sample upside-down in the oven at 65 &#176;C for 2 h.</li>
		<li>Take out samples from the oven and clean the bottom of the glass with 70% ethanol or isopropanol to remove any PDMS residue. 
		NOTE: The protocol can be paused here. To store the sample, place a lid on the substrate and wrap the device in aluminum foil to avoid exposure to the light. The sample is shelf-stable at room temperature indefinitely. The divider utilized in this method is in a 96-well format; however, researchers may employ other formats (384-well, 2-well, 4-well, 8-well, etc.) depending on desired experiment setups and availability of dividing structures. Some further optimization may be required.</li>
	</ol>
	</li>
</ol>
2. Surface functionalization
<ol>
	<li>Dissolve 80 &#181;L of a Sulfo-SANPAH aliquot in 40 mL of 0.1 M HEPES buffer (pH 7-9). 
	NOTE: Prepare Sulfo-SANPAH aliquots by dissolving 100 mg of Sulfo-SANPAH powder in 2 mL of sterile dimethyl sulfoxide (DMSO). Prepare 0.1 M HEPES buffer by diluting 50 mL of HEPES in 450 mL of sterile deionized water and filter through a 0.22 &#181;m pore filter.</li>
	<li>Add 200 &#181;L of diluted Sulfo-SANPAH solution to each well of the 96-well plate.</li>
	<li>Expose the plate to UV (300-460nm) light at appropriate distance and duration. 
	NOTE: After UV exposure, the color of the solution should be darker. UV exposure distance and duration depend on the UV lamp power. In our application, we expose for 10-15 min at a distance of 5 cm.</li>
	<li>Remove the Sulfo-SANPAH solution from the wells and add 200 &#181;L of 5 &#181;g/mL fibronectin solution to each well. 
	NOTE: Researcher-specified protein can be used for surface coating. Some commonly used proteins are collagen, fibronectin, and laminin. We have found Sulfo-SANPAH to be the most effective method, and plasma cleaning while sometimes employing in PDMS is discouraged as it creates a silicon dioxide layer and visibly damages the surface.</li>
	<li>Incubate the plate at 4 &#176;C overnight. 
	NOTE: Different incubation methods can be applied depending on the protein used for coating.</li>
	<li>Remove the fibronectin solution and wash each well with phosphate-buffered saline (PBS)&#160;twice.</li>
	<li>Place a lid on the sample.</li>
	<li>Add 200 &#181;L of PBS to each well. 
	NOTE: The protocol can be paused here. The fibronectin-coated samples can be stored at 4 &#176;C for up to 2 weeks.</li>
</ol>
3. UV sterilization
<ol>
	<li>Sterilize the sample under UV light in a biological safety cabinet for 30 min. 
	NOTE: Longer UV exposure times may negatively impact the bead fluorescence. All subsequent steps must be performed under sterile conditions.</li>
</ol>
4. Cell culture
<ol>
	<li>Remove PBS from each well and add 200 &#181;L of cells suspended in culture media to each well.
	<ol>
		<li>Plate the cells at the desired cell density. Cell density depends on the desired experiment. For single cell studies, cells should be minimum of 50 &#181;m apart and cells near the edges of the imaging window should be not be included in the TFM measurement. For monolayer cells, the imaging window should have the viewing field covered with a confluent layer of cells.</li>
		<li>Prepare the complete growth culture media for NMuMG cells by supplementing DMEM with 5% FBS, 10 mM HEPES, 10 &#181;g/mL insulin, 1% penicillin-streptomycin, 1 mM L-glutamine, and 0.5 &#181;g/mL amphotericin B.</li>
		<li>Prepare insulin stock by reconstituting in acidified water (2.5 mL of glacial acetic acid in 130 mL of deionized water) to a concentration of 10 mg/mL. Store the stock solution at 4 &#176;C. Wait until the solution is clear, and then filter through a 0.22 &#181;m pore filter.</li>
	</ol>
	</li>
	<li>TGF-&#946; addition
	<ol>
		<li>To prepare of TGF-&#946; stock, dissolve 2 &#181;g of TGF-&#946; in 100 &#181;L of 10 mM citric acid (pH 3.0) and filter sterilize with 0.22 &#181;m pores. Vortex the tube and aliquot into the desired volumes. Store the aliquots at -80 &#176;C.</li>
		<li>Add 1.5 &#181;L of TGF-&#946; stock solution to 10 mL of the complete cell culture media to constitute the cell culture media with the final TGF-&#946; concentration of 3 ng/mL. 
		NOTE: To make 10 mM pH 3.0 citric acid, dilute the acid in water and adjust pH to 3.0 by adding HCl.</li>
	</ol>
	</li>
</ol>
5. Data acquisition
<ol>
	<li>For each position, acquire at least one image of fiduciary particles and cells. Focus on the bead layer. 
	NOTE: Pixel size should be optimized based on size of the fiduciary particles and image processing method being used. In this application, the authors use a 10x 0.4 NA objective, and images are acquired with 1024 x 1024 resolution, with 455 nm/pixel. In general, it is helpful to retain a resolution of at least approximately 1-5 pixels per bead; here, beads are polydisperse and have an individual size of 300-500 nm. It is critical that the fluorescent fiduciary beads be in focus for images to be used for TFM calculations. The focus and imaging quality of the beads should be prioritized over imaging the cells themselves. There should be no cross-talk between different channels, particularly any fluorescence not from the fiduciary beads which appears in the imaging spectra of the beads.</li>
	<li>Once all the positions of interest have been recorded, add detachment solution to each well to acquire force-free reference images of the fiduciary particles.
	<ol>
		<li>To prepare cell detachment solution, mix an aqueous solution containing 2% TritonX-100, 50 mM sodium azide, and 500 mM potassium hydroxide. 
		NOTE: The above is provided as an example of an effective detachment solution. Different detachment solutions at researchers&#39; discretion may be used to detach the cells off the substrate surface.</li>
	</ol>
	</li>
</ol>
6. Image analysis
<ol>
	<li>Perform appropriate image analysis as desired. 
	NOTE: Analysis software was developed in-house. Image analysis may be done with custom-made software or software available online.</li>
</ol>
7. Bead synthesis
NOTE: The following protocol is based on the synthesis method described by Klein et al.10.
<ol>
	<li>Under a fume hood, prepare the three-neck flask with a water-cooled reflux condenser. 
	CAUTION: Set up a synthesis in a well-ventilated chemical fume hood.</li>
	<li>Add 0.5 mL of PDMS stabilizer and fluorophore to the flask.</li>
	<li>Equip one neck with a rubber septum with a nitrogen inlet needle and an outlet needle and equip the other neck with a rubber septum for adding reagent with a syringe.</li>
	<li>Add 100 mL of anhydrous hexane in 250 mL to the flask and add a small magnetic stir bar.</li>
	<li>Place the flask in the mineral oil bath at 75 &#176;C and purge it with nitrogen gas for 1 h.</li>
	<li>Add 6 mL of methylmethacrylate to a 25 mL round bottom flask.</li>
	<li>Add 0.100 g of 2,2&#39;-azobisisobutyronitrile (AIBN) to the round bottom flask and purge the mixture with nitrogen gas for 1 h. 
	NOTE: Flush methylamethacrylate through prepacked column to remove inhibitors before use. Add methylamethacrylate and AIBN mixture to the three-neck flask.</li>
	<li>The solution initially becomes cloudy and turns milky. Let the reaction run for 3 h after the solution becomes cloudy.</li>
	<li>After 3 h, place the flask in an ice water bath.</li>
	<li>Vacuum-filter the solution with coarse filter paper.</li>
	<li>Centrifuge the filtrate and re-suspend the particles in hexane. 
	NOTE: The volume of the hexane to be added depends on the desired concentration of the bead solution. Sonication facilitates re-dispersion of the bead particles in hexane solution. Beads produced by the authors have polydisperse diameters of approximately 300-500nm. Due to the use of cross-correlation pattern tracking to determine displacements, monodisperse beads are not required.</li>
</ol>
8. Rheology measurement protocol
NOTE: Rheology is not required for every researcher or experiment, but is necessary to quantify the moduli for new formulations of PDMS. In this protocol, we employ a shear rheometer to measure the effects of crosslinker, frequency, and strain on moduli of PDMS samples. Depending on the available tools and expertise, moduli may also be measured using many other mechanical analysis approaches. Additionally, researchers using this protocol may elect to use our published moduli presented in Table 1, Figure 3 and Figure 4.
<ol>
	<li>Use a rheometer with a 25 mm diameter parallel plate geometry. Other geometries may be used.</li>
	<li>Initialize the system and calibrate the device and measuring system (parallel plate, d = 25 mm). After measuring the zero gap, begin loading the PDMS sample.</li>
	<li>As soon as the PDMS elastomer and crosslinking agent have been mixed, pipet the mixture onto the bottom plate of rheometer.
	<ol>
		<li>Move the spindle down to completely contact the top of the PDMS sample.</li>
		<li>Carefully trim the loaded sample excess from the bottom plate.</li>
	</ol>
	</li>
	<li>In a strain sweep test, apply increasing strains for each composition with different crosslinking density to ensure the polymer structure remains in the linear viscoelastic regime during all shear measurements. 
	NOTE: Strain values relevant for cell studies are typically in the range of 0.1-10%. We have found PDMS to be linear up to approximately 100% strain.</li>
	<li>Measure the dynamic shear storage modulus (G&#39;), and loss modulus (G&#8242;&#8242;) of the PDMS network in a time sweep test with frequency of 1 Hz and oscillatory shear strain of 0.5% at 100 &#176;C.</li>
	<li>To determine the viscoelasticity and time dependency of the final PDMS network, apply a frequency sweep test with frequency ranging 0.1-100 Hz and oscillatory shear strain of 0.5%.</li>
</ol>

<ol>
	<li>Harris, A., Wild, P., Stopak, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicone+rubber+substrata:+a+new+wrinkle+in+the+study+of+cell+locomotion.">Silicone rubber substrata: a new wrinkle in the study of cell locomotion.</a> Science. 208 (4440), 177-179 (1980).</li><li>Oliver, T., Dembo, M., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+forces+in+locomoting+cells.">Traction forces in locomoting cells.</a> Cell Motil Cytoskeleton. 31 (3), 225-240 (1995).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+Cell-to-Substrate+Interface+during+Locomotion+of+Fibroblasts.">Stresses at the Cell-to-Substrate Interface during Locomotion of Fibroblasts.</a> Biophysical Journal. 76 (4), 2307-2316 (1999).</li><li>Yoshie, H., Koushki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+Force+Screening+Enabled+by+Compliant+PDMS+Elastomers.">Traction Force Screening Enabled by Compliant PDMS Elastomers.</a> Biophysical Journal. 114 (9), 2194-2199 (2018).</li><li>Park, C. Y., Zhou, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+modulators+of+cellular+contractile+force.">High-throughput screening for modulators of cellular contractile force.</a> Integrative biology quantitative biosciences from nano to macro. 7 (10), 1318-1324 (2015).</li><li>Kraning-Rush, C. M., Califano, J. P., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+increase+with+increasing+metastatic+potential.">Cellular traction stresses increase with increasing metastatic potential.</a> PLoS ONE. 7 (2), e32572 (2012).</li><li>Agus, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+sciences+network+characterization+of+non-tumorigenic+and+metastatic+cells.">A physical sciences network characterization of non-tumorigenic and metastatic cells.</a> Scientific Reports. 3 (1), 1449 (2013).</li><li>Guo, M., Ehrlicher, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Stochastic,+Motor-Driven+Properties+of+the+Cytoplasm+Using+Force+Spectrum+Microscopy.">Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy.</a> Cell. 158 (4), 822-832 (2014).</li><li>Ngan, E., Northey, J. J., Brown, C. M., Ursini-Siegel, J., Siegel, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+containing+LPP+and+alpha-actinin+mediates+TGFbeta-induced+migration+and+invasion+of+ErbB2-expressing+breast+cancer+cells.">A complex containing LPP and alpha-actinin mediates TGFbeta-induced migration and invasion of ErbB2-expressing breast cancer cells.</a> Journal of Cell Science. 126 (Pt 9), 1981-1991 (2013).</li><li>Klein, S. M., Manoharan, V. N., Pine, D. J., Lange, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+monodisperse+PMMA+microspheres+in+nonpolar+solvents+by+dispersion+polymerization+with+a+macromonomeric+stabilizer.">Preparation of monodisperse PMMA microspheres in nonpolar solvents by dispersion polymerization with a macromonomeric stabilizer.</a> Colloid &amp; Polymer Science. 282 (1), 7-13 (2003).</li></ol>

AJE and RK have interest in Live Cell Technologies, a company which fabricates materials described in this article.

For the success of this method, it is critical to have a uniformly coated sample with a constant thickness of approximately 100 &#181;m. The modulus should be carefully chosen to examine the physical significance of the biological system of interest. When fabricating a top layer, the concentration of the fiducial fluorescent particles should be optimized for accurate analysis of displacement and traction stress. Analyzing isolated single cells requires a denser fiduciary layer than measuring confluent monolayers. Additionally, the surface of the substrate should have stable and uniform coating of adhesion molecules such as collagen, fibronectin, and laminin to ensure proper attachment of the cells to the substrate surface. Particular care must be taken when attaching the multi-well divider; it should be placed without sliding to prevent sealing PDMS glue from being smeared on the culture surface, and the outer edge must be carefully aligned with the glass dimensions to prevent any leaks on the border wells.
To prevent wells from being leaky, an adequate amount of PDMS glue needs to be applied to the well-divider to hold it in place. However, use of excessive amounts will cover cell culture surface.
Sufficient volume of PDMS needs to be added during the spin-coating procedure. When curing, the oven and racks need to be level. Inadequate PDMS or an unleveled oven may lead to uneven PDMS thickness.
Bead density needs to be optimized for proper analysis. When the bead density is not adequate, concentration of the beads in fiduciary PDMS mixture needs to be increased. Additionally, an adequate amount of the mixture needs to be added for spin coating. To ensure proper fluorescent signals of the fiduciary bead particles, curing condition should be carefully monitored. Longer times and higher temperatures than the ones specified in this protocol may degrade the fluorescence. Improper protein coating on the surface of the sample may lead to poor cell adhesion. To prevent this, the surface needs to be cleaned and the expiration of reagents such as Sulfo-SANPAH and ligands needs to be checked. UV exposure time and strength should be optimized. Immunofluorescence or other fluorescent protein construct may be tested to ensure uniform ligand coating.
The device fabricated with this method and set of PDMS formulations is only applicable to substrates with Young&#39;s moduli ranging from 0.4 kPa to 100 kPa. Other moduli may be possible using alternative formulations, however, the current range spans a large set of physiologically relevant values. While this method is specifically for multi-well fabrication, it may also be used to prepare individual slides or other slide geometries, and in these instances further user troubleshooting may be necessary. In this embodiment, a relatively thick (1 mm) glass slide is employed, precluding common high numerical aperture (NA) objectives on an inverted microscope. While it is technically feasible to construct these multi-well TFM dishes using thinner glass, the fragility of these in processing and assembly may result in a high rate of breakage, and can run contrary to the higher-throughput approach. Furthermore, surface coating may be investigated further for wider applications of the device.
Utilizing a multi-well format, high-throughput experiments are possible. Multi-well format enabled us to examine the physical changes of NMuMG cells during EMT with TGF-&#946; treatment with combinations of different concentrations and different incubation times in a single dish. Fabrication of traction stress devices with hydrogels such as polyacrylamide gel have several limitations. With the dominant ingredient being water, hydrogels are sensitive to salt concentrations (osmolarity), temperature, humidity, and pH changes. Changes in any of these properties can lead to the change in mechanical properties of the material, requiring extra care in using samples and interpreting results. Furthermore, having water, hydrogels are more susceptible to infections, requiring extra care. In contrast to water-based hydrogels, silicone-based PDMS is stable and inert. Once fabricated, it can be stored at room temperature indefinitely without requiring any special handling or storage&#160;conditions.
The impermeable nature of PDMS allows us to fabricate a monolithic substrate, ensuring that all wells are truly identical in substrate thickness and composition, while allowing unique biochemistry to be applied in the media, or different cells to be utilized in each well. A hydrogel-based surface allows diffusive factors to permeate and travel through it, necessitating adding the hydrogel to wells that are otherwise partitioned to prevent cross-contamination.
This method allows researchers to study cell contractility, a basic and ubiquitous aspect of cell biology, in a high-throughput manner, enabling more efficient and reproducible data acquisition. This helps to translate Traction Force Microscopy into Contractile Force Screening, allowing researchers to truly use contractility as a metric for cell activity, or efficacy of a compound. As a methodology, this has broad applications across biophysical and biomedical sciences, from understanding the physics of cells, to characterizing and testing pharmaceutical compounds against standardized cell types, or perhaps highly specialized individual cells for personalized medicine.

Acto-myosin contractility is an essential element of active cell mechanics, impacting cell behaviors from motility and proliferation to stem cell differentiation. In tissues, contractility drives activity from polar separation in embryogenesis, to airway constriction and cardiac activity. Critically, to generate tension, cells must first adhere to their extracellular environment. In doing so, this contractility generates traction forces on their surroundings. Traction Force Microscopy (TFM) has emerged in a multitude of forms as a way to quantify these forces from diverse cells under different conditions.
The field of TFM has seen an exceptional breadth of innovation and application, and the results have paved the way for new perspectives in biology, which incorporate mechanics and physical forces. Starting with wrinkling silicone substrates1, researchers have applied various techniques to measure cell traction forces. These approaches have been continuously improved and have now reached a level of resolution on the order of several microns2. However, one principal problem has emerged, which is the difficulty in creating substrates of suitably low moduli using the available silicones. To circumvent this problem, polyacrylamide was adopted as a replacement due to the ease of creating substrates on the order of 1-20 kPa3. We recently implemented very compliant silicones in TFM4, allowing us to fabricate the same range of moduli as polyacrylamide, but with the advantages of inert and robust silicone.
TFM approaches have enabled valuable mechano-biological discoveries, however, a persistent shortcoming is their complexity, often restricting their use to researchers in the engineering or physical sciences disciplines. This is due&#160;in large part&#160;to the detailed calibrations and challenging calculations that are required to quantify contractility. Another significant challenge is that TFM methods are largely low-throughput and therefore ill-suited to study many different conditions or populations simultaneously5. This has presented a bottleneck, which has hampered transfer of TFM from a specialist biophysics setting into broader biological and pharmacological&#160;applications.
We have recently developed a multi-well format TFM plate, which allows researchers to parallelize their TFM measurements for faster quantification of contractility metrics, while exploring the impact of different compounds and also using less reagents4. This methodology has broad utility in diverse mechanobiology studies, from evaluating the effects of compounds on cellular activity, to quantifying the contractile changes in differentiation or disease.
One area of biomedical research that will benefit greatly from TFM is the study of how physical cues impact the malignant phenotypes of cancer cells. Metastasis, responsible for 90% of cancer-related deaths, is characterized by cancerous cells leaving their original tumor site and colonizing a secondary site. For cells to migrate through tissue and pass in and out of the vascular system, they must radically change their shapes to squeeze through these physical barriers while generating substantial forces to pull their way along extracellular matrix or move between other cells. These forces are transmitted to the substrate through focal adhesion interactions2,3, and can be quantified using TFM. While cancers are biochemically exceptionally diverse, with an expanding repertoire of known mutations and protein changes, some common physical changes have been observed; in a variety of cancers, including breast, prostate, and lung cancers, metastatic cells have been shown to exert 2-3 times the traction forces of non-metastatic cells6,7,8. These results suggest that there may be a strong correlation between metastatic progression and the traction forces exerted by cells; however, the detailed time-dependent changes in contractility are difficult to examine.
The epithelial-to-mesenchymal transition (EMT) is a process whereby cells reduce adherens- and tight-junction mediated cell-cell adhesion, becoming more migratory and invasive. In addition to physiological functions that include wound healing and developmental processes, EMT is also a process exploited during metastasis, making it a useful model system to study this process. Using TGF-&#946;, we can induce the EMT in&#160;murine mammary epithelial cells (NMuMG)9 to directly quantify the physical changes during this transformation, and characterize the time and dose-dependent effects of TGF-&#946; on EMT and cell contractility. In this article, we demonstrate the utility of this approach by measuring the changes in contractility during an induced EMT.

<table><tbody><tr><td>&lt;strong&gt;Plate&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GEL-8100</td><td>Nusil Technology</td><td>GEL-8100</td><td>High Purity Dielectric, Soft Silicone Gel kit</td></tr><tr><td>Dow Corning Sylgard 184 Silicone Encapsulant Clear 0.5 kg Kit</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td>curing agent</td></tr><tr><td>Custom Cut Glass&amp;nbsp;</td><td>Hausser Scientific Company</td><td></td><td>109.6mm&amp;plusmn; x 72.8mm&amp;plusmn; x 1mm thickness</td></tr><tr><td>Target 2TM Nylon Syringe Filter</td><td>ThermoFisher Scientific</td><td>F2513-4</td><td></td></tr><tr><td>96-well Stripwell Egg Crate Strip Holder</td><td>Corning</td><td>2572</td><td></td></tr><tr><td>Polystyrene Universal Microplate Lid With Corner Notch&amp;nbsp;</td><td>Corning</td><td>3099</td><td></td></tr><tr><td>Ethyl alcohol</td><td>Greenfield Global</td><td>P016EA95</td><td>0.95</td></tr><tr><td>2-Propanol</td><td>Sigma-Aldrich</td><td>190764</td><td>ACS reagent, &lt;font class=&quot;font5&quot;&gt;&amp;ge;99.5%&lt;/font&gt;</td></tr><tr><td>&lt;strong&gt;Surface Coating&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sulfo-SANPAH Crosslinker</td><td>Proteochem</td><td>c1111-100mg</td><td></td></tr><tr><td>Fibronectin bovine plasma</td><td>Sigma-Aldrich</td><td>F1141-1MG</td><td>solution, sterile-filtered, BioReagent, suitable for cell culture</td></tr><tr><td>PBS, 1X</td><td>Wisent</td><td>319-005-CL</td><td>pH 7.4, without calcium and magnesium</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>472301</td><td></td></tr><tr><td>&lt;strong&gt;Cell Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM, 1X</td><td>Wisent</td><td>319-005-CL</td><td>4.5g/L glucose, with L-glutamine, sodium pyruvate and phenol red</td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Wisent</td><td>080-150</td><td>Premium Quality,&amp;nbsp;Endotoxin &amp;lt;1, Hemoglobin &amp;lt;25</td></tr><tr><td>HEPES</td><td>Wisent</td><td>330-050-EL</td><td>1M, free acid</td></tr><tr><td>Human Insulin Recombinant</td><td>Wisent</td><td>511-016-CM</td><td>USP grade</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Wisent</td><td>450-201-EL</td><td>100 X, sterile filtered for cell culture</td></tr><tr><td>L-Glutamine solution</td><td>Wisent</td><td>609-065-EL</td><td>200mM solution, sterile filtered for cell culture</td></tr><tr><td>Amphotericine B</td><td>Wisent</td><td>450-105-QL</td><td>250&amp;mu;g/ml, sterile filtered for cell culture</td></tr><tr><td>Recombinant Human TGF-&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>HEK293 Derived</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>537020</td><td>Glacial, &amp;ge;99.85%</td></tr><tr><td>Cictric acid</td><td>Sigma-Aldrich</td><td>251275&amp;nbsp;</td><td>ACS reagent, &amp;ge;99.5%</td></tr><tr><td>NMuMG</td><td>ATCC</td><td>CRL-1636</td><td>Mouse Mammary Gland Cell Line</td></tr><tr><td>Sodium azide</td><td>Fisher Schientific</td><td>AC190385000</td><td>99%, extra pure, ACROS Organics</td></tr><tr><td>Potassium hydroxide</td><td>Sigma-Aldrich</td><td>221473</td><td>ACS reagent, &amp;ge;85%, pellets</td></tr><tr><td>TritonX-100</td><td>Sigma-Aldrich</td><td>X100</td><td>laboratory grade</td></tr><tr><td>&lt;strong&gt;Bead Synthesis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1,1&amp;prime;-Dioctadecyl-3,3,3&amp;prime;,3&amp;prime;-tetramethylindocarbocyanine perchlorate (DiI)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>468495-100MG</td><td>97%</td></tr><tr><td>Methyl methacrylate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>M55909-500ML</td><td>contains &amp;le;30&amp;nbsp;ppm MEHQ as inhibitor, 99%</td></tr><tr><td>Inhibitor Remover</td><td>Sigma-Aldrich&amp;nbsp;</td><td>306312-1EA</td><td>Prepacked column for removing hydroquinone and monomethyl ether hydroquinone</td></tr><tr><td>Methacryloxylpropyl Terminated Polydimethylsiloxane</td><td>Gelest&amp;nbsp;</td><td>DMS-R31 (25,000g/mol)</td><td>Polydimethylsiloxane stabilizer, 25,000g/mol, 1,000 cSt</td></tr><tr><td>2,2&amp;prime;-Azobis(2-methylpropionitrile) (AIBN)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>441090-25G</td><td>98%</td></tr><tr><td>Hexane&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>296090-2L</td><td>anhydrous, 95%</td></tr><tr><td>Hexane, mixture of isomers</td><td>Sigma-Aldrich&amp;nbsp;</td><td>227064-1L</td><td>anhydrous, &amp;ge;99%</td></tr><tr><td>Whatman&amp;nbsp;qualitative filter paper, Grade 1</td><td>Sigma-Aldrich&amp;nbsp;</td><td>WHA1001055</td><td>circles, diam. 55&amp;nbsp;mm,</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Laurell WS-650Mz-23NPPB</td><td>Laurell Technologies</td><td></td><td></td></tr><tr><td>UVP Handheld UV Lamp Model UVGL-58</td><td>VWR</td><td>21474-622</td><td></td></tr><tr><td>Rheometer</td><td>Anton Paar</td><td>MCR 302 WESP</td><td></td></tr></tbody></table>

high throughput traction force microscopy using pdms reveals dose-dependent effects of transforming growth factor-&#946; on the epithelial-to-mesenchymal transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

Before addition of TGF-&#946;, a confluent monolayer of cells has a cobblestone like shape and is tightly packed. Upon TGF-&#946; treatment, cells become more elongated in morphology, enlarging the cell area and acquiring a more mesenchymal phenotype. Utilizing the multi-well device fabricated with soft PDMS elastomers, the physical properties of cells in a total 17 different conditions were studied. The cells were treated with four different TGF-&#946; concentrations (0.5, 1, 2, and 4 ng/mL) and four different incubation times (12, 24, 48, 96 h), and these results are summarized in Figure 5. The cells treated with TGF-&#946; applied larger traction stresses and strain energies than the cells cultured without TGF-&#946;. Cells incubated with TGF-&#946; for 96 h showed the largest traction stresses and strain energies. The cells applied larger stresses and strain energies when treated with higher concentration of TGF-&#946;. The difference in tractions and strain energies were more distinct at longer incubation times.
The surface of the substrate needs to be smooth and uniformly coated with ligands, such as fibronectin or collagen. Scratched surface and/or non-uniform coating of ligands may lead to improper cell attachment, resulting in inaccurate traction measurement. Figure 6 shows the localized traction stresses due to the non-uniform substrate surface.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59364/59364fig1.jpg" /> 
Figure 1: Overview of multi-well plate fabrication. (A) A custom-cut glass slide is the starting point. (B) The glass slide is coated with a thick (~100 &#181;m layer of PDMS). (C) A layer of fiduciary beads (shown in green) are then spin-coated in a ~1 &#181;m thick layer on top of the previous layer. (D) The multi-well divider is carefully placed on top of the fiduciary bead layer. (E) The complete multi-well plate is assembled and ready for use or storage. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59364/59364fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59364/59364fig2.jpg" /> 
Figure 2: Custom chuck for spin-coater. To prevent the bottom glass (where PDMS is coated) from flying off of the standard spin-coater chuck, a custom-made holder is placed onto the chuck. (A) A custom-made holder for the spin-coater chuck. (B) Engineering drawing of the holder with all the dimensions. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59364/59364fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59364/59364fig3.jpg" /> 
Figure 3: Substrate stiffness with changing crosslinker concentration (wt%). PDMS mixtures are prepared as described, with the additional crosslinker percentage increasing the elastic modulus until a max of 100 kPa at 1.8 weight percent (wt%) crosslinker. As a guide, the modulus as a function of crosslinker is fit linearly, which can be used by researchers to formulate their own mixture at a particular desired modulus within this range. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59364/59364fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59364/59364fig4.jpg" /> 
Figure 4: Rheology of PDMS elastomer containing 0, 0.15, 0.36 and 1 wt% of crosslink agents at a temperature of 100 &#176;C. Triangle symbols indicate loss modulus (G&#39;&#39;) and square symbols indicate storage modulus (G&#39;). (A) Oscillatory time sweep at frequency of 1 Hz and shear strain of 0.5% during gelation. (B) Oscillatory frequency sweep of PDMS network at shear strain of 0.5%. (C) Strain sweep of PDMS network at frequency of 1 Hz. All data points were acquired in triplicate. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59364/59364fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59364/59364fig5.jpg" /> 
Figure 5:&#160;Representative results utilizing traction stress device with multi-well format. Monolayer of cells in different conditions were cultured in a multi-well device to measure contractility and strain energy. (A)&#160;Graph of traction stresses with increasing TGF-&#946; incubation time for different TGF-&#946; concentrations.Traction stresses increased with increasing TGF-&#946; concentrations and incubation time. All data are statistically significant with respect to control (i.e., no TGF-&#946;) except the following: 12 h - 0.5 ng, 12 h - 1 ng, 48 h - 1 ng. (B) Graph of strain energy with increasing TGF-&#946; incubation time for different TGF-&#946; concentrations. Strain energies increased with increasing TGF-&#946; concentrations and incubation time. Sample sizes range from n = 7 to n = 15. All data are statistically significant with respect to control (i.e., no TGF- &#946;) except the following: 12 h - 0.5 ng, 12 h - 1 ng, 24 h - 0.5 ng, 24 h - 1 ng, 48 h - 1 ng. Statistical significance was determined using the Kruskal Wallis test, which does not assume a normal distribution. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59364/59364fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59364/59364fig6.jpg" /> 
Figure 6: Representative results from a sub-optimal (A, B) and a satisfactory (C, D) experiment. (A)&#160;Reflection image of the substrate surface. Undesired contaminants, which can include dust or fibers, or material defects such as scratches are present on the substrate surface.(B)Stress map of cell contractility: due to the non-uniform surface, irregular and inaccurate traction stresses are observed around the objects.(C)Reflection image of the substrate surface. No apparent contaminants are visible.(D)Stress map of cell contractility: no artifact discontinuities are visible. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59364/59364fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Crosslinker concentration (wt%)&#160;</td>
			<td>G&#39; (Pa)</td>
			<td>Standard Deviation</td>
			<td>E (kPa)</td>
			<td>Standard Deviation</td>
		</tr>
		<tr>
			<td>0</td>
			<td>0.135</td>
			<td>0.014</td>
			<td>0.405</td>
			<td>0.043</td>
		</tr>
		<tr>
			<td>0.15</td>
			<td>1</td>
			<td>0.1</td>
			<td>3</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>0.36</td>
			<td>4.027</td>
			<td>0.245</td>
			<td>12.081</td>
			<td>0.73</td>
		</tr>
		<tr>
			<td>0.75</td>
			<td>16.01</td>
			<td>0.49</td>
			<td>48.03</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>1</td>
			<td>18.44</td>
			<td>0.989</td>
			<td>55.32</td>
			<td>1.94</td>
		</tr>
		<tr>
			<td>1.5</td>
			<td>27.638</td>
			<td>0.93</td>
			<td>82.91</td>
			<td>1.64</td>
		</tr>
		<tr>
			<td>1.8</td>
			<td>33.986</td>
			<td>0.88</td>
			<td>101.94</td>
			<td>1.088</td>
		</tr>
		<tr>
			<td>2</td>
			<td>33.36</td>
			<td>0.67</td>
			<td>100.08</td>
			<td>1.1</td>
		</tr>
	</tbody>
</table>
Table 1:Substrate stiffness with changing crosslinker concentration (wt%). By changing additional crosslinker concentrations, PDMS substrates of the desired Young&#39;s moduli are fabricated. PDMS shear moduli were measured with a rheometer and Young&#39;s moduli were determined. For each data point, 3 independent preparations were done and the standard deviation is given.

Watch this Scientific Journal Video about High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-Î² on the Epithelial-to-Mesenchymal Transi

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

high-throughput-traction-force-microscopy-using-pdms-reveals-dose

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.4K Views.  McGill University. We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-β dependent increase in contractility during the epithelial-to-mesenchymal transition.

Video: High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicine

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Developmental Biology

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Cancer Research

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

Biochemistry

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Biology

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition

Summary

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Chapters in this video

Spheroid Assay to Measure TGF-β-induced Invasion

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Induction and Analysis of Epithelial to Mesenchymal Transition

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling

TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing