Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.
Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user’s specific biological and technical needs.
The rigidity of the tumor-associated ECM has been identified as a significant factor in driving malignant behavior by increasing actomyosin contractility1-3. While this effect has primarily been demonstrated with breast cancer cells, matrix rigidity has been found to alter invasive properties of cells derived from a variety of cancers4-8 suggesting that tumor rigidity may play a role in other type of cancers. To penetrate cross-linked tissues during invasive migration, cancer cells utilize actin-rich adhesive protrusions known as invadopodia that localize proteinases to focally degrade the ECM9. Invadopodia are considered a hallmark of invasive cells and have been implicated in tumor cell invasion and metastasis10,11. Previous work has shown that matrix rigidity can regulate invadopodia numbers and associated ECM degradation4,12 through myosin II activity and mechanosensitive proteins12. Given the correlation between tumor density and cancer aggressiveness13,14, these results suggest a mechanism by which cancer cells may respond to rigid tumor tissues to drive invasion and metastasis through actomyosin contractility.
In vitro ECM rigidity and in vivo tissue density have been shown to regulate invasive behavior of cancer cells1,15-17. While actomyosin contractility appears to be important in this process, current studies conflict as to whether metastatic capacity is correlated to increased or decreased contractile forces6,18-20. Furthermore, it remains unknown whether these forces directly mediate invadopodia activity21. We recently found that cancer cell contractile forces were dependent on matrix rigidity and were predictive of ECM degradation by invadopodia5. These results suggest that cellular forces may play an important role in cancer progression by mediating invadopodia activity in response to the mechanical properties of the tumor microenvironment.
In order to correlate invasive and contractile properties of cancer cells5, we modified a protocol for creating PAAs with different rigidities that was previously used to investigate rigidity-dependent invadopodia activity4,12,22. By chemically crosslinking human plasma fibronectin throughout the PAAs, these modified hydrogels can be used as the basis for both invadopodia and traction force assays to ensure that cells experienced the same rigidities in both experiments5. In the invadopodia assays, the fibronectin provides a natural binding domain for gelatin to link the overlaid ECM to the PAAs to detect matrix degradation. In the traction force assays, the fibronectin provides a ligand for direct cellular adhesion to detect microsphere displacements used to calculate cellular traction forces. This method results in what we have called soft, hard, and rigid PAAs that are bound to glass bottom dishes and have elastic moduli, E, of 1,023, 7,307, and 22,692 Pa5 which span the range of mechanical properties reported for normal and cancerous tissues23.
1. Preparation of Glass Coverslips for PAAs
2. Preparation of PAAs for Invadopodia Assays
PAA (1 ml) |
40% AA (μl) |
2% BIS (μl) |
Ultrapure Water (μl) |
1 mg/ml FN (μl) |
10 mg/ml NHS Ester (μl) |
100 mg/ml APS (μl) |
TEMED (μl) |
Elastic Modulus (Pa) |
Soft | 200 | 25 | 574 | 200 | 1 | 5 | 2 | 1023 |
Hard | 200 | 175 | 409 | 215 | 1 | 5 | 2 | 7307 |
Rigid | 300 | 300 | 169 | 230 | 1 | 5 | 2 | 22692 |
Table 1: Ingredient volumes and resulting elastic moduli for soft, hard, and rigid PAAs used in the invadopodia assays. AA = acrylamide, FN = fibronectin, APS = ammonium persulfate.
3. Preparation of PAAs for Traction Force Assays
PAA (1 ml) |
40% AA (μl) |
2% BIS (μl) |
Ultrapure Water (μl) |
1 mg/ml FN (μl) |
Micro-spheres (μl) |
10 mg/ml NHS Ester (μl) |
100 mg/ml APS (μl) |
TEMED (μl) |
Elastic Modulus (Pa) |
Soft | 200 | 25 | 566 | 200 | 8 | 1 | 5 | 2 | 1023 |
Hard | 200 | 175 | 401 | 215 | 8 | 1 | 5 | 2 | 7307 |
Rigid | 300 | 300 | 161 | 230 | 8 | 1 | 5 | 2 | 22692 |
Table 2: Ingredient volumes and resulting elastic moduli for soft, hard, and rigid PAAs used in the traction force assays. AA = acrylamide, FN = fibronectin, APS = ammonium persulfate.
4. Preparation of ECM for Invadopodia Assays
5. Preparation and Imaging of Invadopodia Assays
6. Preparation and Imaging of Traction Force Assays
In the invadopodia assay, invadopodia are typically identified by colocalization of markers like actin and cortactin at punctate structures within the cell body (Figure 1). Both actively degrading and non-degrading invadopodia can be counted and are differentiated by whether these structures are colocalized with black areas lacking fluorescent signal in the FITC-labeled fibronectin (Figure 1). Invadopodia are manually counted, and ECM degradation per cell is determined by manually thresholding these black areas within outlines of the cells.
In the traction force assay, four images are captured at each cellular location marked by stage position (Figure 2). First, “phase” and “stressed” images are taken of all of the cells of interest. A “bottom” image of the PAA is also taken at each position in order to calculate hydrogel thickness. After removing the cells, a “null” image is taken at each marked stage position. Deformations in the PAAs are calculated based on the change in microsphere positions between the “stressed” and “null” images. These displacements and the mechanical properties of the PAAs (E and Poisson’s ratio assumed as 0.5) are then used to calculate traction forces (Figure 2). Several different methods exist for calculating traction forces24 based on some formulation of the Boussinesq solution for an infinite elastic half space25. While the details of these analyses are beyond the scope of this text, we have licensed LIBTRC software from Micah Dembo at Boston University which uses a previously described method for calculating traction forces26. This particular computational method compensates for finite thicknesses in its calculations which requires the thickness of the PAAs at each position which is calculated based on the difference in the z-position of the “stressed” and “bottom” images. Many research groups have similar computer packages available or choose to write their own programs. In addition, other methods exist for calculating traction forces based on different mathematical approaches24.
Figure 1: The invadopodia assay can be used to identify invadopodia and associated ECM degradation. Example wide-field fluorescence images of invadopodia in a SCC-61 (head and neck squamous cell carcinoma) cell on a hard PAA with 1% gelatin and FITC-labeled fibronectin. Invadopodia are typically identified by colocalization of two markers such as actin and cortactin (red and blue in the overlay image, respectively). Invadopodia can be quantitated as both actively degrading (colocalized with black areas lacking FITC signal as denoted by yellow arrows) and non-degrading (denoted by white arrows). Please click here to view a larger version of this figure.
Figure 2: The traction force assay can be used to determine cellular traction forces generated by the actin cytoskeleton by tracking the displacement of embedded microspheres in the PAAs. Example wide-field phase and fluorescence images of a SCC-61 cell on a hard PAA (“phase”), and the microspheres directly under the cell at the top surface of the PAA (“stressed”). An image of the bottom of the PAA can also be taken to later calculate its local thickness (“bottom”). After the cell is removed, an image is once again taken of the microspheres at the top surface of the PAA (“null”). Microsphere positions between the “stressed” and “null” images can be tracked to yield a “displacement field.” Microsphere displacements and PAA mechanical properties can then be used to calculate a “traction vector field.” Please click here to view a larger version of this figure.
We present a method for fabricating PAAs that can be used as the basis for invadopodia and traction force assays to correlate invasive and contractile cellular behaviors. While PAAs have long been used to look at rigidity effects on cells and calculate traction forces18,24,27, this protocol is the first to develop parallel assays based on PAAs with the same rigidities to correlate invasive and contractile cellular behaviors in response to matrix mechanical properties. Properly activating the coverslips of the glass bottom dishes ensures that the PAAs will bind to them and not come off. While we and others have relied on reagents such as sulfo-SANPAH and acrylic acid NHS ester to bind gelatin to the surface of the PAAs in the past4,12,28, we found that these methods did not produce reliably uniform surface layers of fibronectin. Therefore, fibronectin was embedded and crosslinked throughout the PAAs using acrylic acid NHS ester as performed by other groups29,30. However, increasing concentrations of fibronectin were required in order to yield the same ligand density at the surfaces of the soft, hard, and rigid PAAs5. Additionally, incorporation of fibronectin (but not the microspheres) reduced the mechanical properties of the soft, hard, and rigid PAAs from 1,071, 9,299, and 28,283 Pa4 to 1,023, 7,307, and 22,692 Pa5, respectively. However, these reduced elastic moduli values still encompassed the range of normal and cancerous tissues23. Storage moduli of the PAAs can easily be measured by rheometry and converted to elastic moduli1,4.
While the protocol is straight forward, lowering and removing the 12 mm coverslips are the most difficult steps and require practice. The biggest challenge when lowering a coverslip is to ensure that the PAA solution spreads out evenly without any bubbles or splashing. Removing a coverslip requires a steady hand in order to not damage the 12 mm coverslip, the glass bottom 14 mm coverslip, or the PAA. We have tried hydrophobic coatings on the 12 mm coverslip to aid in removal but found that the PAAs are left with patterns in their surfaces. The use of fine tip and thin, paddle-style tweezers for lowering and removing the 12 mm coverslips, respectively, is highly recommended. In addition, the amount of light used to image the cells must be minimized since some cell types are sensitive to concentrated light on the microscope. Also, L-15 medium was used for the traction force assays since our environmental chamber on our microscope is not equipped for CO2. While the same supplements were used in the media for the invadopodia and traction force assays in an attempt to keep conditions the same, DMEM and RPMI 1640 could be used instead of L-15 if CO2 levels can be controlled.
While the volume of PAA solution was chosen to theoretically yield gels with a thickness of 75 µm, their thicknesses typically vary between 30-60 µm. However, this range is well above the value at which cells can sense the underlying rigidity of the coverslips of the glass bottom dishes31. In addition, the thickness of the ECM layer used to detect degradation (gelatin and FITC-labeled fibronectin overlaid on to the PAAs) has been previously reported as approximately 1 µm in thickness12; therefore, this thin layer does not shield the cells from the rigidities of the PAAs. However, solid debris, cracks, and other deformities in either the PAAs and/or ECM layer can affect individual cellular invasive and contractile properties. These problems can be caused by unclean 12 mm coverslips that introduce debris into the hydrogels, excessive drying of the gels and/or gelatin, and incomplete aspiration of sodium borohydride which can leave bubbles that deform the ECM layer. Therefore, care must be taken when selecting cells for imaging to ensure that they are not unduly influenced by local irregularities in the surfaces of the samples.
Relatively high concentrations of fibronectin in both the PAAs (mixed in at 200-230 µg/ml) and the ECM layer (overlaid on the gelatin at 50 µg/ml) have been used; however, the actual concentrations on either surface have not been directly measured. While each surface appears to be saturated with fibronectin, it is unclear whether cells experience the same exact ligand densities between the two assays. For the traction force assays, 200 nm microspheres at a dilution of 1:125 have proven optimal for imaging. However, it is quite common to find other groups using microspheres of different diameters or dilutions. In this system, smaller microspheres displayed Brownian motion within the PAAs, while larger microspheres caused cracks and deformities. These observations are most likely due to the physical properties of the PAAs (i.e., pore size, degree of crosslinking, etc.) that result from the specific amounts of acrylamide and BIS that were used. The microsphere dilution provides excellent resolution for tracking displacements in the PAAs, particularly since cancer cells exert relatively small forces. For improved optical resolution, particularly if using even smaller microspheres, high NA water or oil immersion objectives can be used.
Overall, many of these experimental factors can be adjusted based on the user’s requirements such as PAAs with different mechanical properties, cancer cells with varying invasive and contractile properties, and microscopy systems with other imaging capabilities. The ratio of acrylamide:BIS can be varied to produce PAAs with similar or different elastic moduli and crosslinking27. The sensitivity of the traction force assays can be adjusted to account for different cellular force levels by changing the rigidity and/or microsphere dilution. Different fluorophores can also be chosen for immunofluorescence and fibronectin labeling based on microscope specifications. While FITC does bleach, it is quite bright making ECM degradation easily identifiable. However, fluorescence imaging of invadopodia and small microspheres typically requires high NA objectives for optimal resolution. In addition, both assays can be performed on glass coverslips in well plates. However, glass bottom dishes are much easier to prepare and use throughout the experimental process. In the future, combining these assays into one would allow for direct visualization of both ECM degradation and cellular force generation. However, several technical challenges would arise including live cell imaging for invadopodia and microbead displacements, increased cellular exposure to light, bleaching of the FITC fibronectin, and whether traction forces are completely transduced through the fluorescently labeled and cross-linked ECM layer to the PAA surfaces.
The authors have nothing to disclose.
The authors have nothing to disclose.
3-Aminopropyltrimethoxysilane | Sigma-Aldrich | 281778 | |
Acrylamide (40%) | Bio-Rad | 161-0140 | |
Acrylic acid NHS ester | Sigma-Aldrich | A8060 | prepare fresh in fume hood 10 mg/ml in DMSO |
Alexa Fluor 546 phalloidin | Life Technologies | A22283 | can also use rhodamine |
Ammonium persulfate | Bio-Rad | 161-0700 | prepare fresh 10% solution in 1X PBS |
Aqua Poly/Mount | Polysciences | 18606 | use six drops to fill microwells |
BIS (2%) | Bio-Rad | 161-0142 | |
Bovine serum albumin | RPI | A30075 | make 3% for blocking solution in 1X PBS and store in 4 °C |
Coverslips (12 mm) | Fisher Scientific | 12-545-80 | |
dialysis tubing | Sigma-Aldrich | D9777 | pre-equilibrate in borate buffer for 15-30 min |
DMEM | Cellgro | 10-013-CV | use to make invadopodia medium |
DMSO | Sigma-Aldrich | D8418 | use to make acrylic acid NHS ester solution |
Epidermal growth factor | Life Technologies | PHG0311 | use to make invadopodia medium |
Ethanol | PHARMCO-AAPER | E200 | dilute with ultrapure water to 70% |
FBS | Thermo Scientific | SH30070.03 | use to make invadopodia medium |
FITC | Sigma-Aldrich | F7250 | protect from light |
Gelatin | Polysciences | 00639 | typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 °C (preheat PBS to dissolve gelatin easily) |
Glass bottom dishes (35 mm coverslips) | MatTek | P35G-0-14-C | coverslips are uncoated |
Glutaraldehyde (25%) | Polysciences | 01909 | dilute with 1X PBS to 0.5% |
goat anti-mouse Alexa Fluor 633 antibody | Life Technologies | A21050 | |
Human plasma fibronectin | Life Technologies | 33016-015 | add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles |
KH2PO4 | EMD Millipore | PX-1565-1 | use to make 10X PBS stock |
mouse anti-cortactin 4F11 antibody | EMD Millipore | 05-180 | |
Na2HPO4 | EMD Millipore | SX-0720-1 | use to make 10X PBS stock |
NaCl | RPI | S23020 | use to make 10X PBS stock and borate buffer |
NaOH (1 N) | Sigma-Aldrich | S2770 | dilute with ultrapure water to 0.1 N |
Nu-Serum (low-protein serum) | BD Biosciences | 355500 | use to make invadopodia medium |
Paraformaldehyde | Acros | 416785000 | typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl) |
PBS (sterile) | Cellgro | 21-040-CV | use for cell culture |
RPMI 1640 | Cellgro | 10-040-CV | use to make invadopodia medium |
Sodium borohydride | Sigma-Aldrich | 452882 | prepare fresh in fume hood 1 mg/ml in 1X PBS |
sodium metaborate tetrahydrate | Sigma-Aldrich | S0251 | use to make borate buffer |
Sucrose | RPI | S24060 | typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 °C (preheat PBS to dissolve gelatin easily) |
TEMED | Bio-Rad | 161-0800 | |
Triton X-100 | Alfa Aesar | A16046 | make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining |