Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Summary

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.

Abstract

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.

Introduction

The rigidity of the tumor-associated ECM has been identified as a significant factor in driving malignant behavior by increasing actomyosin contractility^1-3. 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 cancers^4-8 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 metastasis^10,11. Previous work has shown that matrix rigidity can regulate invadopodia numbers and associated ECM degradation^4,12 through myosin II activity and mechanosensitive proteins¹². Given the correlation between tumor density and cancer aggressiveness^13,14, 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 cells^1,15-17. 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 forces^6,18-20. 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 activity^4,12,22. 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.

Protocol

1. Preparation of Glass Coverslips for PAAs

1. Clean 12 mm coverslips with low lint wipes.
2. Flame the 12 mm coverslips and the 14 mm coverslip in the microwell of each 35 mm glass bottom dish by passing them through a Bunsen burner flame using tweezers.
3. Treat the microwells with 200 µl of 0.1 N NaOH for 5 min at room temperature.
4. Aspirate and air dry the microwells for 30 min.
5. Treat the microwells with 50-100 µl of 3-aminopropyltrimethoxysilane for 10 min at room temperature in the fume hood. This chemical reacts with plastic; therefore, use glass pipettes and do not fill the microwells completely to avoid contact with the dish plastic.
6. Wash the microwells with ultrapure water for approximately 10 min until the 3-aminopropyltrimethoxysilane becomes clear.
7. Rinse the microwells with ultrapure water twice using a squeeze bottle.
8. Wash the microwells with 2 ml of ultrapure water at room temperature on a rocker set at a medium speed (~1 Hz) for 10 min.
9. Aspirate and air dry the microwells for 30 min.
10. Treat the microwells with 2 ml of 0.5% glutaraldehyde solution at room temperature on a rocker set at a medium speed (~1 Hz) for 30 min.
11. Wash the microwells with 2 ml of ultrapure water at room temperature on a rocker set at medium speed (~1 Hz) for 10 min. Repeat two more times for a total of 30 min.
12. Dry microwells at a steep angle (60º or greater) for 30 min.
    Note: Dishes can be stored for 2 months in a dessicator.

2. Preparation of PAAs for Invadopodia Assays

1. For a 1 ml solution of the soft PAA (8% acrylamide and 0.05% BIS), combine 200 µl of 40% acrylamide, 25 µl of 2% BIS, and 574 µl of ultrapure water (Table 1).
2. For a 1 ml solution of the hard PAA (8% acrylamide and 0.35% BIS), combine 200 µl of 40% acrylamide, 175 µl of 2% BIS, and 409 µl of ultrapure water (Table 1).
3. For a 1 ml solution of the rigid PAA (12% acrylamide and 0.60% BIS), combine 300 µl of 40% acrylamide, 300 µl of 2% BIS, and 169 µl of ultrapure water (Table 1).
4. Degas the solutions for 15 min. Add 200, 215, and 230 µl of 1 mg/ml human plasma fibronectin in ultrapure water to the soft, hard, and rigid PAA solutions, respectively. For all solutions, add 1 µl of 10 mg/ml acrylic acid N-hydroxysuccinimide (NHS) ester, 5 µl of a 100 mg/ml ammonium persulfate, and 2 µl of N,N,N′,N′-tetramethylethylenediamine (TEMED; Table 1). Mix with gentle pipetting and avoid creating bubbles in solutions.

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.

5. Pipette 8.48 µl of the PAA solution on to the center of each microwell.
   Note: This volume theoretically yields a PAA of 75 µm in thickness.
6. Gently lower the flamed side of the 12 mm coverslip on to the droplet in the microwell using tweezers.
7. Allow the sandwiched PAA solution to polymerize for 15-30 min. Verify polymerization by checking the leftover solution.
8. Add 2 ml of 1x PBS to each dish and remove the 12 mm coverslips with tweezers.
   Note: 10x PBS is made in the laboratory and composed of 0.011 M KH₂PO₄, 1.54 M NaCl, and 0.056 M Na₂HPO₄.
9. Wash the microwells with 2 ml of 1x PBS at room temperature for 5 min. Repeat two more times for a total of 15 min.

3. Preparation of PAAs for Traction Force Assays

1. For a 1 ml solution of the soft PAA (8% acrylamide and 0.05% BIS), combine 200 µl of 40% acrylamide, 25 µl of 2% BIS, and 566 µl of ultrapure water (Table 2).
2. For a 1 ml solution of the hard PAA (8% acrylamide and 0.35% BIS), combine 200 µl of 40% acrylamide, 175 µl of 2% BIS, and 401 µl of ultrapure water (Table 2).
3. For a 1 ml solution of the rigid PAA (12% acrylamide and 0.60% BIS), combine 300 µl of 40% acrylamide, 300 µl of 2% BIS, and 161 µl of ultrapure water (Table 2).
4. Degas the solutions for 15 min. Add 200, 215, and 230 µl of 1 mg/ml human plasma fibronectin to the soft, hard, and rigid PAA solutions, respectively. Sonicate 8 µl of 200 nm red fluorescent microspheres (excitation/emission of 580/605 nm) for 30 sec. For each solution, add 8 µl of 200 nm red fluorescent microspheres, 1 µl of 10 mg/ml acrylic acid NHS ester, 5 µl of a 100 mg/ml ammonium persulfate, and 2 µl of TEMED (Table 2). Mix with gentle pipetting and avoid creating bubbles in solutions.

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.

5. Pipette 8.48 µl of the PAA solution on to the center of each microwell.
   Note: This volume theoretically yields a PAA of 75 µm in thickness.
6. Gently lower the flamed side of the 12 mm coverslip on to the droplet in the microwell using tweezers.
7. Allow the sandwiched PAA solution to polymerize for 15-30 min. Verify polymerization by checking leftover solution.
8. Add 2 ml of 1x PBS to each dish and remove the 12 mm coverslips with tweezers.
9. Wash the microwells with 2 ml of 1x PBS at room temperature for 5 min. Repeat two more times for a total of 15 min.

4. Preparation of ECM for Invadopodia Assays

1. Heat the gelatin solution (1% gelatin and 1% sucrose) to 37 °C.
2. Treat the PAAs with 150 µl of the gelatin solution for 1 min then carefully aspirate from the bottom of the microwells when holding the glass bottom dishes at a 45° angle.
3. Dry the remaining thin layer of gelatin on the PAAs in the microwells at a steep angle (60° or greater) to optimize drying for 60 min.
4. Treat the microwells with 2 ml of a chilled 0.5% glutaraldehyde solution on ice for 15 min followed by room temperature for 30 min.
5. Wash the microwells with 2 ml of a 1x PBS for 5 min. Repeat two more times for a total of 15 min.
6. Treat the microwells with 2 ml of a sodium borohydride solution for 1 min. Gently tap dishes on bench to remove bubbles that form on the surface of the gelatin.
7. Aspirate and wash the microwells with 2 ml of a 1x PBS for 5 min. Repeat two more times for a total of 15 min.
8. Dilute a FITC-labeled human plasma fibronectin solution to 50 µg/ml with a 1x PBS and centrifuge at 175,000 x g at 4 °C for 15 min. Either purchase commercially available labeled fibronectin or label it as follows:
   1. Dissolve 5 mg of fibronectin in 10 ml of borate buffer (170 mM sodium metaborate tetrahydrate and 40 mM NaCl) and place in dialysis tubing.
   2. Label the fibronectin by placing the tubing in 200 ml of borate buffer containing 6 mg of dissolved FITC at room temperature on a magnetic stirrer set at the lowest setting for 1.5 hr in the dark.
   3. Dialyze with 500 ml of a 1x PBS on a magnetic stirrer set at the lowest setting for 3 days in a dark cold room (4 °C) with two volume changes a day.
   4. Calculate FITC-labeled fibronectin concentration based on the optical density of diluted aliquots (1:200) at 280 nm and 493 nm as [OD₂₈₀ – (0.36 x OD₄₉₃)] / 1.4 times the dilution factor of 200 times 2 (which accounts for the glycerol concentrating the fibronectin in the next step) resulting in mg/ml units.
   5. Dialyze overnight in a 50% glycerol solution on a magnetic stirrer set at the lowest setting overnight in a dark cold room at 4 °C.)
9. Treat the microwells with 150 µl of the FITC-labeled fibronectin solution at room temperature for 60 min in the dark.
10. Carefully aspirate the FITC-labeled fibronectin solution from the bottom of the microwells when holding the glass bottom dishes at a 45° angle and then fill the glass bottom dishes with 70% ethanol solution at room temperature for 10 min in a dark cell culture hood for sterilization. Also fill glass bottom dish lids or wipe clean with low lint wipes soaked in 70% ethanol.
11. Wash glass bottom dishes by filling them with 1x PBS for 5 min. Repeat two more times with 2 ml of 1x PBS for a total of 15 min.

5. Preparation and Imaging of Invadopodia Assays

1. Add 25,000 cancer cells in 2 ml of invadopodia medium (1:1 DMEM and RPMI 1640 with 5% low-protein serum, 10% fetal bovine serum, and 20 ng/ml of freshly added epidermal growth factor) to the glass bottom dishes and incubate overnight.
2. Aspirate and treat the microwells with 2 ml of a 3.7% paraformaldehyde solution at room temperature in the dark for 20 min to fix the cells.
3. After two quick washes with 2 ml of 1x PBS, treat the microwells with 2 ml of a 0.1% Triton X-100 solution at room temperature in the dark for 5 min to permeabilize the cells.
4. After one quick wash with 2 ml of 1x PBS, treat the microwells with 3% bovine serum albumin blocking solution at room temperature in the dark for 60 min to block the cells.
5. Incubate the microwells with 150 µl of mouse anti-cortactin antibody (1:750) in blocking solution at room temperature in the dark for 60 min.
6. Wash microwells with 2 ml of 1x PBS for 5 min. Repeat two more times for a total of 15 min.
7. Incubate the microwells with 150 µl of goat anti-mouse 633 antibody (1:500) and 546 phalloidin (1:750) in blocking solution at room temperature in the dark for 60 min.
8. Wash microwells with 2 ml of 1x PBS for 5 min. Repeat two more times for a total of 15 min.
9. Add six drops of mounting medium to fill each microwell and cover with 22 x 22 coverslips.
10. Image cortactin and actin to identify invadopodia using excitation and emission filters of 632/647 nm and 556/570 nm, respectively, using a high NA, 40X oil immersion lens on a wide-field inverted microscope. Similarly, image fibronectin to identify ECM degradation using an excitation and emission filter of 492/518 nm.

6. Preparation and Imaging of Traction Force Assays

1. Add 15,000 cancer cells in 2 ml of invadopodia medium to the glass bottom dishes and incubate overnight.
2. Aspirate medium in the glass bottom dishes and replace with 2 ml of L-15 medium, with the same supplements (5% low-protein serum, 10% fetal bovine serum, and 20 ng/ml of freshly added epidermal growth factor), pre-warmed to 37 °C.
3. Incubate the glass bottom dishes in an environmental chamber on an inverted microscope pre-equilibrated to 37 °C with high humidity for 1 hr.
4. Take four sets of images using a high NA, 40X lens on a wide-field inverted microscope with an automated stage to mark cellular positions:
   1. Image cells on top of the PAAs using phase contrast (“phase” image).
   2. Image fluorescent microspheres using excitation and emission filters of 560/645 nm by focusing slightly under the cells until the first layer of microspheres at the top of the PAAs comes into focus (“stressed” image).
   3. In addition, focus to the bottom of the PAAs (using the microspheres as markers) and take an image (“bottom” image). Note: The purpose of this image is to record the z-position to get the actual PAA thickness at each position for traction force calculations as described in Representative Results.
   4. After these images have been acquired at multiple positions, gently mix in 220 µl of 10% Triton-X solution to remove cells. Image microspheres at the top of the PAAs at each position as previously described (“null” image).

Results

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 thresh...

Discussion

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 forces^18,24,27, 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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 6 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