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....

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 cont....

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