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

Summary

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

Abstract

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 β, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor α. 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.

Introduction

Fibrosis is involved in the pathogenesis of various chronic progressive diseases, such as interstitial pulmonary disease, cardiac fibrosis, liver cirrhosis, terminal renal failure, systemic sclerosis, and autoimmune disease¹. Among interstitial lung diseases, idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease and shows poor prognosis. Pathological hallmark of IPF is the development of fibroblastic foci consisting of activated fibroblasts and myofibroblasts that are associated with the prognosis. The origins of such pulmonary fibroblasts are proposed to be derived from several mesenchymal cells, including originally resident pulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, epithelial-mesenchymal transition (EMT) has been proposed to be associated with the formation of mesenchymal cells², and to contribute to the pathogenesis of fibrotic disorders.

It is thought that EMT plays important roles in the process of fetal development, wound healing, and progression of cancer, including tumor invasion and metastasis³. Following the process of EMT, epithelial cells obtain the ability of mesenchymal cells by loss of epithelial markers, such as E-cadherin, and by expression of mesenchymal markers, such as vimentin, and α-smooth muscle actin (SMA)^4,5. Previous studies showed the evidence that EMT process has been associated with the development of tissue fibrosis in the kidney⁶ and lung⁷. Additionally, chronic inflammation promotes fibrotic disease⁸; furthermore, such inflammatory cytokines as Tumor necrosis factor superfamily member 14 (TNFSF14; LIGHT), tumor necrosis factor (TNF)-α, and interleukin-1β, have been shown to enhance EMT^9-12.

Collagen gel contraction assay, a collagen-based cell contraction assay in which fibroblasts are embedded in type I collagen gel three-dimensionally, is an ideal in vitro model for the evaluation of contractility. Contractility is one of the characteristic functions of fibroblasts and contributes to normal wound repair and fibrosis¹³. In this assay, it is thought that the attachment of fibroblasts to type I collagen through integrin-dependent mechanisms is supposed to produce mechanical tension under some conditions, and consequently lead to tissue contraction.

Here, the development of the gel contraction assay is reported to be adapted to evaluate the acquisition of contractile function in the cells that underwent EMT. This report demonstrates that this modified assay is suitable for evaluating contractility in mesenchymal cells that underwent EMT.

Protocol

1. Preparations and Culture of Lung Epithelial Cells

1. Culture A549 human lung epithelial cells (adherent cell line) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin.
2. Remove and discard the cell culture media from culture dish, and wash once with 5 - 10 ml of phosphate buffered saline (PBS). After washing, immediately aspirate the PBS.
3. Add 2 ml Trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05%) and incubate at 37 °C and 5% CO₂ for 3 min.
4. Collect the detached cells in centrifuge tubes containing cell culture medium, centrifuge the tubes at RT for 4 min at 150 × g.
5. Resuspend the cells pellet in 2 ml of cell culture medium and remove a sample for cell counting. Count the number of the cells using a hemocytometer with Trypan blue stain to check cell viability.
6. Seed A549 cells on 10 cm polystyrene plates at a density of 0.5 - 1.0 × 10⁶ cells/dish with 10 ml of medium for gel contraction assay. Seed the cells on 6 well polystyrene plates at a density of 0.5 - 1.0 × 10⁵ cells/well with 2 ml of medium for EMT confirmation. Incubate the cells at 37 °C and 5% CO₂ for 24 hr.

2. EMT Procedure

1. Add 10 µl of TGF-β1 (5 µg/ml) and 10 µl of TNF-α (10 µg/ml) to the plate for gel contraction assay seeded in step 1.6. Add 2 µl of TGF-β1 (5 µg/ml) and 2 µl of TNF-α (10 µg/ml) to the plate for EMT confirmation seeded in step 1.6. Incubate at 37 °C and 5% CO₂ for 48 hr.

3. Confirmation of EMT Procedure by PCR and Western Blotting

1. Confirm change in morphology (from cobble stone-like to spindle shape) of treated cells using phase contrast microscopy.
   Note: Normal A549 cells have cobble stone-like and triangular shaped appearance that is a characteristic of epithelial cells, but after stimulation with TGF-β1 and TNF-α, the cells appear long and spindle shaped that is similar to mesenchymal cells^14,15.
2. Evaluate the expression of an epithelial marker, such as E-cadherin, and mesenchymal markers such as N-cadherin, vimentin, and α-smooth muscle actin using PCR or Western blotting.
   1. Extract total RNA from the cells using RNA extraction kit¹⁶ and synthesize cDNA using reverse transcriptase¹⁷ according to the manufacturer's protocol.
   2. Measure expression of mRNA levels using real time PCR system¹⁸ and monomeric cyanine dye PCR kit according to the manufacturers' instructions. The specific primers for GAPDH (glyceraldehyde 3-phosphate dehydrogenase), ACTA2 (alpha-actin-2), CDH1 (cadherin-1; E-cadherin), and VIM (vimentin) are shown in Table 1.
   3. Lyse the cells using a lysis buffer (Table 2) solution containing 1% protease inhibitor cocktail. Measure all sample protein concentrations using a protein assay kit^19,20, and apply the same amounts of protein to a polyacrylamide gel.
      1. Perform SDS gel-electrophoresis and semi-dry transfer of the proteins to PVDF membrane²¹. Incubate the membrane with primary antibodies in blocking buffer for 1 - 2 hr. After incubation, wash twice the membrane with wash buffer and incubate the membrane 1 hr with second antibodies.
         Note: See the Materials/Equipment Table for antibodies and dilutions used in these studies. See Table 2 for the components of blocking buffer.
      2. Wash the membrane with wash buffer twice. Take pictures of the membrane with the Western blotting detection kit²² using a cold CCD camera^11,23.

4. Gel Contraction Assay for Evaluating EMT

1. Aspirate the conditioned media from the cell culture vessel, and wash well with 5 - 10 ml of PBS to remove dead cells. After washing, immediately aspirate the PBS.
2. Add 2 ml of 0.05% Trypsin/EDTA and incubate at 37 °C and 5% CO₂ for 3 min.
3. Collect the detached cells in centrifuge tubes containing DMEM supplemented with trypsin inhibitor (1 mg/ml), centrifuge the tubes at RT for 4 min at 150 ×g.
4. Mix type 1 collagen gel with distilled water, and 4× concentrated DMEM to adjust the volumes to achieve a collagen concentration of 1.75 mg/ml, and 1× DMEM concentration. Be sure to keep the gel medium on ice during this step.
   Note: To make 6 ml of gel medium, mix well 3.5 ml of type 1 collagen gel (3 mg/ml), 1.5 ml of 4× concentrated DMEM, and 1ml of distilled water.
5. Resuspend the cell pellet in 500 µl of PBS and remove a sample for cell counting. Count the number of the cells using a hemocytometer with trypan blue stain to check cell viability.
6. Add the gel medium to adjust the volumes to achieve a cell density of 3.0 × 10⁵ cells/well (6.0 × 10⁵ cells/ml) and gently but quickly mix it by pipetting without gelation.
7. Dispense 0.5 ml of the mixture into each well of a 24-well non-treated plate quickly and carefully to make neat cylindrical form. Be careful not to allow any air bubbles to contaminate the gels (Figure 1A).
   Note: The gel medium containing the cells is viscous and can easily gel and form crescent shape in the well.
8. Incubate the plate for 15 min in a cell incubator at 37 °C, 5% CO₂ and 95% humidity to gel completely.
9. Detach gels from the plate without breaking by moving a sterilized spatula in a manner to draw a circumference in one direction. Using a spatula, gently transfer the gels to 60 mm tissue culture dishes containing 5 ml of DMEM/1% FBS with or without TGF-β1 (5 ng/ml) and TNF-α (10 ng/ml).
10. Gently shake the dishes to ensure gels are floating on the medium. Incubate in a cell incubator at 37 °C, 5% CO₂ and 95% humidity.

5. Measurement of Gel Size

1. Measure the collagen gel size after 0, 24, 48, and 72 hr using an image analysis system.
   1. Turn on the gel documentation system (Figure 2A), and put dishes into the light shielding cabinet. Then take off the lid of dishes in the cabinet.
   2. Open the related gel analyzing software. Click the "image acquisition button" in the menu-bar to show the image of the gels in the cabinet. Then, click "acquire" in the menu bar to take pictures of the gels (Figure 2B).
   3. Click the "detection button" in the menu bar (Figure 2C) and adjust the measurement region (yellow circle in Figure 2C) by dragging the mouse. Then, click the "auto-detect button" in the menu bar (see button in Figure 2D). The software automatically detects the gels showing a heatmap (Figure 2D).
   4. Click "OK" to extract the outline of the gels by image processing and to calculate areas surrounded by the outline (Figure 2E). After the area is calculated, the calculated area is displayed in separate pop-up window.
      Note: If the gels overlap each other during imaging, move gels gently using a sterile pipette tip.

Results

During EMT, epithelial cells lose epithelial markers, like E-cadherin, and gain the expression of mesenchymal markers, such as vimentin and α-smooth muscle actin^4,5. Incubation of A549 human lung epithelial cells with TGF-β1 and TNF-α induces EMT. The appearance of normal A549 cells are cobble stone like shape and triangle shape that is a characteristic of epithelial cells (Figure 3A), but after stimulated with TGF-β1 and TNF-α, the app...

Discussion

The protocol developed in this study comprises two steps. The first step is performed to induce EMT, while the second step is the gel contraction assay. Since it is important to confirm that cells underwent EMT, step 2 provides an excellent complement to the morphological and gene expression changes. Previous studies showed that EMT of A549 cells was induced by TGF-β1 only²⁴; however, as we have reported previously¹⁰, TNF-α treatment enhances EMT and the acquisition of mesenchymal cell mar...

Materials

Name	Company	Catalog Number	Comments
DMEM	sigma aldrich	11965-092	For A549 medium
FBS	GIBCO	10437
Transforming Growth Factor-β1, Human, recombinant	Wako Laboratory chemicals	209-16544
Recombinant Human TNF-α	R&D systems	210-TA/CF
E-Cadherin (24E10) Rabbit mAb	Cell Signaling Technology	#3195	1:3,000 dilution
Vimentin (D21H3) Rabbit mAb	Cell Signaling Technology	#5741	1:3,000 dilution
Anti-α-Tubulin antibody	sigma aldrich	T9026	1:10,000 dilution
Monoclonal Anti-Actin, α-Smooth Muscle antibody	sigma aldrich	A5228	1:10,000 dilution
Anti-N-cadherin antibody	BD Transduction Laboratories	#610920	1:1,000 dilution
Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)	GE Healthcare	NA931-100UL	1:20,000 dilution
Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)	GE Healthcare	NA934-100UL	1:20,000 dilution
Blocking reagent	GE Healthcare	RPN418	2% in TBS-T
6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid	corning	#353046
100 mm Cell culture dish	TPP	#93100
DMEM, powder	life technologies	12100-046	For 4× DMEM
Type 1 collagen gel	Nitta gelatin	Cellmatrix type I-A
24 Well cell culture plate	AGC TECHNO GLASS	1820-024
Gel Documentation System	ATTO	AE-6911FXN	Gel imager
Gel analyzing software	ATTO	Densitograph, ver. 3.00	analysing software bundled with AE-6911FXN
Trypsin-EDTA (0.05%), phenol red	life technologies	25300054
24 Well Plates, Non-Treated	IWAKI	1820-024
Trypan Blue Solution, 0.4%	life technologies	15250-061
RNA extraction kit	Qiagen	74106
Reverse transcriptase	life technologies	18080044
Real time PCR system	Stratagene	Mx-3000P
SYBR green PCR kit	Qiagen	204145
Protease Inhibitor Cocktail (100x)	life technologies	78429
PVDF membrane	ATTO	2392390
Protein assay kit	bio-rad	5000006JA
Polyacrylamide gel	ATTO	2331810
Western blotting detection reagent	GE Healthcare	RPN2232
Cold CCD camera	ATTO	Ez-Capture MG/ST
Trypsin inhibitor	sigma aldrich	T9003-100MG
Polyoxyethylene (20)Sorbitan Monolaurate	Wako Laboratory chemicals	163-11512
Polyoxyethylene (9) octyiphenyl ether	Wako Laboratory chemicals	141-08321