Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

Summary

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

Abstract

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Introduction

HIV-Tat-interactive protein 60 kDa (TIP60) is a lysine acetyltransferase that can acetylate both histone and non-histone proteins^1,2. Its functions are found to have implications in multiple signaling pathways, including transcription, DNA-damage repair, and apoptosis^{1,3,4,5,6,7,8}. Furthermore, TIP60 is often downregulated in cancers, and its downregulation is correlated with cancer metastasis and poor survival rates^{9,10,11,12,13,14}. Tumor metastasis is the major cause of cancer-related death. It is a multi-step process, and the initial step of metastasis involves the migration and invasion of tumor cells into adjacent tissues^15,16. In order to do so, tumor cells first must detach from the primary tumor mass, either in the form of a collectively invading cell sheet or as detached single cells¹⁷. During this process, tumor cells often undergo epithelial to mesenchymal transition (EMT), resulting in changes in morphology and cell adhesion capability^17,18.

A few techniques have been developed to study the migration and invasion ability of tumor cells in vitro. Among them, the wound-healing assay is the most efficient and economical¹⁹. First, this method involves the creation of an artificial gap on a confluent monolayer of cells, thus allowing cells to migrate and close the gap. Second, images of the gaps can be captured at the beginning and end of the experiment. Finally, a comparison of gap closure is used to determine the rate of cell migration. A phase-contrast microscope is usually used to capture images for conventional wound-healing assays. Another advantage of the wound-healing assay is that it partly resembles in vivo tumor cell migration. Similarly to in vivo tumor metastasis, cell migration in wound-healing assays shows both the collective migration of epithelial sheets and the migration of detached single cells.

However, cell migration is known to be dynamic, and there is a need to document the movement of cells in real time. For instance, a conventional wound-healing assay does not allow a researcher to analyze single-cell movement. On the other hand, cells cultured for wound-healing assays are often serum-starved to inhibit cell proliferation. This is done to rule out the possibility of gap closure due to cell proliferation. Nonetheless, there will still be some proliferation and cell death, and phase-contrast images taken before and after the experiment are unable to differentiate between them.

The live-cell imaging technique addresses this limitation of conventional wound-healing assays. Using a live-imaging microscope combined with a CO₂ incubator and appropriate temperature control, researchers can measure the gap size and its rate of closure over time while tracing the movement of actively migrating cells located at the tips of the invasive front. Hence, the implementation of live-cell imaging to monitor cell migration will not only provide better visualization of cell migration, but will also allow for the possibility to differentiate between cell migration and cell proliferation, thus providing a more reliable analysis of cell migration.

In this study, MCF10A breast epithelial cells were used to demonstrate the combination of the wound-healing assay and live-cell imaging to study the role of TIP60 in cell migration. The depletion of TIP60 results in increased migration abilities in MCF10A cells.

Protocol

1. Preparation

1. Prepare MCF10A culture medium: Dulbecco's Modified Eagle Medium (DMEM)/F12 (1:1) with 5% horse serum, 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 µg/mL insulin.
2. Prepare serum-free medium: DMEM/F12 (1:1) with 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 µg/mL insulin.
3. Use the following siRNA sequence:
   siControl:
   Forward: 5'-CGUACGCGGAAUACUUCGAdTdT-3'
   Reverse: 5'-UCGAAGUAUUCCGCGUACGdTdT-3'
   siTIP60:
   Forward: 5'-UGAUCGAGUUCAGCUAUGAdTdT-3'
   Reverse: 5'-UCAUAGCUGAACUCGAUCAdTdT-3'
4. Use 10-cm cell culture dishes and 24-well plates.

2. Transient Depletion of TIP60 Using siRNA

1. On day 1, aliquot 15 µL of transfection reagent (e.g., Lipofectamine RNAiMAX) and 2 mL of reduced serum medium (e.g., Opti-MEM) and mix them with 10 µL of each siRNA (10 µM stock), respectively.
2. Incubate the mixture at room temperature for 20 min.
3. Seed 1 x 10⁶ MCF10A cells together with 3 mL of culture medium in a 10 cm dish. Count the cells using a hemocytometer.
4. Add the siRNA mixture drop by drop to the 10 cm dish.
5. Shake the dish to ensure that the cells are equally distributed before putting it into the 37 °C incubator.
6. After 6 h of incubation, replace the siRNA-containing medium with 8 mL of fresh culture medium.
7. On day 2, prepare another batch of siRNA mixtures and incubate them at room temperature for 20 min.
8. Aspirate out 8 mL of the culture medium added on day 1 and replace it with 3 mL of fresh culture medium.
9. Add the second batch of siRNA mixture to the dishes drop by drop. Shake the dishes to ensure that the reagents are fully mixed before putting them into the incubator.
10. After 6 h, replace the siRNA-containing medium with 8 mL of fresh culture medium.

3. Seed Cells for the Wound-healing Assay

1. On day 3, discard the medium and wash the cells once with 2 mL of phosphate-buffered saline (PBS) buffer. Discard the PBS buffer.
2. Trypsinize the siControl and siTIP60-treated cells by adding 1 mL of 1x trypsin-EDTA buffer to the plate. Put it back in the incubator for 20 min.
3. Add 4 mL of complete culture medium to each plate, resuspend the cells by pipetting, and transfer them to a 15-mL tube.
4. Centrifuge at 200 g for 5 min and remove the supernatant.
5. Re-suspend the cells with 5 mL of serum-free medium and count the cells using a hemocytometer.
6. Prepare a cell suspension of 6 x 10⁵ cells/mL for both the siControl and siTIP60; ensure that the cells are well-mixed. Add 500 µL of the cell suspension to one well of a 24-well plate to ensure 100% confluency. Seed 5 to 6 wells each for siControl and siTIP60.
7. Gently shake the plate back and forth and then side-to-side to ensure even distribution. Avoid a circular motion. Leave the plate in the incubator for 24 h.
8. Harvest the remaining cells to check the knockdown efficiency of TIP60. To do so, centrifuge the remaining cell suspension at 200 g for 5 min and remove the supernatant.
9. Isolate RNA using a commercial reagent, following the manufacturer's protocol, and proceed to real-time quantitative PCR to evaluate the knockdown efficiency.

4. Create the Wound

1. Check under a phase contrast microscope to ensure that the cells are fully attached. Observe the cells under the microscope (4X, 10X, and 20X magnification) to ensure that the cells are fully attached that a confluent monolayer of cells has formed.
   NOTE: siControl cells usually attach better than siTIP60 cells. This is expected because the depletion of TIP60 makes the cells more mesenchymal-like²⁰. Here, a normal benchtop white-light phase contrast microscope is used.
2. To generate the wound, gently scratch a straight line across the center of the well with a 200 µL pipette tip; the length of the wound generated is same as the diameter of the well. Repeat this step for all remaining wells. Use a new pipette tip for each well.
3. Gently wash each well 5 times using serum-free medium to remove the detached cells.
4. Add 1 mL of serum-free medium to each well and proceed to live-cell imaging.

5. Setup of Live-cell Imaging

1. Turn on the microscope and the temperature control at least 1 h before setting up live-cell imaging to ensure that the temperature in the chamber reaches 37 °C.
2. Turn on the carbon dioxide (CO₂) gas supply before starting the live-cell imaging. Set the CO₂ concentration to 5%.
3. Place the 24-well plate on the stage of the microscope and select the objective lens with 10X magnification for imaging. Direct the emitted light to the eyepieces instead of the camera and adjust the focus to locate the wound and the cells around it.
   NOTE: In this case, white light with phase contrast was used.
4. Use a live-cell observer system to mark the position of the wound in each well.
   NOTE: The automated stage of the live-cell imaging system allows for image acquisition at multiple positions. Images are taken on every marked position once every interval.
5. Set up the time condition: 1 h interval and 72 h duration; according to this setting, the system will take pictures at each marked position every hour for 72 h. Set up any desired time interval and period for monitoring.
   NOTE: Here, a duration of 72 h is best because these cells start dying after 72 h in serum-free medium.

6. Tracking Single-cell Movement

1. Export the data after 72 h. Export the data both as images in JPEG format and as videos in AVI format.
   NOTE: This is important because ImageJ software can only recognize videos in AVI format. The different cell migration speeds between siTIP60 and siControl cells can be clearly observed in the video.
2. Open the AVI video in the ImageJ software.
3. Open the MTrackJ plugin (the plugin must be downloaded and installed separately): Plugins > MtrackJ.
4. Add a track for single cells chosen from the video by clicking the "Add" tab in the MtrackJ toolbar. Choose a cell at the tip of the invasive front and a cell that can migrate on its own (single-cell migration). Follow the movement of the chosen cells on the video and trace them using MtrackJ.
5. Measure the distance of movement by clicking the "measure" tab on the toolbar.
   NOTE: Here, almost no cells in the siControl moved as single cells, making it difficult to measure single-cell movement, whereas many of the siTIP60 cells moved as single cells. However, the movement of cells located at the invasive front can still be traced and calculated in both siControl and siTIP60 cells.
6. Save the video with the tracks in AVI format.

7. Quantification of Cell Migration Ability

1. For quantification, select images at 0 h and images at "n" h, where "n" refers to the timepoint when either the siTIP60 or siControl cells have completely closed the wound.
   NOTE: Here, 48 h was chosen, as the wounds in siTIP60 cells were almost completely closed.
2. Quantify the percent migration using ImageJ using the following formula: (area of wound at 0 h - area of wound at "n" h) / area of wound at 0 h x 100.

Results

General Schema of Live-cell Imaging-based Cell Migration Assay
Figure 1A is a schema of this protocol. MCF10A cells transfected with siControl showed typical epithelial morphology. After the depletion of TIP60, MCF10A cells were more mesenchymal compared to control cells (Figure 1B).

Examination of TIP60 Knockdown Efficiency

Discussion

It is known that in the process of cell migration, cells can either move in the form of adherent sheets; loose clusters; or single, isolated cells. The mode and dynamic of cell invasion can vary from one condition to another, and the phenotype can change within hours. The traditional wound-healing assay is based on snapshot pictures taken from a microscope with a long time interval, such as 12 or 24 h. This makes it difficult to capture the dynamic of wound closure and the pattern of cell movements.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MCF10A cell	ATCC	CRL-10317TM	Breast epithelial cell
DMEM/F12 media (1:1)	Gibco	11330-032	MCF10A culture media
Epithelial growth factor (EGF)	Peprotech	AF-100-15	Supplements for MCF10A culture media
Cholera Toxin	Sigma-Aldrich	C-8052	Supplements for MCF10A culture media
Hydrocortisone	Sigma-Aldrich	H-0888	Supplements for MCF10A culture media
Insulin	Sigma-Aldrich	I-1882	Supplements for MCF10A culture media
House serum	Gibco	16050-122	Supplements for MCF10A culture media
Trypsin	Gibco	25200-056	For MCF10A detach from plate
Hemacytometer	Fisher Scientific	267110	For counting cell
Opti-MEM reduced serum media	Gibco	31985-070	For siRNA mixture
Lipofectamine RNAiMAX	Invitrogen/Life Technologies	56532	Transfect reagent
Trizol reagent	Invitrogen/Life Technologies	15596-026	For mRNA extraction
iScript cDNA Synthesis Kit	Bio-Rad	170-8891	For cDNA generation
iTaq Universal SYBR Green Supermix	Bio-Rad	172-5125	For real time qPCR
7500 Fast Real Time PCR system	Biosystems		Real time qPCR mechine
10-cm dish	Greiner bio-one	664160	For Knockdown experiment
24-well plate	Cellstar	662160	For wound healing assay
siControl siRNA	Self designed	Self designed	CGUACGCGGAAUACUUCGAdTdT
siTIP60 siRNA	Self designed	Self designed	UGAUCGAGUUCAGCUAUGAdTdT
Live cell observer	Zeiss		Zeiss inverted Cell Observer for live cell experiments