A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Summary

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Abstract

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

Introduction

Cell migration and invasion are important biological processes that enable normal functions in the human body, such as wound closure, invasion of placenta into the uterus and mammary gland morphogenesis^1,2,3. The human body has precise and strict control of these biological events; however, there are some exceptions. Malignant tumors, for example, are able to escape this safeguard, exhibit abnormal proliferation and invade into neighboring tissue, which is called metastasis. Metastasis is the major cause of cancer-related mortality⁴.

Breast cancer is the most commonly diagnosed cancer in women, and is the second-highest cause of cancer-related death among women in developed countries worldwide⁵. Breast cancer originates from ducts or lobules that consist of one or more layers of epithelial cells. In the normal breast, epithelial cells adhere to one another and to the basement membrane through membrane proteins such as E-cadherin and integrins⁶. However, invasive breast cancer cells have lost their polarity and cell-cell adhesion, and classically undergo epithelial mesenchymal transition (EMT) and gain the ability to move. After extravasation, these cells move across the extracellular matrix (ECM) and enter the blood vessel or the lymphatic system, followed then by intravasation and metastatic growth⁷. Understanding the mechanisms by which this occurs is of great significance, since metastasis is the most common cause of cancer-related mortalities and is closely related to cancer cell migration/invasion. To visualize the movement of cancer cells, migration and invasion assays are ideal models to study 2D and 3D cell movement, respectively. Migration directly assesses the movement of the cells whereas invasion involves interaction with the microenvironment and the ability to degrade biological barriers. The two processes are not fully independent of one another, as migration is a requirement of invasion.

Several methods have been developed to study migration and invasion. As reviewed by Kramer et al., migration assays such as wound healing, fence and micro-carrier assays generate a cell-free area to allow cells to move into, assessing the change of area; whereas, transwell and capillary assays are based on the number of cells that move toward an attractant⁸. For invasion assays, an ECM environment has to be set up with ECM gel or collagen for instance, and 3D movement can be assessed by monitoring the invasion area, distance and cell counts (e.g. transwell assay, platypus assay)⁸. Another type of invasion assay is to combine the invasive cells with non-invasive cells and assess the behavior of the invasive cells (e.g. spheroid assays). The above methods have their pros and cons, and a way that is easy to approach, easy to repeat, and to combine the migration assay and invasion assay in a similar workflow is preferential in experimental design.

This protocol describes the measurement of cell migration and invasion using a live-cell imager. It is a real-time cell monitoring system installed in a standard cell culture incubator. It takes high definition images according to the set scanning intervals and measurements by applying appropriate masks to the cells or fluorescent targets. The module of migration/invasion assay includes using a 96-pin scratch tool, which is suitable for making homogeneous scratch wounds on a cell monolayer in a 96-well plate. The mechanism is based on in vitro wound healing assays, monitoring 2D cell movement on a plastic or coated surface. Invasion or 3D movement across an additional ECM within the scratch wound can also be assessed. A brief workflow is illustrated in Figure 1.

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Protocol

NOTE: Two cell lines should be handled separately. The following procedures should be applied to one single cell line if not specified.

1. Optimize Cell Density Prior to Wounding

1. Culture adherent cells in T75 cm² tissue culture flasks to about 80% confluence in phenol-red free Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine and 2 µg/mL insulin at 37 °C with 5% CO₂ (standard incubation conditions for most cancer cell lines, culture formulations are cell line-dependent).
2. Remove the culture media by pipetting off into a waste container. Pipet 2 mL of pre-warmed 0.1% trypsin-EDTA to briefly rinse the cell monolayer and pipet off. Add another 2 mL of 0.1% trypsin-EDTA and place the flask under standard incubation conditions at 37 °C for 5 min.
3. Gently tap the flask to ensure detachment of the cells and then add 10 mL of pre-warmed culture media to stop the proteolytic reaction.
4. Transfer the cell suspension into a 15 mL centrifuge tube and spin down at 200 x g for 5 min at room temperature (RT). Carefully remove the supernatant without agitating the cell pellet and resuspend with another 10 mL pre-warmed culture media.

2. Counting Cell Number Using an Automated Cell Counter (or Any Counting Methods)

1. Dilute 200 µL of resuspended cell suspension with 800 µL of 1x Dulbecco's Phosphate buffered Saline (DPBS) in a 1.5 mL centrifuge tube.
2. Attach a 60 µm cell count sensor to the cell counter, hold down the toggle and merge the tip into the cell suspension. Slowly release the toggle until the cell suspension is successfully drawn into the sensor. Cell concentration is displayed in cells/mL.
3. Calculate the cell number in the 10 mL cell suspension.

3. Cell Plating

1. Plate cells at a range of cell densities (40,000-90,000 cells/well) in triplicate in a 96-well plate.
2. Place the plate into the live-cell imager, and schedule scanning every 2 h for 24 h.
   1. In the software, click Schedule scans from the task list. In the drawer setup pane, determine the position of the plate, click Add vessel and choose the plate type. In the Scan Setup pane, choose or edit the scan pattern according to the experimental plate setup, and set Scan Type as Standard.
   2. Right click on the Timeline and select Set Intervals. Set Add Scans Every to 2 h at a 24-h schedule. Click Apply.

4. Determine the Optimal Cell Seeding Density for the Migration Assay

1. Stop scanning after 24 h, apply the confluence processing analysis tool to the HD-phase contrast images automatically collected, and generate a cell proliferation curve against time.
   1. Determine 3-6 representative images and place them in a new Image Collection.
   2. Determine a proper mask as Processing Definition.
   3. Launch an analysis job.
   4. Determine optimized cell density according to confluence against time (approximately 100% confluence within 6 -18 h depending on when the migration assay commences).
      NOTE: The amount of time it takes to grow the cells to confluence varies depending on the seeding dilution.

5. Days 1 and 2: Preparation for Migration and Invasion Assays

1. On Day 1, coat the plate for invasion assay.
   NOTE: ECM gel should always be handled on ice and with tips that have been placed in the fridge overnight.
   1. Dilute ECM gel with ice-cold culture media to 100 µg/mL and add 50 µL of diluted ECM gel/media into designated wells.
   2. Place the plate at standard incubation conditions overnight.
2. On day 2, gently aspirate the excess media. Plate cells with optimized cell densities into 2 96-well plates designated for the migration assay (uncoated) and the invasion assay (coated) in triplicates following sections 1-3 in the late afternoon (in the current protocol, optimized seeding densities for ZR75-1 and MDA-MB-231 were 90,000/well and 50,000/well, respectively).
3. Place plates at standard incubation conditions overnight.

6. Day 3: Wound Scratch

1. Spray and wipe the scratch tool and 2 washing boats with 70% ethanol before placing them in the biosafety cabinet. Fill the washing boat 1 and 2 with exactly 45 mL of sterile (autoclaved) distilled water and 70% ethanol respectively.
2. For sterilization, place the scratch tool pin block (top) on washing boat 1 and 2 for 5 min each.
3. Start with the migration assay plate.
   1. Move the plate containing cells from the incubator and make sure no well is dry to avoid damage of the scratch tool. Remove the plate cover and insert into the base plate holder of the scratch tool, and carefully place the top part onto the base part by guiding dowels. Press and hold the black lever, and meanwhile carefully lift the pin block. Gaps in each well are usually visible with the naked eye and under the microscope.
   2. Quickly soak the pins in water; this is sufficient to clean the scratch tool prior to scratching the invasion assay plate if plate setup is identical. Otherwise, repeat the sterilization steps with sterile distilled water and then 70% ethanol for 5 min each.
4. Wash the plate 1 or 2 times with pre-warmed culture media to avoid detached cells or cell sheets reattaching to the well.
5. Add 100 µL of fresh warm media into designated wells with or without treatments.
6. Additional steps for invasion assay.
   1. Place the invasion assay plate at 4 °C for 5 min to equilibrate and carefully aspirate the cold media.
   2. Dilute ECM gel with ice-cold culture media to 5 mg/mL, add 50 µL of diluted ECM gel into designated wells, and place under standard incubation for 30 min.
   3. Add 100 µL of warmed-up media with or without testing compounds.
7. Place the plate into live-cell imager and let it equilibrate for 5 min. Choose Vessel Type as imagelock plate. Set Scan Type as Scratch Wound, choose or edit Scan Pattern according to the experimental plate setup (1 image/well and wide mode) and schedule 24-h repeat scanning every 1-2 h for 72 h until the wounds are healed.
8. To clean the scratch tool, put the top pin block in each of the following solutions (45 mL in washing boats) for 5 min: 0.5% detergent 1 (see Table of Materials), 1% detergent 2 (see Table of Materials), sterile distilled water and 70% ethanol. Place the scratch tool back onto its base plate and store in a dust-free environment.

7. Data Analysis

1. Stop scanning the designated plate after all the wounds have healed by choosing the experimental plate on the Drawer Setup and clicking Remove Vessel.
2. Collect 3-6 representative images spanning a range of sound percentages, including images right after the wound has been made, wound closure by 10% and 50%.
3. To determine a proper processing definition, use Segmentation Adjustment, Cleanup and Filters to apply appropriate Scratch Wound Mask and Confluence Mask. Use Preview current/all to view the accuracy of the masks.
4. Launch the analysis job.
5. Data can be analyzed within the software or exported for further analysis. Three metrics provided by the software can be used to evaluate the HD-phase images: wound width (µm), wound confluence (%) and relative wound density (%). Comparisons will be discussed in the following section.

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Results

This migration/invasion assay is based on the wound healing assay, which evaluates the rate of the cells moving into a cell-free area created by the 96-pin scratch tool. The difference between the migration and invasion assays are that migration assays measure cells moving on the tissue-culture treated plastic surface and invasion measures cells moving across ECM gel.

The scratch tool is designed to make consistent scratch wound...

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Discussion

Migration and invasion are important parameters to assess the mobility of cancer cells. By using the 96-pin scratch tool, it is possible to conduct wound healing assays in 2D and 3D simultaneously. Apart from facilitating automatic scanning, providing a stable cell culture environment with minimum disruption, the scratch assay conducted using the 96-pin scratch tool provides consistent scratch wounds, enabling experiments that are more robust and reproducible. The 96-well plate format gives additional options of either i...

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Materials

Name	Company	Catalog Number	Comments
0.5% trypsin-EDTA solution (10x)	ThermoFisher Scientific	30028-02	Dilute to 2x in DPBS
Countess II FL Automated Cell Counter	ThermoFisher Scientific	AMQAX1000	Automated cell counter
Detergent 1 (Alconox)	Sigma-Aldrich	242985	0.5% working concentration
Detergent 2 (Virkon S)	VetProduct DIRECT		1% working concentration
Dulbecco’s Modified Eagle Medium, no phenol-red	ThermoFisher Scientific	21063-045	Supplimented with 10% FBS, 200 mM L-glutamine, 2 μg/ml insulin
ECM Gel (matrigel)	Sigma-Aldrich	E6909	Growth-factor reduced, phenol red free
Essen ImageLock 96-well plate, flat bottom	Essen	4379
EVE Counting slides	BioTools	EVS-50
Fetal bovine serum (FBS)	Bovogen Biologicals	SFBS-F-500ml
IncuCyte 96-well scratch wound cell invasion accessories	Essen	4444	Including CoolBox, 2x CoolSink
IncuCyte Cell migration kit	Essen	4493	Including the 96-well pin block, 2x wash boats and the software
IncuCyte ZOOM	Essen		Live cell analysis system
Insulin solution human	Sigma-Aldrich	19278-5ML
L-glutamine solution (100x)	ThermoFisher Scientific	25030-091
Tissue culture flask, 75 cm2 growth area	Greiner Bio-One	658175