Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Summary

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Abstract

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Introduction

Macrophages are the first line of immune defense and play a role in orchestrating immune responses against pathogens and foreign materials, including cancer cells. They are a specialized phagocyte that destroys and gets rid of unwanted particles in the body. Macrophages contribute defensive functions such as the clearance of apoptotic cells and microorganisms and the recruitment of other immune cells¹. Macrophages can differentiate into two different types, M1 and M2 macrophages, in response to environmental signals². M1-polarized macrophages (i.e., classically activated macrophages) are activated by the cytokine interferon-γ (IFN-γ) and lipopolysaccharides (LPS) and are involved in the inflammatory response, pathogen clearance, efficient phagocytosis, and tumoricidal immunity^3,4. The M2 macrophages are closely related to tumor-associated macrophages (TAMs) and have anti-inflammatory and tumor promotion properties⁴.

Phagocytosis, derived from Ancient Greek (phagein), meaning "to devour," (kytos), meaning "cell," and -osis, meaning "process"⁵. Phagocytosis is a receptor-mediated process where phagocytes (including macrophages, monocytes, and neutrophils) kill and engulf invading pathogens, clean up foreign particles, and clear apoptotic cell debris. Tumor-associated macrophages (TAMs) are found in the stroma in different tumors, including breast cancer, and have pro-tumor functions^6,7, resulting in resistance to phagocytosis. The detailed mechanism of tumor cell phagocytosis by macrophages is not yet understood.

This study presents a two-step method: 1) 4T1 mouse mammary carcinoma cells and M1-polarized macrophages are cocultured, and 2) the phagocytic activity of the macrophages is assessed using live-cell video microscopy. CFSE fluorescent dye was used to stain the 4T1 mouse mammary carcinoma cells. The stain labels 4T1 cells to distinguish them from the cocultured M1 macrophages within a single imaging dish. RAW 264.7 macrophages are polarized with LPS and IFN-γ into an M1 phenotype. To ensure a complete polarization, immunostaining with anti-iNOS antibody conjugated to FITC was performed. Subsequently, a multipoint series of time-lapse images was acquired to observe multiple events, including phagocytosis, within the coculture.

A better understanding of the interactions between tumor cells and macrophages may lead to potential cancer immunotherapy. Live-cell imaging offers a detailed view of cellular dynamics in a real-time setting and has been used to study cell migration, phenotypic screening, apoptosis, and cytotoxicity^8,9 in neuroscience, developmental biology, and drug discovery. Although the proposed tumor in this study is breast cancer, the method can also be applied to multiple target cells and distinct effector cells.

Protocol

NOTE: Sections 1−6 describe the coculture model of 4T1 mouse mammary carcinoma cells and RAW 264.7 mouse macrophages. Section 7 describes the time-lapse assessment of M1 macrophages phagocyted 4T1 cells.

1. Culturing 4T1 mouse mammary carcinoma cells and RAW 264.7 mouse macrophages

1. Thaw a vial of cryogenically preserved 4T1 and RAW 264.7 passage 6 cells by gentle agitation in a water bath at 37 °C or the normal growth temperature for the cell lines.
   NOTE: Thawing should be done rapidly, approximately within 2 min. To avoid contamination, remove the vial from the water bath and decontaminate it by spraying with 70% ethanol. To avoid genetic drift, especially for the macrophages, use a low passage number (i.e., less than 20).
2. Dilute the thawed cells in 10 mL of complete DMEM medium in a 15 mL conical tube and centrifuge the cells at 600 x g for 5 min to obtain a cell pellet.
   NOTE: Complete DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS), 200 U/mL penicillin/streptomycin, and 2 mM L-glutamine is used throughout the protocol.
3. Carefully aspirate the media, resuspend the cells in 8 mL of complete medium and transfer into a 25 cm² tissue culture treated flask. Culture both 4T1 and RAW 264.7 cells in complete DMEM medium at 37 °C with 5% CO₂.
   NOTE: Resuspend the cell pellets gently and add the cell suspension into the culture flask drop by drop to avoid killing the cells.
4. After 1 week of culturing to allow cell adherence, monitor the flask daily and add 8 mL of complete DMEM medium as needed.

2. Immunostaining of M1 polarized RAW 264.7 macrophages

1. As the confluency of RAW 264.7 cells reaches 70−80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS.
   NOTE: Before seeding RAW 264.7 cells, make sure no cell differentiation has occurred (i.e., dendrite-like phenotype and/or increased cell size). If cell morphology changes are visible, discard the culture and thaw a new cell line from the earlier passage vial.
2. Prepare the macrophages for collection by gently dislodging the adherent cells using a cell scraper and transfer them into a 15 mL conical tube.
3. Count the cells using a hemocytometer and prepare a cell suspension of 1 x 10⁵ cells/dish using complete DMEM medium.
4. Seed 2 mL of the macrophages suspension into two imaging dishes. Incubate the cells 2−3 h at 37 °C with 5% CO₂ to allow cell adherence.
5. Prepare M1 polarization media by adding 100 ng/mL LPS and 20 ng/mL IFN-γ into complete DMEM medium^10,11.
6. Discard the culture supernatant from the macrophages and carefully wash the monolayers in the dish 2x with at least 1 mL of PBS.
7. Add 2 mL of M1 polarization media into one of the imaging dishes, allow the RAW 264.7 cells to induce the M1 macrophage phenotype, and incubate 2–3 h at 37 °C with 5% CO₂.
   NOTE: Seed RAW 264.7 macrophages in an imaging dish with DMEM supplemented with 10% FBS as a control for the anti-iNOS antibody staining. Label the imaging dishes RAW (control) and M1 macrophages (LPS and IFN-γ polarized), respectively.
8. Before immunostaining, fix the RAW 264.7 and M1 macrophages cells using 2 mL of 4% paraformaldehyde and incubate for 15 min at 37 °C (room temperature, RT).
9. Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.
10. Quench with 2 mL of 50 mM ammonium chloride in PBS and incubate for 15 min at RT.
11. Permeabilize using 2 mL of 0.3% Triton X-100 in PBS for 15 min at RT.
12. Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.
13. Block using 2 mL of 10% FBS in PBS for 30 min at RT.
    NOTE: The blocking step in immunostaining is to minimize the non-specific binding of antibody within the cell.
14. Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.
15. Incubate with 2 mL of anti-iNOS-FITC in PBS and 1% FBS overnight at 4 °C.
    NOTE: Label the cells using the appropriate antibody dilution according to the manufacturer's recommendations. Protect the dishes from light by covering them with aluminum foil from this point on.
16. Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.
17. Incubate 1 mL of 300 nM 6-diamidino-2-phenylindole (DAPI) into imaging dishes for 10 min in 37 °C, in the dark by covering with aluminum foil.
18. Wash cells with 1 mL of PBS at least 3x and add 1 mL of complete DMEM medium into the imaging dishes.
    NOTE: The cells are now ready for fluorescence imaging. The microscope setup will need an inverted stage and excitation filters for the chosen stains (in this case, FITC and DAPI).
19. Turn on the microscope and load the imaging software. Mount the imaging dish on the microscope and adjust the focus to observe RAW 264.7 and M1 macrophages. Optimize the appearance of phase-contrast FITC and DAPI images by adjusting the transmitted light and exposure times.
    NOTE: Begin imaging when all points are in focus and the channels are optimized.
20. Capture phase-contrast FITC and DAPI images (Figure 1).

3. Seeding 4T1 mouse mammary carcinoma cells

1. As the confluency of 4T1 cells reaches 70−80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS. Add 1 mL of prewarmed trypsin to dissociate the cells and incubate for up to 20 min at 37 °C.
   NOTE: Observe the cells under a microscope. Detached cells will be rounded.
2. Once the cells detach, transfer them to a 15 mL conical tube and add 2 mL of prewarmed complete DMEM medium to inactivate the trypsin. Gently disperse the medium by pipetting the cell layer surface to recover the cells. Then, centrifuge at 600 x g for 5 min to obtain a cell pellet.
3. Remove the supernatant and resuspend the pellet in 10 mL of prewarmed complete DMEM medium.
4. Count the cells using a hemocytometer and seed 1 x 10⁵ 4T1 cells in 2 mL of complete DMEM medium in each of two 35 mm glass-bottom imaging dishes treated with tissue culture. Incubate the cells overnight at 37 °C with 5% CO_2.
   NOTE: Culture 4T1 cells in one of the imaging dishes with M1 polarization media and label 4T1-Inducer (control). This is to ensure normal growth of 4T1 cells when exposed to the inducers.

4. Labeling living 4T1 mouse mammary carcinoma cells using CFSE staining

1. First, remove the existing media from the 4T1 cells imaging dishes. Wash the cell monolayer at least 2x with 1 mL of PBS.
2. Prepare 5 µM of CFSE staining solution by diluting CFSE in 1 mL of PBS. Add 1 mL of 5 µM CFSE staining solution into each imaging dish and incubate the cells for 20 min at RT or 37 °C, in the dark.
   NOTE: Label the cells using appropriate CFSE working concentration according to the manufacturer's recommendations and preliminary experiments to obtain the optimum CFSE staining concentration. Labeling efficiency will be higher if CFSE is diluted in PBS.
3. Quench the staining by adding an equal volume of complete DMEM medium containing 10% FBS and staining solution to the cells and incubate for 5 min at RT in the dark.
4. Discard the CFSE-containing solution and wash the cells 1x with an equal volume of culture media.
   NOTE: 4T1 cells are now fluorescently labeled.
5. Return the 4T1-CFSE cells to standard culture conditions at RT with 5% CO_2.

5. Seeding RAW 264.7 mouse macrophages and coculture with 4T1

1. As confluency of RAW 264.7 reach 70−80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS.
2. Prepare the macrophages for collection by gently dislodging the adherent cells using a cell scraper and transfer them into a 15 mL conical tube.
3. Count the cells using a hemocytometer and prepare a cell suspension of 1 x 10⁵ cells/mL by diluting the remaining solution with a complete DMEM medium according to the seeding density.
   NOTE: Overly confluent cells in cocultures may severely reduce phagocytosis. In this study, the seeding ratio of macrophages: cancer cells was adjusted to a 1:1 ratio. The ratio can be adjusted depending on the aggressiveness of the tumor cells and the origin of the macrophages.
4. Before coculturing, discard the culture supernatant from the 4T1 cells seeded a day before (step 4.5) and carefully wash the monolayers 2x with at least 1 mL of PBS.
5. Seed 1 x 10⁵ RAW 264.7 cells per 2 mL of complete DMEM medium into one of the 4T1 imaging dishes. Incubate the cells 2−3 h at 37 °C with 5% CO₂ to allow cell adherence. Label the imaging dish 4T1-M1 coculture.
   NOTE: The 4T1 (control) cells were not cocultured with macrophages.

6. M1 polarization of RAW 264.7 macrophages

1. Prepare M1 polarization media by adding 100 ng/mL LPS and 20 ng/mL IFN-γ into complete DMEM medium supplemented with 10% (v/v) FBS^10,11.
2. Discard the culture supernatant from the macrophages incubated before (step 5.5) and carefully wash the monolayers in the dish 2x with at least 1 mL of PBS.
3. Then, add 2 mL of M1 polarization media into the imaging dish and allow the RAW 264.7 cells to induce into the M1 macrophage phenotype by incubating at 37 °C with 5% CO₂.
   NOTE: M1 macrophage cells may take up 2−3 h of incubation to achieve complete polarization. Preliminary experiments need to be done to ensure the suitable incubation period to allow complete polarization of macrophages.

7. Live-cell video microscopy of phagocytosis

NOTE: Many factors need to be considered when performing live-cell imaging to obtain a producible video, including optimization of image exposure, time measurement, and automatic focus correction.

1. Before the experiment, set up the cell culture incubator by turning on thermostat at 37 °C and supplying 5% CO₂.
   NOTE: The microscope setup requires an inverted stage, a stage top incubator, humidity control (95%), and gas incubation system. This setup is crucial to maintain the cells for long-term live-cell video microscopy assessment.
2. Turn on the microscope including the piezo stage and load the microscope imaging software.
3. Position the seeded coculture cells in a 35 mm glass-bottom imaging dish in the center of the stage and gently screw on the top chamber. Use the joystick to motorize the stage by selecting the position to be imaged.
4. To view the sample, select the phase-contrast filter on the turret. After initially viewing the sample, locate the appropriate fields and bring the sample into focus.
   NOTE: Imaging software allows use of fully automated objective selection, Perfect Focus System, time-lapse series, multichannel image acquisition, and multipoint image acquisition.
5. To set up multichannel fluorescence for CFSE labeling and differential interference contrast acquisition, open the imaging software acquisition dialog box and select the [Lambda] tab checkbox (Figure 2).
   NOTE: For a 13 h time-lapse imaging, a phase-contrast channel should be used.
6. Select [Lambda] tab | [Optical Configuration] and select [10X DIC] for differential interference contrast and [GFP-R] for the green fluorescence filter checkbox. Under the [Lambda] | [Focus] column, select the [10X DIC] checkbox to set as the focus reference.
7. Open the imaging software acquisition dialog box and click the [XYZ Time …] button and select the [XY] tab.
8. Move to the image acquisition point using the joystick on the motorized stage while checking the live image or select a different position through the eyepieces. Then click on the checkbox under the [Point Name] column to set each point in each location for image capture (see Figure 3).
   NOTE: The automated stage allows multipoint image acquisition of different XY coordinates to capture multiple fields.
9. Fine-tune the focus for each sample viewed on the screen. Use the controls on Auto-focus correction.
10. Use the software to set up time-lapse image capture. In the imaging software acquisition dialog box, check the [Time] tab (see Figure 4).
11. Determine the [Interval] (the delay between the beginning of one time point to another time point) and [Duration] (the total length of time of the experiment). Units of time vary and can be selected in milliseconds (ms), seconds (s), minutes (m), or hours (h) from the dropdown menu.
    NOTE: The imaging period length for time-lapse imaging of fluorescence and DIC time-lapse multichannel acquisition may vary depending on the fluorescent dye used in this assay. Photobleaching may occur if the labeled cells are exposed for too long.
12. Check the [Save to File] checkbox to save acquired images. Under the [Time schedule] tab click the [Run now] button to acquire multipoint time-series images (see Figure 5).
    NOTE: In the imaging software, images are saved in nd2 file format. Each time-lapse can be individually saved in an MP4 file format later.

Results

The time-lapse two-dimensional (2D) images of the coculture model of 4T1 mouse mammary carcinoma cell lines show the 4T1 cells being engulfed by M1 macrophages during a 13 h period. It is important to ensure a complete polarization of the M1 macrophages by performing immunostaining. The results (Figure 1) show that the concentration of 100 ng/mL lipopolysaccharides (LPS) and 20 ng/mL IFN-γ polarized RAW 264.7 macrophages into the M1 state. Labeling the targeted cells with a fluorescent dye a...

Discussion

The protocol described requires two steps: 1) coculture of 4T1 mouse mammary carcinoma cells and M1-polarized macrophages, and 2) assessment of the macrophage phagocytic activity using time-lapse microscopy. Live cell coculture is widely used in phagocytosis and migration assays. The live cell coculture model here is a simple, adaptable in vitro procedure (Figure 2) that utilizes a fluorescent dye, CFSE, which is used to label the 4T1 targeted cells. This is used for proper tracking of the c...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-INOS antibody conjugated FITC	Miltenyi Biotec	REA982	150 µg in 1 mL
Carboxyfluorescein succinimidyl ester (CFSE)	Invitrogen	C1157	25 mg
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen	D1306	10 mg
DMEM/high glucose	HyClone	SH30003.04	670.0 g
Fetal bovine serum	Tico Europe	FBSEU500	500 mL
Lipopolysaccharides	Sigma	L4516-1MG	1 mg
Mouse recombinant interferon-gamma	Stemcell Technologies	78021	100 µg
Penicillin-streptomycin solution	Cellgro	30-003-CI	100 mL
Phosphate buffered saline	Sigma-Aldrich	P5368-10PAK	10 pack
Trypsin EDTA	Cellgro	25-052-CI	1X, 100 mL
1000 µL pipette tips	WhiteBox	WB-301-01-052	5000 tips/case
2 mL serological pipette	JET BIOFIL	GSP012002	Non-pryogenic
200 µL pipette tips	WhiteBox	WB-301-02-302	20,000 tps/case
25 cm2 cell culture flask	Corning	CLS430639	Tissue culture treated
Bench top centrifuge	Dynamica	FA15C	Model: Velocity 14R
Biological safety fume hood	Nuaire	NU-565-400	Model: Home/ LabGard® ES TE NU-565 Class II, Type B2 Biosafety Fume Hood
CO2/air mixer	Chamlide (Live Cell Instrument)	FC-R-20	FC-5 (CO2/Air Mixer) with the flow meter
Cell scrapper	NEST	710001	220 mm
CO2 cell incubator	Panasonic	N/A	Model: MCO-19M(UV)
Confocal microscope	Nikon Instruments Inc.	N/A	Nikon Ti-Eclipse
Glass bottom dish	Ibidi	81218-200	35 mm
NIS elements software	Nikon Instruments Inc.	Available online download
Pipette controller	CappAid	PA-100	CappController pipette controller, 0.1-100ml
Thermostat	Shinko	Discontinued	JCS-33A 48 x 48 x 96.5mm