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.
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.
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 immunity3,4. 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 functions6,7, 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 cytotoxicity8,9 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.
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
2. Immunostaining of M1 polarized RAW 264.7 macrophages
3. Seeding 4T1 mouse mammary carcinoma cells
4. Labeling living 4T1 mouse mammary carcinoma cells using CFSE staining
5. Seeding RAW 264.7 mouse macrophages and coculture with 4T1
6. M1 polarization of RAW 264.7 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.
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 and leaving the effector cells unstained allows for a live-cell coculture model (Movie 1). Throughout the 13 h and 15 min interval, the phagocytic activity of the M1 macrophages in coculture with the 4T1 cells was documented (Movie 2). The six multipoint videos (A−F in Movie 2) were recorded to assess multiple events within a single 35 mm glass-bottom imaging dish. Movie 3 shows the 4T1 cells phagocytosed by M1 macrophages. Movie 4 was chosen as an example of an in-depth video filmed from this experiment and Movie 5 shows the uptake of 4T1 cells by M1 macrophages.
Figure 1: Immunofluorescence staining with anti-iNOS-FITC in green, nuclei marker DAPI in blue, and enhanced versions of merged images. These panels represent RAW 264.7 macrophages (control) and M1 macrophages (LPS and IFN-γ stimulated) immunostained with anti-iNOS-FITC (M1 marker) in green and counterstained with nuclei marker, DAPI in blue. (A) DAPI stain can be observed both RAW 264.7 and M1 macrophages. (B) Anti-iNOS-FITC (green) only fluoresce on M1 macrophages. (C) Merged images of DAPI stain and anti-iNOS-FITC. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 2: Setting up fluorescence and DIC image acquisition in imaging software acquisition dialog control. [1] select the [Lambda] tab checkbox, [2] select [Optical Configuration] and select [10X DIC] and [GFP-R] for the green fluorescence filter checkbox, [3] select [10X DIC] | [Set this channel as the focus reference]. Please click here to view a larger version of this figure.
Figure 3: Setting up multipoint image acquisition in the imaging software acquisition dialog control. [4] Select the [XY] tab and next [5] select the checkbox under the [Point Name] column to set each point for the image capture. Please click here to view a larger version of this figure.
Figure 4: Setting up intervals and duration for time-lapse measurement image capture. The imaging software acquisition dialog control to set up time-lapse image capture. To set up the time-lapse image acquisition [6] check on the [Time] tab, [7] determine the [Interval] (by sec, min, or hour) and [8] the [Duration] (by sec, min, or hour). Please click here to view a larger version of this figure.
Figure 5: Setting up multipoint movie panels for time-lapse measurement image capture. The preview of six multipoint movies panels combined after completion of 13 h of coculture. Please click here to view a larger version of this figure.
Movie 1: A representative movie of unstained M1 macrophages and 4T1 CFSE-stained mouse mammary carcinoma cells in coculture captured using multichannel acquisition of fluorescent and DIC microscopy. Results showcasing CFSE in the green-labeled cell wall of 4T1 mouse mammary carcinoma cells cocultured with unstained M1 macrophages. The time-lapse images were acquired for a 13 h coculture period with 15 min time intervals using multichannel acquisition (step 7.3, see Figure 2). (A) Differential interference contrast, (red circle) shows small, round M1 macrophages adhering to the 4T1 cells, which have an epithelial morphology. (B) A fluorescence microscopy image of 4T1 cells stained with CFSE before phagocytosis by macrophages. (C) Merged DIC and fluorescence microscopy images of CFSE-stained 4T1 cells being engulfed by unstained M1 macrophages. The blue arrows show unstained M1 macrophages fluorescing green as they engulf CFSE-labeled 4T1 cells. Scale bar = 50 µm. Please click here to download this video.
Movie 2: Live-cell video microscopy movies from multiple stage points in a single imaging dish showing phagocytosis of 4T1 mouse mammary carcinoma cells by induced M1 macrophages. Results represent six different points (Movie 2A−F) set up and coordinated using imaging software (step 7.4, see Figure 3−Figure 5). M1 macrophages have an irregular, pancake-like morphology with a porous cytoplasm, whereas 4T1 mouse mammary carcinomas have an epithelial morphology. M1 macrophages and 4T1 cells were cocultured for 13 h inside the stage incubator at 37 °C with 5% CO2 before being observed for live-cell video microscopy. Images were captured at 15 min time intervals for 13 h. Scale bar = 50 µm. Please click here to download this video.
Movie 3: Live-cell video microscopy movie of a single coordinate point (see Movie 2) chosen to show the phagocytosis of 4T1 mouse mammary carcinomas by induced M1 macrophages in detail. The red arrow shows phagocytosis. The yellow circle shows that the number of 4T1 cells that quenched the engulfment of 4T1 cells. Scale bar = 10 µm. Please click here to download this video.
Movie 4: A detailed video to further visualize the phagocytosis process of 4T1 cells by M1 macrophages. This panel shows live M1 macrophage cells with a porous cytoplasm phenotype. Scale bar = 10 µm. Please click here to download this video.
Movie 5: Macrophages moving towards 4T1 cells to establish cell-to-cell contact, followed by the uptake of 4T1 cells by M1 macrophages (see Movie 2A). The phase-contrast microscopy time-lapse video was imaged in 15 min time intervals for 13 h of coculture inside a stage top incubator at 37 °C with 5% CO2. Scale bar = 10 µm. Please click here to download this video.
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 cell types during live-cell imaging. Time-lapse live-cell imaging was used to assess the phagocytic activity of M1 macrophages using multipoint time-lapse 2D imaging before the 13 h of coculture.
The live cell coculture model was established using 4T1 mouse mammary carcinoma cells and M1 mouse macrophages. To distinguish the cells from one another, 4T1 cells were stained using a CFSE fluorescent dye (see section 4). CFSE allows for tracking the cells and monitoring cell division. CFSE can passively diffuse into cells, making CFSE staining a suitable cell tracker to visualize phagocytosis of targeted cells. As phagocytic macrophages digest and engulf cancer cells, they take up the CFSE dye and eventually become fluorescent12,13. It is essential to seed 4T1 cells in the imaging dish before the M1 macrophages. This is due to the different sizes and shapes of both cells. The 4T1 cells have an epithelial morphology that proliferates in small clusters. Also, the 4T1 cells must be stained with CFSE before coculture with the unstained M1 macrophages. Before macrophage polarization using LPS and IFN-γ, the murine macrophages RAW 264.7 are round, small, and usually grow as single cells. After LPS and IFN-γ polarization, induced M1 macrophages have a relatively flattened, pancake-like shape, with a porous cytoplasm14,15. To ensure proper polarization of RAW 264.7 towards M1 state, 100 ng/mL LPS and 20 ng/mL IFN-γ stimulated macrophages were immunostained with an M1 marker, anti-iNOS antibody conjugated to FITC (Figure 1). This protocol is performed before coculturing the macrophages and targeted cells to ensure that the macrophages are completely polarized into the M1 state.
This study describes a method for fluorescent microscopy to visualize the phagocytic activity of living macrophages. To minimize photobleaching and to provide better fluorescence imaging, fluorescent microscopy can be combined with other imaging techniques that are nondestructive to the fluorochrome, similar to DIC. The present protocol describes the materials and methods necessary for the assessment of the phagocytosis of mouse mammary carcinoma cells by LPS and IFN-γ stimulated macrophages using fluorescent and DIC time-lapse microscopy. Nonetheless, fluorescent microscopy studies have limitations when studying live-cell imaging compared to phase-contrast time-lapse imaging. During fluorescent live-cell imaging, prolonged exposure to a high amount of excitation light can induce serious cell damage16. Photobleaching can cause significant problems in live-cell imaging. The high-intensity illumination used in live-cell imaging can reduce the ability of CFSE dye to fluoresce. Nevertheless, a 13 h phagocytosis assessment was achieved in the second part of this study using phase-contrast time-lapse 2D imaging (Movies 2–5).
Time-lapse microscopy offers advantages such as generation of data from a single experiment at any time point throughout the desired duration period and significantly, multiple stage points within a single imaging dish can be captured for a single experiment. This allows observation of various positions in a single experiment. The protocol can also be used using multiple wells from culture plates (e.g., 6, 24, 96 well plates). Among the most critical technical challenges to perform successful live-cell imaging experiment includes maintaining the cells in a healthy condition by using a stage top incubator to provide a stable temperature of 37 °C, 95% of humidity, and 5% CO2. Because the experiment took 13 h, ensuring that the microscope functions normally throughout was crucial.
During this study, six different points in the imaging dish were captured throughout a 13 h duration for 15 min intervals per point (Movie 2). The points can be adjusted depending on the event of the cell under investigation. Considering there are multiple stage points to be captured throughout this experiment, it is significant to make sure that each point has a stable focus before performing the live-cell video recording. The time interval for investigation must be optimized. A decreased time interval will have more detailed points that can be imaged; however, the file size will be very large. Increasing time interval will result in a lengthy video and will make the movie lose continuity.
The authors have nothing to disclose.
This work was financially funded by Geran Putra Berimpak (GBP), Universiti Putra Malaysia: 9542800. We would like to thank the Agro-Biotechnology Institute, Malaysia (ABI) laboratories, and microscopy imaging facility. Special thanks to Mohd Daniel Hazim for help with editing and recording the experimental video for this work.
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 |