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 interfe....

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 ....

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