Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Summary

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Abstract

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

Introduction

Tumor-associated macrophages (TAM) and their role in chronic inflammation, the onset of cancer, and tumor development are important targets in recent research^1,2. Peripheral blood monocytes that are recruited to the tissue microenvironment of the developing tumor differentiate into macrophages and can be polarized into two main subtypes of macrophages³. The classically activated macrophage represents the primarily pro-inflammatory M1-like phenotype and the alternatively activated M2-like subtype shows predominantly anti-inflammatory characteristics⁴. Macrophages can switch dynamically between these two main phenotypes depending upon their cellular metabolism, with intermediate subtypes having both inflammatory and anti-inflammatory attributes⁵. TAM represents a heterogeneous population of both phenotypes. A tumor-promoting function and poor prognosis in different types of cancers is, however, particularly associated with M2-like macrophages^6,7,8.

The functional profiles of macrophages and their interaction with other cells within the tumor microenvironment are complex and challenging to capture in a continuously changing environment during ongoing tumor development. Cell lines can provide a homogenous cell population with stable viability in culture, which can facilitate the process of demonstrating defined cellular and intercellular mechanisms. The monocyte-like THP-1 cell line is a legitimate model system for primary human monocytes⁹. This spontaneously immortalized cell line has been obtained from the peripheral blood of a one-year-old infant with acute monocytic leukemia^9,10. The differentiation and polarization of THP-1 cells have been reported by several studies and have been performed in multiple different ways^11,12,13,14. Activation and, therefore, the polarization of macrophages into an M1-like phenotype is followed by a compensatory anti-inflammatory rebound mechanism, promoting an M2-like phenotype through cytokines produced by inflammatory macrophages, such as interleukin 6 (IL-6) or itaconate^15,16. This might serve as a break mechanism to attenuate an overshooting inflammatory response following cell activation¹⁷. The process of differentiating and polarizing monocytes and THP-1 monocyte-like cells into an anti-inflammatory M2-like phenotype is itself also accompanied by pro-inflammatory stimuli that must be overcome. An inflammatory cytokine response can be caused by mechanical stress¹⁸, such as changing media to refeed the cells, or adding chemical compounds to differentiate THP-1 cells, such as phorbol 12-myristate 13-acetate (PMA), and induce production of tumor necrosis factor α (TNFα), interleukin 1β (IL-1β) or IL-6¹⁹. This altered cytokine expression profile as a response to PMA can affect and prevent subsequent macrophage polarization²⁰. Adequate resting periods, as reported before after PMA treatment, allow these inflammatory responses to decrease and facilitate cell polarization into a distinct M2-like phenotype²¹.

This protocol demonstrates a method to differentiate and polarize THP-1 monocyte-like cells into an M2-like phenotype of macrophages within 14 days.

Protocol

NOTE: An overview of the steps described in this protocol is shown in Figure 1. The human monocyte-like leukemia cell line THP-1 was purchased. Short tandem repeat analysis was performed to authenticate the THP-1 cell line. Perform all steps under sterile conditions. The THP-1 monocytic cell line grows in suspension and does not attach to cell culture surfaces. Adherence can be induced by differentiating monocytes into macrophage-like cells through, e.g., mechanical stress or specific treatment with PMA.

1. Culturing and maintenance of THP-1 monocyte-like cells

1. Set a timer for 150 s. Remove the frozen vial containing the THP-1 cell line (Table of Materials) from the liquid nitrogen and thaw it immediately in a clean water bath (37 °C). Start the timer as soon as the vial is put into the water bath. Loosen the cap to release the pressure that is building up due to the thawing process but ensure that the tube opening does not contact the water, to avoid contamination. The optimal time period for thawing the cells lies between 120-150 s. Continue thawing the cell suspension until an ice chip of the size of about 4 mm is left within the vial; then, proceed to the next step immediately.
2. Transfer the liquid phase of the cell suspension to a 15 mL tube containing 9 mL of warm (37 °C) growth media (Table of Materials). Then, transfer 1 mL of the warm medium-cell suspension into the THP-1 vial and back into the 15 mL tube to melt the remaining ice chip and flush the vial to assure that no cells are left behind.
3. Mix the suspension gently by pipetting up and down with a 1000 µL pipette. Remove a small sample (approximately 10 µL) to count the cells for viability (using trypan blue for exclusion) while they are spun. Spin down the warm cell suspension at 200 x g for 7 min at 37 °C.
4. Remove the supernatant completely and resuspend with a certain volume of warm growth medium to achieve a cell density of 5 x 10⁵/mL. Mix the suspension gently and transfer 22 mL of volume into T-75 cell culture flasks (Table of Materials). Store flasks upright in an incubator at 37 °C with 5% carbon dioxide (CO₂) concentration. Exchange the growth media every 3-4 days.

2. Seeding of THP-1 cells and differentiation into M0 macrophages

1. Prepare the cell containing growth medium with the respective cell density to seed cells at a density of 3 x 10⁵/mL/well into 24-well cell culture plates (Table of Materials). Mix the medium gently and prepare aliquots of 26 mL, each put into a 50 mL tube. Use each 26 mL aliquot for seeding the cells into a respective plate.
2. Transfer 1 mL of the cell-containing medium into each well of a 24-well plate. Mix the media gently by pipetting up and down between transfers.
3. Prepare a stock solution of PMA (dissolve 1 mg of PMA in 100 µL of Dimethyl Sulfoxide (DMSO) = ~16 mM solution of PMA in DMSO) and dilute it with cold Phosphate Buffered Saline (PBS) to a final working concentration of 10 ng/µL right before cell treatment (Table of Materials). Keep the solution on ice and use it immediately. Do not refreeze. Add 100 ng of PMA per well. Let each cell plate sit in the incubator without any further treatment for 72 h.
4. After 72 h, remove the growth medium and replace it with 1 mL of fresh growth medium. Do not touch the bottom of the wells with pipette tips. Let the cells rest for another 96 h in the incubator.
5. After 96 h, repeat step 2.4 (media change) and let the cells rest for another 24 h.
   ​NOTE: The M0 macrophages are now ready to be used for experiments (Figure 2). Immediately prior to treating the cells as part of further experiments, consider a media change with RPMI only (Table of Materials) since growth media supplements can cause interference with reagents that are added for cell treatment. In case M2-like macrophages are needed, proceed with section 3.

3. Polarization of M0 macrophages into M2-like macrophages

1. Prepare a stock solution of IL-4 and IL-13 (dissolve 20 µg of IL-4 or IL-13 in 200 µL of nuclease-free water) and dilute it to a final working concentration of 2 ng/µL with PBS immediately prior to cell treatment. Keep the solution on ice and use it immediately. Do not refreeze.
2. Remove the growth medium and replace it with 1 mL of fresh growth medium. Add 20 ng of interleukin 4 (IL-4) and 20 ng of interleukin 13 (IL-13) per well. Let the cells rest for 48 h in the incubator.
3. After 48 h, repeat step 3.2. Let the cells rest for another 48 h in the incubator.
4. Remove the growth medium and replace it with 1 mL of fresh growth medium. Let the cells rest for 48 h in the incubator.
   NOTE: M2-like macrophages are now ready to be used for experiments (Figure 2). Immediately prior to treating the cells as part of further experiments, consider a media change with RPMI only (Table of Materials) since growth media supplements can cause interference.

4. Detaching and harvesting macrophages for flow cytometry

NOTE: Use a mechanical method combining cold shocking and cell scraping to detach and harvest the polarized macrophages from plates for flow cytometry.

1. Remove the warm cell medium and replace it with a mixture of ice-cold PBS (without calcium and magnesium) and 5% fetal bovine serum (FBS), 1 mL per well. Immediately after this, place the cell plate on ice for 45 min. Do not place the cell plate on ice before the warm cell medium is removed, since this will decrease cell viability significantly. Keep the cells on ice only after inducing cold shock with ice-cold PBS/5% FBS mixture.
2. After 45 min on ice, scrape off the cells using mini cell scrapers (Table of Materials). Gently transfer the detached macrophages in cold PBS/5% FBS into a 15 mL tube. Keep the tube on ice at all times until cells are stained.
   NOTE: Pool eight wells of cells to reach adequate cell counts for staining.

Results

M2-like macrophages were characterized, and M2-polarization was validated using flow cytometry for Cluster of Differentiation markers (CD) CD14, CD11b, CD80 (M1-like marker), and CD206 (M2-like marker). Flow cytometry staining was performed according to the manufacturer's instructions. Macrophages were washed with PBS/5% FBS and incubated with Fcγ-receptor block to avoid unspecific binding. Cells were then stained with FITC-conjugated mouse anti-human CD14 and CD80 antibodies, with PE-conjugated mouse anti-human...

Discussion

This protocol on differentiating and polarizing THP-1 monocyte-like cells within 14 days provides a method to obtain macrophages with a distinct M2-like phenotype due to long treatment incubation of cells with adequate resting periods between steps.

Certain steps are critical to this protocol. The doubling time of THP-1 monocytes is approximately 26 h. Cells can be split at a cell density of 9 x 10⁵/mL and should be seeded at a density of 3 x 10⁵/mL during every split. Th...

Materials

Name	Company	Catalog Number	Comments
0.4% trypan blue	VWR, Radnor, USA	152-5061
1.5 mL microcentrifuge tube	USA Scientific, Ocala, USA	1615-5510
10 mL serological pipet	VWR, Radnor, USA	89130-898
1000 μL TipOne pipet tips	USA Scientific, Ocala, USA	1111-2821
15 mL  Centrifuge tube	VWR, Radnor, USA	89039-664
20 μL TipOne pipet tips	USA Scientific, Ocala, USA	1120-1810
200 μL TipOne pipet tips	USA Scientific, Ocala, USA	1120-8810
25 mL serological pipet	VWR, Radnor, USA	89130-900
5 mL serological pipet	VWR, Radnor, USA	89130-896
50 mL Centrifuge tube	VWR, Radnor, USA	89039-662
Accutase solution 500 mL	Sigma, St. Louis, USA	A6964
Antibiotic Antimycotic Solution (100x), stabilized	Sigma, St. Louis, USA	A5955-100 mL	with 10,000 units penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per mL, sterile-filtered, BioReagent, suitable for cell culture
Binder CO2 Incubator	VWR, Radnor, USA	C170-ULE3
CytoOne T-75cm flask with filter cap	USA Scientific, Ocala, USA	CC7682-4875
Dulbecco’s Phosphate Buffered Saline (PBS)	Sigma, St. Louis, USA	D8537-500 mL	PBS without calcium chloride and magnesium chloride should be used, since both can alter macrophage polarization
Eppendorf Centrifuge 5804 R (refrigerated)	Eppendorf, Enfield, USA	-
Ethyl alcohol (70%)	-	-
FACSCalibur flow cytometer	BD Biosciences, San Diego, USA	-	The flow cytometer operates with CellQuest software (BD Biosciences)
Falcon 24-well plate	VWR, Radnor, USA	353504
Fetal Bovine Serum (FBS)	ATCC, Manassas, USA	30-2020
FITC Mouse Anti-Human CD14	BD Biosciences, San Diego, USA	555397	Flow cytometry, myeloid cell marker (100 tests)
FITC Mouse Anti-Human CD80	BD Pharmingen, San Diego, USA	557226	Flow cytometry, M1 marker (100 tests)
FITC Mouse IgG1 κ Isotype Control	BD Pharmingen, San Diego, USA	555748	Flow cytometry, isotype control for CD80 (100 tests)
FITC Mouse IgG2a, κ Isotype Control	BD Biosciences, San Diego, USA	553456	Flow cytometry, isotype control for CD14 (100 tests)
Human BD Fc Block	BD Biosciences, San Diego, USA	564220	Flow cytometry, Fc block (0.25 mg)
Human interleukin 13 (IL-13)	R&D, Minneapolis, USA	IL-771-10 μg
Human interleukin 4 (IL-4)	R&D, Minneapolis, USA	SRP3093-20 μg
Labconco Biosafety Cabinet (Delta Series 36212/36213)	Labconco, Kansas City, USA	-
L-Glutamine Solution, 200 mM	ATCC, Manassas, USA	30-2214
Lipopolysaccharide (LPS) from E. coli 0111:B4	Sigma, St. Louis, USA	L2630-100 mg
Mini Cell Scrapers	Biotium, Fremont, USA	22003
Neubauer hemocytometer	Fisher Scientific, Waltham, USA	02-671-5
Nikon Eclipse inverted microscope TS100	Nikon, Melville, USA	-
Nuclease-free water	Invitrogen, Carlsbad, USA	AM9937
Olympus Light Microscope RH-2	Microscope Central, Feasterville, USA	40888
P10 variable pipet- Gilson	VWR, Radnor, USA	76180-014
P1000 variable pipet-Gilson	VWR, Radnor, USA	76177-990
P200 variable pipet- Gilson	VWR, Radnor, USA	76177-988
PE Mouse Anti-Human CD11b	BD Biosciences, San Diego, USA	555388	Flow cytometry, myeloid cell marker (100 tests)
PE Mouse IgG1, κ Isotype Control	BD Biosciences, San Diego, USA	555749	Flow cytometry, isotype control for CD11b (100 tests)
PE-Cy 5 Mouse Anti-Human CD206	BD Pharmingen, San Diego, USA	551136	Flow cytometry, M2 marker (100 tests)
PE-Cy 5 Mouse IgG1 κ Isotype Control	BD Pharmingen, San Diego, USA	555750	Flow cytometry, isotype control for CD206 (100 tests)
Phorbol 12-myristate 13-acetate (PMA)	Sigma, St. Louis, USA	P8139
Powerpette Plus pipettor	VWR, Radnor, USA	75856-448
Precision Water bath (model 183)	Precision Scientific, Chicago, USA	66551
RPMI-1640 Medium	ATCC, Manassas, USA	30-2001
THP-1 cell line, American Type Culture Collection (ATCC)	ATCC, Manassas, USA	TIB-202