In Vitro Assay to Study Tumor-macrophage Interaction

Summary

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Abstract

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

Introduction

Macrophages are immune cells with high phenotypic and functional heterogeneity¹. They play important roles in the host defense systems, tissue repair, development and tumor progression¹. TAMs are macrophages in the microenvironment of solid tumors. Certain TAMs can promote tumor growth through inhibiting T cell-mediated cytotoxic activity, modulating the tumor microenvironment (TME), promoting angiogenesis, invasion and metastasis^2,3,4,5. TAMs are among the most abundant cell types in the TME and higher number of TAMs generally correlates with worse patient survival in many types of solid tumors⁶. The distinct genetic signatures of the tumor cells affect their ability to recruit macrophages. In GBM, an aggressive brain tumor with no cure, macrophages can represent up to a half of the tumor mass⁷. Co-amplification of the epidermal growth factor receptor (EGFR) and its truncation mutant EGFRvIII is frequently observed in GBM, which confers tumor growth advantages⁸. Cells co-expressing EGFR and EGFRvIII attract more macrophages compared to cells expressing EGFR or EGFRvIII singly⁷.

Chemokines are a family of small cytokines that play significant roles in regulating immune composition in the TME^6,9. Human cells express more than 50 cytokines¹⁰. Immune infiltration in the tumors is largely realized by the interaction between cytokines and cytokine receptors¹¹. Each type of immune cells expresses distinct chemokine receptors/chemokines and can be recruited by cells secreting specific chemokines/chemokine receptors¹². Cancer cells can increase expression of certain chemokines to recruit immune cells such as TAMs, regulatory T cells and myeloid-derived suppressor cells (MDSCs)⁶. Blockade of specific chemokine secreted by the tumors can be a promising way in inhibiting infiltration of immune cells into the tumor mass.

Here, we describe a protocol that allows in vitro evaluation of tumor-macrophage interaction, using conditioned media from the tumor cells containing chemokines and macrophage cell lines.

Protocol

1. Medium Preparation

1. Preparation of the serum-free stem cell medium
   1. Thaw the 50x B27 supplement, the epidermal growth factor (EGF, 20 μg/mL in 10 mM acetic acid with 0.1% BSA), and the fibroblast growth factor (FGF, 20 μg/mL in 10 mM acetic acid with 0.1% BSA).
   2. Add 500 μL of EGF (final concentration 20 ng/mL), 500 μL of FGF (final concentration 20 ng/mL), 10 mL of 50x B27 to 500 mL of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) and 5 mL of 100x Penicillin/Streptomycin (Pen/Strep). Invert the bottle 4-6 times to mix, filter sterilize through a 500 mL 0.1 μm filtration cup. Mark the bottle and store at 4 °C.
      NOTE: The medium is good to use for up to a month at 4 °C.
2. Prepare the MV-4-11 culture medium: Add 50 mL of fetal bovine serum (FBS) to 500 mL of Iscove's Modified Dulbecco's Medium (IMDM). Invert the bottle 4-6 times to mix, filter sterilize through a 500 mL 0.1 μm filtration cup. Mark the bottle and store at 4 °C.
   NOTE: The medium is good to use for up to a month at 4 °C.
3. Prepare the tumor cell maintenance medium: Add 50 mL of FBS to 500 mL of Dulbecco's Modified Eagle Medium (DMEM). Invert the bottle 4-6 times to mix, filter sterilize through a 500 mL 0.1 μm filtration cup. Mark the bottle and store at 4 °C.
   NOTE: The medium is good to use for up to a month at 4 °C.

2. Cell Preparation

Day 1:

1. Thawing tumor cells
2. Warm up the tumor cell maintenance medium (as described in step 1.3) at a 37 °C water bath for 20 min.
3. Take the frozen U87, U87:EGFR, U87:EGFRvIII and U87:EGFR+EGFRvIII cells from the liquid nitrogen tank. Thaw the cells at the 37 °C water bath for 2 min.
   CAUTION: Be careful when taking the cells from the liquid nitrogen tank. Wear gloves with thermal protection and safety goggles as needed.
4. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels.
5. Spray the cryogenic vials containing cells and the medium bottle with 70% ethanol, wipe off 70% ethanol on the surface with paper towels, and bring them into the tissue culture hood.
6. Pipette 5 mL of tumor cell maintenance medium into a 15 mL sterile centrifuge tube. Invert the tube containing tumor cells 4-6 times to mix. Pipette all the cells into the 15 mL centrifuge tube. Invert the centrifuge tube 4-6 times to mix.
7. Centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
8. Wash the cells with 10 mL of phosphate buffered saline (PBS). Centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
9. Resuspend the cells in 12 mL of tumor cell maintenance medium (as described in step 1.3). Pipette up and down 3-5 times to mix. Transfer the cells into a T75 cell culture flask.
10. Grow the cells in a 37 °C incubator with 5% CO₂. Put the tumor cell maintenance medium back to the 4 °C fridge.

Day 2:

2. Check the cells under the microscope. Change the medium if needed. Cells should grow in a monolayer in culture.

Day 3:

3. Thawing MV-4-11 cells
   1. Warm up the MV-4-11 culture medium (as described in step 1.2) at a 37 °C water bath for 20 min.
      NOTE: MV-4-11 can be changed to primary macrophages or other macrophage cell lines, depending on the need of the specific study.
   2. Take the frozen MV-4-11 cells from the liquid nitrogen tank. Thaw the cells at a 37 °C water bath for 2 min.
      CAUTION: Be careful when taking the cells from the liquid nitrogen tank. Wear gloves with thermal protection and safety goggles as needed.
   3. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels.
   4. Spray the cryogenic vials containing cells and the medium bottle with 70% ethanol, wipe off 70% ethanol on the surface with paper towels, and bring them into the tissue culture hood.
   5. Pipette 5 mL of MV-4-11 culture medium (as described in step 1.2) into a 15 mL sterile centrifuge tube. Invert the tube containing tumor cells 4-6 times to mix. Pipette all the cells into the 15 mL centrifuge tube. Invert the centrifuge tube 4-6 times to mix.
   6. Centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
   7. Wash the cells in 10 mL of PBS and centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
   8. Resuspend the cells in 12 mL of MV-4-11 culture medium (as described in step 1.2). Gently pipette up and down 3-5 times to mix. Transfer the cells into a T75 cell culture flask.
   9. Grow the cells in a 37 °C incubator with 5% CO₂.
4. Splitting tumor cells
   1. Warm up the serum-free stem cell medium (as described in step 1.1) at a 37 °C water bath for 20 min.
   2. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Take the cell detachment solution from the fridge. Spray the medium and cell detachment solution bottles with 70% ethanol, wipe off 70% ethanol on the surface with paper towels and bring them into the tissue culture hood.
      NOTE: Do not pre-warm the accutase cell detachment solution.
   3. Transfer the tumor cell culture from the incubator into the tissue culture hood. Aspirate the medium, add 2 mL of cell detachment solution into the flask.
      NOTE: Rinsing with PBS before adding accutase is optional here.
   4. Set the flask in the hood. Check if the tumor cells round up every minute. Once the tumor cells round up, gently tap the side of the flask to help the cells detach from the flask.
   5. Pipette 8 mL of serum-free stem cell medium into the flask, pipette up and down 3 times to mix, and transfer the cells into a 15 mL sterile centrifuge tube. Centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
   6. Wash the cells in 10 mL of PBS. Pipette up and down 3-5 times to mix. Centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
      NOTE: This step is optional.
   7. Resuspend the cells in 10 mL of serum-free stem cell medium (as described in step 1.1). Pipette up and down 3-5 times to mix. Take 10 μL of the cells and mix with 10 μL of Trypan Blue solution. Pipette 10 μL of the mixture into the counting slide, quantify living cell number using an automatic cell counter.
   8. Adjust the cell density to 2.5 x 10⁵ cells/mL with serum-free stem cell medium (as described in step 1.1), and seed 2 mL of the cells into each well of a 6-well cell-culture plate. For each type of cells, seed 3 wells (triplicate sample). At the same time, prepare 6 wells with 2 mL of serum-free stem cell medium only (no cells).
      NOTE: These samples will be used: 1) as negative controls and 2) for adjusting conditioned medium volume based on cell numbers.
   9. Put the 6-well plates in a 37 °C incubator with 5% CO₂. Allow the cells to grow for 24 h.

3. In Vitro Macrophage Attraction Assay

1. Preparation of conditioned media
   1. Warm up the tumor cell maintenance medium (as described in step 1.3) at a 37 °C water bath for 20 min.
   2. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Take the cell detachment solution from the fridge. Spray the bottles of the medium and the cell detachment solution with 70% ethanol, wipe off 70% ethanol on the surface with paper towels and bring them into the tissue culture hood.
   3. Take the 6-well plates out from the incubator. Carefully pipette the conditioned media into 15 mL sterile centrifuge tubes. Put the tubes on ice. Pipette the media in the “media only” well into a separate 15 mL sterile centrifuge tube.
      NOTE: Before this step, make sure to check under the microscope to see if the cells attach. If not, one can use coated plates at step 2.4.8 to allow better attachment or include the floating cells in the counting step.
   4. Add 0.5 mL of cell detachment solution into each well of the 6-well plate. Check if the tumor cells round up every minute. Once the tumor cells round up, gently tap the side of the plate to help the cells detach.
   5. Pipette 2.5 mL of tumor cell maintenance medium into the well, pipette up and down 3 times to mix and transfer the cells into a 15 mL sterile centrifuge tube. Take 10 μL of the cells and mix with 10 μL of Trypan Blue solution. Pipette 10 μL of the mixture into the counting slide and quantify the number of living cells using an automatic cell counter.
   6. Adjust the volume of the conditioned media according to cell number using the serum free stem cell medium (37 °C incubation overnight). For example, if Well A has 3x as many live cells as the well with the least number of cells, dilute its conditioned media 1:3. This step is used to control difference caused by cell proliferation rate.
      NOTE: The reason to use serum free stem cell medium with 37 °C incubation overnight is to control the possible change in the media with prolonged 37 °C exposure.
   7. Filter the conditioned media through 0.45 μm filters. Put the conditioned media on ice.
      NOTE: This step removes cells and cell debris in the conditioned media.
2. Preparation of MV-4-11 cells
   1. Warm up the IMDM medium (without FBS) at a 37 °C water bath for 20 min.
   2. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Spray the medium bottle with 70% ethanol, wipe off 70% ethanol on the surface with paper towels and bring them into the tissue culture hood.
   3. Take the flask of MV-4-11 cells into the tissue culture hood. Pipette 10 mL of cells into a 15 mL sterile centrifuge tube. Take 10 μL of cells and mix with 10 μL of Trypan Blue solution. Pipette 10 μL of the mixture into the counting slide and quantify the number of living cells using an automatic cell counter. Centrifuge the cells at 200 x g for 5min at 4 °C. Aspirate the supernatant.
   4. Wash the cells with 10 mL of PBS and centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
   5. Resuspend the cells in IMDM medium (without FBS) for a final cell density of 1x10⁶ cells/mL.
      NOTE: The cell number of macrophages used here can be adjusted.
3. Transwell assay
   NOTE: For this step, use a cell migration assay kit or purchase the reagents and insert separately. For MV-4-11 cells, choose Transwell inserts with 5 μm pore size. To detect macrophage migration, directly quantify the number of macrophages in the lower chamber or use colorimetric/fluorimetric methods. Here, we demonstrate the assay using the colorimetric method.
   1. Bring the 24-well plate, the insert and the reagents to room temperature.
   2. Add 250 μL of MV-4-11 cells prepared above into each insert.
   3. Add 400 μL of conditioned medium/medium only (with overnight incubation at 37 °C, these samples serve as the negative control) to the lower chambers of the 24-well plate. For each tumor line or control, use triplicate samples. Avoid bubbles between the insert and the conditioned medium. Bubbles will inhibit macrophages from migrating to the bottom wells.
   4. Incubate the plates in a 37 °C incubator with 5% CO₂ for 4 h.
      NOTE: The incubation time can be adjusted. Cells migrating into the lower chambers can be seen under the microscope.
   5. Gently tap the insert on the inner wall of the same well and discard the insert. As MV-4-11 cells grow in suspension, cell detachment solution or trypsin treatment is not needed here.
   6. Gently pipette the cells in the wells up and down 3 times to mix. Transfer 225 μL of cell suspension to a black-walled 96-well plate suitable for fluorescent measurement.
   7. Dilute the CyQuant dye 1:75 with 4x lysis buffer. Vortex briefly and spin down the solution. Add 75 μL of solution to each well of the 96-well plate. Incubate 15 min at room temperature.
   8. Read fluorescence using the 480/520 nm filter set with a fluorescence plate reader.
   9. Analyze data by subtracting the blank and normalizing to the control tumor cell line.

Results

The results are usually showed via bar graphs (example shown in Figure 1). Samples with high 480/520 values indicate that the conditioned media has high capacity to recruit macrophages. Depending on experimental need, additional controls can be included. For example, one can use neutralizing antibodies to treat the conditioned media to abolish the macrophage chemotaxis, and perform the same assay. One can also add extra chemokines (i.e., CCL2) to the conditio...

Discussion

In this protocol, there are several key steps: 1) selection of the Transwell insert. For the MV-4-11 cell line, 5 μm Transwell inserts work well. However, for other cell lines such as the commonly used monocyte cell line THP-1, a different pore size might work better. 2) As different cell lines grow at different speeds, it is important to adjust the volume of the conditioned media according to cell numbers. For this purpose, cell-free media incubated under the same experimental conditions can be used to dilute the c...

Materials

Name	Company	Catalog Number	Comments
0.1 μm filtration cup	Thermo fisher	566-0010
0.45 μm filter unit	Millipore	SLHA033SS
10 mL serological pipettes	Olympus plastics	12-104
15 mL sterile centrifuge tubes	Olympus plastics	28-103
1 mL pipette tip	ART molecular bioproducts	2779-RI
2 mL aspirating pipet	Falcon	357558
24-well plate	Millipore	ECM507	Part of ECM507, or can be purchased separately
4x lysis buffer	Millipore	ECM507	Part of ECM507, or can be purchased separately
5 μm Transwell insert	Millipore	ECM507	Part of ECM507, or can be purchased separately
75 cm2 flask	Corning	430641U
Accutase	Innovative cell technologies	AT-104
B27	Gibco	12587-010
CyQuant Dye	Millipore	ECM507	Part of ECM507, or can be purchased separately
DMEM	Gibco	11965-092
DMEM:F12	Gibco	10565-018
EGF	Peprotech	AF-100-15
FBS	Gibco	26140
FGF	Peprotech	100-18B
IMDM	Gibco	12440-053
PBS	Gibco	14190-144
Pen Strep	Gibco	15140-122
Trypan blue	Biorad	1450021