Tumor-associated macrophages play important roles in the tumor microenvironment. Understand how different genetic mutations in the tumor cells affect macrophage recruitment is critical for designing personalized treatment for cancer patients. This assay provides an easy and robust way to assess the interaction between tumor cells and macrophages in vitro.
This assay can be modified to assess the interactions between the tumor cells and other immune cells in vitro. To begin this procedure, warm up the serum-free stem cell medium by placing it into a water bath at 37 degrees Celsius for 20 minutes. Spray the tissue culture hood surface with 70%ethanol and wipe down the sprayed surface with paper towels.
Next spray the medium and cell detachment solution bottles with 70%ethanol and wipe them down with paper towels. Transfer the cleaned bottles into the tissue culture hood. Retrieve the tumor cell and transfer it into the tissue culture hood.
Aspirate the medium and wash the cells with 10 milliliters of PBS. Then add two milliliters of cell detachment solution to the flask. Set the flask down in the hood, checking every minute to see if the tumor cells have rounded up.
Once the tumor cells round up, gently tap the side of the flask to help the cells detach. After this, pipette eight milliliters of serum-free cell medium into the flask and pipette up and down three times to mix. Transfer this cell suspension to a 15 milliliter centrifuge tube and centrifuge at 200 times g and at four degrees Celsius for five minutes.
Aspirate the supernatant. Then wash the cells in 10 milliliters of PBS, making sure to pipette up and down three to five times to mix. Centrifuge the cells at 200 times g and at four degrees Celsius for five minutes.
Aspirate the supernatant, and resuspend the cells in 10 milliliters of serum-free medium. Pipette up and down three to five times to mix. Mix 10 microliters of this cell suspension with 10 microliters of Trypan Blue solution.
Next pipette 10 microliters of the Trypan Blue and cell mixture into a counting slide, and use an automatic cell counter to quantify the living cell number. Use serum-free cell medium to adjust the cell density to 2.5 times 10 to the fifth cells per milliliter. Then seed two milliliters of the cells into each well of a six-well culture plate.
At the same time, prepare six wells with only two milliliters of serum-free stem cell medium. After this, incubate the six-well plates at 37 degrees Celsius with 5%carbon dioxide for 24 hours. First spray the tissue culture hood surface with 70%ethanol and wipe down the sprayed surface with paper towels.
Next spray the medium and cell detachment solution bottles with 70%ethanol and wipe them down with paper towels. Transfer the cleaned bottles into the tissue culture hood. After this, retrieve the six-well plates from the incubator.
Carefully pipette the conditioned media into 15 milliliter sterile centrifuge tubes, and place the tubes on ice. Add 0.5 milliliters of cell detachment solution into each well of the six-well plates, and check every minute if the tumor cells have rounded up. Once the tumor cells round up, gently tap the side of the plate to help detach the cells.
Next pipette 2.5 milliliters of tumor cell maintenance medium into the well and pipette up and down three times to mix. Transfer the cells into a 15 milliliter sterile centrifuge tube. Mix 10 microliters of cells with 10 microliters of Trypan Blue solution, and transfer 10 microliters of this mixture into the counting slide.
Use an automatic cell counter to quantify the number of living cells, and use the serum-free stem cell medium that was incubated at 37 degrees Celsius overnight to adjust the volume of the conditioned media according to the cell number. Filter the conditioned media through 0.45 micrometer filters and place the conditioned media on ice. To begin, warm up the IMDM medium in a water bath at 37 degrees Celsius for 20 minutes.
Clean the tissue culture hood with 70%ethanol and clean the bottle of medium and transport it into the hood as previously described. Next transfer the flask of MV-4-11 cells into the hood. Pipette 10 milliliters of cells into a 15 milliliter centrifuge tube.
Use Trypan Blue and an automatic cell counter to quantify the number of living cells as previously described. Then centrifuge the cells at 200 times g and at four degrees Celsius for five minutes. Aspirate the supernatant and wash the cells with 10 milliliters of PBS.
Centrifuge again at 200 times g and at four degrees Celsius for five minutes. Aspirate the supernatant, and resuspend the cells in IMDM medium at a final cell density of one million cells per milliliter. First bring the necessary materials to room temperature.
Add 250 microliters of the prepared MV-4-11 cells to each insert. Next add 400 microliters of either the conditioned medium or medium only to the lower chambers of the 24-well plate, making sure to add the samples in triplicate. Incubate at 37 degrees Celsius with 5%carbon dioxide for four hours.
Then gently tap the insert on the inner wall of the same well and discard the insert. Gently pipette the cells in the wells up and down three times to mix. Transfer 225 microliters of this cell suspension into the wells of a black-walled 96-well plate suitable for fluorescent measurement.
After this, dilute the CyQUANT dye with 4x lysis buffer at a ratio of one to 75. Vortex briefly and spin down the solution. Transfer 75 microliters of this solution to each well of the 96-well plate and incubate at room temperature for 15 minutes.
Using a fluorescence plate reader, read the fluorescence and analyze the data as outlined in the text protocol. In this study, an in vitro assay is used to study tumor macrophage interaction. Samples with high fluorescence values indicate that the conditioned media has a 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 way. One can also add extra chemokines to the conditioned media, which serve as a positive control.
It's important to control cell number differences for this assay because different cell types can grow at different speeds. In vivo confirmation of the in vitro results are critical. One can inject tumor cells into mice and analyze the tumors with flow cytometry, immunostaining, and CyTOF to confirm the results.
