The overall goal of this experiment is to model the interaction of glioblastoma cells with microglia and macrophages during invasion in an in vitro assay. So this method can help answer key questions in the tumor microenvironment field, such as which compounds or treatments can interfere with cancer interaction with macrophages, a cell type of the microenvironment during the progression to malignancy. One of the main advantages of this technique is that it's relatively easy to perform and can be completed at a reasonable time and seems to accurately model glioblastoma invasion in vivo.
Begin by differentiating the cell line of interest using phorbol myristate acetate, as follows. First, plate two to three times 10 to the fifth THP-1 cells in 1.5 milliliters of medium in a six well culture plate. Immediately after plating, add 100 nanomolar of PMA and incubate at 37 degrees Celsius and 5%CO2 for 48 hours.
After 48 hours, remove the PMA by aspirating the media, wash once with one-X PBS and add fresh media. Incubate for another 48 hours until cells are ready to use in the assay. Then equilibrate matrix pre-coated chambers by placing them in a well containing 500 microliters of media without serum and then adding 500 microliters of serum free media to the top.
Incubate the chambers for two hours at 37 degrees Celsius and 5%CO2. Next, gently aspirate the media from the culture vessels containing the fluorescently-labeled glioblastoma cell lines and the microglia and macrophages. Then add 500 microliters of two millimolar EDTA and PBS and incubate for five to 10 minutes in the tissue culture incubator.
Collect the cells in five milliliters of media containing 0.3%bovine serum albumin and centrifuge for five minutes at 120 times G.Following the centrifugation, aspirate the supernatant and resuspend the cell pellet in appropriate media to a concentration of one times 10 to the sixth cells per milliliter. Use the hemocytometer to count the cells. Next, add 500 microliters of the appropriate media containing 0.3%BSA to the wells of a 24 well plate.
Then remove the media from the equilibrated chambers and place the chambers in the wells of the 24 well plate. If inhibitors are being used to study their effect on macrophage or microglia stimulated glioma invasion, add the appropriate concentration to both the bottom and top portions of the chamber. Depending on the cell line used, mix glioblastoma cells and mouse microglia or glioma cells, and macrophage cells according to the details in the written portion of the protocol.
Add the cell mixture to the top chamber and incubate for 24 to 48 hours, depending on cell type. After the incubation, remove the cells from the top of the chamber by gentle aspiration. Then place the chambers in 3.7%formaldehyde in PBS to fix.
Allow the chambers to remain in fixative for 15 minutes at room temperature. Finally, remove the fixative by gentle aspiration and replace with 500 microliters of one-X PBS. First, thaw the matrix mix at four degrees Celsius overnight.
Next, prepare a 10 milligrams per milliliter concentration of matrix in the appropriate media on ice. Then, harvest the labeled cells for suspension in matrix as previously shown. Then pipette 1.5 times 10 to the fifth GL261 cells in a 150 microliter volume plus five times 10 to the fourth microglia cells in 50 microliters into a 1.5 milliliter microfuge tube.
Add two times 10 to the fifth GL261 cells in a 200 microliter volume into a separate 1.5 milliliter microfuge tube. Spin the cells in a microfuge for five minutes at 120 times G.Then, resuspend the cells in 200 microliters of cold matrix on ice. For this step it is crucial to have ice tray ready in the hood, keep tubes on ice, tips in the refrigerator, make sure everything is chilled in order to prevent polymerization of the mature gel prematurely.
After resuspension, plate 50, 000 cells in a 50 microliter volume onto the center of the top compartment of an eight micron pore size chamber insert. Then incubate it at 37 degrees Celsius for 30 minutes to allow polymerization to occur. After 30 minutes, add 200 microliters of serum free medium to the upper chamber and 700 microliters of serum containing cell growth medium to the lower well.
After incubating for 48 hours, remove the cells from the top part of the chamber by gentle aspiration. Fix the chambers as before and replace the fixative with 500 microliters of one-X PBS before imaging the cells. This representative image shows invading mCherry expressing GL261 cells in the absence of microglia.
The cells were plated on matrix coated chambers and incubated for 48 hours. The images were taken using a 10X objective and the scale bar represents 400 microns. Here the GL261 cells were combined with microglia.
The number of GL261 invasive cells that cross the filter were quantified. As seen here, coculturing the mouse glioblastoma cell line GL261 with microglia enhances its invasion by up to 10 fold in the 2D assay. This image shows the effect of pharmacological inhibitors on microglia stimulated GL261 invasion compared to DMSO control.
Data shown are from six independent experiments. This assay was successfully employed using human cell lines as well. U87 cells plated on matrix coated chambers and stained with CMFDA green are shown after a 24 hour incubation.
Here the U87 cells were plated with differentiated THP1 macrophages. This image shows quantitation of the assay counting only invasive U87 cells. The human macrophage cell line THP1 stimulated U87 glioblastoma cell invasion.
Data shown are the mean values from 10 independent experiments. This image from a 3D invasion assay of GL261 and microglia coculture is a composite from a Z-series of images and shows GL261 cells stained with CMFDA green embedded in matrix alone. Here the GL261 cells were cocultured with murine microglia labeled with CMPTX red.
Here GL261 cells on the underside of the filter, which are determined to be invasive, are shown. The total number of invading GL261 cells was determined and normalized to that of GL261 cells in monoculture. Data shown represent the mean of three independent experiments performed in duplicate.
So once mastered, this technique can be completed from start to finish in two hours or so. And the final results from the experiment can be obtained after 48 hours. So while performing this technique, it's very important to have everything chilled and at four degrees including the tubes and the pipettes.
After watching this video, you should have a good understanding of how to establish glioblastoma in macrophage microglia coculture invasion assays.
