The overall of this protocol is to utilize conditional knockdown of gene expression in cancer cells to study the recruitment of monocytes and macrophages to the tumor microenvironment in vitro. This method can help answer key questions in the tumor microenvironment field regarding the nature of the cross stock between the cancer cells and the cells of the immune system. The main advantage of this technique is that the system used here requires single vector cloning, allows quick generation of multiple clones, and exhibits very low levels of thickness.
Also demonstrated is a method for purification of human monocytes without direct contact with antibodies which avoids accidental activation while resulting in more than 95%pure population of human monocytes. To produce viral particles, transfect 70%confluent 150 millimeter dishes of HEK-293 cells with 25 micrograms of pLKO-tet-on shRNA, 25 micrograms of the psPAX packaging plasmid, and five micrograms of the envelope-expressing plasmid pMD2. G using transfection reagent as per standard protocols.
Add mix to the 150 millimeter dishes. The next day, induce Lentiviral production in the cells by changing the medium to DMEM supplemented with 10 millimolar sodium butyrate and 20 millimolar HEPES. Culture for eight hours at 37 degrees Celsius and 5%carbon dioxide.
After eight hours, wash the cells with PBS and add 25 milliliters of fresh DMEM medium with 20 millimolar HEPES and without sodium butyrate. After incubation for 48 hours, carefully collect the virus-containing medium with a pipette. To generate stable cell lines, start by seeding HCT-116 colon cancer cells and MDA-MB-231 breast cancer cells into a 12-well plate and 50, 000 cells per well.
Incubate at 37 degrees Celsius and 5%carbon dioxide. When the cells are 60-70%confluent, wash the cells with PBS and add one milliliter of virus-containing medium to each well. The next day, carefully remove the virus-containing medium by aspiration.
Then after washing the cells with PBS, add medium containing tetracycline-free FBS. After 72 hours, wash the cells as before and add medium supplemented with one microgram per milliliter puromycin for the selection of virus-transfected cells. Select virus-transduced cells by culturing cells in the presence of puromycin for three to 14 days.
Observe partial killing of the wells by puromycin in the cells with virus-transduced cells. Non-transduced cells will not grow in the presence of puromycin. When these control cells have died, continue culture of the conditionally regulated cell lines for two weeks in the presence of antibiotics to create the conditionally regulated cell lines.
Verify the efficiency of the conditional knockdown in the puromycin-resistant HCT-116 colon cancer and MDA-MB-231 breast cancer cells by adding medium with different concentrations of doxycycline and culturing the cells for 72 hours. After preparing cell lysate, verify the level of knockdown by western blot analysis. Levels of PAI-1 are shown here.
To isolate peripheral blood monocytes, first prepare three 50 milliliter tubes containing 15 milliliters of density gradient solution. Then use a pipette controller set on gravitational flow to slowly layer 30 milliliters of diluted blood over the density gradient solution. Centrifuge in a swing rotor at 400 g at room temperature without breaks for 25 minutes.
Following the centrifugation, dispose of the top layer and transfer the middle layer containing peripheral blood mononuclear cells to a fresh 50 milliliter tube and add FBS supplemented PBS up to 50 milliliters. Centrifuge at 120 g's at room temperature for 10 minutes. After the spin, remove the containing platelet-containing supernatant.
Following the final centrifugation and collection, resuspend the pellet in 50 milliliters of FBS supplemented PBS and count the peripheral blood mononuclear cells using a hemocytometer. After centrifuging the cells at 120 g's, resuspend the pellet in FBS supplemented PBS containing one millimolar EDTA to a final concentration of 50 million cells per milliliter. Then begin to enrich for monocytes by adding 50 microliters of negative selection antibody cocktail for each milliliter of cell suspension and mix.
Incubate for 10 minutes at room temperature. After the incubation, add 50 microliters of magnetic beads per milliliter, mix and incubate for another 10 minutes. Then add PBS with FBS and EDTA up to 25 milliliters and place the peripheral blood mononuclear mixture in a magnet that can accommodate a 50 milliliter tube.
After incubating for 10 minutes at room temperature, the magnetic beads will adhere to the walls of the tube and sequester the non-monocytic cells from the solution. Use a 25 milliliter pipette to remove solution containing monocytes from the tube while being careful not to touch the sides of the tube with the pipette. After centrifuging and removing the supernatant, resuspend the pellet containing monocytes in RPMI medium containing 10%TET-free FBS and 1%pen-strep.
Begin the assay by seeding 40, 000 HCT-116 or MDA-MB-231 cells in a 0.5 milliliter volume in the wells of a 24-well plate in triplicate. The next day, add a filter to the well. Then plate 8, 000 monocytes in a 0.3 milliliter volume onto a BSA-treated porous membrane insert placed on the top of the cancer cells and incubate at 37 degrees Celsius and 5%carbon dioxide.
Collect the inserts after 48 hours. Shake off the excess medium and precisely swipe the inner surface with a cotton-tipped applicator to remove cells that did not migrate. Stain the outer side of the insert using the Wright-Giemsa method.
After mounting the filters with the migrated macrophages side up, image the slides using a light microscope with a 20 power objective. Take pictures of nine fields per filter and count migrated macrophages in all fields. The Boyden Chamber Assay was used to quantify the migration of monocytes towards cancer cells.
In the presence of MDA-MB-231 breast cancer cells, the migration of monocytes was inhibited by 33%after addition of doxycycline to the co-culture to down regulate PAI-1. This inhibition of monocyte migration was more pronounced in the presence of HCT-116 colon cancer cells where knockdown of PAI-1 by doxycycline administration reduced monocyte migration by 74%Similar results were observed when conditions medium for doxycycline-treated cancer cells was placed in the bottom of the wells as a source of chemoattractants. After watching this video, you should have a good understanding on how to use a functional tet-on system for gene expression knockdown in human cancer cell lines to study the chemoattractant effect of cancer-derived secreted protein PAI-1 on the human monocytes.
