The overall goal of the following experiment is to observe in real time and high throughput manner the effect of potential agents on tumor cell dormancy and or emergence from tumor dormancy using the following in vitro assay to model tumor dormancy or the switch to metastatic growth. This is achieved by first culturing dormant and or metastatic tumor cells in a three dimension culturey basement membrane extract, or BME system adapted to a 96 well plate platform. When cultured in the 3D system, metastatic cells enter a transient, dormant, or quiescent phase.
In contrast, tumor cells that are dormant in vivo enter a prolonged quiescence state. In the 3D culture system, metastatic cells can then be treated with candidate agents to prevent their switch from quiescence to proliferation. Whereas dormant tumor cells can be treated with candidate agents that may induce their switch from quiescence to proliferation.
The proliferative behavior of the dormant and metastatic cells as a result of treatment will be scored by adding cell titer 96 aqueous one solution cell proliferation assay kit at the desired time points for analysis by an ELIZA plate reader. Results are obtained that show the effect of treatment on cell proliferation of either dormant or metastatic tumor cells based on a cell proliferation assay in the 3D system. This method can help answer key questions in the metastasis field, such as what factors and cellular mechanisms maintain disseminated tumor cells in a dormant state and what factors and cellular mechanisms induced the switch from tumor dormancy to clinical metastasis Thaw.
Qualtrics growth factor reduced BME in a four degree Celsius refrigerator overnight the day before the proliferation assay is to be performed. Then on the day of the assay, select either a dormant or metastatic tumor cell culture for the experiment. Next place, a 96 well plate on a tray of ice inside a lemon or hood.
Then use a dispenser with a syringe to coat each well of the plate with 50 microliters of the thawed ice cold.BME. Make sure no bubbles form in the wells place the 96 well plate coated with BME in a humidified incubator with 5%carbon dioxide at 37 degrees Celsius for 30 minutes. Then place a bottle of trypsin at 37 degrees Celsius to warm while the BME coated plate is incubating.
Aspirate the media from the tumor cells. Rinse the culture plates with 10 milliliters of PBS. Then aspirate the PBS and add two milliliters of the prewarm trypsin to the culture plates.
Incubate the plates for five minutes. Add five milliliters of DMEM high glucose, supplemented with 10%FCS and antibiotics to a 15 milliliter conical tube. During this time.
After the incubation, transfer the detached cells to the 15 milliliter tube and count the cells. Then spin down the total cell number to be cultured in a tissue culture centrifuge for five minutes at 1500 G at room temperature. Carefully aspirate the sain.
Leave some media behind, as in most cases the pellet is not visible. Then flick the bottom of the tube to ensure that a single cell suspension is obtained. Next, resuspend the pellet with 100 microliters of assay media for every two times 10 to the third cells.
T tri rate the cells many times with a five milliliter pipette to ensure that a single cell suspension is maintained. Plate 100 microliters of cell suspension per well into the 96 well BME coated plate for control Background devaluation plate an additional 100 microliters per well of assay media to only a few wells of the 96 well BME coated plate. Then incubate the plates, aspirate the old media and feed the cells with new assay media every four days.
To evaluate cell proliferation, add 20 microliters of cell titer 96 aqueous one solution from a cell proliferation assay kit per well to each 96 well plate being analyzed at the desired experimental time point. Incubate the plates for two hours. Then using an ELI a plate reader record the absorbance of both the experimental and background wells at 490 nanometers in preparation for immunofluorescent.
Staining of cell signaling molecules and tumor cells thaw A BME in a four degree Celsius refrigerator overnight the day before the staining is to be performed on the day of staining. Place an eight chamber glass slide system on a tray of ice inside a lemon or hood. Next, use a 200 microliter pipette to coat each well of the glass slide system with 50 microliters of ice cold BME.
Make sure the BME is spread evenly and that no bubbles form in the wells. Incubate the coated slides for 20 minutes. Select either a dormant or metastatic tumor cell line.
Harvest the cells using trypsin as described earlier. Then pool the total number of cells to be cultured in a 15 milliliter conical tube. Now spin down the cells, aspirate the supernatant carefully again, leaving some media behind.
For the pellet flick the bottom of the 15 milliliter conical tube to obtain a single cell suspension. Resuspend the pellet with 400 microliters of assay media for every five times 10 to the third cells. Tri rate the cells many times with a five milliliter pipette to ensure that the single cell suspension is maintained plate 400 microliters of cell solution per well to each of the eight BME coated chambers.
Then incubate the glass slide system, aspirate the old media and feed the cells with new assay media every four days at the desired experimental time points. Aspirate the upper layer of the media and add 200 microliters of fixative supplemented with tritton and incubate at room temperature for five minutes. Then aspirate the triton supplemented fixative and add 200 microliters of fixative only and incubate at room temperature for an additional 25 minutes.
Aspirate the fixative again and now add 400 microliters of PBS to each. Well incubate the plate for 10 more minutes at room temperature. Then aspirate the PBS and add 400 microliters of PBS containing 0.05%between 20 and incubate for another 10 minutes.
Also at room temperature. Now block the fixed cells with 200 microliters of blocking solution. Then incubate the cells at room temperature for one hour after the incubation, aspirate the blocking solution and add 200 microliters of the primary antibody diluted in blocking solution to each.
Well incubate the slides with the primary antibody overnight at four degrees Celsius the next day. Aspirate the antibody, then wash the wells three times by adding 400 microliters of PBS for 15 minutes and then aspirating the solution afterward each time. After aspirating the PBS from the last wash, add 200 microliters of a secondary antibody conjugated to rod domine red.
Then cover the eight chamber slide system with aluminum foil and incubate for one hour at room temperature. Next, wash the wells three times with PBS as before after the third wash mount. The slides in beta shield mounting medium with dpi.
Finally dry the slides for 40 minutes at room temperature in the dark. An example of a proliferation analysis of the dormant D 2.0 R and metastatic D two A one tumor cells in the 3D culture is shown here. D 2.0 R cells are dormant through the entire experimental 14 day culture period, whereas the highly metastatic D two A one cells remain dormant only for four to six days of culture, after which they begin to proliferate during the initial dormant phase.
Many cells remain solitary in the 3D culture as shown here in a representative photo of a day four culture, whereas other non proliferating cells for multicellular steroids. The transition of D two A one cells from a dormant to proliferative state in 3D culture is shown here in this photo of a day 12 culture. It is associated with dramatic changes in cell morphology in this figure.
An example of an agent preventing D two A one cells to transition from a dormant to proliferative state is shown treatments of the D two A one cells with a specific inhibitor against myosin light chain kinase. Maintain D two A one cells in a dormant state as illustrated in this figure. A significant increase in myosin light chain phosphorylation in D two A one cells in red, followed by reorganization of the F actin filaments forming actin stress fibers in green occurs during their transition from dormancy days one to four to proliferation.
Day seven. However, blocking myosin light chain kinase activity in D two A one cells by SHRA or by the drug ML seven, keeps D two A one cells in a dormant state and results in the inhibition of myosin light chain phosphorylation, an F act, and stress fiber organization as seen here. After watching this video, you should have a good understanding how to set up the in vitro model system to study tumor dormancy and the switch to metastatic growth.
