This technique enables the identification of the cells with different invasive capacities under the influence of secreted factors from co-cultured stromal cells. The technique assesses the impact of secreted factors from co-cultured stromal or immune cells on cell invasion. It also allows the recovery and analysis of invasive cell subpopulations present in heterogeneous cell mixes.
The technique can determine which tumors harbor invasive cell subpopulations that lead to metastasis. The capture and analysis of invasive cancer cells may inform therapy decisions. To begin the cell culture, wash the adherence cell cultures having approximately 70%confluency with 1X phosphate buffered saline.
Then add 0.05%trypsin and EDTA solution to lift off the cells. Neutralize the trypsin solution with cell culture media containing serum and count the cells using an aliquot of the cell suspension. Place all three sterile chambers in the tissue culture hood.
Locate the knob on the short side of the lower chamber and orient the lower chamber so that the knob is facing the experimenter. Add 30, 000 to 50, 000 cells in 90 microliters of media to each well from the lower chamber without forming any bubbles. Use 5%of fetal bovine serum supplemented media in two lower chambers as a positive control for cell motility, and use 0%serum supplemented media as a negative control.
Rotate the lower chamber at 90 degrees after letting it settle for 10 to 15 minutes in the hood. Then place the middle chamber on top so that the knob on the lower chamber slides into the notch on the middle chamber. Push the chamber vertically downward until a click is heard from each of the long sides of the assembly.
Add 160 microliters of serum-free media to all the wells of the middle chamber. Make sure a dome-shaped meniscus is visible after wells are filled and place the top chamber with electrodes facing down onto the middle chamber to align the blue dots on the middle and top chambers. Push the chamber vertically down until a click sound is heard from each of the long sides of the assembly.
Add 20 to 50 microliters of serum-free media to the top chamber. Mount the assembly on the dual-purpose cell analyzer in the tissue culture incubator and wait 30 minutes before measuring the background. Open the cell analyzer software, then select the cradle to be used, followed by a click on the message tab, and make sure it says Connections okay"to ensure the array is well placed in the cradle and the electrodes are well aligned with the sensors.
Click on the experiment notes tab and fill in all the possible information about the experiment. Then click on the layout tab and fill in the description of the array layout. Then click on the schedule tab and add two steps from the steps menu, a background step with one sweep, and a test step with 100 sweeps.
Once the array has been in the dual-purpose cell analyzer incubator for 30 minutes, click on the play button to start background measurement. After the background measurement is done, remove the array from the cradle and place it back in the cell culture hood. Then add 30, 000 to 50, 000 cells in 100 microliters of serum-free media to each well of the top chamber.
These are the cells that the electrode will detect once they successfully migrate through the membrane. Let the assembly stand in the hood for 30 minutes before mounting on the dual-purpose cell analyzer for impedance measurement. Place the array back into the dual-purpose cell analyzer and check the message tab for the Connections okay"message.
Click on the play button to start impedance measurement and then click on the plot tab to monitor the progress of the signal. If the endpoint is reached before 25 hours, click on the abort step from the execute dropdown menu. To export data, right-click on the graph, choose copy in the list format, and then paste the data into a spreadsheet.
Monitor the migration rate in real time on the dual-purpose cell analyzer to determine the stopping point of interest. Once achieved, unmount the assembly from the dual-purpose cell analyzer and place it in the tissue culture hood. Prepare an appropriate number of 1.5 milliliters of microcentrifuge tubes to collect the cells from the wells of interest.
Place the assembly in a 10 centimeter dish to contain liquids when the chambers detach. Push the flexible snapping ends on the long side of the middle chamber inward until a click sound is heard, followed by dismantling the top chamber and inverting it into a new 10 centimeter dish. Use a cell lifter with a 13 millimeter blade to collect the cells from all the wells harboring the same experimental condition.
Rinse or dip the blade in 1X phosphate buffered saline to collect the cells in 1.5 milliliter microcentrifuge tubes. Then spin down the cells at 500 times G for five minutes and propagate these collected cells or perform endpoint analysis such as single-cell RNA-sequencing. Assessment of invasion of the cells in the presence or absence of stromal cells showed that MDA-MB-231 cell invasion was enhanced when irradiated Swiss-3T3 fibroblasts J2 strain were seated in the bottom chamber for the exchange of factors between the two cell lines.
Interestingly, MDA-MB-231 invasion increased when 3T3 J2 cells were doubled in number. On the other hand, the invasion rate of an invasive clone of MCFDCIS cells, DCIS-Delta Four, appears to be inhibited by the crosstalk with 3T3 J2 cells, suggesting the valuable application of the three chamber array to measure varying effects of the stroma. In this case, fibroblasts on cell invasion.
Human umbilical vein endothelial cells were more invasive in response to factors secreted by MDA-MB-231 cells, unlike those secreted by DCIS cells, which is consistent with the ability of invasive tumors to recruit endothelial cells for blood vessel formation and later dissemination into the circulation. Patient-derived xenograft cells invasion from the top chamber increased in response to co-cultured human bone marrow immune cells. Interestingly, the presence of 2%serum in the bottom chamber with the human bone marrow immune cells was essential for patient derived xenografts invasion.
Wells can be coated with an extracellular matrix to mimic the basement membrane barrier. Therapeutic agents can be added to the media in the wells to monitor their effect on invasion.
