The overall goal of this procedure is to assess the dose dependent proportion change of tumor cells in a culture containing both tumor and non-tumor cells. This method provides a genetic based tool for assessing in vitro effect and specificity of anti-cancer drugs. It has application potential in drug discovery and in personalized cancer care.
The major advantage of this method is that when anti-cancer drug is tested in a culture containing both tumor and non-tumor cells the response of the tumor cells can be separated from that of the non-tumor cells. Demonstrating this procedure will be Eena a technician from my laboratory. Begin this procedure by culturing both tumor cells and fibroblasts in standard DMA medium at 37 degrees Celsius and 5%carbon dioxide overnight.
At subconfluence, harvest the cells with 0.05%trypsin and count them using a hemocytometer. Afterward, mix 400 tumor cells and 1, 600 non-tumor cells together in each well of a 96-well culture plate with 0.2 mL of medium and set up four replicates for each drug concentration. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide overnight.
Then, prepare the stock solutions of Nilotinib in DMSO at 0, 2, 4 and 20 mM. Dilute each of them by 200 fold in 5 mL of DMA medium giving two-fold concentrated drug solutions of 0, 10, 20 and 100 uM. Next, remove 0.1 mL of the medium from each well.
Add 0.1 mL of medium containing the drug and continue to incubate the cells for five days. At the end of the treatment, inspect the attached cells using a face contrast microscope and take photos. Aspirate the medium and wash the survived adhesive cells once with 0.2 mL of PBS.
To lyse the cells using HotSHOT method, add 50 uL of alkaline lysis solution to each well. Seal the wells with foil. Then incubate the plate at 95 degrees Celsius in an oven or on a heating plate for 30 minutes.
After 30 minutes, check under a microscope to make sure that no more intact cells remained at the surface of the wells. In case the cells did not lyse completely increase the heating time or add detergent. Freeze them once at 20 Celsius degrees also enhances breaking cells.
Subsequently, add 50 uL of the neutralizing solution to each well then transfer all the lysates into the U Form wells of a PCR plate and dry them in a PCR cycler at 95 degrees Celsius for 10 to 30 minutes. Afterward, reconstitute the lysates with 20 uL of distilled water. To desalt DNA using drop dialysis divide a 25 mm diameter membrane filter into four areas and number the areas.
Float the filters on distilled water with the shiny side up in a well of a 12 well plate. Then Hipot the 20 uL reconstituted lysate carefully onto the filter. Cover the plate and dialyze for 30 to 60 minutes.
Subsequently suck away the water under the filter without flipping over the filter or disturbing the drops. Transfer the lysate drops into the wells of a new PCR plate. Next, dry the DNA drops by heating the plate at 95 degrees Celsius for 10 to 30 minutes in a PCR cycler.
Then reconstitute the lysates in 10 uL of water. Next prepare the master mix to your containing all components except DNA for the duel probe digital PCR reaction. Vortex and spin down the master mixture for 10 seconds at maximum force in the centrifuge and dispense 15 uL of the mixture in each well of a 96 PCR plate.
Add the lysate of each sample to the wells containing the master mixture. Transfer 20 uL of each reaction mixture into the well on the sample side of a cartridge at room temperature. Then, dispense 70 uL of droplet generator oil into each well on the oil side of a cartridge.
Close the cartridge with a gasket and place the whole setting into a droplet generator before starting droplet generation. Subsequently transfer 40 uL of droplet solution into each well of a 96 well PCR plate. Seal the plate with a foil sheet and place the plate into a PCR cycler.
Next, set the lid temperature at 105 degrees Celsius and the ramp rate at 2 degrees Celsius per second. When finished, transfer the plate into a droplet reader and start reading. Alternatively, store the plate at 4 degrees Celsius for up to 24 hours.
Now load the digital PCR data and perform automatic analysis. Inspect the samples manually to make sure that the positive and negative droplets are well separated. Exclude the samples with no clear separation of positive and negative droplets from further analysis.
Copy the resulting ratio of NF1 to RPP30 into an Excel sheet. Next, calculate the proportion of the tumor cells. For each drug concentration, calculate the mean and standard deviation from the four replicates then plot the means and the standard deviations against drug concentrations.
Shown here is the dose dependent reduction of the visible vital cells. The proportion of tumor cells calculated from the ratio of NF1 to RPP30 decreased in the range of 0 to 10 uM. At the highest dose of 50 uM few vital cells were left, likely due to a toxic effect to all cells.
The viability and proliferation of the total cells can be measured using conventional assays, but the proportion of tumor cells can be determined using the procedure demonstrated in the present study. Using the two parameters the total cell response to a drug in a mixed culture can be divided into the tumor cell response and the non-tumor cell response. The whole testing procedure can be done in one week including the treatment period of five days.
While attempting this procedure it is important to remember that the ratio between the target gene and the reversal gene varies. We're seeing a very narrow range which is between 0 and 1 and therefore precision is essential. Evaluation amongst samples and in handling should be reduced as far as possible.
Following this procedure also primary cultures which contain both tumor cells and non-tumor cells can be used to test effect and specificity of anti-cancer drugs. Furthermore, effect of a drug on certain subpopulation of cells with defined genetic alteration can be specifically assessed. I hope that this video will help you understand how to use genetic feature to determine the proportion of tumor cells in a sample or in a culture which contains both tumor cells and non-tumor cells.
