Cancer Side Population Discrimination Assay: A Method to Isolate Cancer Stem-like Cells Using Hoechst Dye Efflux Assay

A tumor contains a small fraction of stem-like cells known as the side population. These cells are chemoresistant, making them responsible for cancer relapse.

To isolate the side population of cancer cells, begin by adding Hoechst - a fluorescent dye solution - to the vial containing cancer cell suspension. Inside normal cancer cells, Hoechst molecules enter the nucleus and bind to the A-T-rich region in DNA, leading to high fluorescence in these cells.

In contrast, the side population contains a high concentration of ABC transporters - efflux pumps that rapidly remove the dye molecules from the cell. This active efflux of dye molecules makes the side population fluoresce at low intensity.

Now, take another vial of Hoechst-treated cancer cells and add Verapamil to it. This vial acts as a control as Verapamil blocks the ABC transporters. This step causes a reduction in Hoechst efflux and retains the dye inside the side population cells.

Next, counterstain the cells in both the vials using propidium iodide. Propidium iodide exclusively enters the nucleus of dead cells and intercalates between DNA bases. This leads to high fluorescence in the dead cells.

Finally, sort the cells using a fluorescence-activated cell sorter or FACS. FACS separates highly fluorescent, Hoechst-retaining tumor cells and dead cells from the low fluorescent side population cells.

To select a side population of cancer stem-like cells by Hoechst dye efflux assay, first, label one conical tube "Hoechst" and one "Hoechst and Verapamil." Next, wash an HNSCC cell culture with sterile PBS, followed by the addition of 1 milliliter of trypsin-EDTA. After 3 minutes at 37 degrees Celsius, stop the reaction with fresh cell culture medium and count the cells. Dilute the culture to 1 times 10 to the seventh cells per milliliter in complete parental cell line medium.

Then, add 100 microliters to the "Hoechst and Verapamil" tube and 4 milliliters of cells to the "Hoechst" tube. Next, gently mix 10 microliters of 5 millimolar Verapamil hydrochloride solution into the "Hoechst and Verapamil" sample, followed by 5 microliters of Hoechst per 1 times 10 to the sixth cells to both tubes. Then, cover the samples with aluminum foil and place them at 37 degrees Celsius. After 90 minutes with gentle mixing every 15 minutes, centrifuge the tubes and resuspend the pellets in 2 milliliters of PBS.

After a second centrifugation, resuspend the "Hoechst and Verapamil" pellet in 500 microliters of propidium iodide in PBS and the "Hoechst" pellet in 4 milliliters of the same. Now, filter the samples through 70-micron cell strainers into FACS tubes to remove any aggregates and place the samples on ice protected from light. Next, load the "Hoechst" sample onto the flow cytometer and open the "Global Worksheet" window in the flow cytometer software. Click on "Dot Plot" to create a graph on the global worksheet and right click to select the forward and side scatter parameters.

Create a side scatter wide by a side scatter height dot plot in the same way and then click "Polygon Gate" to create a P1 region in the second graph to select the single cells and to discriminate the doublets. Next, create a red Hoechst area by a blue Hoechst area dot plot and right click on the cells to highlight the P1 population. Then, create a P2 region to select the negative Hoechst dye side population of cells that appears as a sidearm to the left of the main population of cells and collect 10,000 of the "Hoechst" sample events.

To confirm that the P2 gate representing the side population is positioned correctly, collect 10,000 "Hoechst and Verapamil" sample events as well. The side population should disappear. Then, sort the side population of Hoechst dye negative cells into a 15-milliliter conical tube containing 1 milliliter of complete cancer stem-like cell medium. At the end of the sort, spin down the cells and resuspend the side population pellet in 1 milliliter of fresh complete cancer stem-like cell medium. Then, count the cells and seed them at the appropriate number into a new cell culture flask.