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.