The overall goal of this procedure is to isolate cancer stem cells from head and neck squamous cell carcinoma or HNSCC cell lines. This method can help answer key questions in the cancer treatment field about how to use chemotherapy to overcome tumor radioresistence. The main advantage of this technique is that it uses a combination of markers to achieve a high specificity in cancer stem cells from HNSCC cell lines.
Demonstrating the procedure will be by Prescillia Battiston-Montagne our technician and Marion Gilormini, a PhD from my lab. 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 one milliliter of Tripsyn EDTA.
After three minutes at 37 degrees Celsius stop the reaction with fresh cell culture medium and count the cells. Dilute the culture to one times 10 to the seventh cells per milliliter and complete parental cell line medium. Then add 100 microliters to the Hoechst and Verapamil tube and four milliliters of cells to the Hoechst tube.
Next, gently mix 10 microliters of five millimolar Verapamil hydrochloride solution into the Hoechst and Verapamil sample followed by five microliters of Hoechst per one 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 two milliliters of PBS.
After a center centrifugation, resuspend the Hoechst and Verapamil pellet in 500 microliters of propidium iodide in PBS and the Hoechst pellet in four 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 side arm 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 one 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 one 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.
To select the CD44-high/ALDH-high subset from the sorted side population cells, first label seven sterile 15-milliliter conical tubes as indicated. Next, dilute the sorted cells to one times 10 to the seven cells per milliliter in stem cell medium and aliquot 100 microliters of cells to the unstained CD44-APC and IGG1-APC tubes and four milliliters of cells to the ALDH and CD44-APC tube, all on ice. Spin down the cells and resuspend the unstained CD44-APC and IGG1-APC pellets in 100 microliters of buffer one from the ALDEFLUOR kit.
Then add five microliters of the ALDH inhibitor DEAB to the ALDH and DEAB and ALDH DEAB and CD44-APC tubes. Resuspend the cells in the ALDH and CD44-APC tube in four milliliters of reagent C containing four milliliters of buffer one and 20 microliters of the reagent from the kit and immediately transfer 100 microliters of these cells into the ALDH, ALDH and DEAB, and ALDH DEAB and CD44-APC tubes. Then place all of the tubes in a 37-degrees Celsius water bath for 30 minutes protected from light mixing the samples after 15 minutes by gentle vortexing.
At the end of the incubation, centrifuge all of the samples and discard the supernatants. Then place all of the tubes on ice and resuspend the ALDH and CD44-APC pellet in four milliliters of buffer A, a CD44-APC and ALDH DEAB and CD44-APC pellets in 100 microliters of the same. Resuspend the IGG1-APC pellet in 100 microliters of buffer B and the unstained ALDH and ALDH and DEAB tubes in 100 microliters of buffer one.
After 10 minutes, spin down all of the samples again followed by a wash in 1 milliliter of buffer one for all of the samples except the ALDH and CD44-APC sample, which is washed in four milliliters of buffer one. After the second centrifugation, resuspend all of the pellets in one milliliter of fresh buffer one except for the ALDH and CD44-APC sample, which is resuspended in four milliliters of buffer one. Now load the ALDH and CD44-APC tube onto the flow cytometer and create an APC by fitsy dot plot to visualize the double stained population.
Next, load the ALDH tube to create a gate for ALDH-high cells. The positive cells should disappear when the ALDH and DEAB tube is loaded. In the same graph, create a second gate using the CD44-APC and IGG1-APC tubes to select the CD44-high cells.
The positive cells will disappear when the IGG1-APC tube is loaded. Then load the ALDH and CD44-APC and ALDH DEAB and CD44-APC tubes to create a third gate that includes the CD44-high/ALDH-high cells. Use double-staining ALDH-CD44 tube to position the last gate on the double-positive cells.
Then sort the CD44-high/ALDH-high cells into a 15-milliliter conical tube containing one milliliter of complete cancer stem-like cell medium collecting the CD44-low/ALDH-low cells in one milliliter of complete cell culture medium as desired. After the second sort, plate the CD44-high/ALDH-high cells into an appropriate cell culture flask with complete cancer stem-like cell medium in the cell culture incubator for 18 to 24 hours. When the cells have attached to the bottom of the flask, change the culture medium and return the flask to the incubator.
To confirm the tumor potential of the CD44-high/ALDH-high cells, trypsinize the cancer stem-like monolayer as demonstrated to obtain a single-cell suspension and transfer one times 10 to the six cells to a 15-milliliter conical tube. After spinning down the cells, resuspend the pellet in serum-low medium supplemented with recombinant human epidermal growth factor heparin and B27 and incubate the cells in a low-anchorage petri dish in the cell culture incubator until tumor spheres are observed by optical microscopy. When the sorting is performed for the first time on a new cell line, it is necessary to confirm the tumor potential of the cell line as evidenced by its ability to form tumor spheres in serum-free medium in vitro.
Moreover, QPCR should demonstrate a high expression of beta-catenin and other stem-like cell markers by the CD44-high/ALDH-high cells. The CD44-high/ALDH-high cells should also be able to form tumors in situ when injected in low quantities compared to CD44-low/ALDH-low cells. Once mastered, each cell sorting should be able to be completed in five hours if they are performed properly.
While attempting this procedure it is important to remember to protect the fluorochrome-labeled samples from direct light exposure. Following this procedure, other methods like invasion migration assays can be performed to answer additional questions about the involvement of cancer stem cells in metastatic process. After its development, this technique paved the way for researchers in the field of cancer to explore the mechanisms of tumor escape in HNSCC cancer.
After watching this video you should have a good understanding of how to isolate cancer stem cells from HNSCC cell lines using multiparametric, flow cytometric analysis, and cell sorting. Don't forget that working with tumor cells and intercalating DNA agents can be extremely hazardous and that precautions such as wearing a lab coat and gloves should always be taken performing this procedure.
