This protocol is significant because the presence of cancer stem cells may be associated with relapse or a poor outcome after radiotherapy. Studying the radioresistant stem cells may provide clues to overcome radioresistance. In this technique, cancer stem cells are salted with putative markers by flow cytometry and the self-renewal capacity of salted cells is evaluated by sphere formation assay.
The colony formation assay is performed to assess the radiosensitivity of salted cells. It determines how many cells lost their ability to generate the synthesis that form the colony after a certain dose of radiation. This manuscript provides the initial few steps for the radiosensitivity study of cancer stem cells, which establishes the basis for fuller mechanism study.
Mechanism study may involve proteins related to DNA damage repair, clearance of reactive oxygen species, cell cycle arrest, etc. It will provide important insights into how cancer stem cells get radioresistance and how to overcome the resistance. Demonstrating the radiation procedure will be Chen-Nan Li, a radiation technician from our department.
To begin this procedure, culture A549 cells in RPMI-1640 medium supplemented with 10%FBS in 10 centimeter dishes at 37 degrees Celsius, with 5%carbon dioxide. When the cells reach 80 to 90%confluence, remove the culture medium. Briefly wash the cells with three milliliters of PBS.
Next, add three milliliters of 0.05%trypsin to each dish. Remove the trypsin and leave the residuary trypsin to digest the cells at 37 degrees Celsius for one to three minutes. Check the detachment of the cells frequently to avoid over-digestion.
When the cells become loose and begin to detach from the dishes, add three milliliters of RPMI-1640 medium supplemented with 10%FBS and transfer the cells suspension to a 50 milliliter tube. Centrifuge at 300 times g and at four degrees Celsius for five minutes. Discard the supernatant and resuspend the cells in 10 milliliters of PBS.
Transfer 0.5 milliliters of the cell suspension into a new tube as an isotype control. Centrifuge both the control and experimental tubes at 300 times g and four degrees Celsius for five minutes. Discard the supernatants.
Then, dilute both the fluorescent diconjugated isotype control and antibody in PBS to the optimal concentration titrated to prepare the working solution. Resuspend the control and experimental cells with each working solution and mix gently. Incubate the samples in the dark and at room temperature for 30 to 40 minutes.
Wash the cells by adding 10 milliliters of PBS, mixing gently, and centrifuging at 300 times g and at four degrees Celsius for five minutes. Discard the supernatants and resuspend the cells with 10 milliliters of PBS. Next, place 40 micrometer cell strainers on new 50 milliliter tubes, making sure to prepare a separate tube and strainer setup for the control and experimental cells.
Apply each cell suspension onto the respective strainer and collect the flowthrough. Centrifuge the flowthrough at 300 times g and at four degrees Celsius for five minutes. Discard the supernatant and resuspend the cells with 0.2 to 1.0 milliliters of PBS and transfer each cell suspension into a separate five milliliter tube for flow cytometry.
In a biological safety cabinet, prepare one six well plate for each radiation dose, including the zero gray group. Add 1.5 milliliters of completed 1640 media to each well. Dilute harvested cells with completed 1640 media to a cell density of 1, 000 cells per milliliter.
Then, add the diluted cell suspension to the wells and shake the plates horizontally to evenly distribute the cells in the wells. Record the volume added in each dose group. Culture at 37 degrees Celsius with 5%carbon dioxide.
After the cells have attached to the bottom of the wells, add completed 1640 media to each well until the media height reaches one centimeter. When the plates are transferred between cell culture room and radiation therapy room, wrap the plates with aluminum foil or put them in a clean box to avoid contamination. Hold the plates flat to avoid medium spilling out.
Next, set at 20 centimeter by 20 centimeter radiation field. Place a tissue equivalent bolus of 1.0 centimeter thickness on the treatment couch and place the six well plate on the bolus to keep them in contact. Make sure the whole plate is within the radiation field.
Set the source-skin distance as 100 centimeters and adjust the height of the treatment couch to align the medium surface level to the laser level. Deliver the assigned dose to each plate in sequence. After this, take the plates to the biosafety cabinet in the cell culture room.
Replace the medium in each well with two milliliters of completed 1640 medium. Culture at 37 degrees Celsius with 5%carbon dioxide, making sure to change the medium every three to five days. Seven to 10 days after radiation, remove the medium and briefly wash the cells with PBS.
In a fume hood, add one milliliter of 4%formaldehyde to each well to fix the cells for 10 minutes. Then, remove the formaldehyde and wash each well twice using two milliliters of distilled water for each wash. Add one milliliter of 1%Crystal violet stain solution to each well and stain for 10 minutes.
After this, remove the Crystal violet stain solution and wash the cells three times with distilled water. Remove the water and let the plates air dry for 30 minutes. Count the number of colonies with more than 50 cells.
In this study, both alpha two delta one high and alpha two delta one low A549 cells are sorted. Some markers may show distinct populations and are easy to gate. However, some markers just show high and low expression patterns, rather than distinctive positive and negative populations.
In this situation, an isotype control is very important for gating. The expression of alpha two delta one in sorted cells is validated by qPCR. The expression of CACNA2D1, the gene that encodes alpha two delta one, is higher in sorted alpha two delta one high cells compared with alpha two delta one low cells.
Typical morphology of spheres and the sphere formation efficiency is shown here. Alpha two delta one high cells show higher sphere formation efficiency, suggesting a higher self-renewal capacity. Typical images of colonies and survival curves are shown here.
A colony with about 50 cells can be examined under a microscope and marked as a reference. The number of colonies is counted, then a survival fraction at each dose can be calculated and a survival curve can be plotted. Alpha two delta one high cells are relatively resistant to radiation compared to alpha two delta one low cells.
Any steps related to cell culture should be performed in the biological safety cabinet or laminar clean bench. To avoid contamination, it is recommended to add antibiotics to the culture medium after sorting. When a putative cancer stem cell marker is preliminarily identified by the sphere formation assay, further characterization by in vivo limiting dilution assay can be performed to evaluate the tumorigenic capacity.
From the mechanism study of radioresistance, researchers can examine the protein related to DNA damage repair, clearance of reactive oxygen species, and cell cycle arrest in response to radiation. During cell radiation, please note that the linear accelerator can only be operated by qualified technicians. Keep away from radiation.
While standing, please note that formaldehyde is volatile and hazardous. Use formaldehyde in the fume hood.
