Rhabdomyosarcoma is a rare form of soft-tissue sarcomas characterized histologically by tissue morphology and the expression of myogenic markers. However, given the existence of different subtypes of these tumors and the heterogeneity of the stimuli that contributes to its formation, the identification of the cell of origin has been very challenging. Here we describe reproducible and reliable methods for the identification of the cell of origin of rhabdomyosarcomas starting from freshly harvest tumor tissues from mice and this assay is the tumorsphere formation assay.
The main advantages of tumorsphere formation assay is that it relies on cellular properties that are known to be present in tumor-originating cells and it can also be used for expansion and enrichment of the specific cell population. Furthermore, its spheroid structure mimic the tumor environment, thus they can be used as drug-screening studies. This method has the potential to be applied to others type of tumors because it does not require prior knowledge of molecular markers but it may require optimization of culture conditions.
Tumorsphere may take some time to form, especially if trying to identify a rare cell population within your tumor. Thus, it's not suggested to look at your subculture plate every day, since it might negatively affect the results of your experiments. Start by warming up a 10-centimeter plate with five milliliters of isolation media in an incubator at 37 degrees Celsius.
Weigh 500 to 1, 000 milligrams of the tumor tissue and place it in the plate. Bring the plate with the tumor tissue to a sterile culture hood and mince it with a razor blade. For optimal digestion, make sure that the sizes of the minced pieces are uniform.
Transfer the minced tissue and cell isolation media to a 15-milliliter centrifuge tube. Wash the plate with another four milliliters of media and add that to the tube. Add 700 units per milliliter of collagenase solution to the tube and incubate it in a shaking water bath at 37 degrees Celsius for 1 1/2 hours.
After the incubation, spin down the tissue at 300 times g for five minutes at room temperature. Aspirate the supernatant without disrupting the pellet, then resuspend the pellet in 10 milliliters of second digestion solution and incubate in the shaking water bath for 30 more minutes. After the second digestion, pipette the cell suspension up and down and pass it through a 70-micrometer nylon filter on a 50-milliliter centrifuge tube.
Then wash the filter with 10 milliliters of cell isolation media and spin down the tissue at 300 times g for five minutes. Aspirate the supernatant and resuspend the pellet in 20 milliliters of tumor cell media. Transfer the suspension to a 15-centimeter culture plate and place the cells in a 37 degrees Celsius incubator overnight.
On the next day, change the media to remove debris and dead cells that may negatively influence cell survival. At this point, assess cell confluency and allow the cells to grow in the incubator until it reaches 90%Monitor the cells every day and change the media every two days. For tumorsphere derivation, use cells at Passage P1 or P2 to avoid cell selection through multiple passages.
Wash the cell dish with PBS and then cover them with detachment solution. Place them in the incubator for five to 10 minutes and then confirm detachment by looking at the plate under a brightfield microscope. Once the cells have detached, add tumor cell media to the plate and transfer the cell suspension to a centrifuge tube.
Spin the cells down at 300 times g for five minutes, remove supernatant, and resuspend the cells in tumorsphere media. After resuspending the cells, count them using Trypan blue and calculate cell concentration. Plate the proper number of cells in a 96-well low-attachment plate and place the cells in the incubator until the end of the experiment, taking care not to disturb the plate unless replenishing media.
After completion of the experiment, identify tumorspheres manually under a brightfield microscope or using Celigo software. The number and size of the tumorspheres can be evaluated as a result of this assay. To prepare tumorspheres for allograft transplantation, pull together all tumorspheres obtained from a specific cell type or treatment.
Centrifuge the tumorspheres and carefully remove the supernatant with a one-millimeter pipette followed by a 200-microliter pipette, then wash them with 10 milliliters of sterile PBS. Spin the tumorspheres down again and aspirate the PBS. Add 500 microliters to one milliliter of cell detachment solution on top of the tumorsphere pellet and incubate the cells in a shaking water bath at 37 degrees Celsius.
Check progression of digestion every 10 minutes. The entire process make take up to 30 minutes. If digestion of tumorsphere in the shaking water bath is not enough to obtain a single-cell solution, mechanical disruption through pipetting up and down is suggested.
Note that after spinning the tumorspheres do not form a stable pellet, so aspirating the supernatant should be done carefully with a one-milliliter pipette and 200-microliter pipettes. Once a single-cell solution is obtained, add a volume of tumor cell media and spin it down at 300 times g for five minutes at 4 degrees Celsius. After this centrifugation, all subsequent steps must be performed on ice.
Resuspend the cells in cold tumor cell media and count live cells using Trypan blue exclusion. Determine the proper number of cells for the allograft and dilute it in 50 microliters of cold tumor cell media. Place a pipette tip in cold tumor cell media to cool it and then use it to take 50 microliters of cold ECM solution and add it to the tube with cells, making sure not to remove the ECM tube from ice during this process.
Maintain the cells on ice until transplantation and place a capped, 0.5-milliliter insulin syringe with a 29-gauge needle on ice. After confirming that the mouse has been properly anesthetized, shave the right side of the animal, aspirate the cell solution into the cooled syringe, and inject the cells subcutaneously into the shaved area. If the injection is performed correctly, a visible bump will form under the skin.
This protocol can be used to reliably form tumorspheres for identification of rare cell populations that are responsible for soft-tissue sarcoma development. A fundamental part of this assay is discrimination between tumorspheres and cell clusters, which exhibit clear morphological differences. To determine the optimal concentration at which a protein of interest triggers an effect on tumor cells, it is necessary to assess the level of expression of the protein's downstream targets.
Three of the tested genes showed a dose-dependent response to recombinant protein treatment and one did not. In order to establish an efficient protocol, the effects of transfection on inherent tumor cells were analyzed;two different amounts of reagent were tested and efficiency was assessed with a GFP-reporter plasmid. Lower amounts of reagent resulted in higher transfection efficiency.
A two-step protocol had to be implemented to perform transfection on cells in suspension. Transfection was performed on adherent cells and 24 hours later they were detached and plated in suspension;after seven days, controlled tumorspheres were expressing GFP. After tumorspheres are obtained, treated, and transplanted it is possible to compare the effect of this different treatment in tumor growth in vivo.
These methods will allow scientists to answer the still-standing questions of the cell of origin of rhabdomyosarcomas while at the same time setting the basis for drug screening.
