We have developed a novel 3D spheroids model which offers a platform to study cancer-stroma interactions and to test cancer therapeutics. In my point of view, this 3D system combines cancer cells in a stroma fibroblast to better mimic genuine tumor conditions and can, therefore, be a very powerful tool for drug discovery. In addition, this model is not limited to study Melanoma.
It can be used to investigate other types of cancers. Demonstrating the procedure will be Dr.Hongwei Shao, a senior scientist from my lab. For human melanoma cell culture, throw the cells under conventional adherent cell culture conditions and complete W489 medium at 37 degrees celsius in 5%carbon dioxide.
When the cells reach 90%confluency, split the cells at a one to five ratio. For mouse skin fibroblast culture, wash the harvested tissue pellets in PBS before culturing the specimens and DMEM supplemented with 10 percent fetal bovine serum, and one percent penicillin-streptomycin in the cell culture incubator. Split the cells at a one-to-two ratio when they reach a 90 percent confluency.
Before setting up the cocultures, seed fibroblasts onto a 100 millimeter dish at a concentration such that the cell confluency will reach approximately 60 percent the next day. The next morning, replace the supernatant with a one-to-three to one-to-five concentration of GFP to lentivirus diluted from stock in regular culture medium supplemented with four micrograms per milliliter of polybrene. Place the cells into cell culture incubator for six hours before replacing the supernatant with fresh culture medium.
After two days, observe the GFP signal from the cells on a fluorescence microscope. When both cell cultures have been stably transfected, detach both cell cultures from 0.25 trypsin-EDTA to collect the single cell suspensions by centuvigation. Re suspend the cells at a two times 10 to fourth cells per milliliter of the coculture medium concentration and mix the melanoma cells and fibroblasts at a one-to-one ratio.
Then see two milliliters of cells to each well of a 24 well plate and triplicate for each condition and place the plate in the cell culture incubator for four hours to allow the cells to attach to the plate bottom. For melanoma cell solo culture to evaluate 2D cell cluster formation, see two times 10 to the fourth melanoma cells into each well of a 24 well plate and culture the cells for seven to 10 days in the cell culture incubator. Then photograph the cells on an inverted fluorescence microscope according to standard fluorescent microscopy protocols.
If the time lapse imaging system is off, turn it on at least one hour before the imaging. When the system is ready, carefully transfer the culture plate to the stage of the microscope and slide the incubator and securely lock the door. Open the software of time lapse imaging system and click add vessel to select the plate type and the manufacturer so the microscope can locate the scanning area accurately.
Select a 10 times objective lens and the wells of interest and set the parameters for the scanning area, the interval time between the scans and a starting and ending time. Then record the time lapse imaging from four to 52 hours. At the end of image recording, use the time lapse imaging system software to retrieve the data and to export the collected videos or image sets.
For confocal microscopy imaging of the cocultures, place the plate onto the stage of an inverted fluorescence microscope and select the red and green laser beams. Observe the cells under a five or 10 times objective and select the spheroid to start scanning. Using a one micron z-step, scan from the bottom to top of the spheroid.
Then process the data using the appropriate image processing software to reconstruct a 3D image that can be further rotated and saved as a 3D movie. Here, images of multicellular 3D spheroids formed by cocultured melanoma cells and fibroblasts are shown. Melanoma cells cultured in the absence of fibroblasts do not form typical 3D spheroids, although some melanoma cells form 2D clusters with extended culture.
Using time lapse imaging, fibroblasts and tumor cells can be observed interacting in coculture, starting to from spheroids at about 36 hours. In this movie, the dynamic process of single cultured melanoma cell aggregate formation from four to 52 hours of culture can be observed. Here, a 3D spheroid structure can be observed by confocal microscopy after seven days of culture, while in this movie, a 2D cell cluster can be visualized.
3D spheroids remain suspended in culture medium and are mobile, while 2D tumor cell clusters tend to attach to the culture plate and are immobile. This 3D model serves as a unique platform for studying tumor-stroma interactions. For example, for elucidating how intercellular Notch1 signaling pathway activity and cancer associated fibroblasts regulate cancer's stem and initiating cell and spheroid formation.
In addition, this model can be used to test the drug responses of cancer stem and initiating cells. Although this method is simple and straight forward, take care to use the correct cell density and cell ratio, and to use the appropriate culture plates as demonstrated. This 3D model can also be applied for either sedating crucial intracellular signaling pathway activity for determining the phenotypes of cancer stem cells as well as screening small molecule compounds to which cancer stem cells are highly sensitive.
