The overall goal of this procedure is to describe the isolation of prostate cancer stem cells from human tissue by fax. This is accomplished by first processing prostate cancer tissue harvested from surgical specimens. In the second step, a cell suspension is generated from the tissue sections and the cells are labeled with fluorescent antibodies.
In the final step, the prostate cancer stem cells are sorted by facts. Ultimately, the molecular and functional attributes of the isolated cells can be characterized by xenotransplantation and gene expression profiling. This method can help answer key questions in the prostate cancer field by facilitating the molecular and functional characterization of cancer stem cells from human samples.
Demonstrating the procedure will be Samuel Vidal, an MD PhD student Aiden Quinn, a master's student, and jania. Our research A from the lab Begin by harvesting excess bulk tissue from the cancer samples not needed for clinical diagnostics into a 50 milliliter conical tube containing 15 milliliters of media. Immediately store the tube at four degrees Celsius for processing within 24 hours of resection, then working in a biosafety cabinet with a sterile scalpel and forceps.
Obtain two to four millimeter thick horizontal tissue sections from the macroscopic tumor nodules within the cancer tissue sample. Place these sections into a new 50 milliliter tube containing media. Then separate a portion and fix it in 10%formalin for histological analysis to confirm the presence of prostate cancer tissue to generate cell suspensions from the tumor tissue.
Next, transfer each of the two to four millimeter tissue sections to a 60 by 15 millimeter culture dish containing one milliliter of sterile PBS Mechanically tritrate the samples using a sterile scalpel and forceps and pool the resulting cell suspensions into a single sterile 50 milliliter conical tube. Add another milliliter of PBS to the sample dish, repeating the iteration three to four more times until each tissue section is completely dissociated. And then use a five milliliter pipette to mix the cell solution.
Vortex the cell solutions at maximum speed for one minute, and then filter the resulting cell slurry through a 35 micrometer cell strainer into a second sterile 50 milliliter conical tube. Spin down the filtered cells for 10 minutes at 450 times G at room temperature. Then resuspend the pellet in five milliliters of red blood cell lysis buffer.
After five minutes, spin down the cells again. Resus suspending the red blood cell free pellet in a small amount of PBS supplemented with 5%FBS for counting by trian blue exclusion. After quantifying the number of viable cells, dilute the cells to a one times 10 to the six cells per milliliter suspension, and store them on ice for no longer than one hour.
To isolate the prostate cancer stem cells begin by labeling five 15 milliliter conical tubes as shown to exclude the hematopoietic and endothelial cells during sorting. Divide the cell suspension generated from the tumor tissue sections between the five tubes, and then incubate the cells with the appropriate antibodies at a one to 250 dilution for 30 minutes on ice. Next, spin down the cells and then wash the pellets in sterile PBS supplemented with 10%FBS After the wash, incubate the pellets in 10 micrograms per milliliter of DPI and filter the final solutions through 35 micrometer strainer caps into 12 by 75 millimeter polystyrene tubes.
Use the first four tubes to set the gates for forward scatter side scatter dpi, fite, and pe. Finally, use tube five to collect the HLA Class one negative and HLA class one positive populations into individual sterile 15 milliliter conical tubes containing two milliliters of RPMI supplemented with 10%FBS. Then spin down the sorted cell suspensions and resuspend the pellets in 200 to 500 microliters of media for quantification of the viable cells by trian blue exclusion.
The demonstrated protocol facilitates the isolation of HLA Class one negative prostate cancer stem cells from human surgical specimens. These images illustrate the grossly visible macroscopic tumor nodules that can be processed and then confirmed by microscopy. As demonstrated the viable cells are isolated by their negative dapi staining the frequency of the HLA Class one negative prostate cancer stem cells can then be determined.
The numbers of HLA Class one negative prostate cancer stem cells varies between patients, but generally accounts for 0.5 to 8%of the total live population Following this procedure. Other methods like gene expression profiling and xenotransplantation can be used in order to better characterize the mechanisms that contribute to chemotherapy resistance and disease progression.
