Cancer stem cells or CSCs have the capacity for long-term self-renewal and the ability to produce differentiated progeny. CSCs have been identified in a growing number of malignancies, including pancreatic adenocarcinoma in which they can be identified based on aldehyde dehydrogenase activity and expression of cell surface antigen, CD 44, CD 24, and CD 1 33. Here tumors are isolated from mice harboring human pancreatic cancer.
Xenografts and single cell suspensions are generated. The cells are then stained with eldor reagent, which identifies cells with high aldehyde dehydrogenase activity and cell surface antigen CD 44 and CD 24. Cancer stem cell populations are then isolated using fluorescence activated cell sorting.
The isolated cells can be used in various downstream functional and analytical assays. The stem cell hypothesis is an emerging area in cancer biology. In essence, the hypothesis states that tumors contain distinct phenotypic and functional subsets of cells.
Certain subsets of cells, term tumor initiating cells or cancer stem cells can generate tumors within immunodeficient mice or colonies within in vitro congenic assays. More recently, cancer stem cells are also thought to play an important role in development of metastasis as well as therapeutic resistance. Originally, cancer stem cells were studied in myeloid leukemias using either peripheral blood or bone marrow samples.
However, with the emergence of stem cell biology and solid tumors, a challenge has emerged to generate single cell suspensions that can be flow sorted than studied for downstream applications. My post O fellow Zan will now demonstrate this procedure. I will visually demonstrate several critical steps during this protocol.
Beginning with the generation of a single suspension of tumor cells from a human pancreatic cancer xenograft. Then I will stain cells for aldehyde dehydrogenase activity and several cell surface antigens. Finally, I will sort cells using a fluorescence activated cell sorter.
The mice used for this protocol harbor subcutaneous human pancreatic cancer xenografts. These mice were previously generated as follows. Freshly collected tumors from patients that underwent surgery for pancreatic adenocarcinoma at the Johns Hopkins Hospital were minced into two to three millimeter pieces and implanted subcutaneously into the flanks of six week old athymic mice.
When the tumors were approximately 1.5 cubic centimeters, they were excised and transplanted into secondary recipient mice to expand the tumor pool. This protocol begins with the harvesting of a subcutaneous tumor from the flank of an anthemic mouse by blunt dissection. Once the tumor has been removed, immediately place it in cell dissociation buffer.
Next, weigh the tumor, then place it back into 10 milliliters of cell dissociation buffer per one gram of tumor tissue working in a sterile biosafety cabinet. Transfer the tumor into a 100 by 20 millimeter culture dish containing one milliliter of cell dissociation buffer using sterile razor blades and forceps. Mechanically mince the tumor.
Transfer the tumor cell suspension to a 50 milliliter conical tube containing the remaining volume of the cell dissociation buffer. Next tri rate the tumor cell suspension through a five milliliter serological pipette 10 times. The tumor pieces should be small so that they do not clog the five milliliter serological pipe pipette and then vortex the suspension at maximum speed for one minute.
Then incubate the tumor cell suspension at 37 degrees Celsius for two hours, vortexing for one minute every 20 minutes. After the two hour incubation, vortex the cell suspension at maximum speed for one minute. Pass the cell suspension through a 70 micron filter and collect it in a fresh 50 milliliter conical tube.
Adjust the final volume to 15 milliliters with DMEM containing 10%FBS and pen strip to further separate viable cells from dead cells and tissue debris. Pipette 15 milliliters of fial pack plus slowly below the cell suspension to prepare a fial gradient for separation by density centrifugation. Take care not to mix the two layers centrifuge at 500 G for 30 minutes at room temperature with the break turned off following the centrifugation, the viable cells will be at the interface of the fial pack plus and cell dissociation buffer.
Transfer them to a new 50 milliliter conical tube. Add fresh DMEM supplemented with 10%FBS to the cell suspension to dilute it one to three centrifuge the isolated cells at 450 G for 10 minutes at room temperature after centrifuging resus, suspend the cell pellet in one milliliter of alor buffer, which is provided in the alor staining kit and will be used in the subsequent cell staining steps. Next, dilute 10 microliters of the cell suspension with 10 milliliters of trian blue and count the viable cells using a hemo cytometer.
If a large number of dead cells or tissue debris are noted during this step, repeat the cell. Separation by density centrifugation cells will be stained for aldehyde dehydrogenase activity. Using the alde Fluor reagent cells will also be stained for several cell surface antigens using specific fluoro four conjugated antibodies.
Label seven 12 by 75 millimeter polystyrene test tubes and place them on ice. Dilute the cell suspension in two milliliters of eldor buffer to a final concentration of five to 10 million cells per milliliter and keep it on ice. Then according to the instructions in the accompanying a written protocol, prepare the following samples to set compensation controls on the fax area.
Flow cytometer first non-ST stained control cells. Second cell stained with CD 44 A PC.Third cell stained with CD 24 PE and fourth cell stained with mouse CD 31 biotin mouse lineage, biotin mouse, H two KD, biotin and streptavidin per cp. Also prepare a fifth sample containing ALOR only and a sixth sample for setting the A LDH positive and CD 44 positive CD 24 positive gates.
This sample will contain ELDOR DEAB, an inhibitor of aldehyde dehydrogenase isotopic control antibodies. IgG two B kappa a PC IgG two a kappa PE mouse CD 31 biotin mouse lineage, biotin mouse, H two KD, biotin and streptavidin per cp. Then in the final tube, stain cells with alor CD 44 A PC CD 24 PE mouse CD 31 biotin mouse lineage, biotin mouse, H two KD, biotin, and streptavidin per cp.
This is the actual specimen to be sorted once all of the staining has been performed, keep the tubes on ice in the dark until cell sorting is performed. This sorting protocol will lead to the isolation of A LDH positive CD 44, positive CD 24 positive pancreatic CSCs. Just prior to sorting, add two milligrams per milliliter propidium iodide to the A L DH positive CD 44 positive CD 24 positive gating tube to gate out non-viable cells using the non-ST stained CD 44 A PC CD 24 PE mouse lineage and alor cells set the compensation controls on a FIA flow.
Cytometer generate gates based on forward and side scatter parameters to collect cell singlets. Use the non-ST stained and mouse lineage tubes to create a gate for non-use per CP negative and viable propidium iodide negative cells using the FL three channel. Most human pancreatic cancer xenografts contain 20 to 60%mouse derived cells using the mouse CD 31 mouse lineage cocktail and mouse H two kd.
Biotin conjugated antibodies using the DEAB treated cells generate gates in the FL one channel for A LDH positive cells. The frequency of the A L DH positive population from different xenografts is variable, but is generally one to 4%of the total cell population. Then place the cells for sorting into the fax port and using these gates.
Collect A LDH positive cells into 12 by 75 millimeter polystyrene test tubes containing one milliliter of DM EM supplemented with 10%FBS. Next generate gates created using cells stained with eldor IgG two B kappa a PC and IgG two a kappa PE in the FL four and FL two channels. Then collect CD 44 positive CD 24 positive, which generally are 0.2 to 5%of the total population after each cell population has been sorted.
Centrifuge the cell suspension at 450 G for 10 minutes and resuspend the cells in 500 milliliters of fresh DMEM supplemented with 10%FBS count the number of sorted cells using a hemo cytometer. This procedure will result in the isolation of viable pancreatic CSCs. These cells can be used for a number of analytical and functional assays requiring viable cells.
We've used them to measure in vitro clon genic growth and invasion. We've used them to measure in vivo tumor initiation and metastasis formation, and we've used them to perform comparative genetic analyses. Once mastered, this technique can be performed in approximately seven hours.
There are several steps that are critical to obtaining a pure population of viable cells. First xenografts that are less than one centimeter in greatest diameter will have the least amount of necrotic tissue within the tumors. This will ensure the least amount of non-viable cells and tissue debris contaminating your final population of isolated cells.
Second, while generating a single suspension of tumor cells, it is important to work quickly and maintain the cells or tumor in media supplemented with 10%serum to enhance cell viability. Third, once cell staining has been initiated with the alde Fluor reagent and antibodies, it is important to maintain the cells on ice and to keep them in the alor buffer so as to reduce the amount of influx of the alor reagent from the cells. Additionally, it is important to maintain the cells in the dark as much as possible so as to reduce the amount of photobleaching of the fluoro fours.
Finally, manually counting the cells using a hemo cytometer is important. Since the flow cytometer will often give inaccurate cell counts.
