Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

The limiting dilution cell transplantation assay is the gold standard for assessing self-renew potential in experimental animal models. In this demonstration, one cell stage syngeneic zebrafish embryos are injected with rag two MIC and RAG two GFP to create transgenic zebrafish that will develop fluorescently labeled T-cell acute lymphoblastic leukemia. By 60 days of life, the primary leukemia cells are isolated from zebrafish and then purified using fluorescence activated cell sorting.

Next, the cells are transplanted at limiting dilution into the peritoneal cavity of adult zebrafish, and the fish are monitored for tumor growth for up to 90 days. Finally, the extreme limiting dilution analysis statistical software program is used to quantify the frequency of leukemia propagating cells within the original tumor. Limiting dilution cell transplantation analysis allows investigators to accurately assess the number of self-renewing cells contained within the bulk of the tumor mass.

It is these self-renewing cells that drive cancer formation and are ultimately responsible for relapse. The main advantage of doing limiting dilution transplantation assays in the zebra fish model is that many fish can be transplanted for each experiment, resulting in better precision when determining the frequency of self-renewing tumor propagating cells. This method provides insight into tumor propagating cells in T-cell, a acute lymphoblastic leukemia.

It can also be applied to understand other zebrafish cancer models. Jessica Blackburn, a fellow in the lab and Sally Liu, a talented technician, will be demonstrating the procedure for you today To linearize Rag two CM and RAG two G-F-P-D-N-A Constructs digest 10 micrograms of each plasmid with restriction enzyme, not one at 37 degrees Celsius overnight. After phenyl chloroform extraction and ethanol precipitation, we suspend each plasmid in 20 microliters of water.

Determine DNA concentrations on a 1%agro gel by running a one-to-one, one to five, and one to 10 dilution of each digested plasmid with 20 microliters, 10 microliters, and five microliters of a high range DNA ladder. Now dilute the DNA to a final concentration of 60 nanograms per microliter for injection into CG one strain syngen. A zebrafish embryos inject 250 nanoliters of 30 nanograms per microliter rag two CM and 30 nanograms per microliter.

Rag two GFP into one cell stage zebrafish embryos carefully targeting DNA directly into the cell and not into the yolk. Store the injected embryos at 28.5 degrees Celsius and remove the dead embryos after 24 hours at five days of life, transfer animals to large tanks approximately 28 days after injection. Anesthetized larval zebrafish by adding 200 microliters of four nanograms per milliliter trica S to 25 milliliters of fish system water in a Petri dish.

Examine the larvae for development of fluorescently labeled TALL by using an epi fluorescence microscope to detect GFP fluorescence. Separate tumor positive larvae from those that are negative to ensure that no leukemic fish are missed in the analysis. Negative larvae can be examined at a later time point once tumors may have grown larger Monitor fluorescently labeled leukemic fish at least once a week using the epi fluorescence microscope to track tumor growth.

When GFP positive leukemia cells have overtaken greater than 50%of the animal, add one milliliter of four nanograms per milliliter. Trica S in a Petri dish containing nine milliliters of fish system water to sacrifice the fish, place the fish in a new Petri dish containing one milliliter of 5%fetal bovine serum in 0.9 XPBS macerate the fish with a razor blade, pipette the mixture to dissociate large cell clumps. Then pass the cells through a 40 micrometer mesh strainer into a 50 milliliter tube.

Pipette 500 microliters of the cells into a four milliliter polystyrene tube. Add one microliter of one milligram per milliliter, peridium iodide to label dead cells vortex, then keep the cells on ice. Also prepare a wild type CG one fish to collect normal blood cells, which serve as a carrier for the tumor cells during transplant at four milliliters of 5%FPS in 0.9 X PB S to the 50 milliliter tube for a total volume of five milliliters.

Count the normal blood cells dilute to three times 10 to the fifth cells per milliliter in 5%FBS in 0.9 x pbs then pipette 100 microliters per well of a 96 well plate using a fluorescence activated cell sorter sort GFP positive PI negative tumor cells into the 96 well plate containing blood cells at indicated doses. Additionally, sort 10, 000 cells into a well without normal blood cells and reanalyze using facts to assess the purity and viability of the sorted cells centrifuge. The 96 well plate at 2000 GS for 10 minutes to pellet the cells.

Remove 95 microliters of the snat and resuspend the cells in the remaining five microliters. Inject the cell suspension into the peritoneal cavity of greater than 60 day old adult syngeneic zebrafish using a 26 and a half gauge micro syringe. Zebrafish do not have to be anesthetized before transplant.

Transplant 45 fish with 10 leukemia cells, 20 fish with 100 cells, seven fish with 1000 cells and five fish with 10, 000 TALL cells approximately 28 days after transplant. Examined the recipient zebra fish with an epi fluorescence microscope using a 4 85 20 excitation wavelength and 5 30 25 emission filter. To detect GFP fluorescence.

Record the number of positive fish per total number of fish injected. Re-examine the fish every 14 days for up to 90 and record the number of positive fish. When a tumor positive fish becomes more abundant.

Sacrifice the animal by trica US overdose, input the results into a table and upload the data into the web-based extreme limiting dilution analysis software to determine the frequency of self-renewing leukemia cells within the primary TALL CG one strain embryos were injected with RAG two cmic and RAG two GFP larva were screened for primary TALL After 28 days consistent with previous work, approximately 5%of injected fish have GFP positive T cells within their thymus, and 100%of these fish will develop leukemia. Here, fluorescently labeled TALL cells were sorted from 65 day old diseased animals and used in the limiting dilution cell transplantation assay. First, a gait was drawn to select single cells.

Then propidium iodide negative cells were selected. And finally a gait was drawn to select only the GFP positive leukemia cells for sorting. In this example of TALL transplanted fish at day 28 early stage tumors were seen as an area of GFP positive cells near the injection site.

While some TLS were more progressed, having expanded to fill the peritoneal cavity for these experiments, over 220 animals were transplanted, which can be easily accomplished by one person within several hours. Data analysis using elda software showed that on average one in 135 T all cells were self-renewing leukemia propagating cells. After watching this video, you should have a good understanding of how to transplant tumor cells at limiting dilution into peritoneal cavity of syngenetic zebrafish, and how to use the resulting data to determine the frequency of tumor propagating cells within a given tumor.

Further studies utilizing genetically modified animals may help to identify the mechanisms governing self-renewal of these tumor propagating cells.