Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining

Leukemia stem cells are a rare subpopulation of cells within the leukemia that are responsible for long-term disease, maintenance, and relapse. Our goal is to better characterize these cells to find ways to target and eliminate them and improve patient outcomes. Cancer stem cells, including leukemia stem cells, exist in a state of quiescence or slow growth, which may enable them to escape anti-proliferative cancer treatments.

Understanding cellular quiescence will help identify potential vulnerabilities of leukemia stem cells and new ways to target them. Using this protocol, we can identify and isolate quiescent leukemia cells from a zebra fish model of T-cell acute lymphoblastic leukemia. Those cells can be used for downstream applications, such as in vivo drug screening, transcriptomic profiling, and proteomics analysis.

To begin thaw vial containing one milliliter of frozen GFP labeled zebra fish TALL cells in a 37 degree Celsius water bath with gentle shaking. Slowly pipette the contents of the vial into a 15 milliliter conical tube containing four milliliters of fish media on ice. Spin down the cells at 2, 500 G for five minutes at four degrees Celsius and discard the supernatant before resuspending the pellet in 0.5 milliliters of fish media.

Add 10 microliters of cell suspension into the micro centrifuge tube containing 10 microliters of tripan blue dye. Mix well and transfer 10 microliters onto a hemo cytometer and count the cells under the microscope. Next, hold the anesthetized adult CG1 zebra fish with ventral side up.

Using a Hamilton micro syringe, inject five to six microliters of cell suspension into the intraperitoneal space. Approximately 21 to 28 days post-transplant, assess the percentage of the GFP labeled leukemia cells from anesthetized zebra fish. To harvest leukemia cells, move the euthanized zebra fish into a new Petri dish and add one milliliter of fish media to serve as a buffer for the cells.

Using a razor blade, first decapitate the zebra fish then macerate the tissue. Pipette up and down to dislodge large clumps of cells. Pass the cell suspension through a 40 micron cell strainer into a 50 milliliter conical tube to dissociate into a single cell suspension.

To begin, collect one time 10 to the power of six fluorescently labeled zebra fish TALL cells in 1.5 milliliter micro centrifuge tubes. Centrifuge the cells at 2, 500 G for five minutes at four degrees Celsius and resuspend the pellet in one milliliter of 0.9 XPBS. Place 250 microliters of cell suspension into a micro centrifuge tube.

Then add 250 microliters of 0.9 XPBS to bring the volume up to 500 microliters. Add the required volume of CTFR dye stock solution, and incubate for 20 minutes at 37 degrees Celsius, protecting from light. Centrifuge the cells at 2, 500 G for five minutes at room temperature.

Add 500 microliters of fish media to remove the excess dye, and centrifuge again before resuspending the pellet in 25 microliters of fish media. Stain the cells with tripan blue, and examine them under the microscope for viability. Using the optimized CTFR concentration, stain the zebra fish cells and leave some tumor cells unstained.

Using a Hamilton micro syringe, inject five microliters of stained and unstained cell suspension into the intraperitoneal cavity of anesthetized zebra fish. For the preparation of the donor plate transfer 500, 000 wild type CG1 zebra fish cells into a 15 milliliter conical tube. Add five milliliters of fish media to dilute the cell stock to 100 cells per microliter.

Dispense 50 microliters of the cell suspension into each well of the 96 well plate and place the plate at four degrees Celsius until sorting. Prepare no stain and single color controls for the setup of the sorting experiment. Pass the cell suspension of the controls and the samples through a 35 micron filter cap into a fax tube on ice.

Centrifuge the 96 well plate at 2, 500 G at 4 degrees Celsius. Carefully remove 45 microliters of the supernatin from each well leaving five microliters of liquid behind. Resuspend the cells with a 20 microliter pipette and using the Hamilton micro syringe, inject the cell suspensions into the desired number of CG1 Zebra fish.

After transplanting CG1 zebra fish with sorted cells for the limiting dilution analysis, the CT-FR high cells had a fourfold higher leukemic stem cell frequency compared to CT-FR low cells. Representative images of zebra fish at 28 days post-transplant showed that 89%of CTFR high zebra fish developed leukemia compared to 20%in the CT-FR low group. CT-FR high zebra fish had a significantly shorter latency to leukemia.

CT-FR high zebra fish had a significantly lower overall survival compared to the CT-FR low zebra fish.