preparation and sorting of ct-fr-stained zebrafish t-all cells for transplantation and tumor tracking

Estimating LSC Frequency in Zebrafish T-ALL Using Cell Proliferation Dye Sorting

Adult stem cells are responsible for the regeneration of differentiated cell types in a given organ and are predominantly present in a dormant, non-dividing state1,2. For example, hematopoietic stem cells (HSCs), which maintain the blood, largely remain quiescent, and only a small fraction enters the cell cycle to self-renew or differentiate to generate mature blood components3. Similarly, in cancers, a rare subpopulation of cells called cancer stem cells (CSCs) possess the self-renewal ability and are responsible for the long-term maintenance of the malignancy4. Cancer stem cells exist in vivo in a state of quiescence or slow growth, which may enable them to escape the anti-proliferative cancer treatments5, evade clearance by the immune system6, reduce oxidative stress, and enhance their DNA repair pathways7. Even a low number of CSCs left behind after treatment can potentially repopulate the tumor, resulting in a patient&#39;s relapse8. Accordingly, understanding cellular quiescence holds great promise for the identification of potential vulnerabilities of CSCs and the development of new ways to target them.
Cell proliferation dyes, such as the carboxyfluorescein succinimidyl ester (CFSE) stain and its derivatives, are commonly used to track the frequency of cell divisions9. The dye permeates across the cell membrane and, once inside the cell, undergoes activation by intracellular esterases into a fluorescent product. The resultant fluorescent compound is retained inside the cell through the covalent amide bonds formed between the succinimidyl moiety and the amine functional groups of intracellular proteins10. With each cellular division, the fluorescent compound is divided equally between the two resulting cells, causing a two-fold signal dilution. This dye enables the detection of up to 10 cell divisions through flow cytometry analysis11.
This approach has been previously utilized to enrich CSC populations in vitro by identifying slow-cycling populations of cells with high retention of the dye11,12. In T-ALL, CFSE has been used to track tumor growth in vivo in patient-derived xenografts in mice. Following cell labeling and three weeks of transplant, flow cytometry analysis showed a rare population of cells that still retained CFSE fluorescence. This population was associated with stemness, treatment resistance, and high similarity to relapse-causing cells in patients13. Accordingly, this dye provides a useful tool for the study of leukemia stem cell (LSC) phenotypes in T-ALL.
The aim of the work is to extend the application of the cell proliferation dye to study quiescence in vivo using a zebrafish T-ALL model. In particular, the rag2:Myc-driven zebrafish T-ALL model14 provides an excellent venue for the study of self-renewal due to the high frequency of LSCs compared to mouse models and human disease15. In addition, the use of zebrafish allows for large-scale transplantation studies, which can be done at a much lower cost of care and maintenance compared to their mouse counterparts16. Zebrafish are also excellent for live imaging applications, as fluorescently labeled tumor cells can be readily viewed using a simple fluorescence microscope to estimate the rate of tumor development16.
In this protocol, we describe the workflow for staining&#160;zebrafish T-ALL cells with cell proliferation dye followed by&#160;in vivo propagation of stained cells in syngeneic CG1 zebrafish. Upon leukemia development, we describe the sorting of cells that retained the dye and their use for a subsequent limiting dilution transplantation experiment to quantify rates of LSC self-renewal. This protocol can be extended for additional applications, including in vivo drug screening of potential compounds for the targeting of quiescent LSCs. In addition, collected cells can be used for different downstream analyses, such as transcriptomic profiling, proteomics, and metabolomics, offering unique insights into the behavior of quiescent LSCs in T-ALL.

Author Spotlight: Identification and Isolation of Quiescent Leukemia Stem Cells from Zebrafish T-ALL

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 zebrafish 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.

Preparation and Sorting of CT-FR-Stained Zebrafish T-ALL Cells for Transplantation and Tumor Tracking

For the preparation of the donor plate transfer 500, 000 wild type CG1 zebrafish 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 four degrees Celsius.Carefully remove 45 microliters of the supernatant 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 zebrafish. After transplanting CG1 zebrafish 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 zebrafish at 28 days post-transplant showed that 89%of CT-FR high zebrafish develop leukemia compared to 20%in the CT-FR low group. CT-FR high zebrafish had a significantly shorter latency to leukemia. CT-FR high zebrafish had a significantly lower overall survival compared to the CT-FR low zebra fish.

preparation-sorting-ct-fr-stained-zebrafish-t-all-cells-for

Research

JoVE Journal

Cancer Research

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry.
Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments.
Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect ...

In Vivo Propagation and Harvesting of GFP-Labeled Zebrafish T-ALL Cells

To begin, thaw vial containing one milliliter of frozen GFP-labeled zebrafish T-ALL 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 supernate before resuspending the pellet in 0.5 milliliters of fish media.Add 10 microliters of cell suspension into the microcentrifuge tube containing 10 microliters of trypan blue dye. Mix well and transfer 10 microliters onto a hemocytometer and count the cells under the microscope. Next, hold the anesthetized adult CG1 zebrafish with ventral side up.Using a Hamilton microsyringe, 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 zebrafish. To harvest leukemia cells, move the euthanized zebrafish 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 zebrafish, 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.

Optimization and In Vivo Propagation of CellTrace Far Red (CT-FR) Stained Zebrafish T-ALL Cells

To begin, collect one time 10 to the power of six fluorescently labeled zebrafish, T-ALL cells in 1.5 milliliter microcentrifuge 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 microcentrifuge tube.Then, add 250 microliters of 0.9 XPBS to bring the volume up to 500 microliters. Add the required volume of CT-FR 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 trypan blue and examine them under the microscope for viability. Using the optimized CT-FR concentration, stain the zebrafish cells and leave some tumor cells unstained.Using a Hamilton microsyringe, inject five microliters of stained and unstained cell suspension into the intraperitoneal cavity of anesthetized zebrafish.