Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.
Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment.
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.
1. DNA microinjection of zebrafish embryos
2. Screening for primary T-ALL in zebrafish larvae
3. Isolation and purification of fluorescently-labeled primary leukemia cells
4. Limiting dilution transplantation into adult zebrafish
5. Analysis to determine leukemia-initiating cell frequency
6. Representative Results:
DNA microinjection into embryos from syngeneic fish often results in a low survival rate of the injected embryos, with <60% of embryos surviving for 24 hours. Additionally, survival of the injected syngeneic fish is generally poor during larval development, with often <20% surviving for 28 days. It is therefore important to inject a large number of embryos in order to create transgenic fish that will develop T-ALL.
As an example, CG1 strain embryos were injected with rag2::cMyc+rag2::GFP. The transgenes co-segregate in the developing embryo, so that every cell that expresses GFP will also express cMyc. Larvae were screened for primary T-ALL after 28 days (Figure 1). Previous work has shown that approximately 5% of injected fish will have GFP-positive T-cells within their thymus (Figure 1), and 100% of these fish will develop leukemia as the T-cells become transformed11. While a small proportion of zebrafish may have a more advanced leukemia that is very easy to detect at day 28, most will only have a GFP-positive thymus (Figure 1), so larvae should be examined individually under high magnification to ensure that all positive fish are selected. GFP-positive T-ALL cells will overtake >50% of the animal between days 45-70.
In the example provided, zebrafish were sacrificed at 65 days of life, and leukemia cells were isolated and FACS sorted for limiting dilution transplantation. Single cells that were propidium iodide negative and GFP-positive (Figure 2) were sorted into a 96-well plate. Reanalysis was done on 10,000 sorted cells in order to assess viability (ideally >95%) and purity (ideally >92%, Figure 2). Transplanted T-ALLs generally arise before 28 days, but some tumors may take more than 60 days to develop. In this example, at day 28, early stage tumors were seen as an area of GFP-positive cells near the injection site (Figure 3B), while some T-ALLs were more progressed, having expanded to fill the peritoneal cavity (Figure 3C). Data from limiting dilution cell transplantation assays from three different primary T-ALLs are shown in Figure 3D. 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, 1 in 135 T-ALL cells were self-renewing leukemia-propagating cells (Figure 3E).
Figure 1. Zebrafish larvae injected at the one-cell stage with rag::-cMyc+rag::-eGFP were screened for primary leukemia growth 28 days post-injection. Fish (*) had GFP-positive T-cells within the thymus; this fish will develop T-ALL as the T-cells become transformed over time. This stage is very common at day 28. One fish (**) had an advanced T-ALL that has spread into the soft tissue. The remaining two fish are negative for tumor growth. The image was taken at 16X magnification.
Figure 2. Fluorescently-labeled T-ALL cells were sorted from diseased animals and used in the limiting dilution cell transplantation assay. First, a gate was drawn to select single cells (upper left panel), then propidium iodide negative cells are selected (middle left panel). Finally, a gate was drawn to select only the GFP-positive leukemia cells for sorting (lower left panel). Sorted cells should be reanalyzed to assess viability and purity before transplant (right panels).
Figure 3. Zebrafish were examined for leukemia growth 28 days after transplant. Fish are either tumor negative (A), have a small tumor growing at the injection site (B), or have a progressed leukemia (C). The images were taken at 7X magnification. The total number of leukemia-positive fish per total number of fish transplanted at each dilution is recorded, as in (D). The data are input into the web-based ELDA program to calculate the number of self-renewing leukemia cells and the upper and lower 95% confidence intervals (E).
A major strength of using zebrafish in cancer research is that large numbers of animals can be used at relatively low cost. This is especially important in limiting dilution cell transplantation assays, where the proportions of transplanted animals that develop tumors to the total number transplanted are used to determine tumor-initiating cell frequency. In the method presented here, over 70 transplant recipient animals are used per assay, providing an accurate estimate of tumor-propagating cell number. Both the number of animals used and the doses of tumor cells transplanted should be optimized for a given cancer model; for example, if initial experiments show tumor-initiating cells are rare, useful transplant doses may be 100,000, 50,000, 10,000 and 1,000 cells per transplant.
Syngeneic zebrafish strains are useful in limiting dilution analysis. However, these strains are not yet commonly used in all laboratories, and some zebrafish cancer models exist only in allogenic fish. Limiting dilution cell transplantations can be performed in wild-type strains that have undergone immune ablation, although the self-renewing cell frequency may be calculated as 10-100 fold higher than if syngeneic zebrafish were used8. It is important to note that greater doses of cells should be used for transplants into non-immune matched, irradiated recipients, as some tumor cells will not survive transplant into a foreign microenvironment. Additionally, many tumors will begin to regress after 21 days when the recipient animal’s immune system recovers, so animals should be assessed for tumor growth every 7 days after transplant.
The methods presented here are suitable for use with any zebrafish cancer model, and have thus far been used to determine tumor-initiating cell frequency in both T-cell acute lymphoblastic leukemia8, and a solid tumor model, rhabdomyosarcoma16. These tumors were fluorescently labeled by co-injection of embryos with both an oncogene and fluorescent marker; because the DNA co-segregates, any cell expressing the oncogene will also express the fluorescent protein17. While fluorescence is recommended because it facilitates tracking of tumor growth without the need to sacrifice the animal and provides a straight-forward way to isolate tumor cells by FACS, it is possible to label tumor cells with antibodies to tumor-specific makers to facilitate their purification, though antibody staining in zebrafish may require optimization.
Finally, once the incidence of tumor-propagating cells has been determined for a given tumor type, it is possible to use these assays to identify the mechanisms that govern their cell frequency. For example, primary tumor cells can be treated with pathway specific inhibitors before limiting dilution transplant, or transplanted fish themselves may be dosed with drugs. Additionally, the genetics of the tumors themselves may be manipulated by injecting the one-cell stage fish with a gene of interest, so that the resulting primary tumors will over-express the gene. In these experiments, any effect on self-renewing cell frequency will be observed in the limiting dilution assays.
The authors have nothing to disclose.
Funding has been provided by NIH grants 5T32CA09216-26 (for J.S.B.), and K01 AR055619-01A1 and 3 K01 AR055619-03S1 (for D.M.L.), as well as by the Alex’s Lemonade Stand Foundation, the Harvard Stem Cell Institute and the American Cancer Society.
Name of the Reagent | Company | Catalog Number |
---|---|---|
NotI restriction enzyme | New England Biolabs | R0189 |
Phenol:Chloroform | EMD Biochemicals | 6805-OP |
5M KCl | Sigma-Aldrich | P9541 |
100X Tris-EDTA | Sigma-Aldrich | T9285 |
High Range DNA ladder | Fermentas | R0621 |
Syngeneic zebrafish | References6-8 | |
Fetal Bovine Serum | Gibco | 16000-036 |
Propidium Iodide | Sigma-Aldrich | P4864 |
26G/2″ Microsyringe | Hamilton | 80366 |
40μm mesh cell strainer | BD Falcon | 352340 |