Zebrafish provide many advantages over mouse models to study cancer self-renewal. For example, large numbers of zebrafish can be housed in a relatively small space, and husbandry costs are 20 times less compared with mice. Additionally, cell transplantation assays can utilize large numbers of animals that are unparalleled in mouse studies. Limiting dilution cell transplantation experiments in mice commonly use three to five animals per dilution and assess only three dilutions per tumor.11,31,32,45
Cell transplantation experiments in zebrafish routinely use 10–12 animals per dilution at four dilutions, greatly facilitating accurate assessment of tumor-initiating potential (Smith et al.
, unpublished). For example, 300+ adult zebrafish can be transplanted by intraperitoneal injection in 1 day, showing the massive numbers of adult animals that can be used for these experiments. Such large-scale cell transplantation experiments in mice are possible, but not economically feasible for most labs because of both excessive per diem
charges and space constraints. Low numbers of tumor cells can be transplanted and can induce tumors in recipient zebrafish. In a transgenic model of ERMS, a subset of tumors engrafted disease into irradiated adult fish with as few as 10
The ability to transplant tumors with few engrafting cells makes it possible to design experiments to study clonal evolution and its effects on tumor-initiating potential.
Fluorescent protein expression within cancer cells greatly facilitates the tracking of tumor formation and can be used to quantify transplant engraftment into recipient animals (). Transgenic zebrafish models of leukemia that label tumor cells with fluorescent proteins have been used to quantify the numbers of tumor-initiating cells contained within the bulk of the leukemia mass. In these experiments, limiting dilution cell transplantation analysis of primary zebrafish T-cell leukemias established that 1 in 103
to 1 in 2
cells are capable of tumor engraftment into irradiated recipient animals.41,46
Remarkably, these numbers are in keeping with published reports from a mouse model of Pten deficiency-induced T-cell leukemia which suggest that tumor-initiating cell number may be low in these leukemias.34
However, we caution that experiments in zebrafish have utilized nonimmune-matched, irradiated recipient animals and likely severely underestimate true leukemia-initiating potential because of incomplete suppression of the immune system following gamma irradiation (Smith et al.
, unpublished). Further experiments will be required to better address self-renewal in zebrafish (see below).
FIG. 3. Heritable T-cell malignancies are transplantable and can be easily visualized by fluorescent protein expression in leukemic cells (modified from Frazer et al.41). Green fluorescent protein (GFP+) leukemias from shrek (srk; A–C), hulk (hlk; D–F (more ...)
Heterogeneous tumor cell populations can be identified using transgenic lines that express fluorescent proteins in distinct cancer cell subpopulations. Subsequently, FACS and limiting dilution cell transplantation experiments can be used to isolate specific tumor cell types and assess if they are responsible for tumor regrowth and self-renewal. This strategy was used to identify CSCs in embryonal rhabdomyosarcoma.37
The RAS-induced ERMS stem cells expressed rag2-dsRED2
but not the mature muscle marker alpha-actin–green fluorescent protein (GFP
) (). Other ERMS cancer cells failed to engraft disease as robustly as the dsRED+
cell population. Molecular analysis established that the ERMS CSCs were most similar to the normal activated, muscle satellite cells. Together, our results showed that ERMS follows either a CSC model or the hierarchy model of self-renewal. In total, these three published reports were the first to quantify the extent to which self-renewal was found in zebrafish cancer.37,41,46
Importantly, they lay the foundation for new studies to better refine the rules of self-renewal in both leukemias and solid tumors.
FIG. 4. The dsRED2+ cell population from double transgenic rag2-dsRED2/alpha-actin-GFP animals contains the serially transplantable cancer stem cell in zebrafish embryonal rhabdomyosarcoma (ERMS). (A–D) Primary transplanted tumors from alpha-actin-GFP (more ...)
Fluorescent reporter lines are invaluable for detecting tumor growth and engraftment into recipient zebrafish; however, their use is still limited by directly visualizing tumor cells through translucent zebrafish. In Medaka, see-through fish were generated by creating genetic strains of fish that lack both iridophores and melanocytes.47
In these experiments, Wakamatsu et al.
directly visualized organ formation and used a transgenic GFP reporter line to follow gonad growth in vivo
. Building on these observations, White et al.
have recently created a see-through zebrafish—creatively called “casper”—that also lacks iridophores and melanocytes.48
This breakthrough facilitated the tracking of GFP-labeled blood cells following cell transplantation into irradiated recipient fish and the visualization of melanoma regrowth after injection into casper mutant animals. Remarkably, the authors were able to track melanoma dissemination in these animals over time and suggested that individual cells can be visualized within whole adult fish by laser-scanning confocal microscopy. Such approaches will greatly facilitate the identification of transplant engraftment into recipient animals and will likely aid in identifying tumor niches where self-renewing cells reside. Together, optically clear adult zebrafish provide many advantages over existing vertebrate models of cancer to visualize cancer development and progression.
Syngeneic zebrafish were recently created and were successfully used to engraft liver and pancreatic tumors into transplant recipients.49
The two clonal lines described by Mizgireuv and Revskoy were created by squeezing eggs from a single female, fertilizing them with ultraviolet-inactivated sperm, and then applying 2
min of heat shock at 41.4°C prior to the first cleavage (~13
min after fertilization). The exceedingly small fraction of animals that survived this procedure was raised to adulthood. Eggs were obtained from gynogenetic diploid female fish and subjected to a second round of heat shock. The resulting progenies were incrossed to create the CB1 and CW1 clonal fish lines. Importantly, carcinogen-induced liver and pancreatic tumors that developed in these lines could be transplanted into syngeneic recipient animals (). Although a pioneering study, the potential for these and other clonal zebrafish lines has yet to be fully realized. In fact, limiting dilution experiments using these lines should correctly assess and quantify true tumor-initiating cell number in a variety of cancers. Creating clonal fish lines that are see-through will revolutionize the types of experiments that can be completed in zebrafish cell transplantation. Finally, developing truly immune-deficient zebrafish will provide a much needed tool to effectively beat the immune system. Recent advances in targeted gene disruption using zinc finger nucleases should facilitate the development of fully immune-suppressed zebrafish.50–52
IL2-gamma receptor-deficient fish would provide a universal recipient line for both zebrafish cancer and xenograft transplantation studies.
FIG. 5. Tumors from clonal zebrafish can be transplanted into syngeneic recipient animals. Advanced stages of tumor development after intramuscular transplantation of moderately differentiated hepatocellular carcinoma zt34 (A, B) compared with normal liver ( (more ...)
Human and mouse cells can be transplanted into larval fish or immune-suppressed adult fish (). Xenograft transplantation experiments in zebrafish have been recently reviewed by Stoletov and Klemke,54
but we will summarize several key findings from this work. Stoletov et al.
showed that adenocarcinomas (MDA-435), fibrosarcomas (HT-1080), and melanomas (B16) can engraft into 30-day-old dexamethasone-treated fli1-GFP
and established that vascular reorganization can be easily visualized in engrafted animals56
(). In these experiments, RAS family homolog member C (RHOC) played a critical role in cell movement and could partially regulate the early stages of metastasis. Additional experiments by Nicoli et al.
demonstrated that tumorigenic FGF2-overexpressing mouse aortic endothelial cells could be transplanted into 2-day-old embryos prior to establishment of the acquired immune system.57
These experiments capitalized on the use of immune-incompetent zebrafish embryos as recipients and showed that FGF2-expressing cells were able to undergo new vascular growth. Cell transplantation of human C8161 melanoma cells into blastula stage embryos showed that the nodal pathway was active in melanomas, despite these fish never developed robust engraftment of tumors.58,59
By contrast, introduction of human metastatic melanoma WM-266-4 cells into the yolk of 2-day-old fish formed observable tumors by 7 days postinjection60
(). Additionally, human U251 glioblastoma cells when transplanted into the yolk sac of blastula-stage embryos proliferated, formed tumors (), and recruited blood vessels. Engrafted glioblastoma cells were more sensitive to radiation treatment when treated in combination with temozolomide.61
Lally et al.
also used U251 glioblastoma cells to identify novel small molecule sensitizers and identified a novel drug NS123 (4′-bromo-3′-nitropropiophenone) that enhanced the growth-inhibitory effect of U251 cells to ionizing radiation.62
Although xenograft transplantation into zebrafish is a firmly established method for assessing recruitment of vasculature, response to therapy, and early metastatic potential, their use in determining self-renewal capacity has yet to be described. Xenograft cell transplantation experiments that assess self-renewal potential will likely require fully immune-compromised fish such as those outlined earlier.
FIG. 6. Human cancers can engraft into zebrafish embryos and larvae. Hematoxylin and eosin-stained cross section of a juvenile zebrafish that had been transplanted with MDA-435 adenocarcinoma cells. Cells were injected intraperitoneally and imaged at 5 days posttransplantation (more ...)