For nearly a decade, researchers in the field of pediatric oncology have been using zebrafish as a model for understanding the contributions of genetic alternations to the pathogenesis of neuroblastoma (NB), and exploring the molecular and cellular mechanisms that underlie neuroblastoma initiation and metastasis.
Over the past ten years, zebrafish have become an increasingly popular tool for scientists conducting biomedical studies and other research. The species’ high fecundity rate, low cost of maintenance, and the ease of observation and genetic manipulation all contribute to its increasing use as an alternative and valuable vertebrate model system to study human disease. The expanding community of researchers using zebrafish has brought advanced technologies to the model, as well as a rapidly expanding inventory of transgenic and mutant lines that can be applied to different research niches. Cancer research using the zebrafish model can be traced back to 1965, when Dr. Mearle Stantion performed pioneered work to induce hepatic neoplasia in zebrafish with Diethylnitrosamine [1]. In 2003, the first zebrafish genetic cancer model was reported by Drs. David Langenau and Thomas Look, in which the MYC oncogene was overexpressed under control of the rag2 promoter, resulting in the development of T cell leukemia in the transgenic animal [2]. Since then many more zebrafish cancer models have been developed to understand the pathogenesis of leukemia, melanoma, rhabdomyosarcoma, hepatocellular carcinoma and many other tumor types [3][4][5][6]. In particular, the zebrafish model has also shown exceptional promise in dissecting the contributions of genetic alterations that were identified from integrative genomic analyses of neuroblastoma (NB) to the pathogenesis of this devastating pediatric cancer.
NB is the most common extracranial solid tumor in children and accounts for ~10% of all childhood cancer-related deaths [7]. It is derived from transformed neural crest progenitor cells in the developing peripheral sympathetic nervous system (PSNS) [8][9]. High-risk patients with amplified MYCN and over 18 months of age are often presented with widespread metastasis at diagnosis. Over the past few years, the five-year event-free survival rate for children with high-risk disease remains lower than 50% [10][11]. Very recently, a Phase III trial of immunotherapy, consisting of Dinutuximab, granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-2 (IL2), showed significantly increased five-year overall survival rate of patients with high-risk NB to ~70% [12][13]. This immunotherapy has been approved by FDA for the treatment of patients with high-risk NB who achieve at least a partial response to prior first-line multiagent, multimodality therapy [12]. Although the improved outcomes are observed with the inclusion of Dinutuximab as part of treatment regimens for newly diagnosed NB, the prognosis for the relapsed disease remains poor (<10% progression-free survival) [14][15]. Therefore, better understanding of the pathogenesis of this disease and developing novel and more effective therapies are needed.
As an important member of the MYC proto-oncogene family identified from NB patients [16], MYCN amplification accounts for ~25% of NB cases and is associated with poor disease outcome [17][18][19]. MYCN is a bHLH transcription factor and is homologous to c-MYC structurally and functionally. It can promote neoplastic transformation of cultured mammalian cells and rat embryo fibroblasts [20][21]. In 1997, Dr. William Weiss developed the first animal model of NB by overexpressing MYCN under control of Tyrosine Hydroxylase (TH) in transgenic mice, which is by far still the most popular model for NB research [22]. Following Dr. Weiss’s effort, several genetically modified mouse (GEMM) lines with direct, conditional, inducible overexpression, knock-in or knockout of NB-relevant genes, including mutationally activated ALK (Anaplastic Lymphoma Receptor Tyrosine Kinase) [23][24][25], LIN28B (Lin-28 Homolog B) [26][27], SV40 large T antigen (Simian Vacuolating Virus 40 TAg) [28][29][30] and others [31][32] were subsequently developed. These models demonstrated a sufficient induction of NB in mice, which resemble the features of human NBs [24].
Although the mouse model provides valuable molecular insights on NB pathogenesis and opened the door for NB research, it has some disadvantages when compared to the zebrafish model. Neuroblastomas are different from adult tumors, in that they arise early in development; identifying the early onset of tumorigenesis in mice without euthanizing the animals is difficult and creates challenges in dissecting the molecular and cellular mechanisms underlying early onset tumor initiation. Zebrafish, by contrast, are translucent and develop from externally fertilized eggs, which allows for early detection of tumor onset in live animals. The zebrafish model is also more practical than GEMM, less expensive, and does not require sacrificing these animals to track tumor initiation and visualization of tumor growth. Therefore, the zebrafish model can serve as an alternative for the commonly used mouse model to conduct genetic research.
In 2012, the first zebrafish model of NB was generated and published by Zhu et al. [33]. Two oncogenes, MYCN and mutationally activated ALK (the most commonly mutated genes in primary neuroblastoma [34][35][36][37] and an attractive candidate for targeted therapy [38][39]), were expressed under control of the dopamine-beta-hydroxylase (dβh) promoter [33]. Following this initial effort on modeling NB in zebrafish, many new transgenic fish lines were developed, uncovering additional novel genetic alterations that cooperate with MYCN or c-MYC during NB pathogenesis. The evolution of zebrafish NB models has revealed the complexity of this disease at the molecular level and demonstrates the robustness of the model system in deepening our understanding of the molecular and cellular basis underlying NB pathogenesis. An overview of the NB zebrafish disease model workflow is illustrated in Figure 1.
Of course, the advances in understanding NB have not been achieved without obstacles and challenges, some of which appear daunting. In this paper, we aim to:
Zebrafish | Mouse | Fly | Worm | ||||||
---|---|---|---|---|---|---|---|---|---|
New Models Developed | Drugs Tested in the Zebrafish Models | Drugs applied in NB Treatment, Clinical Trials or other Animal Models | |||||||
Transparency | Fully transparent at embryonic stage and remain translucency through adulthood. PTU can be used to inhibit pigmentation during early embryonic development. Mutant fish line without pigments are available. |
Not transparent | Transparent in larva stage and some parts of the adults | Transparent No pigmentation |
|||||
Amsterdam, A. et al., 2009 [40] | Retroviral-mediated mutagenesis | Hagoromo Mutants | N/A | N/A | |||||
Offspring size per mating | Up to 100 | ~3–12 | Up to 500 | Hermaphrodites, varies | |||||
Zhu, S. et al., 2012 [33] | I-SceI meganuclease mediated transgenesis | Tg(dβh:EGFP-MYCN) and Tg(dβh:EGFP; dβh:ALKF1174L) transgenic fish lines | N/A | N/A | Genetic similarity (humans genome as reference) |
71% | 85% | 50% | 52% |
Pei, D. et al., 2013 [41] | Morpholino-mediated gene knockdown & transient overexpression of structure variants | Embryos with gain or loss of function of phox2b/PHOX2B | 13–cis retinoic acid (at 1~100 nM) treatment of embryos | Applied to patients with high-risk NB as maintenance therapy after consolidation therapy [42][43] | Immune System | Underdeveloped adaptive immune system in larvae | |||
He, S. et al., 2016 [44] | I-SceI meganuclease mediated transgenesis | Intact | Does not possess acquired/adaptive immunity | Does not possess acquired/adaptive immunity | |||||
Tg(dβh: GRD; dβh:mCherry | ) transgenic fish line | Isotretinoin (13-cis retinoic acid, at 1~2 µM) and Trametinib (MEK inhibitor, at 10~40 nM) treatment of juvenile fish | Trametinib is in clinical trials for the treatments of various types of cancers, including high-risk NB (see NCI clinical trial information). | Tumor visualization | Directly visualized in vivo by microscopy | Cannot be easily visualized inside the body | Directly visualized in vivo by microscopy | Directly visualized in vivo by microscopy | |
Zhang, X. et al., 2017 [45] | I-SceI meganuclease mediated transgenesis | Tg(dβh:Gab2wt;dβh:EGFP) and Tg(dβh:ptpn11E69K-EGFP) transgenic fish lines | CBL0137 (FACT inhibitor, at 4 mM) and Trametinib (MEK inhibitor, at 2 μM) treatment of tumor-bearing fish | CBL0137 is in a clinical trial for the treatment of patients with advanced extremity melanoma or sarcoma with metastasis (see NCI clinical trial information). In TH-MYCN tumor-bearing mice, CBL0137 combined with panobinostat can ablate tumor completely (Oncology Times: December 20, 2018) |
Gene editing tools | ||||
Morpholino | |||||||||
Zhu, S. et al., 2017 [46] | I-SceI meganuclease mediated transgenesis | Tg(dβh:LMO1;dβh:mCherry) transgenic fish line | N/A | N/A | Established | Feasible but very limited | |||
Radic-Sarikas, B. et al., 2017 [47 | Possible but not done yet | ] | Drug treatment | Possible but not done yet | |||||
N/A | Lapatinib (EGFR inhibitor, at 2 µM) and YM155 (ABCB1 blocker, at 6.5 nM) treatment of tumor-bearing adult fish | Lapatinib is in clinical trials for the treatments of various types of cancers (see NCI clinical trial information). | Retroviral insertion mutagenesis screen | Feasible | Established | dβh:MYCNFeasible | ) transgenic fish linesFeasible | ||
N/A | N/A | DNA co-injection (I-SceI) Transgenesis | Established, high efficiency | Hypothetical and not efficient | Hypothetical | ||||
Zimmerman, M. W. et al., 2018 | Possible | ||||||||
[49 | CRISPR/TALENs | Established | Established | Established | Established | ||||
Tao, T. et al., 2017 [48] | I-SceI meganuclease mediated transgenesis | Tg(dβh:mCherry;dβh:DEF) and Tg(dβh:EGFP;] | I-SceI meganuclease mediated transgenesis | Tg(dβh:c-MYC; dβh:mCherry) transgenic fish line | N/A | N/A | |||
Shen, J. et al., 2018 [50] | Injection of tumor cells into the yolk sac of zebrafish embryos | Zebrafish embryos xenografted with SK-N-BE(2)-C human NB cell line | Crizotinib (ALK/MET inhibitor, at 8 μM) and 20a (histone deacetylase inhibitor, at 100 μM) treatment of embryos transplanted with SK-N-BE(2)-C human NB cells. | Crizotinib is in clinical trials for the treatments of various types of cancers, including high-risk NB (see NCI clinical trial information). | Tumor transplantation/Xenograft application | Efficient | Moderate to difficult | N/A | N/A |
Aveic, S. et al., 2018 [51] | Injection of tumor cells into the duct of Cuvier of zebrafish embryos | Tg(fli1:GFP) zebrafish embryos transplanted with NB3 and SH-SY5Y NB cell lines | TP-0903 (multi-kinase inhibitor) treatment of embryos transplanted with NB3 and SH-SY5Y NB cell lines | TP-0903 is in a clinical trial for the treatment of FLT3 mutated acute myeloid leukemia (see NCI clinical trial information). | Chimeric animal development | Mouse-zebrafish Chimeric | Human-mouse Chimeric | N/A | N/A |
Seda, M. et al., 2019 [52] | Compound screen using Tg(sox10:gfp) transgenic larvae | N/A | Leflunomide was one of the top hits identified from a library of 640 compounds to regulate cartilage remodelling and NB cell viability. | Leflunomide is approved by FDA for the treatment of active rheumatoid arthritis. It is also in clinical trials for the treatments of various types of cancers (see NCI clinical trial information). | Syngeneic model | Yes | Yes | Yes | N/A |
Drug screening | Established, high-throughput | Established, low-throughput | Established, high-throughput | Established, high-throughput |
Publications | Approaches | |||
---|---|---|---|---|
Koach, J. et al., 2019 | ||||
[ | ||||
53 | ||||
] | ||||
Tol2 transposon- mediated transgenesis | ||||
Tg(dβh:PA2G4) | ||||
transgenic fish line | ||||
WS6 (175.4 mg/kg, 5 μL) treatment of tumor-bearing fish | ||||
WS6 can also suppress tumor growth in the | TH | - | MYCN | mouse model and mice xenografted with human NB cell lines [53]. |
Tao, T. et al., 2020 [54] | I-SceI meganuclease mediated transgenesis | Tg(dβh:EGFP;dβh:LIN28B_WT) and Tg(dβh:EGFP;dβh:LIN28B_MU) transgenic fish lines | N/A | N/A |
Shi, H. et al., 2020 [55] | CRISPR/Cas9-mediate gene knockout | arid1aa and arid1ab knockout fish lines | N/A | N/A |
Dong, Z. et al., 2021 [56] | TALEN-mediate gene knockout | gas7 knockout fish line | N/A | N/A |
Since topics related to PSNS development in zebrafish and mammals and NB genetics have been previously covered in detail by ourselves and others [57][58], we will not be addressing these subjects in this review.
TH-MYCN
[22]
TH-MYCN
MYCN
LMO1
[46]
[61]
[62]
[46]
gas7
[56]
[63]
[64]
[65]
[69]
MYCN
LMO1
[46]
dβh-MYCN
MYCN
[47]
MYCN
MYCN
PA2G4
[53]
MYCN
MYCN
GAB2
MYCN
GAB2
[45]
nf1
MYCN
[44]
[72]
[73]
[74]
fgf8
[40]
MYCN
ALKF1174L
[33]
TH-MYCN
TH
dβh
[33]
dβh-EGFP-MYCN
[33]
dβh-EGFP
dβh-mCherry
dβh
[33]
[33]
EGFP
[33]
mCherry
[46]
ALKwt
[33]
PTPN11
[45]
GAB2
[45]
LIN28B WT
[54]
LIN28B_MU
[54]
LMO1
[46]
DEF
[48]
MYCN
dβh
[48]
dβh-EGFP-MYCN
dβh-MYCN
dβh-EGFP
TgMYCN_TT
TgMYCN_TT
MYCN
MYCN
[77]
MYCN
TgMYCN_TT
[48]
TgMYCN_TT
MYCN
[46]
c-MYC
MYCN
[49]
[80]
[81]
arid1aa
arid1ab
MYCN
ARID1A
arid1aa
arid1ab
MYCN
[55]
[82]
gas7
MYCN
[56]
nestin
[83]
[84]
[85]
[86]
[87]
[88]
2.3. High Throughput Transplantation, Patient-Derived Xenograft (PDX) and In Vivo Drug Screening Using Zebrafish Larvae
To understand disease pathogenesis and screen or validate drug efficacy in vivo, scientists have successfully transplanted tumor cells with different genetic alteration(s) or manipulated gene expression, as well as patient-derived tumor cells into zebrafish at embryonic stage or adulthood [89]. Several features of zebrafish larvae make them uniquely suited for these studies, including: (i) transparent bodies that allow for easy tumor cell injection; (ii) ability to use trackable fluorescent-tagged cells following transplantation; (iii) an immature immune system during early embryonic development, which reduces the chance of the immune rejection of transplanted tumor cells; and (iv) availability of large clutches of embryos for transplantation. Multiple injection sites, such as the perivitelline space, pericardial space, yolk, retro-optical region, and brain, have been explored in a variety of studies to understand the mechanisms of tumor metastasis, angiogenesis, cellular intravasation/extravasation [90][91][92].
Drug screening on zebrafish transplants or xenografts is another exemplary usage of this model. Both embryos and adults can be used in high-throughput drug-screening assays. Embryos are relatively easy to work with due to their permeability of small molecules [93]. Researchers have already performed small-molecule drug screening using zebrafish embryos transplanted with neural crest stem cells (NCSCs) [52]. Since NB is derived from the sympathoadrenal lineage of neural crest cells, small molecules that inhibit NCSC induction might be potentially useful for the NB treatment. Among the 640 FDA-approved drugs applied in this screen, one drug, leflunomide, was identified to inhibit NCSC induction. Leflunomide, as an inhibitor of dihydroorotate dehydrogenase (DHODH) and an immunosuppressive agent for the treatment of patients with rheumatoid arthritis, has already been shown to reduce proliferation and induce apoptosis in NB cells both in vitro and in vivo [94]. Hence, this result further demonstrates the important application of zebrafish as an unbiased in vivo system for effective drug screening. Recently, zebrafish transplanted with human NB cells have been used to demonstrate the effect of a new multi-kinase drug, TP-0903, on reducing extravasation and inducing tumor cell death, suggesting the therapeutic potential of this compound for the NB treatment [51].
Although PDX mouse models are considered the gold standard for the in vivo validation of drug efficacy, the studies led by Drs. Ferreira and Fior, have demonstrated that the patient-derived zebrafish xenografts (zPDX, also called cancer “avatars”) can be used to sense cancer behavior and screen for potential novel therapies. Using a panel of zebrafish xenografts with patient-derived colorectal cancers, Ferreira and Fior rapidly screened the available therapeutic options for the colorectal cancers and predicted the treatment outcomes [92][95], which set the groundwork for using zPDX as a rapid in vivo screening platform for future personalized cancer treatments. Following these efforts, a high-throughput zebrafish xenograft assay of neuroblastoma was performed to confirm cannabinoid receptor 2 (CNR2) and Mitogen-activated protein kinase 8 (MAPK8) as promising candidates for the treatment of high-risk NB and to identify the drugs GW405833 and AS601245 as the most effective and well-tolerated CNR2 and MAPK8 targeted compounds to inhibit the growth of xenografts in zebrafish [96].
To better mimic the cytokine-enriched microenvironment found in human patients for xenotransplantation, Dr. Berman’s group generated the first humanized zebrafish by overexpressing transgenes encoding human hematopoietic-specific cytokines, such as GM-CSF, stem cell factor (SCF), or stromal cell-derived factor 1α (SDF1α). Transgenic lines with overexpression of each of the individual gene mentioned above were developed first using Tol2 transposon-mediated transgenic approach and then incrossed to generate a compound transgenic fish line with overexpression of all of the aforementioned cytokines (GM-CSF, SCF, and SDF1α) (designated GSS fish) [97]. Patient-derived leukemias transplanted into the GSS zebrafish exhibit improved survival, self-renewal ability and broader clonal representation. Therefore, the GSS fish establish a new standard for zebrafish xenotransplantation that more accurately recapitulates the human context for evaluating personalized treatment [97].