healthmedicinet

Danio rerio, better known as zebrafish, have emerged as a popular model for studying developmental
processes and human disorders. Zebrafish share a high level of genetic and physiologic
homology with humans, including brain, digestive tract, musculature, vasculature,
and an innate immune system 1]–6]. Moreover, approximately 70 % of all human disease genes have functional homologs
with the species 7]. Zebrafish are prolific reproducers with the potential to produce over 100 embryos
per clutch. Their extrauterine development is rapid; the major organs of the zebrafish
are fully developed by 24 hours post fertilization (HPF), and they are ready for use
in larvae experiments by 3 days post fertilization (DPF). Zebrafish larvae are transparent
during the early stages of life (through to 7 DPF), and this phase can be extended
to 9–14 DPF by the addition of melanin synthesis inhibitor 8]. Zebrafish are small in size and require inexpensive food. It is easy, therefore,
to maintain thousands of larvae in a laboratory at a reasonable cost.

Due to the advantages of genetic homology, physiology, and developmental similarity,
zebrafish have increasingly become a desirable tool for studying the development and
modeling of human disease 9], 10]. In the transparent embryo and larvae, clear time-lapse non-invasive imaging and
protein/cell marker tracking significantly aid the observation of biological and disease
processes 11], 12]. Several types of gastrointestinal disorders, such as inflammatory bowel disease,
non-alcoholic fatty liver disease (NAFLD), and alcoholic liver disease, can be modeled
in zebrafish 13]–17]. Zebrafish have also been used in the analysis of complex brain disorders and muscle
disease, including depression 18], autism 19], psychoses 20], and muscular dystrophies 21]. In addition, the ability to regenerate both fins and cardiac tissue make zebrafish
particularly suitable for studying the wound healing response to various injuries
22].

Because of these advantages, zebrafish have proved to be superior for use in cancer
research over the last decade. There are several long-standing methods for establishing
a cancer model in zebrafish, including carcinogenic treatment, transgenic regulation,
and the transplantation of mammalian tumor cells 23]. By inducing different gene mutations or activating signaling pathways through the
use of chemicals, tumors can be induced in a wide variety of organs in zebrafish,
such as the liver, pancreas, intestinal canal, skin, muscle, vasculature, and testis
24]–28]. Transgenic technology enables the formation of specific types of tumor by the overexpression
of particular oncogenes. The xenotransplantation of mammalian tumor cells into zebrafish
provides a novel way of studying the interactions between the transplanted tumor cells
and the host’s vasculature. Zebrafish have also been exploited for the investigation
of tumor angiogenesis, which represents a critical step in tumor progression and is
a target for anti-tumor therapies. The vascular system in a zebrafish embryo bears
a strong resemblance to that in humans, and rapidly forms a single blood circulatory
loop at 24 HPF. In zebrafish, the vascular endothelial cells can be stained by a fluorescent
protein so that the neovascularization in the tumor microenvironment can be observed
in the earliest stage. Tumor metastasis has also been modeled in zebrafish. The fluorescent-stained
tumor cells are highlighted in the transparent zebrafish embryos and larvaes, so that
the process of metastasizing tumor cells can be accurately tracked at the cellular
level. The novel casper zebrafish line, a generation of double pigmentation mutant, even has a completely
transparent body in adulthood. This superior generation of zebrafish, in conjunction
with fluorescent imaging techniques, allows the noninvasive tracing of stained tumor
cells in adult fishes 8]. It is worth mentioning that cancer stem cells account for only a small fraction
of tumor cells and are too few in number to be feasibly transplanted in a mammalian
model in order to assess metastasis. However, only a very small number of cancer stem
cells are required in zebrafish for this purpose because of their small size. Additionally,
the high fertility and low maintenance costs of zebrafish makes them suitable for
the large-scale screen of antineoplastic drug efficacy and toxicity.

This paper focuses attention on the wide application of zebrafish as a superior model
in cancer research, particularly with regard to establishing tumor models, and studying
angiogenesis, metastasis, and antineoplastic drug screens.

Cancer model establishment in zebrafish

Neoplasia was rarely found in wild zebrafish. Using a combination of chemical treatment,
genetic technology, and tumor cell xenotransplantation, the vast majority of human
tumors can be modeled in zebrafish 29]. Carcinogenic chemical treatment is commonly used in inducing tumorigenesis. Several
carcinogenic compounds are able to induce canceration in a number of organs, such
as dimethylbenzanthracene (DMBA) 30], diethylnitrosamine (DEN) 23], N-nitrosodimethylamine (NDMA) 31], N-ethyl-N-nitrosourea (ENU) 24], and N-methyl-N
¹
-nitro-N-nitrosoguanidine (MNNG) 32]. The induced tumors cover a wide spectrum of tumors found not only in the digestive
system (i.e. liver, pancreas, and intestinal canal) but also in the skin, muscle,
vasculature, and testis 24]–28]. As reported, exposure of the vhl^+/?
zebrafish to DMBA revealed an increase in the occurrence of hepatic, bile duct, and
intestinal tumorigenesis at 2 months following treatment 33]. Exposure to DEN results in different types of hepatocellular carcinomas, hepatoblastomas,
hepatoma, cholangiocarcinoma, and pancreatic carcinoma in zebrafish 26]. Exposure of zebrafish to NDMA for 2 months leads to cholangiolar tumors (cholangiocarcinomas
and cholangiomas) and hepatocellular tumors (hepatocellular carcinomas and adenomas)
31]. And exposure of zebrafish to ENU and MNNG results in liver and testis tumorigenesis
24], 27].

A number of reverse genetic tools have been developed for the study of gene functions
in zebrafish. Morpholinos are usually injected at the 1–4 cell stage of embryos to
provide transient knockdown of the target gene expression 34]. Another targeted genome modification technology, called TILLING (Targeting Induced
Local Lesions IN Genomes), is highly dependent on large-scale traditional post-transcriptional
forward genetic screens expression 35]–38]. Moreover, engineered endonucleases, including ZFNs (zinc finger nucleases), the
CRISPR-Cas system, and TALENs (transcription activator-like effector nucleases), provide
efficient strategies to disrupt site-directed genes by inducing double strand breaks
in the target genes 39], 40].

Several types of tumor have been generated by inducing mutants in known tumor suppressor
genes. The knockout p53 gene in zebrafish, for example, was found to result in an increase of malignant peripheral
nerve sheath tumors (MPNST) 41]. In addition, the APC gene mutant in zebrafish leads to colon adenoma initiation and progression, suggesting
an association with the activation of the Wnt signaling pathway 42]. Several other gene mutants were found to be related to different types of tumors
in zebrafish. As reported, mutants in the NF1 gene lead to high-grade gliomas and MPNSTs 43], those in BRCA2, mybl2, and espl1 lead to testicular neoplasias 44], 45], those in the pen/lgl2, bmyb and cds genes cause epidermal neoplasia 32], 46], 47], and GSTT1 deletion related to lymphoma progression 48], and vhl mutants lead to an increase in hepatic and intestinal tumors 33]. The immune and hematopoietic system in zebrafish is similar to that in humans, which
means that not only solid tumors but also hematologic malignancies can be modeled
6]. The most frequent mutant in the tumor suppressor pten in zebrafish was related to an increasing morbidity of T-cell acute lymphoblastic
leukemia (T-ALL) and hemangiosarcoma 49], 50].

Through the transgenic expression of human or mouse oncogenes, several cancer models
have been established in zebrafish. T cell acute lymphoblastic leukemia was the first
cancer induced by transgenic technology in zebrafish, which was induced by the Myc
transgenes 51]. Subsequently, overexpression of the oncogenes xmrk, Myc and KRAS^(V12)
in zebrafish was found lead to hepatoma formation in both juvenile and adult transgenic
fish 52]–55]. Amplification of MYCN and fgf8 expressions markedly promotes the formation of neuroblastoma 56]. Rhabdomyosarcoma has also been induced in zebrafish by using a specific up-regulate
oncogenic KRAS^(G12D)
expression 57]. Overexpression of Akt1 enhances lipoma formation 58]. In combination with the p53 mutant, overexpression of some oncogenes in zebrafish leads to different tumor phenotypes,
such as scr (hepatoma) 59], NRAS (melanoma) 60], BRAF (melanoma) 41], and EWS–FIL1 (Ewing’s sarcoma) 61]. Additionally, the co-activation of the hedgehog and AKT pathways promotes tumorigenesis,
suggesting that a transgenic approach is a useful tool for studying the interaction
of oncogenes and oncogenic pathways in zebrafish 62].

Xenotransplantation represents a novel method to establish tumor models in zebrafish.
One of the great strengths of xenotransplantation is that the transplanted tumor cells
can be marked by fluorescent staining to enable them to be distinguished from normal
cells in order to allow clear observation of the development process of the tumor
63]. The first human xenotransplant assays in zebrafish began in 2005. By injecting 1?~?100
melanoma cells into 3.5?~?4.5 HPF embryos, the migration in the developing larvae
was clearly observed 64]. Transplantation of different types of tumor cells in zebrafish was carried out subsequent
to this innovative work. Microinjecting glioma stem cells into the embryonic yolk
sac region in 2 DPF embryos resulted in an observable invasion in the embryos via
the vessels 65]. Hepatocellular carcinoma (HCC) was also modeled for the identification of the curative
effect of anti-cancer molecules 66]. Several other types of tumor, such as lung cancer 67], pancreatic cancer 68], ovarian carcinomas 69], breast cancer 70], prostate cancer 71], retinoblastoma 72], and leukemia 73], have also been transplanted in zebrafish.

All the methods and types of induced tumor are combined in Table 1. The induced tumors are mainly located in the digestive and reproductive systems,
and then the nervous system and epithelium.

Table 1. Summary of the methods used and the types of tumor induced in zebrafish

Tumor angiogenesis in zebrafish

Angiogenesis is considered a key factor in tumor growth and subsequent metastasis.
Tumor vessels play an important role in transporting oxygen and nutrients to support
the growth of tumor cells. For this reason, the capability of blood vessel formation
within the tumor not only determines the malignancy of the cancer but also influences
the therapeutic effects and prognosis. Both in research evidence and clinically, angiogenesis
inhibitors in combination with chemotherapy improved the outcomes in cancer patients
74]. However, it is difficult to detect the original vascularization in traditional mammalian
models because such models only permit the capture of static images, which probably
relate to the late stage of the tumor. The lack of observation at the earliest stages
of tumor formation means that the mechanism of vascularization is still not fully
understood.

Human umbilical vein endothelial cells (HUVEC) are widely used in the investigation
of angiogenic mechanisms in vitro. The system of angiogenesis can be evaluated by
the cellular responses of HUVEC, such as cell proliferation, cell cycle, tube formation,
cell migration, and cell adhesion to matrix proteins 75]. Several other quantitative angiogenesis assays, for instance the matrix implant
assay and microcirculatory preparations such as the chicken chorioallantoic membrane
and corneal micropocket assay, provide continuous monitoring of the angiogenic response
76]. However, the physiological status of angiogenesis may be quite different when translated
to the area of cancer research. Indeed, angiogenesis in the tumor microenvironment
relys on a distinct signaling pathway and displays large alterations in morphology
and function when compared with normal vasculogenesis. Thus, in vitro research may
be not suitable for modeling angiogenesis in tumor organization.

Zebrafish provide an ideal in vivo model for the research of tumor angiogenesis. The
physiology and pathology of tumor angiogenesis in zebrafish is similar to that in
humans because the tumor microenvironment in zebrafish is strikingly similar 77]. Additionally, the zebrafish vasculature grows rapidly (a single blood circulatory
loop in zebrafish is fully developed in 24 HPF) and the transparent body allows for
high-resolution in vivo non-invasive imaging 77]. The addition of PTU (a tyrosinae inhibitor that prevents melanin synthesis) to water
can lengthen the transparency of the larvae to 9–14 DPF 78].This years, a pigmentation mutant casper line with a completely transparent body has allowed the non-invasive imaging of the
vasculature across the whole body 8]. Real-time observation of vessels in larvae can be achieved after microinjection
of chemical dyes into the vascular system 79]. Additionally, taking advantage of the Tg(flk1: EGFP) zebrafish, a transgenic fish
line with a green fluorescent protein tissue-specific expression in the vasculature,
individual cell growth and vessel formation can be easily detected under confocal
microscopy 80]. In red fluorescent tumor tissues, the green fluorescent protein marked neovascularization
is highlighted and enables the observation of angiogenesis in the initial stages.

Gene identification plays a key role in the exploration of angiogenesis and in discovering
novel therapeutic targets for anti-angiogenesis drugs. Zebrafish are compliant to
genetic manipulation at low cost and within a short time. In this manner, a number
of signaling pathways for angiogenesis and various targets for drug treatment have
been identified over the past few years. Targeted gene knockdown of TNFRSF1B in zebrafish
was found to promote the apoptotic program, and knockdown of TNFRSF1A, or up-regulation
of NF-?B, prevented endothelial cell apoptosis, suggesting that TNFRSF1A and TNFRSF1B
were involved in the signaling pathways of angiogenesis 81]. In another study, a silencing of LIM kinases in pancreatic cancer tissues resulted in a decrease of angiogenesis in zebrafish
82]. These data suggest new therapeutic targets for the control of the tumor-driven angiogenesis
process.

Compared with other angiogenesis models such as the chorioallantoic membrane of the
chicken embryo, zebrafish show their superiority with regard to modeling the in vivo
environment, compliance in genetic manipulation, and allowing clear observation of
the interaction between tumor cells and neonatal micrangium.

Tumor metastasis in zebrafish

The large amount of evidence from various studies has clarified that metastasis is
a dynamic, complex, and multi-step process that includes tumor cells penetrating into
the circulatory system, spreading to distant tissues, engrafting in the parenchyma,
and developing in the graft area 83]. An insight into the mechanism of tumor metastasis is conducive to the discovery
of anti-tumor drugs and the improvement of clinical treatments. Much of the previous
analysis of metastasis conducted in in vitro cell systems had obvious weaknesses because
the complete metastasis process cannot be abstracted away from the in vivo environment
and vascular system. In vivo mouse models also have significant disadvantages: 1)
it is difficult to evaluate the early stage of metastasis; 2) the complete process
of metastasis in a mouse requires a long period of time; 3) real-time imaging of minute
tumor lesions in deep tissues is impossible without termination and autopsy; 4) immunodeficiency
mice may still have a residual anti-tumor competence that can prevent tumor cell metastasis
84]; 5) mice require feeding at high cost throughout the experiment.

The cancer model of zebrafish overcomes the drawbacks of murine xenograft models and
shows several exceptional strengths. The adaptive immune system in zebrafish larvae
is not completely developed until 14 DPF so that most transplanted cancer cells can
survive and metastasize 85]. The transparent body of zebrafish enables the clear observation of tumor metastasis
under the microscope. In the transparent casper line, the dynamic and spatial characteristics of micrometastases can be real-time
imaged at the single cell level 8]. In order to highlight metastasis in zebrafish, tumor cells can be stained by a chemical
dyestuff (such as CM-Dil) or labeled by red fluorescent protein (RFP) 86]. By injecting red fluorescent mammalian tumor cells into the Tg(fli1: EGFP) transgenic zebrafish, in which vascular endothelial cells are labeled by green
fluorescent protein, both the process of tumor cell metastasis and changes in the
vascular system can be clearly seen throughout the body. In addition, cancer stem
cells are too few in number to be transplanted in mammalian models but zebrafish are
small enough for such xenografting, and the rapid progress of metastasis in zebrafish
is able to be observed within 2 days after injection 65].

Zebrafish provide an experimentally tractable animal model for the identification
of suppressing or promoting factors in metastasis. By transplanting RFP expressing
U87 glioma stem cells (GSCs) into the yolk sac of Tg(fli1:EGFP)
^y1
zebrafish embryos, the different invasive stages of GSCs, such as approaching, clustering,
invading, migrating, and transmigrating, can be clearly observed at 2 days post-injection
65]. In this experiment, invasive GSCs were found to have MMP-9 high expression in common
and treatment with the MMP-9 inhibitor significantly decreased the percentage of invasive
cells in the embryos 65]. In an experiment on hypoxia, DiI-labeled tumor cells were injected into the perivitelline
space of 48 HPF embryos, which were subsequently placed in hypoxic water for 3 days.
A significant increase in metastasis and angiogenesis was detected using a fluorescent
microscope at the single-cell level 87], 88]. Tumor cells and immune cells have been co-implanted in the same zebrafish to investigate
the interactions in the tumor microenvironment 89]. The co-implanting of DiI-labeled tumor cells and DiD-labeled tumor-associated macrophages
(TAM) relates to an increase in metastasis in zebrafish, and their association could
be detected in overlapping colors 89].

The signaling pathway for metastasis has been evaluated in the zebrafish model. The
technology of target knockdown of proteins involved in the signaling pathway with
chemical inhibition or small interfering RNA is not new in zebrafish. The TGF-beta
signaling pathway was found to control human breast cancer metastasis in zebrafish.
After treatment with the TGF-beta signaling pathway inhibitor, the invasion and metastasis
processes in zebrafish were inhibited significantly 70], 90].

Drug screening in zebrafish

The effects of molecule antineoplastic drugs have often been detected by biochemical
assays or in cell line models, but the outcomes were unsatisfactory. Because of a
lack of a complete biologic context in the screening process, the identified active
compounds were often ineffective when applied in a vertebrate model. At this point,
a whole animal screen sheds valuable information on anti-tumor effects, organ toxicity,
and pharmacokinetic data based on the entire organism 91]. However, mice are fiscally prohibitive for large-scale screen. Zebrafish, on the
other hand, have emerged as a powerful platform for use in high-throughput antineoplastic
drug screening on the strength of the following advantages. A pair of zebrafish produce
hundreds of embryos a week, and larvae have a small size that can be arrayed in a
96-well plate, which greatly decreases the cost of maintaining them in the laboratory.
Drug treatments can be easily achieved by merely adding the medicine to the aqueous
environment. In addition, the transparent zebrafish body enables the real-time non-invasive
imaging of anti-tumor effects and drug toxicity.

Most types of cancer can be modeled in zebrafish, thus zebrafish can be used to assess
the anti-tumor effects of chemotherapeutic drugs. The growth of tumor cells and degree
of invasion are the main concerned outcomes. Over the past 5 years, several large
chemical screens have been performed in zebrafish. The anti-melanoma chemical genetic
screen is one of the best representations. To our knowledge, the propagation of melanoma
is critically related to the neural crest lineage. 2,000 chemicals were screened to
identify inhibitors of the neural crest lineage in zebrafish embryos, and the selected
chemicals were tested for effects in melanoma. Leflunomide, an inhibitor of dihydroorotate
dehydrogenase, was found to inhibit the development of both neural crest and human
melanoma. This screen shed light on the important role of zebrafish in antineoplastic
drug discovery 92].

An anti-leukemia compound screen was performed in zebrafish in 2012. Zebrafish show
a striking similarity in the hematopoietic system development with humans, and almost
all human adult blood lineages have corresponding homologous cell lines in zebrafish.
For this reason, effective hematopoietic drugs in zebrafish may serve the same function
in humans. More than 25,000 small compounds were identified in this drug screen and,
finally, a compound called lenaldekar (LDK) was found to be able to induce long-term
remission in adult zebrafish with T-cell acute lymphoblastic leukemia (T-ALL). A subsequent
study showed that LDK had a generalized anti-leukemia effect not only to T-ALL but
also to several diverse leukemias such as B-ALL and CML 93], 94].

Anti-angiogenesis drugs have been screened in zebrafish. Following a screen of 288
new compounds, two kinase inhibitor compounds were found to have anti-angiogenic properties
and a phosphorylase kinase subunit G1 (PhKG1) was identified as the kinase target
95]. In a similar way, rosuvastatin was identified as inhibiting the angiogenesis in
developing zebrafish embryos 96]. Anti-lymphatic drug compounds were also identified in zebrafish. Four compounds
previously used in humans were found to have anti-lymphatic activity in zebrafish
97]. These studies demonstrate that zebrafish provide an effective utility platform for
large-scale antineoplastic drug screens and medicine efficacy detection.

Zebrafish have been used to identify compounds that work in the genetic signaling
pathways of carcinogenesis. The bmyb gene is important for controlling the mitotic checkpoint and is connected with cancer
susceptibility 32]. In order to identify the drug function of small molecules in the bmyb pathway, 16,000 compounds were tested. A compound named persynthamide was noted to
have an inhibiting effect in bmyb-dependent mitotic defects and reduced the incidence of tumors in zebrafish 39].

Antineoplastic drug toxicity can be observed over a short period because of the rapid
development of zebrafish. In a screen for detecting the inner ear hair cell toxicity
of anti-tumor drugs, 13 out of 88 anti-tumor drugs, and 5 out of 10 drug combinations,
were authenticated ototoxic. In addition, dose–response studies were performed on
these detected drugs 98]. Several outcomes were usually detected to assess the toxic effect of antineoplastic
drugs, such as cell damage, development process, and vitality. Drug toxicity screening
has an important significance in selecting the appropriate therapy in clinical cancer
treatments.