Zebrafish cancer and metastasis models for in vivo drug discovery

TECHNOLOGIES

DRUG DISCOVERY

TODAY

Zebrafish cancer and metastasismodels for in vivo drug discoveryJennifer Tat1,2,*, Mingyao Liu2,3, Xiao-Yan Wen1,2

1Zebrafish Centre for Advanced Drug Discovery, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto,

Ontario M5B 1T8, Canada2Department of Physiology, Faculty of Medicine, University of Toronto, Canada3Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada

Drug Discovery Today: Technologies Vol. 10, No. 1 2013

Editors-in-Chief

Kelvin Lam – Simplex Pharma Advisors, Inc., Arlington, MA, USA

Henk Timmerman – Vrije Universiteit, The Netherlands

Model organisms as in vivo screens for promising therapeutic compounds

There is a great need for more efficient methods to

discover new cancer therapeutics, as traditional drug

development processes are slow and expensive. The

use of zebrafish as a whole-organism screen is a time

and cost-effective means of improving the efficiency

and efficacy of drug development. This review features

zebrafish genetic and cell transplantation models of

cancer and metastasis, and current imaging and auto-

mation technologies that, together, will significantly

advance the field of anti-cancer drug discovery.

Introduction

Cancer is a leading cause of death worldwide and there is a

substantial need for development of therapeutic agents cap-

able of targeting the processes and pathways responsible for

its genesis and progression. The classical method of new drug

development is arduous, and the low success rate of tradi-

tional cell-based screens demonstrates a need for whole-

organism screening strategies [1]. Zebrafish models have

numerous qualities that are well-suited to drug discovery

and high-throughput screening (HTS): accessibility for

manipulation and observation, high fecundity, small size,

rapid development, and physiology and pharmacology that is

analogous to other vertebrates [2]. Furthermore, the effect of

drugs, their metabolites and potential toxicities can be

assessed simultaneously. The topics featured in this review

*Corresponding author: J. Tat ([email protected])

1740-6749/$ � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ddtec.2012

Section editor:Isabelle Draper – Tufts Medical Center, Boston, MA, USA

highlight the promising role of zebrafish in vivo screens in the

development of anti-cancer drugs.

Key technologies

Zebrafish cancer and metastasis models

In the past decade, zebrafish has emerged as a promising

experimental system for modeling human cancers through

genetic manipulation or cell transplantation [3]. Performing

drug screens using in vivo models recapitulating key processes

of cancer development can help identify new drugs that may

treat cancer more effectively.

Oncogene overexpression models

The use of forward and reverse genetic tools has produced

several transgenic human cancer models in zebrafish, includ-

ing melanoma, multiple myeloma, leukemia, pancreatic and

liver cancer [4�]. These genetically engineered models repro-

duce many key features in human tumourigenesis with high

penetrance. The first transgenic cancer model in zebrafish is a

leukemia model with overexpression of the c-Myc oncogene

under the zebrafish T-cell specific promoter rag2 [3]. While

tissue-specific oncogene overexpression permits fluorescent

tracking of tumor growth, transgenic lines are often difficult

to maintain and there is a long latency of tumor formation.

Genetic techniques have advanced considerably to allow for

tight spatial and temporal control of transgene expression.

An early onset model of melanoma has been established

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http://dx.doi.org/10.1016/j.ddtec.2012.04.006


mailto:[email protected]


Drug Discovery Today: Technologies | Model organisms as in vivo screens for promising therapeutic compounds Vol. 10, No. 1 2013

using the Gal4-UAS system expressing the oncogene HRAS in

melanocytes of zebrafish larvae [5]. This model faithfully

reproduces the phenotypes of human melanoma while its

early onset eases manipulation and large-scale screening with

zebrafish larvae [5]. Another model uses a mifepristone indu-

cible LexPR system to dose-dependently induce fluorescent

tumor growth in the liver of KRAS-transgenic embryonic and

adult zebrafish [6��]. Several known inhibitors targeting

downstream effectors of RAS were able to decrease RAS-

induced tumourigenesis in the livers of 7-day-old larvae

[6��]. Inducible systems circumvent oncogenic toxicity thus

allowing for the generation of stable transgenic lines while

maintaining high penetrance and short latency of tumor

development. Furthermore, as demonstrated in the RAS

model, oncogene-based zebrafish models of cancer can be

applied to perform disease- and pathway-specific drug screen-

ing [6��].

Models of cancer-related processes

Some transgenic zebrafish lines that can be employed in anti-

cancer drug screens do not develop cancer but rather model

cancer-related pathophysiological processes, such as angio-

genesis or the inactivation of specific tumor suppressor genes

[7]. These models generate distinct phenotypes that can be

distinguished from wild-type fish. The application of these

lines has led to the identification of inhibitors of angiogen-

esis, a key feature of tumourigenesis [8�]. Tg(Flk1:EGFP) and

Tg(Fli1:EGFP) zebrafish have their entire vascular network

labeled with green fluorescence while Tg(Gata1:DsRed) zebra-

fish have red fluorescent blood cells. A comparison of fluor-

escent images of compound-treated and untreated fish allows

for assessments of vessel development and functionality.

Gene mutant models can also be employed to identify new

therapeutic agents that target genetic predispositions to can-

cer [9]. TP53 is the most common tumor suppressor gene

mutated in human cancer [9]. TP53 mutant zebrafish

embryos exhibit abnormal apoptosis and cell-cycle regula-

tion following temperature induction [9]. Screening com-

pounds using this model may lead to the identification of

small molecules that can restore TP53 function and prevent

subsequent tumourigenesis [9]. A variety of transgenic lines

such as those containing mutations in TP53 [9,10], APC [11]

and PTEN [12] will serve as tools for discovery of drugs that

affect specific features of cancer pathophysiology.

Xenotransplant models

Cell transplantation models in zebrafish have been used to

study tumor angiogenesis, cancer cell invasion and metasta-

sis by engrafting dyed or fluorescently-labeled tumor cells.

Angiogenesis is a critical feature of tumor growth and pro-

pagation while metastasis is the main cause of cancer patient

death. Therefore, targeting these processes is a valid strategy

for treating cancers. Nicoli et al. (2007) demonstrated that an

e84 www.drugdiscoverytoday.com

angiogenic response induced by the injection of human and

murine cancer cells into the yolk sac of 2-day-old zebrafish

embryos can be abrogated by the addition of chemical inhi-

bitors targeting vessel formation [13]. A typical screen using

the embryonic xenograft neovascularization model can be

completed in less than a week (Fig. 1). Variations can be used

to investigate specific processes – genetic modification of

tumor cell lines or gene-specific knockdown zebrafish can

be implemented to investigate a specific phenotype or signal-

ing pathway. For example, Marques et al. stimulated injected

cancer cells with TGF-b to model tumor invasion and metas-

tasis [14]. Nicoli et al. developed a similar but more complex

tumor angiogenesis model by injecting cells overexpressing

FGF2 into morpholino (MO)-treated zebrafish [13]. MO

knock down of the vascular cell adhesion gene VE-cadherin

abrogated tumor graft vascularization, thus demonstrating

the potential for MO-based gene identification in zebrafish

xenograft models [13]. Lee et al. devised a hypoxia model in

2-day-old embryos that permits the study of tumor cell

neovascularization, dissemination and metastasis approxi-

mately a week after engraftment in the yolk sac [15]. With

the hypoxia model, the investigators demonstrated that che-

mical and MO inhibition of VEGF significantly reduces vas-

cular-dependent metastasis [15]. Stoletov et al. investigated

extravasation, a late phase of the metastatic cascade whereby

tumor cells exit the vasculature, by injecting tumor cells

overexpressing the pro-metastatic gene Twist directly into

the circulation of Tg(Fli1:EGFP) zebrafish embryos [16].

The limitation of embryo xenograft experiments is that

they can only be studied for up to a week due to the devel-

opment and activation of the immune system, subsequent

tumor cell death and embryo lethality [17]. Adult zebrafish

permit longer-term evaluation of tumor development. White

et al. have established a transparent adult zebrafish line that

permits in vivo assessment of tumor grafts for up to 5 weeks

post-transplant [18]. However, adults must be immunosup-

pressed and are not as amenable to high-throughput proces-

sing due to their size. Overcoming one of these drawbacks,

Smith et al. demonstrate an allograft leukemia model in

syngeneic zebrafish that does not require immune suppres-

sion and leukemia develops as early as 30 days after injection

in juveniles [19]. Transplant models allow for flexible study of

various aspects of tumourigenesis and metastasis, and

increasingly sophisticated equipment will facilitate drug

screening in these models of cancer.

Drug screening

Regarding the use of genetic and transplant models of cancer

in drug screening, it is critical to assess the advantages and

limitations of each model as well as the amenability to high-

throughput screening. HTS requires simple phenotypic read-

outs such as optical scoring to identify hits among the

thousands of pharmacological agents being tested. For

Vol. 10, No. 1 2013 Drug Discovery Today: Technologies | Model organisms as in vivo screens for promising therapeutic compounds

Imag

e

Micr

oinj

ect c

ance

r cel

ls an

d ad

d dr

ug

Trea

t em

bryo

s with

PTUCol

lect

egg

s

0 hpf

12 hpf

2-3 dpf

2 dpi

SIVs

Array and dose

Analyze SIV

Drug Discovery Today: Technologies

Figure 1. Workflow of drug screening using embryonic tumor xenograft model. Newly fertilized eggs are collected following natural breeding of

Tg(Fli1:nEGFP) fish [2]. At 12 hpf, PTU may be added to inhibit the development of embryonic pigmentation. At 2–3 dpf, embryos are microinjected with

cancer cells and dispensed into 96-well plates. Drugs, compounds or controls are then added into the water within the wells. After 2 dpi, the embryos are

imaged using fluorescence microscopy and the SIVs/endothelial cell numbers are counted to assess tumor angiogenesis. hpf: hours post fertilization. PTU:

propylthiouracil, dpf: days post fertilization, dpi: days post injection, SIV: sub intestinal vessels.

instance, the readout for tumor burden in a transplant model

may be complicated by variations in injected cell number and

multiple sites of tumor growth. Comparatively, models such

as the mifepristone KRAS model and the Tg(Flk1:EGFP) angio-

genesis model generate precise and reproducible readouts (i.e.

tumor growth in the liver and vessel number in the trunk,

respectively). All of these models involve zebrafish embryos,

which are advantageous compared to mammalian models for

their small size, transparency and immature immune system

but must be considered for differences in developmental

physiology and time course. Currently, no immunocompro-

mised zebrafish lines exist though immune suppression is

routinely achieved in adults using sub-lethal doses of radia-

tion [19]. The aforementioned models represent only a few of

the numerous zebrafish cancer models available. Several

reviews cover zebrafish genetic [3,7] and transplant models

[17,20] in greater detail.

Imaging technologies

One of the strengths of the zebrafish model is its tissue trans-

parency, which facilitates in vivo visualization of the stages of

cancer progression: proliferation, neovascularization, intra

and extravasation, dissemination and growth at secondary

sites. The natural transparency of zebrafish embryos can be

extended to several weeks by adding propylthiouracil (PTU) to

the water to prevent pigment production [21], while the crea-

tion of a non-pigmented zebrafish line prolongs transparency

throughout adulthood [18]. Transparent and transgenic zebra-

fish can be easily visualized using widely available brightfield

and fluorescence stereo- and confocal microscopy. Many alter-

native imaging modalities have been developed to provide

additional levels of analysis. Here we discuss a few key tech-

nologies, while a more detailed review of imaging modalities

relevant to HTS has recently been covered elsewhere [22,23].

Confocal microscopy is capable of high resolution cellular

imaging with well-developed software analysis tools, but its

application is limited by tissue penetration and the transpar-

ency and size of the zebrafish being analyzed (only larvae or

adult sections) [22,24��]. The light-emitting diode (LED)

fluorescence macroscope allows imaging of a whole Petri dish

of up to 30 live adult zebrafish, with the capacity to detect up

to five fluorophores at once [19]. Using LED lights and

webcams equipped with optical filters, the macroscope pro-

vides a cost, labor and time efficient means of identifying

fluorescently-labeled tumors at the expense of resolution.

MicroMRI has been applied to detect and characterize mel-

anomas in adult zebrafish [25], and microscopic ultrasound

has been utilized to assess liver tumors in adult zebrafish in

response to chemotherapeutic treatment [26]. Live imaging

technologies can be employed for effective longitudinal and

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real-time assessment of tumor development following phar-

macotherapy. While valuable, the technical demands (e.g.

special equipment and personnel) and time scale of image

acquisition and processing (ranging from minutes to hours

per sample) for these technologies results in poor throughput

potential. Thus, these are likely modes of secondary analyses

rather than components of the screening process. Synchro-

tron micron-scale tomography (microCT) is capable of rapid,

whole animal imaging at a cellular resolution not possible

with light microscopy [22]. Histological arrays are available

for rapid scoring of phenotypic analyses of adults, larvae and

embryos [22]. However, samples must be fixed in both

microCT and histology. While these imaging technologies

are becoming increasingly efficient, substantial resources are

required in terms of robotics and computer directed screens

to reach its full potential [22].

An ideal imaging technique would have high resolution

and sensitivity, allowing accurate in vivo analysis of complex

(a)Zebrafish model

Drug treatment

Sort, array and dose

Imaging preparation

Imaging

Phenotypicanalysis

Autom

Flu

Acquisition and scoring

(d)

(e)

(g)

Figure 2. New technologies developed to increase the throughput of drug scr

[Sciclone G3] and (C) fluorescence sorting by flow cytometry [Copas XL] can fac

be improved using (D) microfluidics [21] and (E) specialized multiwell plates [2

software such as MetaMorph and CNT [Definiens] can greatly accelerate imag


developmental and physiological processes. Speed, field of

view, resolution, cost and ease of use must all be considered

when selecting an imaging modality to employ. Although

existing light-based imaging of larval or transparent models

of zebrafish are effective for rapid, simple analysis, alternative

imaging modalities like microMRI and CT are available for

more detailed characterization of cancer pathophysiology.

The key to HTS: automation

Drug discovery can be greatly streamlined by automating the

laborious phases of drug screening; sorting, sample proces-

sing, loading, image acquisition, analysis and interpretation

(Fig. 2). The COPAS XL (Union Biometrica) is a large particle

flow cytometer that is capable of sorting embryos and hatchl-

ings based on optical light and fluorescence (up to three

fluorophores at once) and dispensing them into multiwell

plates [23]. This device can significantly accelerate embryo

selection and sorting for drug treatment in genetic and

HTS Technologies

Microfluidic capillary systemSpecialized multiwell plates

ated microinjectionLiquid handlingorescence sorting

High-content microscopyAnalysis software

(c)

(f)

(h)

(b)

Sciclone

LEDs Steppermotor

Steppermotor

Capillary

Photodetector

Computerizedsyringe

Drug Discovery Today: Technologies

eens in zebrafish. (A) Automated microinjection [26], (B) liquid handling

ilitate embryo preparation, sorting and drug dosing. Image preparation can

9]. Lastly, (G) high-content microscopy [ImageXpress] and (H) analysis

e acquisition and scoring.


transplant models by identifying embryos positive for cell

fluorescence. Throughput can be further increased by auto-

mating slow and tedious tasks, like drug dosing and micro-

injection. Liquid handling robots such as Sciclone G3

(Caliper Life Sciences) can dispense reagents and/or zebrafish

embryos in series or in parallel into multiwell plates [27].

With automated batch microinjection, up to 15 embryos can

be injected per minute with genetic material such as MO

[28,29]. Automated microinjection reduces the variability

between injections and errors due to operator fatigue [28].

Most zebrafish screenings are performed using multiwell

plates, and phenotypic analysis within the plates is possible

using Confocal Laser Scanning Microscopy (CLSM or LSCM),

such as the ImageXpress Ultra (Molecular Devices), with

point-by-point image acquisition allowing for three-dimen-

sional reconstructions. Several methods have been devised to

expedite image processing outside of multiwell plates to

overcome the problems caused by the large working distance

and random movement and orientation of zebrafish [23].

Capillaries [30], agarose-coated plates [31], round-bottom

plates (Corning COSTAR) and rectangular microplates with

prisms (Physical Sciences Inc) are several strategies that have

been conceived to manage embryo orientation for quick

image capture. High-throughput histology is possible for

larval and adult zebrafish using several methods to accelerate

sample handling: agarose arrays, automated tissue processors,

rotary microtomes and automated slide stainers [32]. An

automated imaging system developed by Gehrig et al. com-

bines embryo recognition software with a high-content

microscope such that fluorescent gene expression patterns

in up to 2000 embryos may be acquired within 4 h [31].

Analysis programs have been built to handle the quantity

of data produced by rapid image acquisition technologies

while also reducing the burden of visual scoring and elim-

inating observation bias [33]. Image analysis software such as

Cognitive Network Technology (Definiens) and MetaMorph

software application modules (Molecular Devices) can be

custom-designed to detect and quantify specific structures

such as intersegmental vessel number in Tg(fli1:EGFP) zebra-

fish [33]. The development of these technologies has greatly

increased the efficiency of screening. However, in most zebra-

fish screenings reported thus far, automation is not contin-

uous and embryos must be manually manipulated at several

steps.

The next step to increase throughput is to integrate these

automated components into a highly efficient HTS platform.

Pardo-Martin et al. have developed such a system using glass

capillaries and microfluidics to transport embryos out of

multiwell plates to a correct orientation beneath a high-speed

confocal microscope for subsequent imaging and analysis

[24��]. The entire process of loading, positioning, imaging

and dispensing lasts 16 s compared to the manual average of

10 min per embryo [24��]. A total of approximately 30 min is

required to screen a 96-well plate [24��]. Although this system

is designed for screening retinal mutants and performing

neuronal regeneration assays, it is expected that such a fully

automated HTS system will be adapted to zebrafish cancer

model screens.

Recent drug discoveries using zebrafish for in vivo cancer drug

screening

Although complete automation has yet to be developed for

cancer drug screening in zebrafish, existing technologies have

been quite successful. Zebrafish embryos were used as a

secondary screen of small-molecule inhibitors of the Wnt

pathway, a critical therapeutic target that is implicated in

liver, colon and breast cancer [34]. While only simple wild-

type embryos were employed, this study illustrates the power

of zebrafish for effective in vivo screens; of the 306 hits

identified from the primary screen of a library with 63,040

compounds performed on a reporter cell line, one novel

compound CCT036477 demonstrated in vivo activity against

Wnt target genes [34].

Structure–activity relationships frequently surface from

chemical screens and zebrafish can be used to test analogues

of hits that have been modified to reduce specific side effects

or improve bioactivity [35]. Analogues of dorsomorphin, a

selective inhibitor for bone morphogenic protein (BMP), were

tested in zebrafish embryos in this manner, resulting in the

identification of several selective inhibitors of BMP and VEGF

[35].

The angiogenesis model using vasculature-labeled trans-

genic zebrafish has been widely used by several investigators

to identify small molecules inhibiting angiogenesis. Craw-

ford et al. combined this zebrafish bioassay with analytic

chromatography to isolate angiogenesis inhibitors found in

East African medicinal plants [36]. Wang et al. used this

model to screen the Spectrum Collection library and identi-

fied seven compounds inhibiting zebrafish angiogenesis [8].

One of these compounds, rosuvastatin, is a commonly pre-

scribed statin used to treat high cholesterol. Rosuvastatin was

shown to inhibit angiogenesis and suppressed the growth of

xenografted prostate tumors in mice [8]. Discovering new

applications for existing drugs saves a considerable amount of

time and cost [37]. The compound SKLB1002 was developed

using a de novo design method to target VEGFR2 signaling

[38]. SKLB1002 was demonstrated to inhibit zebrafish angio-

genesis and the growth of xenografted tumors [38].

Nanotechnology has also been tested in zebrafish for drug

delivery applications. Cheng et al. developed carbon nano-

tubes to deliver anti-angiogenic agents and used zebrafish to

assess the biodistribution, biocompatibility and efficacy of

these agents in embryo models of angiogenesis and tumor

xenograft neovascularization [39]. Similarly, Gou et al. used

zebrafish and mice to test the efficacy of biodegradable poly-

meric nanoparticles loaded with curcumin, an anti-cancer



agent found in the spice turmeric [40]. Nano-therapeutics

opens up possibilities for the improved delivery of drugs with

poor water solubility (such as curcumin) and targeted cell or

tissue-specific delivery among many other applications [41].

Nanotechnology has great potential and further implemen-

tation of zebrafish into the screening process will rapidly

accelerate its application to drug development.

Conclusion

Chemical genetic screening in live zebrafish embryos repre-

sents a technological advancement in developmental biology

as well as in the drug discovery industry. In contrast to

traditional in vitro cell culture-based screening, the zebrafish

provides a whole vertebrate system for drug discovery that

combines the biological complexity of in vivo models with the

capability of high-throughput screening. As this is performed

in vivo, drug toxicity can be evaluated at the same time as drug

efficacy (including those attributes of drug metabolites),

allowing for a higher success rate compared to in vitro drug

screens.

Many transgenic or mutant zebrafish models have been

developed for cancer research that either recapitulate human

cancers or its critical processes, such as angiogenesis and

metastasis. These models can be used to screen compounds

for the development of anti-cancer drugs. The increasing

popularity of zebrafish screening has led to the development

of numerous technologies to improve zebrafish handling,

imaging and data processing. The transparency and small

size of zebrafish embryos allow ease of light-based imaging.

Stereo- and confocal microscopies have been widely used in

zebrafish studies. However, several additional imaging mod-

alities, such as microCT, microMRI and ultrasound are also

available as complementary methods.

Novel automation technologies have been developed to

improve the speed and precision of the slow, labor-intensive

steps of the screening processes. These include zebrafish

embryo sorting and dispensing, automated microinjection,

liquid handling and drug dosing, image acquisition and

analysis. Fully automated HTS platforms represent the pin-

nacle of efficiency for drug screening. While they have not yet

been applied for anti-cancer drug discovery, the technologi-

cal foundation is now laid out.

Many compound libraries have been screened in zebrafish

models, leading to the identification of compounds targeting

cancer cells, its metastasis, molecular pathways or cancer

angiogenesis. Efficacy of some lead compounds has been

confirmed in mouse and other models and it is expected that

clinical trials will be initiated for several compounds.

Systematic investigation of potential cancer therapeutics

using zebrafish in a coordinated joint research effort will

further improve the efficiency of anti-cancer drug discovery.

This initiative is already underway in Europe in a consortium

titled ZF-CANCER [42]. Considering the potential of zebrafish


screening and the tools developed to improve its execution,

we envision a promising future for zebrafish pharmacoge-

nomic research in the drug development industry.

Acknowledgement

We would like to thank Mr. David Spillane for critical reading

of the paper. This work is supported by grants from Heart and

Stroke Foundation of Ontario (X.Y.W.) and Canada Founda-

tion for Innovation (X.Y.W.).

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Zebrafish cancer and metastasis models for in vivo drug discovery