The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

Abstract

Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma.

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Journal of Histochemistry & Cytochemistry 2015, Vol. 63(10) 749 –761
© The Author(s) 2015
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DOI: 10.1369/0022155415595670
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Review
Introduction
It is recognized that cancer results from complex interac-
tions of cancer cells with their microenvironment and the
whole organism. Thus, established in vitro cell and tissue
models of cancer must be complemented by in vivo models,
the former aiming at deciphering molecular mechanisms of
tumor progression, and the latter, elucidating multicellular
interactions during tumor progression. Commonly used
mammalian models have several drawbacks in that they are
expensive, time consuming, and ethically questionable; yet
are not necessarily a good approximation of physiological
processes taking place in the human body. In recent years,
steps have been taken towards bridging the gap between
high-throughput in vitro studies on the one hand and animal
cancer models on the other by introducing models with
higher throughput that are less ethically problematic.
Recently, the zebrafish (Danio rerio) and its embryos have
become a popular in vivo experimental model that enables
rapid, medium-throughput studies at low cost and offers the
possibility to image tumor progression directly at single-cell
resolution in real time (Armatruda et al. 2002; Konantz et al.
2005; Mione and Trede 2010; White at al. 2013; Zon and
Peterson 2005).
This review aims to provide a brief overview of the
methodology involved in studying human tumors by xeno-
transplantation into zebrafish embryos and the application
of this model to the study of gliomas, focusing on glioblas-
toma multiforme (GBM), the most common and aggressive
form of glioma (Behin et al. 2003; Ohgaki and Kleihues
2013). Several properties of GBM make these tumors dif-
ficult to treat. Their diffuse growth and invasion into sur-
rounding areas of the brain prevent their complete surgical
removal, whereas the presence of the blood-brain permea-
bility barrier (BBB) limits drug delivery (Claes et al. 2007;
595670JHCXXX10.1369/0022155415595670Vittori et al.Glioma Xenotransplantation in Zebrash
research-article2015
Received for publication April 28, 2015; accepted June 19, 2015.
Corresponding Author:
Miloš Vittori, Department of Genetic Toxicology and Cancer Biology,
National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
E-mail: milos.vittori@nib.si
The Study of Glioma by Xenotransplantation in Zebrafish
Early Life Stages
Miloš Vittori, Helena Motaln, and Tamara Lah Turnšek
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia (MV, HM, TLT).
Summary
Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes
involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of
fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in
vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow
for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of
fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential
for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation
studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for
antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most
common and malignant form, glioblastoma. (J Histochem Cytochem 63:749–761, 2015)
Keywords
cancer models, glioma, intravital microscopy, drug screening

750 Vittori et al.
Persano et al. 2013). The utilization of a complex in vivo
model that enables real-time imaging of cellular interac-
tions during GBM progression and its interactions with the
environment at single-cell resolution can contribute greatly
to the development of methods to improve the treatment of
this devastating disease. We discuss the potential use of
zebrafish xenotransplantation for the discovery of novel
pharmaceuticals and molecular markers with potential diag-
nostic, prognostic and therapeutic value.
The Zebrafish Cancer Model
Zebrafish and Their Early Life Stages in
Experimental Research
The zebrafish (Fig. 1) is a freshwater teleost that has been
extensively studied from developmental and genetic points
of view (Grunwald and Eisen 2002). It is an established
developmental model organism due to the ease of obtaining
and studying its embryos. The fecundity of zebrafish allows
even a small breeding facility to achieve a daily production
of embryos in their hundreds (Kari et al. 2007). Zebrafish
embryos are convenient, as they develop outside of their
parents’ bodies and can thus be monitored easily throughout
their development. The development of zebrafish embryos
is rapid: at 48 hours, an embryo already possesses a well-
developed nervous system and displays a functional circu-
lation as well as motility (Kimmel et al. 1995). In addition,
zebrafish embryos are small and are suitable for mainte-
nance on multi-well plates, making them an in vivo experi-
mental system with relatively high throughput at a
reasonable cost (Geiger et al. 2008; Kari et al. 2007; Zon
and Peterson 2005). Reverse genetic approaches are well
developed in this species (Hwang et al. 2013a, 2013b;
Lawson and Wolfe 2011; Zu et al. 2013), with a perspective
to produce knockout mutants for various genes in its
genome (Kettleborough et al. 2013).
Besides these benefits, the major advantage of zebrafish
embryos and larvae is their potential for in vivo visualiza-
tion of cellular processes at high resolution. This is due to
their small size and optical transparency, making in vivo
observations of developmental processes easy to accom-
plish at single cell resolution (Hendricks and Jesuthasan
2007; Keller et al. 2008). Intravital fluorescence micros-
copy of zebrafish embryos has been further enhanced by the
application of light sheet microscopy (Jung et al. 2012;
Kobitski et al. 2015). Although in vivo fluorescence imag-
ing of engrafted tumors has been performed on mammals
(Yang et al. 2001), they do not offer the high-resolution
imaging that can be performed in zebrafish embryos.
Furthermore, transgenic zebrafish strains with tissue-spe-
cific expression of fluorescent proteins are being produced
and are readily available for use (Distel et al. 2009; Lawson
and Weinstein 2002), enabling fluorescence imaging of
microanatomical structures and gene expression patterns;
this adds greatly to the value of this model.
Zebrafish in Cancer Research and the Tumor
Xenotransplantation Model
The zebrafish has been successfully utilized in cancer
research over the past decade (Feitsma and Cuppen 2008;
Konantz et al. 2012; Mimeault and Batra 2012; Veinotte
et al. 2014). Cancer in zebrafish has been induced by genetic
manipulation (Blackburn and Langenau 2014; Ignatius
et al. 2012; Langenau et al. 2003; Sabaawy et al. 2006) or
Figure 1. The zebrafish (Danio rerio) and its embryo. (A) Adult zebrafish. (B) The anatomy of a zebrafish embryo at 2 days after
fertilization. The areas of cancer cell implantation discussed in text are marked.

Glioma Xenotransplantation in Zebrafish 751
by xenotransplantation of cultured mammalian cancer cells
(Geiger et al. 2008; Haldi et al. 2006; Lee et al. 2005;
Konantz et al. 2012). Gene expression patterns of cancer
cells were shown to be highly similar in zebrafish and
humans (Lam et al. 2006; Zheng et al. 2014). Similar
molecular pathways may lead to tumor development in the
two species (Jung et al. 2013; Mione and Trede 2010) and
tumors in zebrafish were found to be histologically similar
to their mammalian counterparts (Armatruda et al. 2002;
Eden et al. 2014; Stern and Zon 2003). Furthermore, zebraf-
ish cells can respond to human signaling molecules
(Drabsch et al. 2013).
Because of the small size of zebrafish embryos, only a
few hundreds of cancer cells per embryo are generally
implanted (Konantz et al. 2012), and single-cell imaging
enables monitoring of the xenograft’s behavior in the
embryo. It has been proposed that implantation of a low
number of cells mirrors the early stages of tumor develop-
ment (Lal et al. 2012; Nicoli and Presta 2007). This also
makes the zebrafish xenotransplantation model particularly
suitable for the study of cells that are difficult to obtain,
such as cancer stem cells (Yang et al. 2013a).
Nevertheless, there are drawbacks or limitations to the
use of zebrafish in xenotransplantation experiments.
Because of the phylogenetic distance between teleost fish
and mammals, zebrafish might provide the implanted cells
with a different microenvironment than the human body,
especially when orthotopic implantation is impossible due
to the lack of corresponding organs in zebrafish (Konantz
et al. 2012). Furthermore, the use of embryos can be prob-
lematic due to the immaturity of their developing tissues.
For example, myelinated axonal sheaths do not develop in
the zebrafish central nervous system (CNS) until 4–7 days
post-fertilization (dpf) (Brösamle and Halpern 2002), which
may affect the invasion of implanted glioma cells (Lal et al.
2012). Furthermore, the BBB does not develop in zebrafish
embryos until 3 dpf (Xie et al. 2010) and is not mature for
another 7 days (Fleming et al. 2013), an issue important for
glioma drug screening.
Growing Human Tumors Underwater:
Approaches to Zebrafish Xenotransplantation
For cancer cell xenotransplantation, cells isolated from
tumors are grown in culture, labeled in order to distinguish
them from the recipient tissues, and implanted into the
zebrafish, after which the effect of experimental manipula-
tions on tumor progression are monitored (Fig. 2). Since the
zebrafish adaptive immune system matures at 3–4 weeks
after fertilization (Lam et al. 2004; Willett et al. 1999),
xenotransplantation in early life-stages does not necessitate
immunosuppression, making embryos particularly suitable
for xenotransplantation. An additional benefit is that
embryos do not need to be fed, relying on the yolk for
nutrition.
The zebrafish model is relatively novel and therefore
most methodology involved is not yet standardized. A vari-
ety of incubation conditions such as temperature and
Figure 2. Overview of the zebrafish glioma xenotransplantation model workflow. Cells isolated from a patient’s tumor are grown in
culture, fluorescently labeled, and implanted into the zebrafish embryos. After implantation, the embryos are incubated for several days,
during which time the behavior of the implanted cells can be monitored using fluorescence microscopy.

752 Vittori et al.
composition of the medium vary between laboratories, with
no agreement as yet on best practice.
A major issue in the use of zebrafish as recipients of
implanted mammalian cells is that the optimal temperature
for the development of zebrafish embryos is 28°C (Kimmel
et al. 1995), whereas human cells grow optimally at 37°C.
In some studies, some cancer cell lines are able to tolerate
28°C (Nicoli and Presta 2007; Zhao et al. 2009, 2011a). For
the GBM cell line, U251, it has been demonstrated that
varying the incubation temperature between 28°C and 35°C
has no effect on the survival of cells after engraftment
(Geiger et al., 2008). In other cases, the temperature has
been raised to 30°C (Geiger et al. 2008; Haldi et al. 2006;
Lally et al. 2007) or even as high as 35°C (Marques et al.
2009; Yang et al. 2013a, 2013b). Embryos can tolerate up to
35°C, although their survival is reportedly best at tempera-
tures below 33°C (Kimmel et al. 1995).
Implanted cells are generally rendered fluorescent to
visualize them within the body of the recipient embryo.
This can be achieved by staining cells prior to implantation
with stable fluorescent dyes, such as DiI, which are passed
onto the cell progeny (Jung et al. 2012; Lal et al. 2012; Lee
et al. 2009; Rouhi et al. 2010; Teng et al. 2013). The stain-
ing of cells prior to implantation is relatively fast and can
also be performed in primary cultures. In this way, the inva-
sive potential of cancer cells obtained from biopsies has
been assessed in vivo using the zebrafish model (Marques
et al. 2009), but studies on established cell lines have also
been performed with the use of dyes (Jung et al. 2012; Lal
et al. 2012). The disadvantage of using fluorescent dyes is
that their fluorescence can only decrease during the experi-
ment, making the quantification of cell proliferation by
means of fluorescence impossible. Furthermore, fluores-
cent dyes may be transferred to other cells.
An alternative to the fluorescence-labeling approach is
the establishment of cancer cell lines that stably express
fluorescent proteins (Drabsch et al. 2012; Eden et al. 2014;
Geiger et al. 2008; He et al. 2012; Yang et al. 2013a). The
fluorescence of endogenous fluorescent proteins is more
stable and correlates with cell number, enabling the quanti-
fication of proliferation by measuring fluorescence inten-
sity (Geiger et al. 2008; Vittori et al. 2014; Yang et al.
2014).
The crossing of different mutant zebrafish strains led to
the development of the Casper strain, which does not
develop skin pigmentation (White et al. 2008). The absence
of pigmentation aids microscopic observations because pig-
ments in the embryo may conceal the fluorescence emitted
by fluorescently labeled cells and only non-pigmented fish
enable quantitative intravital fluorescence imaging of
implanted cells. Despite its benefits, the Casper strain has
rarely been used in xenotransplantation studies (Corkery
et al. 2011; Eden et al. 2014). Instead, low concentrations of
phenylthiourea (PTU) are often used to inhibit melanin
synthesis (Lee et al. 2009; Yang et al. 2013a, 2013b, 2014).
Numerous studies have also used wild type embryos with
normally developed pigmentation for xenotransplantation
(Kitambi et al. 2014; Lally et al. 2007; Rampazzo et al.
2013).
Although human cells have been successfully engrafted
into zebrafish embryos during the late blastula stage (Geiger
et al. 2008; Lee et al. 2005; Zhao et al. 2009), cells are most
often implanted at 2 dpf, when the embryos develop all of
their major organ systems. Whereas some observations,
especially on angiogenesis and cell invasion, are concluded
at 5 dpf (Nicoli and Presta 2007; Yang et al. 2013a, 2013b),
longer studies, extending into the period of larval develop-
ment, have also been performed (Kitambi et al. 2014;
Pruvot et al. 2011). It is worth noting that the yolk is gener-
ally degraded at 5 dpf (Kimmel et al. 1995). When the yolk
mass is the site of implantation, this should be taken into
account, as the microenvironment of the implanted cells
will change with yolk resorption.
Applications of the Zebrafish Model
Studies on Cell Invasion and Metastasis
In the studies of cell invasion, the yolk sac is the most com-
mon area of implantation (Eguiara et al. 2011; Jung et al.
2012; Marques et al. 2009; Yang et al. 2013a). In this case,
cells are injected into the center of the yolk mass, a syncy-
tium containing nutrients required for embryonic develop-
ment. The movement of engrafted cells from the yolk to
other parts of the embryo can then be observed and quanti-
fied over a period of several days. The number of migrated
cells or the number of embryos in which invasion occurs are
determined (Eguiara et al. 2011; Marques et al. 2009; Yang
et al. 2013a).
It has been argued that quantification of the invasion of
cells from the yolk sac does not reflect their invasive poten-
tial, as cells may be passively transported to other parts of
the body via blood vessels (Drabsch et al. 2013). However,
differences in the capacity of cells to leave the yolk sac have
been demonstrated among cells grown in different culture
conditions (Eguiara et al. 2011; Yang et al. 2013a) and
among different cancer cell lines (Lee et al. 2009; Marques
et al. 2009), indicating that this is a valid model. The cor-
relation between the in vitro invasive potential of cell lines
and their ability to invade the body of the embryos has also
been established for cells implanted into the perivitelline
space, the cavity between the periderm forming the body
wall and yolk (Fig. 1B; Teng et al. 2013).
Alternatively, cells have been injected into to duct of
Cuvier (the cardinal vein of zebrafish embryos; Fig. 1B)
and allowed to spread throughout the body via the blood
circulation (Drabsch et al. 2013; He et al. 2012). Later, their
invasive potential can be assessed by counting cells located

Glioma Xenotransplantation in Zebrafish 753
within the tail fin, a structure that possesses no blood ves-
sels, thus ensuring that the cells had actively invaded the
tissue (Fig. 3A). This approach has been successfully
employed to study the involvement of neutrophils, which
may help to process the collagen matrix to facilitate cancer
cell invasion (He et al. 2012).
Angiogenesis may be involved in cancer cell dissemina-
tion from the injection site. For example, vascular endothelial
growth factor receptor (VEGFR) blockage was shown to
inhibit cell invasion from the perivitelline space (Lee et al.
2009), whereas hypoxia promoted it (Lee et al. 2009; Rouhi
et al. 2010). For the study of hypoxia-induced effects, zebraf-
ish embryos may be maintained in a hypoxic chamber (Rouhi
et al. 2010), although hypoxia may lead to developmental
abnormalities (Lee at al. 2009; Padilla and Roth 2001).
Zebrafish have also been used to study metastatic pro-
cesses. The transparency of embryos and the availability of
transgenic zebrafish embryos (fli1:GFP) expressing GFP in
their vascular endothelium (Lawson and Weinstein 2002)
has enabled the high-resolution imaging of processes
involved in metastasis (Stoletov et al. 2007). The metastatic
potential of cancer cells was associated with the upregula-
tion of specific molecular markers that are involved in
metastasis (Drabsch et al. 2013; He et al. 2012; Stoletov
et al. 2010). With cells implanted in the pericardium (Fig.
1B), cell invasion along the abluminal side of blood vessels
was also observed (Zhao et al. 2011). The results of these
studies show that zebrafish embryos can be used to evaluate
the invasive potential of cancer cells as well as the mecha-
nisms of metastasis.
Quantification of Cell Survival and Cell
Proliferation
The assessment of the effects of different treatments on the
proliferation of cancer cells through fluorescence quantifi-
cation has been successfully implemented (Eden et al.
2014; Geiger et al. 2008; Lally et al. 2007; Yang et al.
2014). An alternative approach is the use of biolumines-
cence imaging of implanted cells genetically modified to
express luciferase (Zhao et al. 2009). The advantage of bio-
luminescence is the low background signal as compared to
fluorescence, but it is less suitable for single-cell visualiza-
tion (Klerk et al. 2007). Alternative approaches for the
quantification of cancer cell proliferation include measur-
ing the projected tumor area on fluorescence micrographs
(Lal et al. 2012) and estimating the volume occupied by
cancer cells (Zhao et al. 2011a); however, these approaches
may not be indicative for proliferation, as an increase in
tumor volume can also result from cell dispersion or the
accumulation of host cells in the tumor (Lal et al. 2012).
Modeling Tumor-Induced Angiogenesis
Zebrafish embryos are particularly suitable for the study of
tumor-induced angiogenesis owing to the simplicity of their
cardiovascular system and its predictable patterning (Seabra
and Bhogal 2010; Stoletov et al. 2010; Tobia et al. 2011;
2013). The possibility to directly visualize the developing
vasculature is facilitated by the transparency of the embryos
and the availability of transgenic strains with fluorescent
Figure 3. Fluorescence observation of processes in zebrafish embryos in vivo. (A) A U87 DsRed cell (arrow) invading the tail fin of a
zebrafish embryo 2 days after implantation from the spinal cord. (B) Visualization of the vasculature in a living transgenic embryo at 1 day
after fertilization (courtesy of Marchien Dallinga). The embryo expresses GFP in the vascular endothelium. Scale, 100 µm.

754 Vittori et al.
blood vessels (Fig. 3B). To study tumor-induced angiogen-
esis, cells are usually implanted into the perivitelline space,
where the effect of implanted cells on the development of
the subintestinal vasculature is then monitored (Nicoli and
Presta 2007; Nicoli et al. 2007; Vitale et al. 2014; Zhao
et al. 2011a). Assessment of this effect can be rapid, as
changes can be observed within two days after tumor xeno-
grafting (Vitale et al. 2014; Yang et al. 2013b, 2014). The
quantification of the xenograft’s angiogenic effect involves
the determination of the percentage of embryos in which
abnormal angiogenesis is observed (Nicoli and Presta 2007;
Yang et al. 2013b) or measurement of parameters such as
vessel length, diameter, or the number of branching points
(Yang et al. 2013; Zhao et al. 2011). Alternatively, ex vivo
quantification of the frequency of tumor-induced angiogen-
esis and the number of generated blood vessels has been
performed using whole-mount alkaline phosphatase activ-
ity staining of the vasculature (Nicoli and Presta 2007;
Nicoli et al. 2007; Tobia et al. 2011).
The Zebrafish Xenotransplantation
Model in the Study of Glioma
Introducing Glioma to Zebrafish
Human glioma cells have successfully been transplanted
into zebrafish, and most of the methodological approaches
outlined above have been applied to study glioma. Unlike
many other mammalian tumors, glioma may be particularly
suitable for orthotopic xenotransplantation experiments in
zebrafish.
The yolk was the initial area of GBM cell implantation,
at first in blastula stage embryos and later in 2 dpf embryos
(Geiger et al. 2008; Lally et al. 2007; Yang et al. 2013a,
2013b). Apart from survival and proliferation, the assess-
ment of the invasion potential of glioma cells may be par-
ticularly important for the development of new treatment
approaches for this highly invasive type of cancer. Due to
the possible invasion of glioma cells into other parts of the
CNS, successful treatment of glioma by surgical removal
and directed radiotherapy is impossible (Claes et al. 2007).
Glioma cell lines that have so far been studied in zebrafish
do not demonstrate a great tendency to invade if implanted
into the yolk sac. Differentiated U87 GBM cells are not
invasive when implanted in this area (Lal et al. 2012; Yang
et al. 2013a, 2014), and invasion of U251 GBM cells has
not been reported (Geiger et al. 2008; Lally et al. 2007).
The inability of implanted cells to leave the yolk sac likely
reflects their low metastatic potential.
On the other hand, implantation into the yolk sac has
provided new insights into the role of glioma stem-like cells
(GSLCs) in tumor progression. One of the hallmarks of
GSLCs is the expression of the cell-surface marker CD133,
which is also used for isolating these cells. GSLCs have
been proposed to play important roles in glioma progres-
sion and radiotherapy resistance, which is attributed to their
more efficient DNA repair mechanisms (Bao et al. 2006;
Campos and Herold-Mende 2011; Hira et al. 2015; Molina
et al. 2014; Persano et al. 2013; Tabatabai and Weller 2011;
Wang et al. 2010). As a result, GSLCs are the focus for
research into targeted therapy, including studies using the
zebrafish model. To this end, CD133+ U87 cells were
obtained by single-cell cloning of U87 cells and growing
them under specific culture conditions (Yang et al. 2013a)
or by obtaining CD133+ cells by flow cytometry-based cell
sorting (Yang et al. 2013b, 2014). The CD133+ subpopula-
tion of U87 cells implanted into the yolk sac of 2 dpf zebraf-
ish embryos invaded the body more frequently than
CD133- cells (Yang et al. 2013a). These findings suggest
that GBM cells with stem cell properties are more invasive
than their differentiated counterparts.
Studies on the proliferation of GBM cells in the yolk sac
have provided somewhat conflicting results, as U87 cells
generally do not proliferate in this area (Lal et al. 2012;
Yang et al. 2014; Vittori et al. 2014) unless injected together
with Matrigel (Yang et al. 2014). In contrast, a study assess-
ing proliferation through bioluminescence showed the pro-
liferation of U87 cells within the yolk sac (Zhao et al. 2009).
Experiments on the cell line U251 also demonstrated mea-
surable proliferation in this area (Lally et al. 2007; Geiger
et al. 2008).
To study GBM-induced angiogenesis in zebrafish
embryos, implantation into the perivitelline space—as has
been suggested for other tumor types (Nicoli and Presta
2007)—may not be necessary, as remodeling of the vascu-
lature has been observed in embryos in which U87 cells
were implanted in the center of the yolk mass (Yang et al.
2013b). The implantation of U87 cells in the yolk resulted
in better survival as compared with their implantation into
the perivitelline space. Angiogenic activity of xenotrans-
planted U251 cells in the yolk mass has also been demon-
strated (Geiger et al. 2008). This approach has been applied
to study transforming growth factor-β (TGF-β) involve-
ment in angiogenesis by pretreating U87 cells with TGF-β
and implanting them in the yolk sac of 2 dpf embryos.
Pretreatment resulted in enhanced xenograft-induced
angiogenesis in the area of U87 cell implantation (Yang
et al. 2013b).
Location Matters: Orthotopic Studies on Glioma
In zebrafish, the CNS begins to form as early as 9 hr after
fertilization and, by 2 dpf, the brain, spinal cord, eyes and
the inner ear are well formed. The CNS is a relatively large
structure within the embryo’s body (Fig. 4), which speaks in
favor of orthotopic xenotransplantation of GBM cells. The
advantages and disadvantages of implanting cells in the
embryonic brain are summarized in Table 1.

Glioma Xenotransplantation in Zebrafish 755
Orthotopic implantation of glioma cells is advanta-
geous over yolk sac implantation, as the CNS represents
an environment that is similar to that of humans. In addi-
tion, orthotopic implantation can improve the survival and
proliferation of glioma cells as compared to implantation
into the yolk sac (Figs. 5, 6; Vittori et al. 2014). Human
GBM cells have been successfully implanted into the
embryonic brain at two days after fertilization (Kitambi
et al. 2014; Vittori et al. 2014). However, most orthotopic
studies have used older developmental stages, such as lar-
vae and juveniles (Eden et al. 2014; Lal et al. 2012;
Rampazzo et al. 2013). Lal et al. (2012) assessed the inva-
siveness of GBM cells implanted into the brain of either
4-day-old embryos or 10-day-old larval zebrafish.
Implanted DiI-labeled U87 cells were shown to disperse in
the larval brain, moving predominantly along the brain
vasculature, which is also a major track for invading GBM
cells in the human brain.
Rampazzo et al. (2013) implanted patient-derived
CD133+ glioma cells, obtained by cell sorting and trans-
fected with the GFP gene, into zebrafish larvae at 7 dpf. In
this study, the implanted CD133+ cells underwent cell
cycle arrest, decreased the expression of stem cell markers
and increased the expression of neuronal markers. It was
suggested that the apparent loss of stemness is linked to
Wnt-signaling. The activity of Wnt signaling was visual-
ized in vivo by using transgenic zebrafish larvae express-
ing a fluorescent protein upon activation of a Wnt-associated
transcription regulator. As demonstrated by Yang et al.
(2013a), the percentage of CD133+ U87 cells was greatly
reduced in the body of embryos at two days after implanta-
tion of a CD133+-enriched population of this cell line into
Figure 4. Confocal image of methyl green staining in a zebrafish embryo at 3 days after fertilization, showing the relative size and basic
anatomy of its central nervous system. The brain is one of the largest structures within the body of the embryo, facilitating orthotopic
implantation of glioma cells. Scale, 200 µm.

756 Vittori et al.
the yolk sac. The mechanism of this reduction is not clear.
Taken together, these results suggest that the zebrafish
embryonal environment may promote differentiation of
GSLCs, which would have great impact on the type of
research that can be applied to GSLCs.
Bigger Fish to Fry: Studying Glioma in Juvenile
Zebrafish
The xenotransplantation approach has also been applied to
older zebrafish that are approximately 30 days old (Eden
et al. 2014; Stoletov and Klemke 2008; Stoletov et al.
2007). The possible advantages and disadvantages of this
approach are summarized in Table 2. It has been argued
that the use of juveniles may provide more similarity to
the native tumor environment. However, the application of
xenotransplantation to older life stages comes at a cost.
The brain is not as easily imaged by optical microscopy in
juvenile and adult fish as in embryos and larvae, even in
the Casper strain (White at al. 2008), making older life
stages less suitable for in vivo imaging. Xenotransplantation
of cancer cells into juvenile fish requires immunosuppres-
sion of the recipient fish through the delivery of dexa-
methasone (Eden et al. 2014; Stoletov and Klemke 2008;
Tobia et al. 2013) or radiation (Taylor and Zon 2009;
Zhang et al. 2014). However, this is not necessarily a dis-
advantage, as GBM patients are often exposed to these
treatments as well.
In the study of Eden et al. (2014), xenografting of
cells derived from mouse brain tumors (including GBM)
was performed in 30-day-old zebrafish. The formation of
secondary tumor nodules elsewhere in the brain was
observed in these experiments, and tumor progression in
zebrafish was found to recapitulate that of the parent
tumor. In addition, the authors demonstrated similarities
between the transcriptomes of zebrafish tumors and their
corresponding mammalian tumors, which is an encour-
aging validation of the orthotopic xenografting approach
in zebrafish.
Application of Zebrafish Studies to Drug
Screening
Zebrafish embryos are an attractive model to study the
effects of novel pharmaceuticals, since small molecules are
taken up directly from the aqueous environment, making
chemical delivery simple and non-invasive (Drabsch et al.
2013; Taylor and Zon 2009; Yang et al. 2014). The simulta-
neous treatment of numerous embryos with small amounts
of chemicals enables a relatively high throughput of such
studies at low cost. Zebrafish embryos and larvae can thus
potentially bridge the gap between high-throughput in vitro
screening and mammalian models, as promising chemical
hits can first be validated in zebrafish and later studied in
greater detail in mammals (Jung et al. 2012; Stern and
Weinstein 2003).
The earliest application of zebrafish embryos to validate
glioma-related cytotoxic drug screening served to identify
potential small-molecule radiosensitizers. In this approach,
drug candidates were identified by in vitro screening and the
zebrafish embryo model was used to validate the in vitro
findings in an in vivo model. The novel radiosensitizer
NS-123, which increases the effects of radiation treatment
on GBM cells in vitro, was validated in vivo by implanting
U251 GBM cells in the yolk of blastula stage embryos (Lally
et al. 2007). This test enabled the simultaneous evaluation of
the potential toxic and teratogenic effects of the chemical.
The study demonstrated that the compound effectively
decreased the survival of GBM cells in the yolk sac follow-
ing radiation treatment without toxic effects to the zebrafish
embryos. Another validation of a therapeutic candidate in
zebrafish was performed with the macropinocytosis-target-
ing compound Vacquinol-1, which is reported to selectively
induce vacuolization and death of GBM cells (Kitambi et al.
2014). Zebrafish embryos at 2 dpf were orthotopically
implanted with fluorescently labeled U3013 GBM cells and
the tumor area was monitored for 10 days. The use of
Islet1:GFP transgenic zebrafish (Higashijima et al. 2000)
with a fluorescent CNS facilitated the visualization of
Table 1. Advantages and Disadvantages of the Yolk Sac or Brain for Glioma Cell Implantation.
Yolk Sac Brain
Advantages Disadvantages Advantages Disadvantages
Studying angiogenic
effects is straightforward
Yolk sac is degraded after a few
days
Is a permanent structure Studying angiogenic effects
may be difficult
Survival and proliferation of
glioma cells are limited
Glioma cells survive and
proliferate better
Limited suitability for studying
cell invasion
Suitable for studying cell invasion
Orthotopic implantation may give
more relevant results

Glioma Xenotransplantation in Zebrafish 757
implanted cancer cells in the CNS. In the study, several drug
candidates were first tested for toxicity in zebrafish embryos,
which helped to narrow down the candidates to a few
compounds. Vacquinol-1 was then demonstrated to most
effectively decrease the survival of GBM cells in the brain of
zebrafish larvae without causing toxic effects.
Figure 5. Time-lapse imaging of U87 DsRed glioblastoma cells
implanted in the brain of a zebrafish embryo at 2 days after
fertilization. Images were obtained at 4-hr intervals. Cells can be
seen invading the spinal cord in the posterior direction (arrows).
Scale, 300 µm.
Figure 6. Orthotopic xenotransplantation of fluorescent glioma
cells. (A) Fluorescent human glioblastoma cells (red) within
the brain of a zebrafish embryo at 3 days after implantation. (B)
Proliferation of implanted U-87 glioblastoma cells expressing the
fluorescent protein DsRed within the brain of a zebrafish embryo
over the course of several days. Day 1 marks the time point of
cell implantation at 2 days after fertilization. (C) Comparison of the
proliferation of implanted U-87 glioblastoma cells within the brain
and within the yolk sac of zebrafish embryos. Approximately 100
DsRed-expressing cells were implanted into the brain or the yolk
sac of zebrafish embryos at 2 days after fertilization, and the embryos
were incubated at 31°C for 3 days. The fluorescence emitted by the
cells was measured daily. Scale (A) 100 µm; (B) 250 µm.

758 Vittori et al.
The validity of zebrafish xenotransplantation in the
search of novel therapeutics for the treatment of glioma has
been shown in various studies. The antiangiogenic effects
of Axitinib, Suntinib and Vatalanib were tested in zebrafish
with U87 GBM cells implanted in the yolk sac. These
chemicals all inhibited tumor-induced vessel formation
(Yang et al. 2014). This study also demonstrated that the
novel anti-tumor compound Nordy inhibited angiogenesis
and increased the antiangiogenic effects of Vatalanib. The
radiosensitizing effects of temozolomide on U251 GBM
cells implanted in the yolk sac of blastula stage embryos
were also confirmed. In addition, temozolomide in concen-
trations that increased the toxicity of radiation to GBM cells
did not have a negative effect on embryo development
(Geiger et al. 2008). In a study on juvenile zebrafish at 30
dpf, the effects of two chemotherapeutics on the survival
and proliferation of implanted cancer cells were assessed:
tyrosine kinase inhibitor erlotinib and the pyrimidine ana-
log 5-fluorouracil (5-FU). In juveniles, 5-FU was adminis-
tered by dissolving it in the fish maintenance water, whereas
erlotinib was administered orally because of its low solubil-
ity (Eden et al. 2014). Quantification of cancer cell fluores-
cence demonstrated that 5-FU and erlotinib inhibited GBM
cell proliferation in vivo. Taken together, these studies dem-
onstrate that the zebrafish embryo model is a good model
for drug screening and preclinical validation.
Issues, Prospects and Conclusions
The zebrafish xenotransplantation cancer model is emerging
from its infancy and holds great promise for the visualiza-
tion of tumor progression and high-throughput therapeutic
compound validation. In this regard, the model fits between
in vitro cell studies and in vivo animal studies (Eden et al.
2014). It offers intermediate throughput, being more time
and labor consuming than in vitro experiments, yet faster
and more ethical than animal experimentation.
The strength of the model lies in its potential for single-
cell visualization and the ease of genetic manipulation; the
transgenic arsenal available for zebrafish has likely not
been fully exploited yet. The use of zebrafish with a fluo-
rescent vasculature is becoming routine in xenotransplan-
tation experiments (Geiger et al. 2008; Lee et al. 2009;
Stoletov et al. 2007; Yang et al. 2014), whereas other
structures, such as the CNS (Higashijima et al. 2000;
Zhang and Gong 2013) can also express fluorescent pro-
teins, enabling high-resolution imaging of implanted cells
and their microenvironment in vivo. In the future, several
genetically coded fluorescent labels may be used simulta-
neously to highlight different structures interacting with
the engrafted cells.
Zebrafish embryos are ideal for the use of fluorescent
reporter genes. For example, the generation of transgenic
zebrafish expressing three different molecular markers that
are characteristic for different levels of muscle cell differen-
tiation linked to three different fluorescent proteins pro-
vided unprecedented insights into the progression of
rhabdomyosarcoma induced by KRAS overexpression
(Ignatius et al. 2012). This methodology can also be applied
to xenotransplants, enabling visualization of gene expres-
sion patterns during cancer progression at single-cell reso-
lution. Indeed, the combination of the visualization of
implanted cells with the expression of fluorescent reporter
genes has been successfully applied to study glioma xeno-
grafts (Rampazzo et al. 2013), showing that this approach
has great potential.
Another possibility for future developments of the
zebrafish model is to study cancer cell heterogeneity
(Blackburn and Langenau 2014). For example, two cancer
cell lines expressing different fluorescent proteins have
been implanted simultaneously in a single embryo in order
to demonstrate the difference in their invasive potential
(Marques et al. 2009). In another study, wild type cells and
cells in which the expression of certain genes was upregu-
lated were labeled with different fluorescent proteins and
simultaneously implanted into zebrafish (Stoletov et al.
2010). This approach not only enables the simultaneous
visualization of the behavior of several cell types, but also
holds potential for the analysis of cell–cell interactions in
tumor progression.
Table 2. Advantages and Disadvantages of Zebrafish Embryos or Juveniles in Xenotransplantation Studies of Brain Tumors.
Embryos Juvenile Fish
Advantages Disadvantages Advantages Disadvantages
Transparency enables high-
resolution microscopic
observations
Experiments last only a few days Longer-term experiments are
possible
Reduced transparency limits
the resolution of microscopic
observations
No need for
immunosuppression
Can accommodate only a few
hundred mammalian cells
Can accommodate more
mammalian cells
Xenotransplantation requires
immunosuppression
No need for feeding Organ systems are still
developing
Most organ systems are mature Require feeding
Blood-brain barrier is developing Blood-brain barrier is functional

Glioma Xenotransplantation in Zebrafish 759
Steps have been taken to increase the throughput of
zebrafish xenotransplantation experiments (Snaar-Jagalska
2009), leading to a proposal of automated approaches to
cell implantation and imaging that focus predominantly on
the quantification of cell invasion from the yolk sac (Ghotra
et al. 2012). The study of this process can be primarily
applied to the screening and validation of anti-angiogenic
and anti-metastatic drugs. Nevertheless, ethical consider-
ations with respect to the use of embryos in large-scale
screens should not be ignored entirely.
Acknowledgments
The authors wish to thank Matjaž Novak, Dr. David Dobnik
(National Institute of Biology, Ljubljana) and Marchien Dallinga
(Academic Medical Center, Amsterdam) for their assistance.
Declaration of Competing Interests
The authors declared no potential competing interests with respect
to the research, authorship, and/or publication of this article.
Author Contributions
MV, HM and TLT all contributed to literature overview and writ-
ing of the manuscript.
Funding
The authors disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
Slovenian Research Agency Program P1-0245; INTERREG EC
Project 2011, Ref. No. 42: Identification of novel biomarkers to
brain tumors - glioma, for diagnosis and as new therapeutic tar-
gets, Acronym: GLIOMA.
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