Research Article
Transplantable Tumor Lines Generated in Clonal Zebrafish
1 2,3
Igor V. Mizgireuv and Sergei Y. Revskoy
Laboratories of 1Genetic Toxicology and 2Endocrinology, N.N. Petrov Research Institute of Oncology, St. Petersburg, Russia and
3
The Asher Center, Department of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Northwestern
University, Chicago, Illinois
Abstract
Transplantable zebrafish tumors are a novel and very
promising model in cancer research. However, further prog-
ress in this field has been contained by a lack of true inbred
lines in zebrafish. To overcome this problem, we generated
two lines of homozygous diploid clonal zebrafish lines (i.e.,
CB1 and CW1), which allowed us to carry out transplantation
of any tissue, including tumors, from one fish to another
within a line without rejection of the graft. The primary
tumors in CB1 fish were induced by N-nitrosodiethylamine
(DEN). The histologic analysis of these tumors revealed
different types of hepatocellular carcinomas, hepatoblasto-
mas, hepatoma, cholangiocarcinoma, and pancreatic carcino-
ma. Four spontaneous acinar cell carcinomas of pancreas
were also found in 10- to 18-month-old CB1 fish. Small pieces
of tissue or cell suspensions of either DEN-induced or
spontaneous tumors were serially transplanted into the
peritoneal cavity of syngeneic fish at different stages of
development from 5-day-old larvae to adult fish. The
development of grossly visible tumors occurred from 2 weeks
to 3 months after tumor grafting and grew either as solitary
smooth nodules or as an amorphous jelly-like mass infiltrating
abdominal organs. The majority of tumors were also
successfully transplanted to isogeneic (F1 generation from
crossing CB1
CW1) fish. At the present time, 19 transplant-
able zebrafish tumor lines have been generated and main-
tained for as long as 3 to 25 passages. This model provides a
novel tool for studying experimental tumor biology and
therapy and will become a cost effective system for high
throughput screening of anticancer drugs. (Cancer Res 2006;
66(6): 3120-5)
Introduction
The zebrafish is currently viewed as a very promising model in
cancer research (1, 2). A number of chemical carcinogens potent in
mammals seemed also to be efficient in induction of zebrafish
tumors (3–7). As in rodent models, transgenic zebrafish that
overexpress c-myc and N-myc oncogenes develop T-cell leukemia
(8) and neuroendocrine tumors (9), respectively. These data
indicate a close similarity between mechanisms of carcinogenesis
in mammals and zebrafish. Therefore, zebrafish should be
considered as an adequate model for investigation of tumor
biology and treatment. However, long and variable latent periods
of tumor progression, asynchronous growth, and <100% tumor
incidence rate make use of these currently available tumor models
Requests for reprints: Sergei Y. Revskoy, Psychiatry and Behavioral Sciences,
Northwestern University, 303 East Chicago Avenue, Ward Building 9-233, Chicago, IL
60611. Phone: 312-908-1771; Fax: 312-503-0466; E-mail: s-revskoy@northwestern.edu.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-3800
Cancer Res 2006; 66: (6). March 15, 2006
for development and screening of new antitumor drugs somewhat
problematic. In the meantime, other standard mammalian tumor
models have not been used in zebrafish until now. In particular,
serially transplanted tumors have been used in investigation of
tumor biology and experimental tumor therapy in rodents for >100
years (10). Generation of transplanted tumor lines in zebrafish
might become an inexpensive alternative to this model in rodents.
Furthermore, transplanted tumor lines would be an indispensable
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supplement for already established models of tumor development
in zebrafish and as a source of transformed cell lines maintained
ex vivo. The ability to transplant tumors from one fish to another
was shown in the viviparous fish Poecilliopsis licuda (11), which is
considered to be a naturally inbred strain (12). Generation of a true
inbred zebrafish line that would permit unconstrained transplan-
tation of tissues between fish at present remains a difficult task
(13). In this study, we proposed to generate transplantable tumors
using clonal homozygous lines of zebrafish established by a
procedure described elsewhere (14). Each such a line consisted of
fish that were exact genetic copies of each other. This makes it
possible to carry out transplantation of any tissue, including
tumors, from one fish to another within a line without rejection of
the graft. Here, we present data showing the successful generation
of transplantable tumors in clonal zebrafish.
Material and Methods
Animals. Zebrafish of the line brass were purchased in a local
pet shop. The wild-type (AB) zebrafish were obtained from Dr. E.
Weinberg (University of Pennsylvania, Philadelphia, PA) and
maintained in the laboratory for >8 years. The golden strain of
zebrafish was obtained from Dr. H.G. Frohnhoefer (MPI for
Developmental Biology, Germany). During the entire study, fish
were maintained in 20-L acrylic tanks connected to a closed water
recirculation system with 50 to 60 fish in each. Fish were
maintained in standard conditions (15) briefly: 14/10-hour light/
dark cycle, T = 26 F 1jC. Tetramin (Tetra, Melle, Germany) and
Ovo-vit (QXL, Poznan, Poland) were used as a basal diet
supplemented with nauplii Artemia salina.
Generation of clonal homozygous zebrafish. Clones of
homozygous zebrafish CB1 and CW1 derived from brass and AB
strains of zebrafish, respectively, were obtained by heat shock
procedure as described previously (14) with some modifications.
Briefly, eggs from female AB and brass strains of zebrafish were
fertilized by UV-inactivated sperm from male zebrafish of golden
and AB strains, respectively. After 13-minute incubation at 28.5jC
eggs underwent a 2-minute heat shock at 41.4jC to block the first
cleavage. This procedure led to the development of a homozygous
diploid fish. Eggs obtained from a raised to maturity homozygous
female fish of either AB or brass strain underwent the second
round of UV-sperm fertilization/heat shock procedure exactly
as described above. The offspring obtained from each homo-
zygous female were genetic copies of each other (i.e., clones of
3120 www.aacrjournals.org

homozygous fish). The further maintenance of homozygous strains
was carried out by crossing fish within each clone. Mating of the
fish that belonged to different homozygous clones led to the
generation of an isogeneic fish strain consisting of genetically
identical but not homozygous fish.
Tumor induction. Sixty-five 2.5-month-old fish from the clonal
CB1 line were used for tumor induction by continuous immersion
in N-nitrosodiethylamine (DEN) solution (100 ppm) during the
8 weeks. During DEN, exposure fish were maintained in 20-L acrylic
tanks equipped with a mechanical filter and an automatic heater.
The filters were cleaned twice weekly. The complete exchange of
water containing the carcinogen was done every 2 weeks. The
water temperature was maintained at 26 F 0.5jC. Following
completion of DEN exposure the fish were transferred to a 20-L
system tank and remained under observation for up to 9 months.
Tumor transplantation. The tumor tissue for further grafting
was obtained from fish with grossly visible neoplasms located in
the abdomen. These fish were euthanized by 0.015% tricaine
(Sigma, Munich, Germany) solution and were opened ventrally with
iridectomy scissors. Tumors were gently separated from surround-
ing tissues and placed into cold (4jC) Ringer solution with
antibiotics (100 units/mL penicillin and 0.1 mg/mL streptomycin)
for 2 to 30 minutes (i.e., until transplantation). In the meantime,
2.5- to 5-month-old syngeneic recipients were placed in 0.015%
tricaine solution in groups of three to six fish. Immediately after
reaching immobility, the fish were transferred individually onto a
wet sponge for further transplantation of tumor tissue. I.p.
transplantation of tumor tissue was carried out by a small punch
using a thick syringe needle ( ; 2 mm) in the left side of abdomen.
A piece(s) of a tumor (1-2 mm3) in 10 to 20 AL of Ringer solution
was injected into abdomen through this punch by a fire-polished
Pasteur pipette. The wound caused by the punch was not stitched.
In i.m. transplantation, pieces of tumor were injected into dorsal
muscles in the area of the dorsal fin by a syringe needle equipped
with a well-fitting metal plunger. After the transplantation, fish
were transferred into dechlorinated tap water containing 10 Ag/mL
methylene blue for 24 hours followed by their return to a system
tank. To transplant tumors to 7- to 14-day larvae, the tumors
removed from the donors were mechanically minced in 200 to 300
AL of cold (4jC) PBS and filtered through 40-Am mesh (Becton
Dickinson Labware, Mountain View, CA). After a short centrifuga-
tion, the pellet consisting of single cells and cell clusters (5-20 cells)
was resuspended in 25 AL PBS and kept on ice until transplan-
tation. Using a flexible syringe needle ‘‘MicroFil’’ (WPI, Inc.,
Tonasket, WA), f5 AL cell suspension was placed into a glass
micropipette with an external diameter at the tip of 30 Am without
a filament and made with a vertical puller. Recipient larvae were
placed into 0.015% tricaine solution until they became immobile.
Then, the larvae were transferred onto a wet filter paper in groups
of 25 animals. After i.p. injection of f50 to 100 nL (depending on
recipient size) of cell suspension containing 2.5 to 3
106 cells per
10 AL, all the larvae were returned to clean tap water. The animals
in the control group were injected with the same volume of Ringer
solution.
Cryopreservation. Large tumors nodules that underwent 5 to
10 passages were extracted from fish abdomens and cut into 2 to 3
parts (f5
5
5 mm). Each part was placed in a 0.5-mL
cryopreservation tube (Sarstedt, Nuembrecht, Germany) contain-
ing 400 AL DMEM supplemented with 20% FCS and 10% DMSO
(Sigma, Munich, Germany) and then was further cut with scissors
into pieces of about 1
1 mm. The tubes were kept at 4jC for
www.aacrjournals.org
Transplantable Tumors in Zebrafish
30 minutes followed by overnight storage at 80jC and then
transferred to liquid nitrogen for a long-term storage. Thawing was
carried out by a quick transfer of the tubes from liquid nitrogen to
a water bath at 30jC to 32jC followed by a replacement of
cryopreservation medium with DMEM without FCS and DMSO.
After thawing, samples were kept at 4jC until transplantation as
described above.
Histology. After 24 to 72 hours of fixation in 4% solution of
neutral paraformaldehyde, all primary and transplanted tumors
together with adjacent abdominal organs underwent standard
histologic processing. Tissue slices (3-4 Am) were stained by H&E
or, if necessary, with other staining methods. Photographs of the
slides were obtained by Nikon Coolpix 4500 digital camera,
mounted with an optical adapter on the microscope BIOLAM-I
(LOMO, St. Petersburg, Russia).
The mean survival time tumor-bearing fish. The mean
survival time of tumor-bearing fish was assessed in two most fast
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growing tumor lines zt23 and s1 implanted i.p. to syngeneic
3.5-month-old fish or to 12-day-old syngeneic larvae (zt23 only).
Each experimental and control group consisted of either 10 adult
females or 29 larvae. During the observation, each group of fish was
kept in a separate 20-L system tanks ( for adults) or in 400-mL
beakers ( for larvae) at 26jC and fed a standard diet or nauplii
Artemia, respectively. The assessment of survival time continued
until the death of the last fish in each experimental group.
Results
Treatment of zebrafish with DEN resulted in 35 primary tumors
localized in the abdominal area and reaching from 3 to 10 mm in
diameter. The grossly visible tumors began to emerge in fish 3 to
7 months after the beginning of exposure to DEN. Four
spontaneous tumors diagnosed as acinar cell carcinomas of the
pancreas were detected in 10.5- to 18-month-old female CB1 fish
(Fig. 1G).
Of 29 transplanted DEN-induced and spontaneous tumors, 19
underwent from 3 to 25 consecutive passages in syngeneic fish. The
other tumors were lost as a result of the recipient’s death or
because of a tumor growth arrest after several passages. In the
majority of cases, i.p. or i.m. transplanted tumors showed almost
synchronous growth in fish inoculated simultaneously. The efficacy
of tumor transplantation to both sites was close to 100% as would
be expected in syngeneic recipients. The main features of the
transplanted tumor lines that are currently being maintained in
the lab are shown in Table 1. The majority of tumors were
characterized by a moderate growth rate and required further
passage approximately every 1 to 2 months. The others tumor lines
remained either slow growing during the entire period of
observation (zt10, zt12, zt16, zt20, and s3) or, by contrast, had a
very high growth rate and required transplantation every 2 to
4 weeks (s1, zt23, and zt34). It is noteworthy that invasiveness also
varied among different transplanted tumor lines. In particular,
tumor lines zt23 and s1 seemed to be the most invasive.
Macroscopically, these tumors were composed of an amorphous
jelly-like mass of milky-white (s1) or yellowish (zt23) color, filling
essentially the entire abdominal cavity (Fig. 1D). The liver, ovaries,
and abdominal wall were the most common sites of invasion of
these tumors. Less invasive tumors typically grew as solitary pink
or yellowish solid smooth nodules up to 1 cm in diameter, well
separated from surrounding abdominal organs (Fig. 1E). These
nodules were usually connected to the host vascular system by a
3121 Cancer Res 2006; 66: (6). March 15, 2006
Cancer Research
blood vessels stalk. The majority of these tumors also showed
invasive growth. However, in this case, the signs of invasion into
surrounding organs could be determined after a microscopic
assessment only (Fig. 2H). ‘‘Minimal deviation’’ hepatoma (zt20)
grew as a relatively benign noninvasive tumor, forming structures
grossly similar to those of normal liver with regard to shape, color,
and consistency (Fig. 1F).
During multiple serial transplantations, all tumor lines retained
a close similarity to the histologic structure of the primary tumors.
Most of the DEN-induced tumor lines were diagnosed as a typical
hepatocellular (zt8, zt9, zt12, zt15, zt16, zt18, zt28, zt29, and zt34;
Fig. 2C) and cholangiocellular (zt10; Fig. 2D) carcinomas. The
morphology of these types of tumors has been described in detail
for various fish species, including zebrafish (7, 16). Hepatoblasto-
mas (zt2, zt6, and zt21) were typically composed of undifferenti-
ated embryonal-like cells, sometimes in combination with
hepatocarcinoma cell areas that often have an arrangement
suggestive of a rosette-like formation. The histology of these
neoplasms resembled tumors observed in Fundulus heteroclitus
from a creosote-contaminated area (17). The ‘‘minimal deviation’’
hepatoma zt20 was composed of essentially normal hepatocytes
with round unimorfic nuclei and vacuolated cytoplasm (Fig. 2E).
This tumor may represent the zebrafish analogue of ‘‘minimal’’
hepatomas described earlier in rats (18). The DEN-induced acinar
cell carcinoma in pancreas (zt23) commonly grew as large fields
consisting of separate strongly basophilic small cells infiltrating
the gaps between adjacent organs (Fig. 1K). In some cases, we
observed liver metastases after i.p. grafting of this tumor. The
Cancer Res 2006; 66: (6). March 15, 2006
Figure 1. Growth of transplantable and
spontaneous tumors in juvenile and adult
zebrafish. Advanced stages of tumor
development after i.p. (A) and i.m. (B)
transplantation of tumors zt6 and zt34,
respectively. Typical enlargement of frontal
abdominal wall as a result of tumor
growth (arrowhead). C, tumor mass
(arrowhead) penetration of abdominal
wall after i.p. transplantation of tumor zt18.
D, infiltrating growth of s1 tumor after i.p.
transplantation. Amorphous tumor mass
fills in the entire abdomen invading
inner organs. E, tumor zt15 after i.p.
transplantation. The tumor is growing as an
isolated nodule pressing on surrounding
tissues. F, formation of liver-like structure
in result of growth of ‘‘minimal’’ hepatoma
zt20 after i.p. transplantation. Age of
transplant is 8 weeks. G, primary
spontaneous acinar cell carcinoma of
pancreas s10 developed in 11-month-old
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female zebrafish of CB1 line (T , tumor
nodules). H, tumor zt34 developed after
i.p. transplantation into isogeneic host fish.
I and J, 23-day-old juvenile fish through
11 days after i.p. injection of either PBS (I)
or zt23 tumor cell suspension (J ). The
tumor-bearing fish reveals enlarged,
compare with control fish, peritoneal cavity
filled with opaque tumor mass (arrowhead)
penetrating abdominal wall (arrow).
K, sagittal histological section of 23-day-old
juvenile fish through 11 day after i.p.
injection of zt23 tumor cell suspension.
Tumor mass (T ) fills in almost the entire
peritoneal cavity, infiltrating abdominal
organs (L, liver; SB, swim bladder).
L, zt23 cell suspension and cell clusters
just before the transplantation (white field
microscopy).
histology of these metastases was similar to the tumor that they
were derived from (Fig. 2G). Spontaneous acinar cell carcinomas in
pancreas (s1, s3, s10, and s11) grew as solid sheets formed by large
polygonal cells with moderately polymorphic nuclei, with prom-
inent nucleoli and basophilic cytoplasm that contained bright
eosinophilic secretory granules (Fig. 2F). In some areas, the
structure of these tumors strongly resembled normal pancreatic
tissue in zebrafish (Fig. 2B). Histologic diagnoses of all tumor lines
were validated by the Registry of Tumors in Lower Animals
(Sterling, VA).
Signs of advanced stages of tumor growth, such as significant
enlargement (often asymmetric) of the abdomen (Fig. 1A),
development of ascit in the abdominal cavity, tumor penetration
of the abdominal wall (Fig. 1C), cachexia, or various combinations
of these symptoms, were as criteria for expediency of the next i.p.
tumor passage in adult fish. By contrast, slow growing (zt10, zt12,
zt16, and s3) or relatively benign (zt20) tumor lines underwent
consecutive passages approximately every 2 to 3 months without
strong dependency upon macroscopic signs of tumor progression.
Tumor size was the main criteria for the next tumor passage after
i.m. transplantation (Fig. 1B).
As expected, we failed to transplant several moderately and fast
growing tumor lines (zt9, zt18, and zt23) to wild-type (AB) zebrafish.
By contrast, transplantation of the majority of tumor lines to
isogeneic zebrafish was successful in all cases (Table 1; Fig. 1H).
However, the growth rate of tumors transplanted to isogenic hosts
was typically f50% slower than that in their syngeneic counter-
parts.
3122 www.aacrjournals.org
 Transplantable Tumors in Zebrafish

Table 1. Main features of transplantable tumor lines in zebrafish

No. Tumor Tumor No. Interval between Duration Invasivenessx Growth in RTLA Histologic diagnosis of
c
line origin passages* passages days of tumor isogenic no.k tumor lines
maintenance fish
b
(mo)

1 zt2 DEN induced 11 45-60 18.0 +/ Yes 7606 Hepatoblastoma and
Hepatocellular Carcinoma
2 zt6 DEN induced 10 45-60 17.5 +/ Yes 7607 Hepatoblastoma and
Hepatocellular Carcinoma
3 zt8 DEN induced 13 30-45 17.3 +/ Yes 7609 Hepatocellular Carcinoma
4 zt9 DEN induced 13 30-45 17.3 +/ Yes 7610 Hepatocellular Carcinoma
5 zt10 DEN induced 7 65-80 17.3 +/ Yes 7611 Cholangiocarcinoma
6 zt12 DEN induced 7 60-90 17.3 +/ Yes 7612 Poorly differentiated
hepatocellular carcinoma
7 zt15 DEN induced 12 30-45 17.3 +/ Yes 7613 Hepatocellular carcinoma
8 zt16 DEN induced 8 50-75 17.0 +/ Yes 7614 Poorly differentiated

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hepatocellular carcinoma
9 zt18 DEN induced 13 40-50 16.7 +/ Yes 7615 Poorly differentiated
hepatocellular carcinoma
10 zt20 DEN induced 8 45-75 16.5  Yes 7616 ‘‘Minimal deviation’’
hepatoma
11 zt21 DEN induced 12 40-50 16.5 +/ Yes 7617 Hepatoblastoma
12 zt23 DEN induced 25 10-20 16.5 + Yes 7618 Pancreatic acinar cell
carcinoma
13 zt28 DEN induced 11 45-60 16.3 +/ Yes 7619 Hepatocellular carcinoma
14 zt29 DEN induced 12 30-45 16.3 +/ Yes 7620 Hepatocellular carcinoma
(with spindle cell pattern)
15 zt34 DEN induced 18 20-30 16.2 +/ Yes 7621 Poorly differentiated
hepatocellular carcinoma
16 s1 Spontaneous 14 20-30 12.5 + Yes 7605 Pancreatic acinar cell
carcinoma
17 s3 Spontaneous 5 50-75 10.0  NT Pancreatic acinar cell
carcinoma
18 s10 Spontaneous 3 30-45 2.0  NT Pancreatic acinar cell
carcinoma
19 s11 Spontaneous 3 30-45 1.5  NT Pancreatic acinar cell
carcinoma

Abbreviation: NT, not tested.

*Number of tumor passages up to date.
cAverage interval between two consecutive tumor transplantations (recipient’s age ranged between 2.5 and 6 mos).
bTotal duration of the tumor line maintenance beginning from the first passage.
x (+), infiltrative growth; (+/), invasive growth detected during histologic analysis only; (), tumor invasion is not detected.

kIdentification no. at the Registry of Tumors in Lower Animals.

The frequency of successful transplantation of tumor lines zt23,
zt34, and s1 recovered after cryopreservation remain as high as
100%, 100%, and 33%, respectively. All tumors that developed after
cryopreservation showed invasive growth and retained a histologic
structure of the corresponding tumor lines.
The mean survival time of adult fish after i.p. tumor grafts was
25 F 4.0 and 44.7 F 6.2 days for tumor lines zt23 and s1, respectively
(Fig. 3A). No fish death occurred in a control group during the entire
period of observation. The mean survival time of 12-day larvae i.p.
injected with zt23 tumor cells was 15 F 4.2 days (Fig. 3B).
Discussion
Transplantable tumor lines remain one of the main models for
investigation of different aspects of tumor biology in rodents (19).
Various attempts have been made to establish transplantable
tumor lines in lower vertebrates, including homologous epitheli-
oma grafting into the anterior eye chamber in catfish (20). More
recently, a possibility of transplantation of carcinogen-induced liver
tumors to intact adult fish through more conventional routes was
shown by M. Schultz and R. Schultz (11) using a naturally inbred
stock of Poeciliopsis licuda. Considering a significant progress in
developmental biology and genetics of zebrafish, this animal hold a
great potential with regard to novel cancer research models. It has
been shown that mMyc-induced leukemia (8), MycN-induced
neuroendocrine tumors (9) and mutated BRAF V600E-induced
melanoma (21) can successfully be transplanted to g-irradiated
zebrafish. In addition, zebrafish embryos were shown to be
potentially suitable for human and mammalian tumor cells grafting
(22, 23). However, further progress in generation of sustainable

www.aacrjournals.org 3123 Cancer Res 2006; 66: (6). March 15, 2006
Cancer Research
transplantable tumors in zebrafish has been contained because of
the lack of a true inbred zebrafish strain as a result of lost of
fertility during the inbreeding (24). To overcome this obstacle, we
generated two clonal homozygous lines of zebrafish whose viability
and fertility was comparable with that of wild-type fish. The lack
genetic difference between individual fish within a clone enabled us
to carry out successful multiple passages of DEN-induced and
spontaneous tumors in zebrafish.
As expected, the majority of DEN-induced tumors in clonal
zebrafish were liver-derived tumors. A carcinoma of pancreas was
the only extrahepatic DEN-induced tumor. This finding was
somewhat surprising because before this study, benign pancreatic
tumors were described in zebrafish treated with N-methyl-NV-nitro-
N-nitrosoguanidine (5) and 7,12-dimethylbenz(a)anthracene (6)
but not with DEN (7). It is noteworthy that four spontaneous
tumors diagnosed as acinar cell carcinomas of pancreas were
detected in 10.5- to 18-month-old female CB1 zebrafish. Considering
the fact that spontaneous tumors of pancreas occur very seldom in
Figure 2. Histologic structure of normal and tumors tissue in zebrafish.
Histologic structure of normal liver (A) and pancreas (B ). C, moderately
differentiated hepatocellular carcinoma zt34. D, cholangiocarcinoma zt10.
E, ‘‘minimal’’ hepatoma zt20 displayed histologic structure that resembles
with normal liver. F, spontaneous acinar cell carcinoma of pancreas s1
containing areas morphologically similar to normal pancreas. G, a metastasis of
zt23 tumor within the lumen of a liver blood vessel (arrowhead). H, destruction
of muscle tissue cause by invasion of zt23 tumor in a 23-day-old juvenile fish
on 11 days after i.p. transplantation of the tumor cell suspension. Bar, 50 Am
(A-F and H ) and 250 Am (G ).
Cancer Res 2006; 66: (6). March 15, 2006 3124
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Figure 3. Lifespan of tumor-bearing fish. A, i.p. transplantation of tumor
zt23 (n) and s1 (E) to 2.5-month-old fish. Mean lifespan: - 25 F 4.0 and 45 F
.
6.2 days in zt23 and s1 tumor-bearing fish, respectively. Control intact fish ( ).
B, i.p. zt23 tumor transplantation (n) to 12-day larvae. Mean lifespan: - 15 F 4.6
.
days. Control intact fish ( ).
zebrafish (25), we suggest that CB1 fish might have genetic
predisposition to development of pancreatic tumors (e.g., because
of a possible loss of heterozygosity of tumor suppressor genes;
ref. 26) as a result of homozygous nature of their genome. This
hypothesis is supported by the recent reports on mutant lines of
zebrafish that are highly susceptible to spontaneous tumors (27–29).
The majority of DEN-induced and spontaneous tumors that
emerged in CB1 zebrafish showed growth after grafting to both
syngeneic and isogeneic but not to wild-type animals. These results
indicate that immunogenicity of transplantable tumors in zebrafish
is determined by their histocompatibility complex (30) and
therefore is similar to that of mammalian tumors (31). Currently,
of 19 transplantable tumors that we continue to maintain in the
laboratory, 12 lines have undergone from 10 to 25 passages. These
tumors retain their morphology and growth rate that are hallmarks
of stable tumor lines. The tumor lines had significant differences
in their growth rate and in other biological features just as occurs
in the rodent model (32). Some transplantable tumors generated in
this study underwent growth arrest after several consecutive
passages (data not shown). In such cases, necropsy did not reveal
visible tumor connection to the recipient’s vascular system. It can
be suggested that such tumors lack an ability to promote
angiogenesis playing a vital role in carcinogenesis (33). The growth
rate of tumors derived from spontaneous (s1) and DEN-induced
(zt23) carcinomas in pancreas was similar to that the most fast
growing tumor lines in mammalians (34).
Methods of quantitative assessment of the development of
transplanted tumors in zebrafish would open an opportunity to
use this tumor model for investigations in experimental cancer
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 Transplantable Tumors in Zebrafish

chemotherapy and screening of antitumor drugs. For these
applications, we propose to use the mean survival time of tumor-
bearing adult fish or 5- to 14-day-old larvae grafted with tumor
cells as a criterion of treatment efficacy. Further refinement of this
technology could be achieved by, for example, injecting tumor cells
constitutively expressing fluorescent tags (8, 35) into embryos and
early larvae. The small size and transparent body of the developing
zebrafish make this approach applicable to high-throughput
screening assays based on a 96- or 24-microwell format (36).
Moreover, some standard methods that were routinely used in
rodents to assess tumor growth might be readily adapted to
zebrafish. In particular, our preliminary experiments showed that
the dynamics of body weight gain in tumor-bearing fish may also
be used for quantitative assessment of tumor growth in zebrafish
(data not shown). A direct measurement of the size of i.m.
transplanted tumors may also be optimized for the zebrafish model
by computer morphometry.
We were not able to detect any specific biological feature of
zebrafish tumors that had not been described earlier in mammals
(19). This indicates a similarity if not an identity of the molecular,
cellular, and immune mechanisms of tumor formation in fish and
mammals. Therefore, the model proposed in this study might be
successfully used in the same areas of experimental cancer
research, where until now mammalian models were the exper-
imental tool of choice. The advantages of using transplanted
tumors in clonal zebrafish are not limited to significantly lower
cost of fish maintenance. The availability of genetically identical
subjects opens new opportunities for investigations in tumor
immunology (37) and cancer genetics (38). In addition, unlike
transplanted zebrafish tumors described elsewhere (8, 9, 21), our
approach does not require sublethal irradiation of recipients to
suppress host immunity and therefore makes our model suitable
for much broader applications, including investigation of host-
tumor interactions.

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The successful transplantation of zt23, zt34, and s1 tumors
recovered after cryopreservation in liquid nitrogen showed the
possibility of long-term storage of zebrafish tumor lines. This
method makes it unnecessary to maintain the tumor lines by routine
serial transplantation in live fish and therefore would reduce labor
and cost of maintenance of large number of tumor lines.
In conclusion, a comparison of transplanted tumor lines in
zebrafish and in rodents shows a striking similarity in their biology.
Acknowledgments
Received 10/21/2005; accepted 1/10/2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Marilyn Wolfe of the Registry of Tumors in Lower Animals and
Christina Wentz and Dr. Keith Cheng of Penn State Milton S. Hershey Medical Center
for the examination of the zebrafish tumor section.

References
1. Amatruda JF, Shepard JL, Stern HM, et al. Zebra-
fish as a cancer model system. Cancer Cell 2002;1:
229–31.
2. Zon LI, Peterson RT. In vivo drug discovery in the
zebrafish. Nat Rev Drug Discov 2005;4:35–44.
3. Beckwith LG, Moore JL, Tsao-Wu GS, et al. Ethyl-
nitrosourea induces neoplasia in zebrafish (Danio rerio).
Lab Invest 2000;80:379–85.
4. Mizgireuv IV, Majorova IG, Gorodinskaya VM, et al.
Carcinogenic effect of N -nitrosodimethylamine on
diploid and triploid zebrafish (Danio rerio). Toxicol
Pathol 2004;32:514–8.
5. Spitsbergen JM, Tsai H, Reddy AR, et al. Neoplasia in
zebrafish treated with 7,12-imethylbenz[a]anthracene by
two exposure routes at different developmental stages.
Toxicol Pathol 2000;28:705–15.
6. Spitsbergen JM, Tsai H, Reddy AR, et al.
Neoplasia in zebrafish treated with N -methyl-N V-
nitro-N-nitrosoguanidine by three exposure routes at
different developmental stages. Toxicol Pathol 2000;
28:716–25.
7. Khudoley VV. Use of aquarium fish Danio rerio and
Poecilia reticulata as a test species for evaluation of
nitrosamine carcinogenicity. J Natl Cancer Inst Monogr
1980;65:65–70.
8. Langenau DM, Traver D, Ferrando AA, et al. Myc-
induced T cell leukemia in transgenic zebrafish. Science
2003;299:887–90.
9. Yang HW, Kutok JL, Lee NH, et al. Targeted expression
of human MYCN selectively causes pancreatic neuroen-
docrine tumors in transgenic zebrafish. Cancer Res
2004;64:7256–62.
10. Loeb L. On trasplantation of tumors. J Med Res 1901;
6:28–39.
11. Schultz ME, Schultz RJ. Transplantable chemically-
induced liver tumors in the viviparous fish Poeciliopsis.
Exp Mol Pathol 1985;42:320–30.
12. Angus RA, Schultz RJ. Clonal diversity in the
unisexual fish Poeciliopsis monacha-lucida : a tissue graft
analysis. Evolution 1978;33:27–40.
13. Davidson AJ, Zon LI. The ‘definitive’ (and ‘primitive’)
guide to zebrafish hematopoiesis. Oncogene 2004;23:
7233–46.
14. Streisinger G, Walker C, Dower N, et al. Production of
clones of homozygous diploid zebra fish (Brachydanio
rerio). Nature 1981;291:293–6.
15. Westerfield M. The Zebrafish Book. Guide for the
laboratory use of zebrafish (Danio rerio). 3rd ed. Eugene
(OR): University of Oregon Press; 1995.
16. Boorman GA, Botts S, Bunton TE, et al. Diagnostic
criteria for degenerative, inflammatory, proliferative
nonneoplastic and neoplastic liver lesions in medaka
(Oryzias latipes): consensus of a National Toxicology
Program Pathology Working Group. Toxicol Pathol 1997;
25:202–10.
17. Vogelbein WK, Fournie JW, Van Veld PA, et al.
Hepatic neoplasms in the mummichog Fundulus
heteroclitus from a creosote-contaminated site. Cancer
Res 1990;50:5978–86.
18. Morris HP. Some growth, morphological, and bio-
chemical characteristics of hepatoma 5123 and other
new transplantable hepatomas. Prog Exp Tumor Res
1963;3:370–411.
19. Klein G. The usefulness and limitations of tumor
transplantation in cancer research: a review. Cancer Res
1959;19:343–58.
20. Lucke B, Schlumberger H. Transplantable epithelio-
mas of the lip and mouth of catfish. I. Pathology,
transplantation to anterior chamber of eye and into
cornea. J Exp Med 1941;74:397–408.
21. Patton EE, Widlund HR, Kutok JL, et al. BRAF
mutations are sufficient to promote nevi formation and
cooperate with p53 in the genesis of melanoma. Curr
Biol 2005;5:249–54.
22. Phylonix Inc., U.S. patent 6,761,876.
23. Lee LM, Seftor EA, Bonde G, et al. The fate of human
malignant melanoma cells transplanted into zebrafish
embryos: assessment of migration and cell division in
the absence of tumor formation. Dev Dyn 2005;233:
1560–70.
24. Trede NS, Langenau DM, Traver D, et al. The use of
zebrafish to understand immunity. Immunity 2004;20:
367–79.
25. Smolowitz R, Hanley J, Richmond H. A three-year
retrospective study of abdominal tumors in zebrafish
maintained in an aquatic laboratory animal facility. Biol
Bull 2002;203:265–6.
26. Nikiforova MN, Nikiforov YE, Biddinger P, et al.
Frequent loss of heterozygosity at chromosome 3p14.2–
3p21 in human pancreatic islet cell tumours. Clin
Endocrinol 1999;51:27–33.
27. Berghmans S, Murphey RD, Wienholds E, et al.
tp53 mutant zebrafish develop malignant peripheral
nerve sheath tumors. Proc Natl Acad Sci U S A 2005;
102:407–12.
28. Amsterdam A, Sadler KC, Lai K, et al. Many
ribosomal protein genes are cancer genes in zebrafish.
PLoS Biol 2004;2:0690–8.
29. Shepard JL, Amatruda JF, Stern HM, et al. A zebrafish
bmyb mutation causes genome instability and increased
cancer susceptibility. Proc Natl Acad Sci U S A 2005;102:
13194–9.
30. Ono H, Klein D, Vincek V, et al. Major histocompat-
ibility complex class II genes of zebrafish. Proc Natl
Acad Sci U S A 1992;89:11886–90.
31. Snell GD. The immunogenetics of tumor transplan-
tation. Cancer Res 1952;12:543–6.
32. Reuber MD. Histopathology of transplantable hepatic
carcinomas induced by chemical carcinogens in rats.
GANN Monogr 1961;6:43–54.
33. Carmeliet P, Jain RK. Angiogenesis in cancer and
other diseases. Nature 2000;407:249–57.
34. Dawe CJ, Potter M. Morphologic and biologic
progression of a lymphoid neoplasm of the mouse
in vivo and in vitro . Am J Pathol 1957;33:603.
35. Lyons SK. Advances in imaging mouse tumour
models in vivo . J Pathol 2005;205:194–205.
36. Love DR, Pichler FB, Dodd A, et al. Technology for
highthroughput screens: the present and future using
zebrafish. Curr Opin Biotechnol 2004;15:564–71.
37. Ostrand-Rosenberg S. Animal models of tumor
immunity, immunotherapy and cancer vaccines. Curr
Opin Immunol 2004;16:143–50.
38. Streisinger G. Attainment of minimal biological
variability and measurements of genotoxicity: produc-
tion of homozygous diploid zebra fish. J Natl Cancer Inst
Monogr 1984;65:53–8.

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