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\begin{document}

\title{Rhabdomyosarcoma xenotransplants in zebrafish embryos}



\author[1]{Jakob Siebert}%
\author[1]{Michaela Schneider}%
\author[2]{Daniela Reuter-Schmitt}%
\author[1]{Julia Würtemberger}%
\author[2]{Annette Neubüser}%
\author[2]{Wolfgang Driever}%
\author[1]{Simone Hettmer}%
\author[1]{Friedrich Kapp}%
\affil[1]{Universitatsklinikum Freiburg}%
\affil[2]{Albert-Ludwigs-Universitat Freiburg Fakultat fur Biologie}%


\vspace{-1em}



  
  \date{May 16, 2024}


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\selectlanguage{english}
\begin{abstract}
Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue
sarcomas. High-risk and metastatic disease continues to be associated
with very poor prognosis. RMS model systems that faithfully recapitulate
the human disease and provide rapid, cost-efficient estimates of
anti-tumor efficacy of candidate drugs are needed to facilitate drug
development and personalized medicine approaches. Here, we present a new
zebrafish-based xenotransplant model allowing for rapid and easily
accessible drug screening using low numbers of viable tumor cells and
relatively small amounts of water-soluble chemicals. Under optimized
temperature conditions, embryonal RMS-xenografts were established in
zebrafish embryos at 3 hours post fertilization (hpf). In
proof-of-principle experiments, chemotherapy drugs with established
clinical anti-RMS efficacy (vincristine, dactinomycin) and the MEK
inhibitor trametinib were shown to significantly reduce the
cross-sectional area of the tumors by 120 hpf. RMS xenograft models in
zebrafish embryos henceforth could serve as a valuable addition to cell
culture and mammalian models of RMS and represent a rapid and
cost-effective solution for pre-clinical candidate drug testing.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Representing approximately 6\% of all pediatric malignancies, soft
tissue sarcomas constitute the third most frequent extracranial solid
malignant tumor entity in children . Rhabdomyosarcomas (RMS) are the
most common pediatric soft tissue sarcomas~ and a heterogeneous group of
cancers. The main subgroups of RMS in children and adolescents are
alveolar and embryonal, which differ in their histology and genetic
underpinnings, but also their clinical behavior. Approximately 80\% of
alveolar RMS (ARMS) are associated with the chromosomal translocations
t(2;13)(q35;q14) or t(1;13)(p36;q14) . These translocations lead to
fusions of the \emph{FOXO1} gene on chromosome 13 to the \emph{PAX3}gene
on chromosome 2 or the \emph{PAX7} gene on chromosome 1. The resulting
fusion genes \emph{PAX3-FOXO1} and \emph{PAX7- FOXO1} are considered
drivers of malignancy in ARMS . Embryonal RMS (ERMS) show a higher
degree of genetic diversity, including frequent RAS pathway gene
mutations and genetic imbalances such as loss of heterozygosity (LOH) at
chromosome locus 11p15 . This genomic region contains several genes that
play important roles in growth control and are therefore associated with
different types of embryonal tumors . There are major differences in
clinical characteristics, response to therapy and outcomes between
embryonal and alveolar RMS. The 5-year overall survival rate of
pediatric patients with RMS is approximately 70\% . ARMS are generally
associated with substantially worse outcomes than ERMS . There is an
urgent need to establish the anti-RMS efficacy of candidate drugs and
improve long-term survival, especially for patients with relapsed or
metastatic disease, for whom survival rates continue to be dismal . In
this study, we sought to establish a rapid RMS research platform in
zebrafish embryos by implanting 3 hours-old zebrafish embryos with RMS
cells. RMS xenografts were shown to shrink in response to exposure to
anti-RMS drugs in proof-of-principle experiments.

\textbf{Methods}

\textbf{Cell culture.} RD \emph{(PAX3:FOXO1} -negative\emph{; NRAS
Q61H)}and Rh30 \emph{(PAX3:FOXO1} -positive\emph{)} cells were
maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher
Scientific) supplemented with 20\% fetal bovine Serum (FBS; Thermo
Fisher Scientific) (Rh30) cells and 10\% FBS (RD), respectively. 1\%
Penicillin/Streptomycin (Thermo Fisher Scientific) was added to the
medium for both cell lines. Cells were cultured at 37\selectlanguage{ngerman}°C in a 5\%
CO\textsubscript{2} atmosphere containing incubator. The identity of the
cell lines was confirmed by short tandem repeat (STR) fingerprinting by
Eurofins

\textbf{Zebrafish husbandry.} Zebrafish were maintained and bred under
standard conditions at the Developmental Biology, Institute for Biology
I, University of Freiburg, as approved by Regierungspräsidium Freiburg.
Experiments were conducted only during the first 120 hours
post-fertilization. The following fish lines were used for the
experiments: \emph{nacre} (\emph{mitfa} mutant, deficient in body
pigment cells) and \emph{sandy} (also known \emph{sdy}
;\emph{tyrosinase} mutant, deficient in melanin dark pigment). All
experiments were carried out in accordance with German laws for animal
care.

\textbf{Optimization of environmental temperatures.} Zebrafish embryos
were maintained at three different temperature levels from 3 to 120
hours post fertilization \emph{(hpf).} The control group (n=48) was
maintained at 28.5°C, which is considered the standard temperature for
maintenance of zebrafish embryos. Experimental group 1 (n=48) was kept
at 33°C from 3 to 48 hpf, followed by a rise in environmental
temperatures to 35°C until 120 hpf. The temperature for experimental
group 2 (n=48) was maintained at 35°C from 3 to 120 hpf. Embryos were
microscopically observed each day and classified as ``abnormal'' if they
showed any visible signs of damage such as pericardial edema, scoliosis,
or absence of heartbeats at 72 hpf.

To examine the effects of different environmental temperatures on RMS
growth \emph{in vitro} , RD and Rh30 cells were plated in 96-well plates
at a density of 500 cells per well. Starting at the time of plating,
cells were maintained at 33°C for two days, followed by a temperature
rise to 35°C until day 5, analogous to the zebrafish experiment
described above. For comparison, control cells were maintained at 37°C
from the time of plating until day 5. Cell counts were determined on the
day of plating as well as 2, 4 and 5 days after plating using a
hematocytometer (4 replicates per day and condition). Mean cell numbers
on day 5 were tested for statistical significance by Students t-tests.

\textbf{Transplantation procedure.} RMS cells at 80\% confluence were
washed in PBS followed by incubation in staining-solution comprised of
10 \selectlanguage{greek}µ\selectlanguage{english}L lipophilic DiI (Thermo Fisher Scientific, V22885) in 2ml serum
free DMEM for 20 minutes at 37\selectlanguage{ngerman}°C. The freshly stained RMS cells then
were washed twice in PBS to remove staining remnants, spun down and
resuspended in 1 ml of PBS supplemented with 2\% FBS and 2\% PVP360 to
prevent the cells from clotting in the injection capillaries. The cells
were stored on ice until transplantation. Immediately before
transplantation, cells were spun down and resuspended to a final
concentration of \textasciitilde{} 1x10\textsuperscript{6} / 100\selectlanguage{greek}µ\selectlanguage{english}L

1\selectlanguage{greek}µ\selectlanguage{english}L of the cell suspension was aspirated into a 100\selectlanguage{greek}µ\selectlanguage{english}M-diameter pulled
borosilicate glass needle without filament (Science Products GmbH),
using an oil-based microinjector (L.S. Starrett). The 3 hours-old
zebrafish embryos were lined into injection molds made from agarose gel.
Approximately 100 cells per embryo were transplanted into the blastoderm
of 1000-cell to high stage zebrafish embryos. Transplanted embryos then
were transferred into petri dishes containing E3 medium and kept at
33\selectlanguage{ngerman}°C. One hour after transplantation embryos were screened for
successful transplantation by fluorescence microscopy to detect the
DiI-labeled xenografts.

Although transplantation and screening were performed at room
temperature (24-28°C), development of embryos implanted with RMS cells
and unmanipulated embryos at 28.5°C progressed without notable
differences. All embryos were euthanized before exceeding 120 hpf.

\textbf{Xenograft imaging and analysis.} Embryos were anesthetized with
0.168 mg tricaine/ml E3 medium at 24, 72 and 120 hpf and placed in
lateral position. Serial images were taken with Zeiss ZEN-Software using
an Axio Examiner D1 (Zeiss) at 5X magnification. The cross-sectional
area of the xenografts \emph{in vivo} was measured sequentially at 24,
72 and 120 hpf to analyze changes in the size of the xenotransplants
over time. For each timepoint, cross-sectional areas of the xenografts
were measured using FIJI software. Xenografts were outlined using the
default algorithm of the FIJI software; the z-plane with the largest
cross-sectional surface area was considered for analyses. To make
changes in tumor size directly comparable, the standardized
cross-sectional area (SCSA) was calculated for each timepoint by
dividing the cross-sectional area measured at any given timepoint (24,
72, 120 hpf) by the value measured at 24 hpf.

\textbf{Histology.} Larvae were euthanized in tricaine at 72-120 hpf
followed by fixation in 4\% PFA overnight. Fixed embryos then were
embedded in paraffin, sectioned into 5\selectlanguage{greek}µ\selectlanguage{english}M thick slices, and stained with
Hematoxylin and Eosin, following standard staining procedures .

\textbf{Toxicity screening.} 24 hours-old embryos were manually
dechorionated and transferred to a 96-well plate containing E3 medium
(one embryo per well). Vincristine, dactinomycin and trametinib were
added to the wells in increasing concentrations as indicated in Figure
3A-C. Control embryos were maintained in standard E3 medium without
exposure to any chemicals. Embryos in the carrier group were exposed to
carrier solution only using the same carrier volume that was necessary
to add the highest drug concentration tested. Embryos were
microscopically analyzed at 120 hpf for visible damage. If they showed
signs of toxicity such as pericardial edema, scoliosis, or absence of
heartbeats at 72 hpf, or if they were already dead, the embryos were
classified as ``did not survive''. Each experimental group contained 16
embryos per experiment; the experiment was repeated 3 times. Maximum
tolerated concentrations of each drug were determined based on
differences in the survival rates of embryos exposed to increasing drug
concentrations.

\textbf{Testing the effects of candidate drugs on RMS-xenografts in
zebrafish embryos.} For drug testing experiments, approximately 100
DiI-labeled RD cells were transplanted into zebrafish embryos at the
1k-cell stage (3hpf). One hour after the transplantation procedure,
embryos were inspected for successful transplantation via fluorescence
microscopy. The embryos were then transferred to a 24-well plate
containing E3 and maintained at 33\selectlanguage{ngerman}°C until they reached 48 hpf, followed
by a temperature rise to 35°C until 120 hpf. Chemicals were added to the
E3-medium at 24 hpf. The following concentrations were tested:
vincristine at 2 and 20 \selectlanguage{greek}µ\selectlanguage{english}M; dactinomycin at 0.1 and 1 \selectlanguage{greek}µ\selectlanguage{english}M; trametinib at
5 and 50 nM. Furthermore, embryos were exposed to 2\selectlanguage{greek}µ\selectlanguage{english}M vincristine and
0.1\selectlanguage{greek}µ\selectlanguage{english}M dactinomycin in combination.

To measure the effects of vincristine and dactinomycin on RD
xenografts\emph{nacre} embryos were used, whereas the effects of
Trametinib on RD-xenografts were tested in transplanted \emph{sdy}
embryos, due to availability of the respective fish lines.

Observations from animals exposed to the same chemicals in 9 independent
experiments were pooled and compared to the control group, which
consisted of 30 \emph{nacre} embryos and 15 \emph{sdy} embryos,
respectively, from 9 independent experiments. The following number of
xenograft-bearing embryos were considered for analyses, after ``dead or
disformed'' ones were removed from the experiments: 17 embryos exposed
to 2 \selectlanguage{greek}µ\selectlanguage{english}M vincristine, 30 exposed to 20 \selectlanguage{greek}µ\selectlanguage{english}M vincristine, 16 to 0,1 \selectlanguage{greek}µ\selectlanguage{english}M
dactinomycin, 27 to 1 \selectlanguage{greek}µ\selectlanguage{english}M dactinomycin, 16 to 2 \selectlanguage{greek}µ\selectlanguage{english}M vincristine and 0,1 \selectlanguage{greek}µ\selectlanguage{english}M
dactinomycin, 11 to 5 nM trametinib, 21 to 50 nM trametinib and 17 to 10
\selectlanguage{greek}µ\selectlanguage{english}l DMSO.

\textbf{Statistical Analysis.} Experiments were analyzed using Prism
GraphPad 9.1.2. All Experiments with two experimental groups were
analyzed using the Student's t-test, whereas experiments with more than
two groups were analyzed using ANOVA followed by Turkey\selectlanguage{ngerman}´s multiple
comparisons test. Differences were considered significant when\emph{p}
-Value was below 0.05\emph{. P} -Values were illustrated in figures
using asterisks: p\textless{}0.05 (*), p\textless{}0.005 (**),
p\textless{}0.0005 (***), p\textless{}0.00005 (****).

\textbf{Results}

\textbf{Optimization of environmental temperatures.} In laboratory
environments, zebrafish embryos commonly are raised at 28.5°C , whereas
human cells are maintained at 37°C . To enable engraftment of RMS
xenotransplants in zebrafish embryos, it was necessary to establish a
temperature level that allows for normal development of both zebrafish
embryos and human tumor cells. To determine optimal environmental
conditions, zebrafish embryos were exposed to different temperatures.
The percentage of surviving embryos at 120 hpf was significantly lower
at 35°C compared to the control group maintained at 28.5°C
(Fig.1A,\emph{p} =0.0016). This is consistent with previously published
observations . Considering the higher mortality rate at 35°C, we tested
a gentler two-step approach using temperatures of 33°C from 3 hpf to 48
hpf, followed by a temperature rise to 35°C until 120 hpf, which did not
have a negative impact on the survival rates of the zebrafish embryos
compared to those in the control group (Fig.1A, \emph{p} =0.6244). Next,
these temperature settings were applied to RMS cells in culture, to
examine possible differences in cell proliferation at 1, 48, 96 and 120
hours after plating\emph{.} Lower temperatures did not affect RD cell
growth, and the number of RD cells kept at 37°C did not differ
significantly 120 hours after plating (Fig.1B, 4750 cells/well at 37°C
vs. 5000 cells/well at 33/35°C, \emph{p} =0.6753). In contrast,
proliferation of Rh30 cells slowed down significantly at lower
temperatures compared to the control cells maintained at 37°C (Fig.1C,
8750 cells/well at 37°C vs. 3438 cells/well at 33/35°C,\emph{p}
\textless{}0.0001).

\textbf{Induction of RMS xenografts in zebrafish embryos.} To analyze
the development of Rh30 and RD cells \emph{in vivo} , DiI-labeled RMS
cells were transplanted into zebrafish embryos at the 1000-cell stadium
(Fig.2). Images of the transplants were taken, and cross-sectional areas
were measured at 24, 72 and 120 hpf (Fig.3A-C). RD xenografts showed
stable growth from 24 hpf until 120 hpf (Fig. 3D), whereas the
cross-sectional areas of Rh30 xenografts decreased during the
observation period~(Fig. 3D). Histology sections from 72-120 hours-old,
successfully transplanted embryos were prepared and stained with
hematoxylin and eosin. The presence of tumor cell nests confirmed
successful engraftment of xenotransplants (Fig. 3E).

\textbf{Toxicity screening.} Possible toxic effects of the candidate
anti-RMS drugs on embryonal and larval development were explored until
120 hpf. Vincristine significantly reduced the proportion of surviving
embryos starting at drug concentrations higher than 20 \selectlanguage{greek}µ\selectlanguage{english}M (Fig. 4A, 94\%
surviving embryos in E3 vs. 69\% surviving embryos in E3 medium with
25\selectlanguage{greek}µ\selectlanguage{english}M vincristine, \emph{p} \textless{}0.0001). Dactinomycin treatment
also led to significantly lower survival rates starting at
concentrations exceeding 1 \selectlanguage{greek}µ\selectlanguage{english}M (Fig. 4B, 88\% surviving embryos in E3 vs.
73\% surviving embryos in E3 medium with 2\selectlanguage{greek}µ\selectlanguage{english}M dactinomycin,\emph{p}
=0.0398). The mitogen-activated protein kinase kinase (MEK) inhibitor
trametinib negatively affected the embryos starting at
drug-concentrations higher than 50 nM (Fig. 4C, 88\% surviving embryos
in E3 vs. 67\% surviving embryos in E3 medium with 100nM
trametinib,\emph{p} =0.0007).

\textbf{Testing the effects of candidate drugs on RMS xenografts in
zebrafish embryos.} Successfully transplanted embryos were exposed to
selected drugs to assess their effect on RD-xenotransplants \emph{in
vivo} . Vincristine concentrations of 2 and 20 \selectlanguage{greek}µ\selectlanguage{english}M reduced the
standardized cross-sectional area (SCSA) to 39\% (Fig 5A;\emph{p}
\textless{}0.0001) and 15\% (Fig. 5A;\emph{p} \textless{}0.0001),
respectively, compared to vehicle-treated RMS xenotransplants. A
statistically significant, dose-dependent effect of vincristine at 2 and
20 \selectlanguage{greek}µ\selectlanguage{english}M could also be observed (Fig. 5A;\emph{p} \textless{}0.0001),
further supporting the observation that vincristine has antitumoral
effects in zebrafish RMS-xenotransplants.

Dactinomycin showed an analogous inhibitory effect on RMS xenografts in
zebrafish with a reduction to 52\% of the SCSA of non-treated
xenotransplants (Fig. 5B; \emph{p} \textless{}0.0001) at a concentration
of 0.1 \selectlanguage{greek}µ\selectlanguage{english}M, and to 24\% of the SCSA of non-treated xenotransplants (Fig.
5B; \emph{p} \textless{}0.0001) at concentrations of 1\selectlanguage{greek}µ\selectlanguage{english}M dactinomycin.

2\selectlanguage{greek}µ\selectlanguage{english}M vincristine and 0.1\selectlanguage{greek}µ\selectlanguage{english}M dactinomycin in combination showed stronger
inhibitory effects compared to 0.1\selectlanguage{greek}µ\selectlanguage{english}M dactinomycin monotherapy therapy
(Fig. 5C; reduction to 28\% of the SCSA of untreated xenografts by 2\selectlanguage{greek}µ\selectlanguage{english}M
vincristine and 0.1\selectlanguage{greek}µ\selectlanguage{english}M dactinomycin in combination vs. 52\% by 0.1\selectlanguage{greek}µ\selectlanguage{english}M
dactinomycin alone; \emph{p} =0.0055). There were no significant
differences in the efficacy of the combination treatment compared to
exposure to 2\selectlanguage{greek}µ\selectlanguage{english}M vincristine alone (Fig. 5C; p=0.4265). Given that RD
cells carry an \emph{NRAS proto-oncogene, GTPase (NRAS)Q61H-} variant
resulting in activation of mitogen-activated protein kinase (MAPK)
signaling, the MEK-inhibitor trametinib was also tested for inhibitory
effects on RMS xenotransplants in zebrafish embryos. At trametinib
concentrations of 5 and 50~nM, we observed a reduction of the SCSA to
57\% (\emph{p} \textless{}0.0001) and 21\% (\emph{p} \textless{}0.0001)
of the SCSA of non-treated RMS-xenotransplants, respectively (Fig. 5D).

\textbf{Discussion}

Rhabdomyosarcomas continue to be a formidable challenge in pediatric
oncology. Especially patients with \emph{FOXO1-} rearranged or
metastatic RMS face extremely poor outcomes. In recent years,
significant research efforts have provided insights into possible new
therapeutic strategies . Also, molecular characterization of tumor
tissue, aimed at identifying targets for individualized treatment of
patients with refractory disease, has proven feasible . There is an
increasing need for RMS model systems that faithfully recapitulate the
human disease and provide rapid, cost-efficient estimates of anti-tumor
efficacy of candidate drugs.

Zebrafish represent an extremely cost-efficient model organism, that is
used in many laboratories all over the world, easy to handle and
well-suited for high-throughput experiments. In optimized conditions,
female zebrafish can lay up to 200 eggs per clutch , allowing for large
numbers of experimental animals and thereby reducing the statistical
risk of incorrect measurements. RMS xenografts in zebrafish were
previously reported in the literature. Chuan et al used ERMS and ARMS
xenografts, established in 2 months-old zebrafish by injecting cells
into the peri-ocular muscle and intraperitoneal spaces, to observe the
effects of drugs, which were administered by oral gavage, at
environmental temperatures of 37\selectlanguage{ngerman}°C over a period of 28 days post
injection. Xenograft induction in zebrafish embryos offers a number of
advantages over xenotransplants in adult fish, including faster
turnaround time, better scalability, easier drug application , and ease
of repetitive imaging including time-lapse microscopy to assess
xenograft dynamics. In this study, we set out to establish RMS
xenografts in zebrafish embryos. Due to the small size of the zebrafish
embryos, only few tumor cells were required to establish xenografts, and
relatively small amounts of drugs were sufficient for testing.

The zebrafish platform established here allows for reliable induction of
RMS xenografts, derived from the embryonal RMS cell line RD, in 3
hours-old zebrafish embryos. To reconcile viability and normal
development of the embryos with engraftment of human cells, we selected
optimized temperature conditions based on our own data (Fig. 1A) and
previous reports . Temperatures higher than 33.5°C during early
embryonic development are associated with abnormal embryonic development
and higher mortality . Post gastrulation, higher temperatures at 35°C
are tolerated more easily by the embryos. Indeed, we successfully kept
embryos at temperatures of 33°C from 3 hpf to 48 hpf, followed by a
temperature rise to 35°C until 120 hpf.

Proof-of-principle experiments revealed dose-dependent inhibitory
effects of vincristine and dactinomycin - two chemotherapy drugs with
long-established anti-RMS efficacy -- on RMS xenografts in zebrafish
embryos, validating the reliability of the model system in assessing
antitumor effects of established anti-RMS drugs. The MEK inhibitor
trametinib also led to a significant reduction in the size of
xenografts, derived from \emph{NRAS Q61H} -mutated RD cells . Of note,
in the experiments reported here, PAX3:FOXO1 fusion-positive Rh30 cells
did not engraft under the conditions described above. This corresponded
to slower proliferation of Rh30 cells \emph{in vitro} at lower
environmental temperatures. Interestingly, lower temperatures did not
exert any adverse effects on the \emph{in vitro} growth of RD cells.
Future experiments will determine if RMS cells, derived from other
PAX3:FOXO1 fusion-positive cell lines or fresh patient-derived tumor
tissue, are capable of engrafting in zebrafish embryos. Also, it will be
interesting to observe possible differences in the location of primary
tumors and patterns of metastases between xenografts derived from
genetically distinct RMS tumors.

Zebrafish embryos as model organisms are generally associated with
certain limitations. While there are substantial functional homologies
between the zebrafish and the human genome , fundamental differences
between these two vertebrate organisms must not be neglected. For
example, the temperature requirements of zebrafish differ from those of
mammalians. The application of water-soluble chemicals to the embryo
medium is remarkably easy, but the exact chemical dose that is absorbed
into the embryo remains unclear, and larger or non-water-soluble
molecules need to be injected into the embryo's yolk sac, necessitating
more complex experimental procedures comparable to drug application
procedures used in mice or older zebrafish. Furthermore, the development
of the mature immune system in zebrafish embryos takes 4-6 weeks .

A xenotransplant model similar to the system described here has recently
been published and is based on injection of ERMS patient-derived
xenograft (PDX) tumor cells into the yolk sac of zebrafish embryos at 48
hpf, with first addition of drugs in the medium at 72 hpf and continued
exposure until 120 hpf. Embryos were kept at 28°C prior to injection of
tumor cells and at 34 °C after injection of tumor cells . These
observations are in line with those obtained from our experiments and
confirm the potential of the zebrafish embryo xenograft platform.
Important differences lie in the timing of transplantation and drug
treatment with our model offering a longer treatment and observation
period. Also, we argue that transplantation into the embryo itself
compared to transplantation into the yolk sac offers the advantage of a
cellular milieu.

In conclusion, the zebrafish embryo-based platform described here is
very fast, cost-efficient, and easy to handle. It takes only 120 hours,
low numbers of viable tumor cells and relatively small amounts of
chemicals to obtain estimates on the anti-tumor efficacy of
water-soluble chemicals. Laboratory requirements are easily accessible,
and regulatory thresholds are low. By contrast, turnaround times range
from weeks to months if xenografts are established in mice/ older
zebrafish or if tumor cells are expanded in tissue culture. These
demands increase when experiments are designed to cover replicate
measurements and therefore require large numbers of cells. We argue that
RMS xenograft models in zebrafish embryos lift rapid pre-clinical drug
testing to a new level and henceforth could serve as a valuable addition
to cell culture and mammalian models of this devastating cancer.

\textbf{Conflict of Interest statement}

The authors declare that they have no conflict of interest.

\textbf{Acknowledgments}

Simone Hettmer was supported by the Berta-Ottenstein-Program for
Advanced Clinician Scientists, Faculty of Medicine, University of
Freiburg.

\textbf{References}

\textbf{Figure Legends}

\textbf{Figure 1:} \textbf{(A)} The survival rate of zebrafish embryos
at different environmental temperatures was examined during the first
120 hpf. Larvae maintained continuously at 35°C showed lower survival
rates compared to the control group kept at 28.5°C. In contrast, embryos
maintained at 33°C to 35°C exhibited similar survival compared to the
control group. In parallel experiments, the development of
\textbf{(B)}RD cells and \textbf{(C)} Rh30 cells was evaluated using the
same temperature conditions. \textbf{(B)} RD cell numbers did not differ
significantly at lower temperatures compared to control cells maintained
at standard temperatures. \textbf{(C)} Rh30 cell numbers counted at 120
hours were significant lower when cells were maintained at lower
temperatures.

\textbf{Figure 2:} Transplantation procedure: fertilized eggs were
maintained at 28.5°C until 3 hpf. RMS cells were injected into the
blastodisc, and environmental temperature was raised to 33°C. Embryos
were screened at 4 hpf for successful transplantation. The embryos then
were transferred to 24 well plates. Maintenance temperature was raised
to 35°C at 48 hpf until the end of the experiments.

\textbf{Figure 3:} Development of RMS xenografts, derived from RD cells,
is depicted at \textbf{(A)} 24 hpf, \textbf{(B)} 72 hpf and
\textbf{(C)}120 hpf. \textbf{(D)} The development of RD and Rh30 cells
xenotransplants was observed until 120hpf. The SCSA of RD tumors
increased, whereas the SCSA of Rh30 tumors decreased during the
observation period. \textbf{(E)} The head of a xenotransplanted
zebrafish embryo was sectioned and stained with hematoxylin eosin. The
arrow marks a nest of tumor cells.

\textbf{Figure 4:} Toxic effects of vincristine, dactinomycin and
trametinib were screened in zebrafish embryos. Embryos tolerated
exposure to \textbf{(A)} vincristine up to concentrations of 20
\selectlanguage{greek}µ\selectlanguage{english}mol/l,\textbf{(B)} dactinomycin up to concentrations up to 2 \selectlanguage{greek}µ\selectlanguage{english}mol/l
and\textbf{(C)} trametinib up to concentrations of 50 nmol/l.

\textbf{Figure 5:} The anti-RMS effects of candidate chemicals on RD
xenografts in zebrafish embryos. \textbf{(A)} Vincristine reduced the
SCSA at concentrations of 2 and 20 \selectlanguage{greek}µ\selectlanguage{english}mol/l, respectively.
\textbf{(B)}Dactinomycin exhibited analogous anti-RMS effects compared
to vincristine. \textbf{(C)} Vincristine and dactinomycin in combination
did not show stronger anti-RMS effects compared to vincristine alone but
had stronger inhibiting effects compared to dactinomycin
alone.\textbf{(D)} The MEK-inhibitor trametinib also exerted
dose-depending anti-RMS effects \emph{in vivo} .\selectlanguage{english}
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