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Tumor Formation and Regulation in Xenopus
when small molecules are administered to the water. This is
already the case for embryos and larvae where compounds
are taken up via the skin or gills or via direct swallowing
(Wheeler and Brändli, 2009) but is likely even more important when treating post-metamorphic animals. A critical
question that arises when adding compounds to the water
is the determination of the dose and the compound refreshment scheme. There is no “gold standard” for this, but it may
evidently be possible to determine the concentration of the
compound in the liver or in targeted organs by performing
mass spectrometry. However, the best test to determine the
required concentration is to look at a biological read-out for
the compound. As an example, we documented H3K27me3
levels in the liver to determine the required concentration
of the previously mentioned EZH2 inhibitor Tazemetostat.
Of note, the required concentration in the frog water was
100 times of what was needed in cell culture experiments.
We refreshed half of the water (with compound) daily or
every second day. For compounds that are not taken up via
the rearing water or that are very expensive, intraperitoneal
injection is relatively easy to perform, and a protocol for oral
gavage has been described for Xenopus laevis ( Du, Mashoof
and Criscitiello, 2012).
21.6. CURRENT AND FUTURE
XENOPUS TROPICALIS CANCER
MODELING METHODOLOGIES
21.6.1. TOOLS FOR IN VIVO MONITORING
OF TUMOR PROGRESSION
Correct follow-up of tumor progression and quantitative
phenotypic analysis are essential to cancer research studies.
While external signs of morbidity associated with cancer
development such as lethargy, cachexia, or a swollen abdomen may be straightforward to observe, they only provide
limited information on the actual pathological status of
the underlying malignancy. Furthermore, when investigating responses on treatments, a longitudinal follow-up of
disease status is paramount to assess the effectiveness of
the treatment. Currently, micro CT scanning has proven
its usefulness for uncovering ectopic calcif ed outgrowths,
such as osteosarcoma, in tp53 heterozygote animals
(Naert, Dimitrakopoulou, et al., 2020). Likewise, magnetic resonance imaging (MRI) of adult Xenopus tropicalis
tumor-bearing animals is a useful tool to follow eff cacy of
compound treatments (Naert et al., 2021). In addition, optical coherence tomography may serve as alternative tool for
monitoring cancer development, which was nicely demonstrated in mice (reviewed by Vakoc et al., 2012). While the
latter method should be applicable to Xenopus ( Boppart
et al., 1997 ), its use may be primarily restricted to tumors in
early tadpoles, since the tissue penetrance depth is limited to
approximately 2 mm. As an alternative, a penetrance depth
of centimeters can be obtained in Xenopus via ultrasound
imaging (Bartlett et al., 2010; Slater et al., 2019) and could
be applicable for monitoring tumor progression, as is done in
mouse studies (Snyder et al., 2009). Finally, the generation
of stable transgenic reporter lines can be a very valuable tool
for following tumor progression. Of note for the detection of
tumors in the internal organs, and given the size of the frogs
and the opacity of the post-metamorphic skin, bioluminescent reporters may be better suited than f uorescent reporter
genes to detect tumor masses.
21.6.2. OVERVIEW OF TUMOR CELL
TRANSPLANTATION POSSIBILITIES
Transplantation experiments are useful tools for investigating the role of the host immune system upon subcutaneous
tumor cell injection in Xenopus tadpoles (Haynes-Gimore
et al., 2015) and are also useful for propagating tumor
cells. In addition, with regard to cancer research, investigation of tumor engraftment potential has already shown its
usefulness in validating zebrafsh leukemia models (Smith
et al., 2010; Borga et al., 2019). Currently, multiple possibilities exist that are suitable for performing Xenopus tumor
transplantation experiments. First, transplantation of tumor
cells in thymectomized Xenopus animals, thus lacking the
main functional T-cell compartment, offers a possibility to
avoid graft rejection (Robert et al., 1997 ). Besides, syngeneic lines (e.g. LG-6, LG-15 . . .) being MHC identical have
proven their usefulness for tumor transplantation purposes
(Hadji-Azimi and Fischberg, 1971; Rau et al., 2002; HaynesGilmore et al., 2014, 2015). Additionally, sublethal gamma
irradiation has been used to allow transplantation of spontaneous lymphoid tumors (Robert, Guiet and du Pasquier,
1995; Rau, Cohen and Robert, 2001) or even for transplantation of leukemic cells derived from GEXMs (Tulkens et al.,
2021). We would also like to mention that a rag2 homozygote knock-out line (lacking mature T- and B-cells), which
has already proved its usefulness in zebraf sh transplantation experiments (Tang et al., 2014), has recently been generated in Xenopus tropicalis and shows engraftment potential
for multiple tumor types (Tulkens et al., 2021). Finally, while
in rag2 knockout animals, rejection of allografted tumors
may still occur by natural killer cells or other populations
of the innate immune system, this potential problem can be
circumvented by the use of fully immunocompetent X. tropicalis inbred lines (Sato et al., 2018).
Recently, zebrafsh larvae and immunocompromised
adults have been used as recipients for xenotransplantation
of human cancer cells and even patient-derived tumor biopsies. These so-called “avatars” can be used for phenotypic
testing of drug responses with the ultimate goal of f nding
patient-tailored molecular therapies (Fazio et al., 2020).
Recent work by the Langenau laboratory described the generation of a semi-transparent prkdc −/− , il2rgα−/− line that
lacks adaptive immune cells and natural killer cells and can
be adapted to 37°C before being engrafted with human cancer cells, after which candidate therapeutics were administered via oral gavage (Yan et al., 2019). While this may
seem an attractive model to introduce in Xenopus, the need
to adapt the animals to 37°C may not be achievable, and one