302
Xenopus
species (Sprague et al., 2008; Noy et al., 2009). Nevertheless,
over the last decades, Xenopus has received increasing interest and recognition for its valuable use as a model for human
disease (reviewed by Blum & Ott, 2018), especially strengthened by its unique features (further described subsequently).
Very recently, Xenopus (in particular the true diploid X. tropicalis) entered the cancer modeling feld as the f rst robust
genetically engineered Xenopus model (GEXM) for familial
adenomatous polyposis (Van Nieuwenhuysen et al., 2015).
With the further expansion of the current genome engineering revolution, we can expect rapid establishment of novel
cancer models in Xenopus that can serve for exploration of
cooperative gene mutations involved in cancer induction or
progression and the identifcation of cancer cell vulnerabilities, which can offer therapeutic opportunities for targeted
cancer treatments (Naert et al., 2021).
21.1.2. A DIPLOID GENOME FAVORS GENETIC
RESEARCH IN XENOPUS TROPICALIS
With its high fecundity, large externally developing embryos,
simple housing demands, and highly conserved developmental pathways, Xenopus has proven to be extremely well suited
for biomedical research (Tandon et al., 2017). However, since
Xenopus laevis harbors an allotetraploid genome, employing genetic engineering to mimic human genetic diseases
or cancer can be challenging for certain genomic regions in
this species (Session et al., 2016). The same applies to some
extent to zebrafsh, which has at least 20% of its genome
duplicated (Postlethwait et al., 2000). In contrast, Xenopus
tropicalis harbors, just like humans and mice, a true diploid
genome displaying high synteny with the human genome
(Hellsten et al., 2010). In addition, despite the obvious evolutionary distance between frogs and humans, from what is
known today, 79% of human disease genes have a Xenopus
tropicalis orthologue (Hellsten et al., 2010; Khokha, 2012).
In conclusion, these special features place X. tropicalis in
a unique position for employing nuclease-based techniques
with regard to disease/cancer modeling.
21.2. PAST OBSERVATIONS
21.2.1. EARLY XENOPUS EMBRYOGENESIS AS A SOURCE
OF INFORMATION FOR STUDYING PATHWAYS
AND CELLULAR PROCESSES INVOLVED IN
CANCER INITIATION AND PROGRESSION
Many mechanisms and hallmarks underlying cancer initiation and progression (Hanahan and Weinberg, 2000, 2011)
are comparable between a wide range of species, including
Xenopus (Hardwick and Philpott, 2018). As cancer development and progression often involve the re-use of particular
embryonic pathways and processes (Pennisi, 1998), Xenopus
serves as an important tool for elucidating these normal
developmental processes in order to understand the parallel mechanisms and molecular players underlying malignant transformation. A considerable amount of research has
indeed exposed high similarities between normal embryonic
developmental processes and particular tumor cell behavior, especially at the level of gene and protein expression,
epigenetic regulation, and cell invasion and migration (Ma
et al., 2010). As an example, Wnt signaling, which is known
to play a key role in embryonic development (e.g. during
body axis formation, cell migration, cell fate determination,
and others), is deregulated in the vast majority of human
colon cancers (Hardwick and Philpott, 2015). Interestingly,
Xenopus researchers have exploited a functional biological assay based on duplication of the dorsal axis upon the
ventral injection of mRNA-encoding mediators of the Wnt
pathway, such as β-catenin, to develop chemical screens
(Kühl and Pandur, 2008). Regarding embryonic morphogenetic processes re-activated during tumor progression,
epithelial to mesenchymal transition (EMT), occurring
for instance during neural crest cell delamination, is a key
process during tumor cell invasion (Yang et al., 2020). The
molecular processes underlying EMT during neural crest
cell delamination have been intensively investigated by several Xenopus research groups (Pegoraro and Monsoro-Burq,
2013). Interestingly, Xenopus anti-EMT compound screens
have been deployed in embryos, which identif ed chemicals
that also affect cancer cell invasion (Tanaka et al., 2016) (see
section 5.2).
21.2.2. NATURALLY OCCURRING TUMORS IN XENOPUS
Spontaneous occurrence of tumors is rather uncommon
in Xenopus, leading to the now-contested belief that frogs
are rather recalcitrant to carcinogenesis. This was further
strengthened by the fact that potent human carcinogens have
shown limited to no effect in Xenopus laevis ( Hardwick and
Philpott, 2018). For example, attempts with N-methyl-Nnitrosourea (NMU), a known human carcinogen, failed to
induce tumors in Xenopus laevis (Goyos and Robert, 2009).
However, cases of spontaneous adenocarcinoma, f broma,
lipoma, lymphosarcoma, and leukemia have been occasionally documented (Balls, 1962; Stacy and Parker, 2004;
Suzuki et al., 2020). Finally, a number of naturally occurring thymic tumors, from which several lymphoid tumor
cell lines could be established, are now being used for elucidating key mechanisms for tumor versus immune system
interactions, especially via transplantation experiments (see
subsequently) (Robert, Guiet and du Pasquier, 1994; Goyos
and Robert, 2009).
21.2.3. INDUCED TUMOR-LIKE STRUCTURES
The very frst report of genetically induced tumors in
Xenopus laevis embryos dates from 1997, where mRNA
encoding human dominant-negative p53 was injected in early
embryos, inducing the formation of embryonic tumors, socalled induced tumor-like-structures (ITLSs) (Wallingford
et al., 1997; Wallingford, 1999). Closely thereafter, similar
approaches were undertaken to model tumors in embryos
upon injection of gli1 or rel (Xrel3) mRNA (Dahmane et al.,
Xenopus
species (Sprague et al., 2008; Noy et al., 2009). Nevertheless,
over the last decades, Xenopus has received increasing interest and recognition for its valuable use as a model for human
disease (reviewed by Blum & Ott, 2018), especially strengthened by its unique features (further described subsequently).
Very recently, Xenopus (in particular the true diploid X. tropicalis) entered the cancer modeling feld as the f rst robust
genetically engineered Xenopus model (GEXM) for familial
adenomatous polyposis (Van Nieuwenhuysen et al., 2015).
With the further expansion of the current genome engineering revolution, we can expect rapid establishment of novel
cancer models in Xenopus that can serve for exploration of
cooperative gene mutations involved in cancer induction or
progression and the identifcation of cancer cell vulnerabilities, which can offer therapeutic opportunities for targeted
cancer treatments (Naert et al., 2021).
21.1.2. A DIPLOID GENOME FAVORS GENETIC
RESEARCH IN XENOPUS TROPICALIS
With its high fecundity, large externally developing embryos,
simple housing demands, and highly conserved developmental pathways, Xenopus has proven to be extremely well suited
for biomedical research (Tandon et al., 2017). However, since
Xenopus laevis harbors an allotetraploid genome, employing genetic engineering to mimic human genetic diseases
or cancer can be challenging for certain genomic regions in
this species (Session et al., 2016). The same applies to some
extent to zebrafsh, which has at least 20% of its genome
duplicated (Postlethwait et al., 2000). In contrast, Xenopus
tropicalis harbors, just like humans and mice, a true diploid
genome displaying high synteny with the human genome
(Hellsten et al., 2010). In addition, despite the obvious evolutionary distance between frogs and humans, from what is
known today, 79% of human disease genes have a Xenopus
tropicalis orthologue (Hellsten et al., 2010; Khokha, 2012).
In conclusion, these special features place X. tropicalis in
a unique position for employing nuclease-based techniques
with regard to disease/cancer modeling.
21.2. PAST OBSERVATIONS
21.2.1. EARLY XENOPUS EMBRYOGENESIS AS A SOURCE
OF INFORMATION FOR STUDYING PATHWAYS
AND CELLULAR PROCESSES INVOLVED IN
CANCER INITIATION AND PROGRESSION
Many mechanisms and hallmarks underlying cancer initiation and progression (Hanahan and Weinberg, 2000, 2011)
are comparable between a wide range of species, including
Xenopus (Hardwick and Philpott, 2018). As cancer development and progression often involve the re-use of particular
embryonic pathways and processes (Pennisi, 1998), Xenopus
serves as an important tool for elucidating these normal
developmental processes in order to understand the parallel mechanisms and molecular players underlying malignant transformation. A considerable amount of research has
indeed exposed high similarities between normal embryonic
developmental processes and particular tumor cell behavior, especially at the level of gene and protein expression,
epigenetic regulation, and cell invasion and migration (Ma
et al., 2010). As an example, Wnt signaling, which is known
to play a key role in embryonic development (e.g. during
body axis formation, cell migration, cell fate determination,
and others), is deregulated in the vast majority of human
colon cancers (Hardwick and Philpott, 2015). Interestingly,
Xenopus researchers have exploited a functional biological assay based on duplication of the dorsal axis upon the
ventral injection of mRNA-encoding mediators of the Wnt
pathway, such as β-catenin, to develop chemical screens
(Kühl and Pandur, 2008). Regarding embryonic morphogenetic processes re-activated during tumor progression,
epithelial to mesenchymal transition (EMT), occurring
for instance during neural crest cell delamination, is a key
process during tumor cell invasion (Yang et al., 2020). The
molecular processes underlying EMT during neural crest
cell delamination have been intensively investigated by several Xenopus research groups (Pegoraro and Monsoro-Burq,
2013). Interestingly, Xenopus anti-EMT compound screens
have been deployed in embryos, which identif ed chemicals
that also affect cancer cell invasion (Tanaka et al., 2016) (see
section 5.2).
21.2.2. NATURALLY OCCURRING TUMORS IN XENOPUS
Spontaneous occurrence of tumors is rather uncommon
in Xenopus, leading to the now-contested belief that frogs
are rather recalcitrant to carcinogenesis. This was further
strengthened by the fact that potent human carcinogens have
shown limited to no effect in Xenopus laevis ( Hardwick and
Philpott, 2018). For example, attempts with N-methyl-Nnitrosourea (NMU), a known human carcinogen, failed to
induce tumors in Xenopus laevis (Goyos and Robert, 2009).
However, cases of spontaneous adenocarcinoma, f broma,
lipoma, lymphosarcoma, and leukemia have been occasionally documented (Balls, 1962; Stacy and Parker, 2004;
Suzuki et al., 2020). Finally, a number of naturally occurring thymic tumors, from which several lymphoid tumor
cell lines could be established, are now being used for elucidating key mechanisms for tumor versus immune system
interactions, especially via transplantation experiments (see
subsequently) (Robert, Guiet and du Pasquier, 1994; Goyos
and Robert, 2009).
21.2.3. INDUCED TUMOR-LIKE STRUCTURES
The very frst report of genetically induced tumors in
Xenopus laevis embryos dates from 1997, where mRNA
encoding human dominant-negative p53 was injected in early
embryos, inducing the formation of embryonic tumors, socalled induced tumor-like-structures (ITLSs) (Wallingford
et al., 1997; Wallingford, 1999). Closely thereafter, similar
approaches were undertaken to model tumors in embryos
upon injection of gli1 or rel (Xrel3) mRNA (Dahmane et al.,