Cancer Letters 387 (2017) 114–120

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Cancer Letters
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Mini-review

Animal models of colorectal cancer with liver metastasis

Bo Young Oh a, Hye Kyung Hong b, Woo Yong Lee a,c, Yong Beom Cho a,c,d,*
a Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
b Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University, Seoul, Republic of Korea
c Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea
d
Department of Medical Device Management & Research, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords:
Liver metastasis is a leading cause of death in patients with colorectal cancer. Investigating the mecha-
Colorectal cancer
nisms of liver metastasis and control of disease progression are important strategies for improving survival
Liver metastasis
Mouse model
of these patients. Liver metastasis is a multi-step process and relevant models representing these steps
Orthotopic model are necessary to understand the mechanism of liver metastasis and establish appropriate treatments.
Genetically engineered mouse model Recently, the development of animal models for use in metastasis research has greatly increased; however,
Xenografts there is still a lack of models that sufficiently represent human cancer. Thus, in order to select an optimal
model for of a given study, it is necessary to fully understand the characteristics of each animal model.
In this review, we describe the mouse models currently used for colorectal cancer with liver metastasis,
their characteristics, and their pros and cons. This may help us specify the mechanism of liver metas-
tasis and provide evidence relevant to clinical applications.
© 2016 Elsevier Ireland Ltd. All rights reserved.

Introduction
Colorectal cancer is the third most common malignancy and a
leading cause of cancer-related death worldwide [1–4]. The sur-
vival of patients with colorectal cancer has improved; however, the
mortality rate still ranks third due to metastasis or recurrence [4,5].
Metastases are found in about 20–25% of patients with colorectal
cancer at diagnosis, and will develop in about 50% of patients during
the course of the disease [6,7]. The liver is the most common site
of metastasis for colorectal cancer, and only 15–20% of patients with
liver metastases are suitable candidates for surgical resection [8–10].
Several adjunctive therapies have been applied in patients with
unresectable liver metastasis, but survival rates remain unfavor-
able [10]. Thus, investigating the mechanisms of liver metastasis and
control of disease progression are important strategies for improv-
ing survival of these patients. Metastasis is a multistep process
which involves an epithelial–mesenchymal transition, suppres-
sion of apoptosis, local invasion and cell migration, angiogenesis and
intravasation, and extravasation into distant organs [11]. During these
processes, tumor cells continuously interact with the microenvi-
ronment of the host cell [12]. Relevant models representing the
metastatic characteristics of tumor cells are necessary in under-
standing these metastatic processes. In particular, organ-specific
metastasis models can help us to specify the mechanism of me-
tastasis and, accordingly, establish appropriate treatments.
* Corresponding author. Tel.: +82 2 3410 0217; fax: +82 2 3410 1655.
E-mail address: gscyb@skku.edu (Y.B. Cho).
Liver metastasis models have been developed both in vitro and
in vivo. In vitro metastasis models are easy to manipulate, inexpen-
sive, and reproducible. These models allow for the manipulation of
each step in metastasis, but there are limitations with respect to a
comprehensive analysis of the whole process of metastasis [13,14].
In contrast, in vivo metastasis models using animals may more ac-
curately represent the metastatic process and can be genetically
manipulated to mimic human cancer. Thus, animal models have been
widely used in metastasis research. However, they are associated
with ethical concerns and cost burdens [15–17]. Currently, a number
of animal models have been developed for the evaluation of the
mechanism of liver metastasis and for the establishment of ther-
apeutic strategies [12,15–19]. Such preclinical studies have provided
evidence relevant to clinical applications and have contributed to
improving the survival of patients with colorectal cancer with liver
metastasis. In this review, we focus on the mouse models cur-
rently used for colorectal cancer with liver metastasis.
Overview of the current understanding of animal models
Ideal animal models need to satisfy several conditions in order
to be suitable for metastasis research. Animal models of metasta-
sis should allow for optimal tumor take of both primary and
metastatic tumors. The tumor take should be predictable and re-
producible. They should recapitulate the entire process of human
metastatic diseases. Furthermore, they should reflect the genetic al-
terations with subsequent changes occurring in the metastatic
process. Ultimately, they should be available to test and validate novel
therapeutics for cancer patients [14,19–21]. However, as no model

http://dx.doi.org/10.1016/j.canlet.2016.01.048
0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.
 B.Y. Oh et al. / Cancer Letters 387 (2017) 114–120
meets all these conditions, models should be chosen according to
the specific experimental purpose.
Several animal models have been used in metastasis research,
with mouse models being most commonly used. Mouse models have
potential advantages such as anatomical and biological similari-
ties to humans, small size, ease of handling, breeding capacity, short
gestation time, cost-effectiveness, and ease of genetic manipula-
tion [14,22–24]. Hence, mouse models have evolved according to
the specific experimental purpose required to study the biology of
human cancer metastasis. Most metastasis models have been ob-
tained by transplanting cancer cell lines or tissues into mice. The
most commonly used types of mice in transplantable models are
nude mice and severely compromised immunodeficient (SCID) mice.
Nude mice have a mutation of the Foxn1 gene leading to an athymic
state. Thus, they exhibit depletion of T cells and impaired T and B
cell function. SCID mice have a mutation of the Prkdc gene, result-
ing in deficiencies in the number and function of both T and B cells.
However, they retain intact innate immune system components such
as natural killer (NK) cells and macrophages [14,19,25–27]. In ad-
dition, SCID-beige mice, non-obese diabetic (NOD)-SCID mice, NOD/
Shi-scid IL2rγnull (NOG) or NOD-SCID gamma (NSG) mice, and
recombination activating genes (RAG) mice have been used as mouse
models. These mice have different genomic mutations leading to
various levels of immunodeficiency. Genetically engineered mice
(GEM), which, unlike transplantable models, are generated through
alterations in the expression of genes of interest, have also been de-
veloped. In these models, organ-specific oncogenes or tumor
suppressor genes are manipulated, leading to the spontaneous de-
velopment of cancer [14,16,27,28].
Transplantable mouse models are divided into syngeneic and xe-
nograft models. Syngeneic models refer to models developed through
the inoculation of murine cancer cell lines or tissues into another
mouse which has an identical genetic background [27,29]. Synge-
neic models are valuable for evaluating the interaction between
tumors and their microenvironments. The graft is seldom rejected
and they are cost-effective and convenient. However, these models
lack the genetic heterogeneity of human tumors [14,27]. In con-
trast to syngeneic models, xenograft models usually refer to models
developed through the inoculation of human cancer cell lines or
tissues into immunodeficient mice [14,19]. These models have been
widely used in human cancer research, although they have limita-
tions such as deficiencies in immune system function and low rates
of engraftment [30–32]. In particular, patient-derived xenograft (PDX)
Fig. 1. Types of mouse models for colorectal cancer with liver metastasis according to the formation method.
115
models have received attention with the rise of personalized med-
icine. PDX models can reflect the characteristics of each patient and
are useful for predicting response to new therapeutics [16,33].
Types of mouse models for colorectal cancer with
liver metastasis
Spontaneous liver metastasis models
There are several ways to establish liver metastasis models (Fig. 1).
The first approach for developing liver metastasis is the implanta-
tion of cancer cell lines or tissues into the colon (orthotopic
transplantation) or subcutaneous layer (ectopic transplantation),
which results in the formation of primary tumors at the injection
site, followed by the spontaneous development of liver metasta-
sis. The most commonly used cell lines are HCT116 and HT29; other
cell lines used include SW480, SW620, and Lovo [12,18,34–37].
Ectopic transplantation is the easiest method for tumor engraft-
ment. However, this model mostly fails to develop liver metastasis
because the subcutaneous microenvironment is quite different from
that of the colon [38–40]. In contrast, the orthotopic transplanta-
tion model is similar to human cancer in terms of histology,
vascularity, gene expression, and the metastatic process [41–43].
Thus, this model is mainly used in spontaneous liver metastasis
models. In this model, colon cancer cell lines or human tumor tissues
can be transplanted into the colonic wall (usually the cecum) of the
immuno-compromised mice by subserosal injection or surgical im-
plantation (Fig. 2A and B) [14,19,27].
To ensure the effective development of liver metastasis, in vivo
selection of highly metastatic tumor cells is an important strate-
gy. The metastatic potential of the engrafted tumor can be increased
by serial passage. In this method, tumorigenic samples are subse-
quently expanded and re-implanted into the cecum for several
generations to obtain higher tumorigenicity and metastatic abili-
ties [19]. Such orthotopic transplantation models with highly
metastatic potential have been demonstrated to effectively devel-
oped liver metastasis [38,44,45]. However, this selection process and
the subsequent in vitro cell culture result in adaptation of tumor
cells to growth in a two-dimensional environment rather than in
the normal three-dimensional environment [27].
A major advantage of the orthotopic spontaneous liver metas-
tasis model is that metastatic dissemination follows a natural course
and utilizes a mechanism like that used in humans. Thus, this model
116 B.Y. Oh et al. / Cancer Letters 387 (2017) 114–120

Fig. 2. Transplantable models for colorectal cancer with liver metastasis. (A) Orthotopic model with serosal injection of cancer cell lines, (B) Orthotopic model with surgi-
cal implantation of cancer tissue, and (C) Experimental model with intra-splenic injection.

is suitable for predicting drug response in human tumors. This model
represents the entire metastatic process from the formation of the
primary site to the developing liver metastasis. Thus, it enables
the investigation of all steps in the metastatic cascade. It mimics
the human tumor microenvironment and provides a realistic level
of heterogeneity among tumor cells. In addition, the number of pas-
sages required to isolate highly metastatic tumor cells in the
spontaneous metastasis model is less than that required for the ex-
perimental metastasis model [14,19,28,46–48].
Disadvantages of this model are the low predictability and re-
producibility of spontaneous metastasis from colon to liver. It is also
difficult to assess the contribution of the immune system to meta-
static progression as this model due to the lack of a functional
immune system in the xenograft. In addition, it takes a long time
to develop liver metastasis, and sometimes resection of the primary
tumor is needed to reduce tumor burden and allow for the forma-
tion of metastasis [18,19,28,30]. Additionally, cecal injection is
technically difficult and carriers a risk of tumor cell leakage or intra-
luminal spillage (Table 1).
Experimental liver metastasis models
The second approach to developing liver metastasis is the in-
jection of tumor cells directly into systemic circulation, which induces
liver metastasis experimentally. Experimental metastases occur in
different organs, depending on the site of injection and the tropism
of the injected cell. In experimental liver metastasis models, colon
cancer cells are directly injected into the spleen or the portal vein
(Fig. 2C). The tumor cells reach hepatic microcirculation without un-
dergoing the steps of primary tumor growth and intravasation, and
are then arrested by size or specific retention factors. These ar-
rested cells undergo cellular extravasation and enter the liver
parenchyma or they may grow out while remaining within the vas-
culature. Some of the injected cells eventually form liver metastases
and the rest disappear without colonization in the liver. To in-
crease the efficiency of liver metastasis, the previously described
serial passage method is necessary in experimental models as in
spontaneous models. There are several reports that highly malig-
nant cells develop liver metastases when human colon cancer cells
are injected into the spleens of immunocompromised mice
[12,14,19,27,49].
Experimental metastasis models provide several advantages for
metastasis research. The tumor cells are injected directly into the
systemic circulation, so it takes a short time for metastases to
develop. Furthermore, these models are highly reproducible and con-
sistently lead to metastasis formation. Metastasis formation in these
models is more efficient compared to the spontaneous metastasis
models. Use of these models enables control over the number and
types of tumor cells introduced into the circulation, which is im-
portant in achieving the experimental endpoint. In addition, the
metastatic organ can be targeted to specific sites [14,27,28,50,51].
A major disadvantage of the experimental metastasis models is
that these models cannot represent the entire metastatic process.
They represent only the late stage of the metastasis cascade, since

Table 1
Advantages and disadvantages of models for liver metastasis according to the type of mouse model.

Model types Advantages Disadvantages

Spontaneous ● Natural course of metastatic dissemination
model ● Suitability for predicting drug response
● Identification of all steps in the metastatic cascade
● Similarity to the human tumor microenvironment
● Similarity to human disease progression
● Maintenance of realistic heterogeneity of tumor cells
● Minimal number of passages required to obtain high metastatic potential
Experimental ● Short time required to develop liver metastasis
model ● High reproducibility and consistency of metastasis formation
● Targetable metastatic organs
● Control over the number of cells delivered
Patient-derived ● Suitability for examining patient-specific drug response
xenograft ● Preservation of intratumoral heterogeneity of original tumor
model ● Effectiveness in evaluating the initiation and progression of metastasis
Genetically ● Identification of specific genetic mutation during carcinogenesis
engineered ● Effectiveness in evaluating the early steps in tumorigenesis
model ● Similarity of defined mutations to those of in human tumors
● Intact immune system of mice
● Species-specific microenvironment
● Low predictability and reproducibility of metastasis
● Functional immune system deficiency in xenograft
● Length of time required to develop liver metastasis
● Possibility of primary tumor resection due to tumor burden
● Technical difficulties
● Asynchronous development of liver metastasis
● Inability to represent the entire metastatic process
● Occurrence of only the late stage of the metastasis cascade
● Necessity of serial passage to enhance tissue-specific ability
● Artificial route of metastatic dissemination
● Low incidence of metastasis
● Length of time required to develop liver metastasis
● High cost and labor-intensive
● Technical difficulties
● Immune system deficiency in xenograft
● Low predictability and reproducibility of liver metastases
● Length of time required for the development of metastasis
● Low incidence of metastasis
● Possibility of embryonic lethality, severe developmental defects,
or sterility
● Difficulty in evaluating therapeutic response
● High cost and labor-intensive
 B.Y. Oh et al. / Cancer Letters 387 (2017) 114–120
they bypass the early stages, including primary tumor growth and
intravasation of tumor cells. Tumor cells should be isolated through
serial passage to enhance their tissue-specific abilities. Metastatic
dissemination follows an artificial route, rather than its natural course
(Table 1) [14,19,27,51].
PDX liver metastasis models
PDX liver metastasis models are established by implanting cancer
cells or tissues from patients into immunodeficient mice, in which
liver metastasis can then be induced spontaneously or experimen-
tally, as described above. Xenografts derived from cell lines are
reproducible, easy to manipulate, and well characterized; however,
they do not exhibit tumor heterogeneity or the histopathologic and
genetic characteristics of the original tumor [52–54]. In contrast,
PDX models better reflect the characteristics and genetic diversity
of the original tumor; thus, these models are currently the best pre-
clinical models for testing drug response, especially for patients with
refractory cancer. In addition, PDX models can be used for further
genomic and pharmacologic studies on personalized treatments
[53,55].
Mouse types commonly used in PDX models include nude mice,
SCID mice, and NOD/SCID humanized mice [14,16]. PDX models typ-
ically have a low engraftment rate, so co-injection of stromal
components like Matrigel or human stromal cells have been used
to improve the success rate [14]. Subcutaneously transplanted PDX
models rarely develop liver metastasis. The most commonly used
PDX liver metastasis model is a patient-derived orthotopic xeno-
graft model [45,56]. Hoffman and colleagues found that histologically
intact colon cancer tissues obtained surgically from patients and
orthotopically implanted into the cecum of nude mice led to the
development of liver metastases in PDX models after 10 passages
[44,45,57]. Other studies also reported that liver metastasis devel-
oped in orthotopic PDX models of colorectal cancer [55,58,59].
A major advantage of PDX metastasis models is that these models
can examine patient-specific therapeutic responses to drugs and
predict disease course, providing crucial information regarding per-
sonalized treatment [16,60]. PDX models also preserve the
intratumoral heterogeneity of the original tumor at the cellular and
genetic levels compared with xenograft models derived from cancer
cell lines [33,52,55]. In addition, PDX metastasis models provide the
opportunity to track the initiation and progression of metastasis [60].
Despite these advantages, PDX metastasis models have several
disadvantages in metastasis research. PDX models show variable en-
graftment rates and low liver metastasis rates [14,60]. The necrotic
area often present in patient tissues makes engraftment success even
more challenging [55]. These models are also time consuming, ex-
pensive, and technically challenging [16]. In addition, the immune
system is impaired in PDX models, which influences cancer pro-
gression. This drawback is partially overcome by using NOD/SCID
humanized mice via injection of peripheral blood or bone marrow
cells, but even in humanized mouse models the immune system
is not fully restored (Table 1) [16,55,60].
Genetically engineered liver metastasis models
The other approach to developing liver metastasis is the use of
genetically engineered mouse models (GEMMs), which may serve
as alternatives to human cancer xenografts. Xenografts have been
used to gain an understanding of human cancer progression.
However, the use of GEMMs in immunocompetent mice has been
suggested depending on the emphasis on the importance of the
tumor microenvironment on tumor progression. GEMMs are gen-
erated through alterations in genomic expression, such as the
activation of oncogenes and the inactivation of tumor suppressor
genes. They recapitulate the histologic, genomic, transcriptomic, and
117
proteomic spectrum of human cancers. Therefore, they provide an
understanding of entire carcinogenic progress and the mecha-
nisms of specific cancer-related genes [23,61–63]. However, they
usually cannot fully reproduce the genetic complexity of human
tumors.
GEMMs of colorectal cancer usually use germline or tissue-
wide genetic modification, which are useful in the study of hereditary
colorectal cancer [17]. However, somatic mutations develop in spo-
radic colorectal cancer, which corresponds to about 80% of all
colorectal cancer. Thus, somatically engineered mouse models are
needed to investigate the carcinogenesis of colorectal cancer [64].
Most GEMMs for colorectal cancer have been generated by induc-
ing mutations of adenomatous polyposis coli (APC) upon loss of
heterozygosity [65]. APC, a tumor suppressor, negatively regulates
beta-catenin concentrations and interacts with E-cadherin [66]. Al-
though APC mutations can lead to the formation of invasive colorectal
cancer, liver metastases have not been demonstrated in these models
[65,67]. Recently, a GEMM of colorectal cancer was established that
develops liver metastases with considerable efficiency. This model
was generated by Adeno-Cre injection into the colon of LSL-KRASG12V/
Apcflox/flox mice. Adenoviral administration of Cre into the colon
induces the loss of the APC tumor suppressor, activation of onco-
genic KRASG12V, and the generation of sporadic colorectal cancer
followed by liver metastases. This model limits the overgrowth of
cancer cells, therefore avoiding premature lethality, and leads to
tumor progression and liver metastases [64].
There are significant advantages to the use of GEMMs. These
models provide useful information about the effects of specific
genetic mutations during carcinogenesis. Compared with trans-
plantable models, GEMMs more accurately represent the natural
course of tumor development and the interaction between tumor
cells and tissue microenvironments [14,16,68]. Moreover, they are
effective for evaluating the early steps in tumorigenesis. In addi-
tion, these models have intact immune systems and species-
specific microenvironments [69].
A major disadvantage of genetically engineered liver metasta-
sis models is that liver metastases seldom develop and their
occurrence is not predictable [19]. Even if liver metastases occur,
they require a long period of time to do so [27,70]. Mutation of spe-
cific genes can lead to embryonic lethality, severe developmental
defects, or sterility prior to the development of metastases [24,71].
A potential limitation of GEMMs is that it is difficult to evaluate ther-
apeutic responses because these models show a low incidence of
and take a long time to develop metastasis [72,73]. In addition, they
are expensive and labor-intensive (Table 1) [16,68].
Monitoring metastasis in animal models
Histologic evaluation
Tumor growth is measured by histologic analysis (Fig. 3). The
extent of tumor growth is determined by measuring the area of liver
tissue that has been replaced by metastatic tumor tissue [74,75].
This method can quantitatively determine the extent of liver me-
tastasis; however, it does have several disadvantages. A major
disadvantage is that it can only be performed postmortem. Thus,
it cannot be used to sequentially evaluate the process of tumor
growth and it is difficult to assess therapeutic response. In addi-
tion, this is a labor-intensive and time-consuming method [15,19].
Histologic analysis is also useful in the molecular characteriza-
tion of liver metastasis. This method can validate similarities in the
genetic or histologic features between the primary and metastatic
tumors. It is particularly suitable for investigating human tumor be-
havior in mice [52,76]. This supports the importance of animal
models as preclinical tools for metastatic biology.
118 B.Y. Oh et al. / Cancer Letters 387 (2017) 114–120
Fig. 3. Monitoring liver metastasis in animal models. (A) Histologic evaluation (macroscopic and microscopic) and (B) Imaging evaluation (MRI, PET, and bioluminescence
imaging).
Imaging evaluation
Several imaging techniques have been developed for use in animal
models and, consequently, the limitations of histologic analysis can
be overcome (Fig. 3). In contrast to histologic analysis, imaging tech-
niques are noninvasive and sensitive, so they can be performed
several times. These techniques allow visualization of the entire
process of metastasis and the response to therapy [14,19]. Imaging
is a relevant method for monitoring metastasis in animal models.
Conventional imaging techniques, such as magnetic resonance
imaging (MRI), computed tomography (CT), positron emission to-
mography (PET), and single photon emission tomography (SPECT),
have been widely used in monitoring metastasis formation in animal
models [77–79]. MRI and CT provide high resolution three-
dimensional anatomical images, while PET provides high sensitivity
and specificity for detecting metastatic lesions. MRI is particularly
useful in monitoring liver metastasis and therapeutic response
[52,78].
Currently, in vivo optical imaging techniques, such as biolumi-
nescence imaging and fluorescence imaging, are being developed.
Bioluminescence imaging allows for the effective detection of tumor
cells expressing firefly luciferase, a light-emitting photoprotein. This
imaging can be performed in a sequential manner to visualize in-
trahepatic tumor growth. In addition, it is very sensitive, fast, and
easy to manipulate [15,19]. Many studies have reported the use-
fulness of bioluminescence imaging in liver metastasis animal models
[15,18,80]. Fluorescence imaging has been used to detect the growth
of tumors expressing the Aequorea victoria green fluorescent protein
(GFP). This fluorescence marker is genetically-encoded and con-
tinuously expressed in tumor cells. Thus, it provides the ability to
follow metastatic cells in real time [14,19,27]. Many studies have
monitored liver metastasis of colon cancer using this method
[81–83]. These optical imaging methods allow for monitoring of
single metastatic cells in vivo and provide new insights into the
biology of metastasis, especially during the early stage of liver
metastasis.
Conclusion
Mouse models for colorectal cancer with liver metastasis, such
as transplantable models (spontaneous and experimental models)
or GEMMs, have contributed to our understanding of the meta-
static progress. The ideal liver metastasis models should permit the
predictable and reproducible establishment of liver metastasis. Fur-
thermore, they should effectively represent the human tumor
microenvironment and be available to validate novel therapeutics
for cancer patients. However, there are still limitations in the gen-
eration of ideal models for human liver metastasis. Fortunately, the
development of sequence-specific genome editing techniques, such
as the clustered regularly interspaced short palindromic repeats
(CRISPR)-Cas system, provides a new means for the development
of liver metastasis models [84–86]. Ultimately, the goal of such
animal models is to design therapeutic approaches for patients with
refractory colorectal cancer. Additional research should be con-
ducted to develop next-generation models of human cancer, as
ultimately these efforts will promote further clinical investigation.
Conflict of interest
None declared.
Acknowledgement
This study was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF)
funded by the Ministry of Science, ICT and Future Planning
(2015R1A2A2A01003225).
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