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Page 12 of 16 Liu et al. Hepatoma Res 2020;6:7 I http://dx.doi.org/10.20517/2394-5079.2019.39

The gut microbiome (and its associated HCC risk) can be transmissible between mice and, interestingly,
this risk can also be transferred via the microbiome across generation to offspring of treated mothers [79,83] .
This opens up another avenue to induce hepatocarcinogenesis alongside GEM, hepatotoxins and dietary
manipulation in future models.

Thus, as we explore the new frontier of gut microbiome, animal models will be crucial for understanding
causality, pathogenesis and testing of therapeutic options targeting the microbiome (e.g., antibiotics,
probiotics, synthetic bile acids and faecal microbiota transplantation). Although mouse and human gut
microbiome communities are dominated by the same set of bacterial phyla, they are on the whole distinct
[78]
from one another . Therefore, experimental findings from microbiome studies in mouse models need
validation in human studies. The emerging use of a humanised gnotobiotic model (human donor stool
[84]
transplanted into germ-free mice) may also improve the applicability of preclinical findings .

CURRENT CHALLENGES AND FUTURE DIRECTIONS
Although, as described above, there are many different animal models for HCC related to alcoholic and
metabolic liver diseases, a single model faithfully recapitulating all features of human disease is lacking
and unlikely to exist. This is partly because human HCC is genetically heterogeneous, consisting of
several subtypes that are clearly different in behaviour, prognosis and response to treatment themselves.
[26]
Clearly, the identification of models that represent different human HCC subsets is required. Yan et al.
argued for combining a chronic injury model (e.g., NASH, CCl or MDR2 knockout) with alterations
4
in oncogenes or tumour suppressor genes found in human HCC which alone are not sufficient to cause
hepatocarcinogenesis (e.g., weak activation of pathways by heterozygous deletion or targeting only a
small percentage of hepatocytes) to achieve a more realistic representation of human HCC. The optimal
combinations for each aetiology are yet to be determined and will be an area of further research. When
achieved, this would not only help improve our understanding of the pathobiology of aetiology-specific
HCC but also improve our preclinical testing of new targeted treatments as we work towards personalised
medicine. Humanised mouse models may be a bridge for translating findings from mouse studies to
humans and presents a promising future strategy. However, several major challenges need to be overcome
not the least of which is the engraftment of a humanised immune system.

The amount of time required for tumourigenesis is another obstacle, as most models take more than
nine months to produce macroscopic HCCs. Furthermore, time is also needed to establish steatosis,
[21]
inflammation, fibrosis and cirrhosis . While implantation HCC models are established within weeks,
they are lacking these biologically important changes in the background liver. Indeed, human liver disease
typically takes decades to progress to cirrhosis and HCC. For example, patients with NASH progress at a
[54]
mean rate of only 0.09-0.14 fibrosis stages per year . Thus, the models most representative of human HCC
may require the most time which is suboptimal for studying response to therapy.

At present, almost all mouse studies assess tumour size and number at the one time point of sacrifice;
however, in clinical practice, HCC is diagnosed and monitored regularly using imaging (CT, magnetic
resonance imaging and ultrasound). Although these imaging modalities give reliable measurements that
correlate with tumour size at sacrifice, they are currently time-consuming and labour intensive (requiring
[21]
scanners, anaesthesia and injection of intravenous contrast agents) . Since tumour development can be
lengthy and their responses to treatment (especially new immunotherapies) are dynamic over time ,
[85]
measurement of experimental tumours on imaging will likely play an increasingly important role in the
future.

Recently, three-dimensional in vitro cell culture systems (organoids) using cells isolated from human
biopsies have been developed to study HCC. These tumour organoids (tumouroids) have been shown to