Table 4.

Available techniques for induction of hepatocellular carcinoma in relation to temporal and technical aspects as well as major advantages and disadvantages (summarized from[92])

Method and specification	Time to HCC short (+) to long (+++)	Technical efforts low (+) to high (+++)	Major “Pros” (+) vs “Contras” (-)
Chemotoxic agents linked models
Diethylnitrosamine	++	+	(+) good combination options with other methods
9,10-dimethyl-1,2-benzanthracene			(-) time to HCC not easily predictable
Direct implantation of tumor cells or tissue
Heterotopic/orthotopic	+	+/++	(+) heterotopic xenografts are often and easily done
			(+) syngeneic orthotopic models better reflect the natural liver microenvironment
Syngeneic/xenografts			(-) xenografts need immunocompromised mice
			(-) orthotopic tumor implants need surgical and imaging experience
Genetically engineered mouse models
Mouse embryo manipulation	++/+++	+++	(+) hepatocarcinogenesis can be analyzed stepwise
Cre-Lox recombination			(-) effects of manipulated gene(s) could have heterogeneous latency and genetic penetrance
Hydrodynamic injection
CRISPR-Cas9
Humanized mouse models
Immunologically humanized mice	+++	+++	(+) immunotherapeutical issues can be studied based on human cell lines in mice
Genetically humanized mice			(-) establishment difficult due to engraftment failure and development of stable stem cell-derived hepatocytes

HCC: Hepatocellular carcinoma.