Development of an orthotopic HCC mouse model for analysis of tumor-specific CD8 T cell responses

Immunotherapy against liver cancer is a promising approach that needs to be further developed for the CD8 T cells.

We have established an orthotopic HCC mouse model in order to study the influence of hepatocellular carcinoma on cytotoxic CD8 T cells.

Hepatic tumors were generated in vivo using the "Sleeping beauty" transposon system.

The transfection of murine hepatocytes with the plasmids that contain cDNA coding for oncogenes was performed by hydrodynamic injection.

Tumor development in the mice livers began after the SB Transposase integrated the oncogenes into the hepatocyte genome.

We have used the oncogenic form of NRAS (G12V). The constitutively active form of RAS promotes continuous signalling for cell growth and proliferation of the hepatocytes after integration into the genome.

The active form of RAS alone is not sufficient to initiate tumor development. As the second oncogene we used myristoylated AKT (myr-AKT), which is involved in cell growth, metabolism, proliferation and survival.

A short hairpin against the mRNA of p53 (shRp53) is used to block the p53 apoptotic pathway.

The development of a model for in vivo monitoring of tumor growth by utilising the firefly luciferase allowed us to observe tumor development without sacrificing the mice.

The cell transformation begins after the genomic integration and was confirmed by histology one week after hydrodynamic injection. The oncogene expression leads to tumor development within four to eight weeks in the livers of C57BL/6J mice.

In order to analyse the CD8 T cell responses we have linked the model antigen ovalbumin (OVA) to the oncogenic NRAS (G12V).

We want to investigate the antitumor response of cytotoxic T cells by using the adoptive transfer of ovalbumin-specific CD8+ T cells (OT-I).