Investigation of exhausted tumor-specific CD8 T cells in orthotopic hepatocellular carcinoma mouse model

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death and the fifth most common kind of cancer worldwide. This cancer has a 5-year survival rate of 10% and its increasing incidence requires the development of efficacious treatments against HCC. Recent advances in immunotherapy demonstrated its capability in treatment of cancer, but there is still potential for further development of immunotherapies against liver cancer. The immune response against cancer is often hampered by upregulation of co-inhibitory receptors on the surface of CD8 T cells. This inhibition leads to emergence of T cell exhaustion, where tumor-infiltrating lymphocytes (TILs) show a reduced proliferative capacity and low production of effector cytokines IFNγ and TNFα, a mechanism that impedes tumor rejection by CD8 T cells.

The aim was the development of an orthotopic HCC mouse model in order to analyse the exhaustion of tumor-specific CD8 T cells and investigate the changes in their transcriptome. We have established an orthotopic HCC mouse model using the “Sleeping beauty” (SB) transposon system. Tumor induction was achieved by the use of plasmids encoding transposon-flanked oncogenes and stable genomic integration of oncogenes was mediated by SB transposase. The transfection of murine hepatocytes was performed by hydrodynamic injection and hepatic tumors were generated by the use of oncogenic NRAS (G12V), in combination with myristoylated AKT (myr-AKT) and a short hairpin against the mRNA of p53 (shRp53). The expression of the oncogenes has led to tumor development within four to eight weeks in livers of C57BL/6J mice. In order to analyse CD8 T cell responses we have linked the model antigen ovalbumin (OVA) to oncogenic NRAS (G12V). Investigation of the anti-tumor response was by means of adoptive transfer of OVA-specific CD8 T cells (OT-I). In mice with OVA liver tumors the expression of co-inhibitory receptors PD-1, TIM-3, CD160, LAG-3, 2B4 was increased on the surface of OVA-specific CD8 T cells, and the tumor-specific CD8 T cells also showed a reduced cytokine production and degranulation capacity. The tumor-specific CD8 T cells were isolated from the liver tumor and total RNA transcriptome microarray was performed to elucidate the molecular cause of tumor-induced T cell exhaustion. Transcriptome data revealed altered expressions of various genes involved in T cell trafficking, acquisition of effector functions, proliferation and cell survival, including increased expression of apoptosis facilitators like BIM, BMF and caspase 3.