The majority of Hepatocellular Carcinomas (HCC) evolves from fibrotic liver parenchyma. So far, there is no experimental model of liver cancer in the background of fibrosis to study intratumoral application of immunotherapies and their impact on disease recurrence after tumor resection. Therefore, the aim of our study was to establish a clinically relevant murine model of single-nodule liver cancer in fibrotic livers facilitating investigations on (neo)adjuvant therapies.
For this purpose, we wanted to apply our established method for local tumor induction in mice by intrahepatic electroporation of oncogenic transposons in the context of liver fibrosis mediated by CCl4-intoxication. At an age of 6-8 weeks, C57BL/6-p53fl/fl mice were electroporated with oncogenic transposons for KRasG12V and an expression plasmid for Cre recombinase. CCl4, resolved in mineral oil was applied twice weekly for a total duration of 4-6 weeks starting before or after electroporation. Tumor development was monitored, liver specimen were investigated by HE- and Sirius-Red-staining, and degree of fibrosis was determined.
Three experimental groups of mice were used. In the first two groups CCl4 injections were started 2 or 3 weeks prior to electroporation whereas in the third group fibrosis induction was started immediately after electroporation. We found that CCl4 treatments for induction of fibrosis before electroporation did not interfere with successful tumor induction. In all groups, tumors were histologically verified in explanted tissue specimen. Liver fibrosis could also be confirmed in all three groups. However, mice starting with CCl4-treatments after electroporation developed only a low to moderate degree of fibrosis before tumor size became critical. In contrast, mice which received the first CCl4 treatments prior to tumor induction showed an average to high grade fibrosis in both groups according to the Desmet/Scheuer staging score once tumors reached a palpable/injectable size. Nevertheless, when applying the longer period of CCl4 injection prior to electroporation the reliability of tumor induction might be negatively affected.
In summary, we have developed an experimental murine tumor model that enables studies on intratumoral application of oncolytic virotherapy in the presence of coexisting fibrosis and its effects on the tumor microenvironment, thereby reflecting the clinical situation.
