Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models

Abstract

Mouse models are commonly used for studying hepatocellular carcinoma (HCC) biology and exploring new therapeutic interventions. Currently three main modalities of HCC mouse models have been extensively employed in pre-clinical studies including chemically induced, transgenic and transplantation models. Among them, transplantation models are preferred for evaluating in vivo drug efficacy in pre-clinical settings given the short latency, uniformity in size and close resemblance to tumors in patients. However methods used for establishing orthotopic HCC transplantation mouse models are diverse and fragmentized without a comprehensive comparison. Here, we systemically evaluate four different approaches commonly used to establish HCC mice in preclinical studies, including intravenous, intrasplenic, intrahepatic inoculation of tumor cells and intrahepatic tissue implantation. Four parameters--the latency period, take rates, pathological features and metastatic rates--were evaluated side-by-side. 100% take rates were achieved in liver with intrahepatic, intrasplenic inoculation of tumor cells and intrahepatic tissue implantation. In contrast, no tumor in liver was observed with intravenous injection of tumor cells. Intrahepatic tissue implantation resulted in the shortest latency with 0.5 cm (longitudinal diameter) tumors found in liver two weeks after implantation, compared to 0.1cm for intrahepatic inoculation of tumor cells. Approximately 0.1cm tumors were only visible at 4 weeks after intrasplenic inoculation. Uniform, focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular, dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells. Notably, metastasis became visible in liver, peritoneum and mesenterium at 3 weeks post-implantation, and lung metastasis was visible after 7 weeks. T cell infiltration was evident in tumors, resembling the situation in HCC patients. Our study demonstrated that orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically, suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.

Fig 1. Systemic injection of Hepa1-6 cells in syngeneic C57BL6 mice.

Hepa1-6 cells (2x10⁶) suspended in PBS were injected into C57BL6 mice intravenously. (a) Morphological examination of tumor nodules in different tissues. The results showed that tumor formation in lung, heart and ribs. (b) Histological assessment of liver tumor nodules in lung, liver and heart (scale bar = 50 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C.

Fig 2. Intrasplenic and intrahepatic inoculation of Hepa1-6 cells in C57BL6 mice.

Hepa1-6 cells (2x10⁶) suspended in PBS were injected into C57BL6 mice intrasplenicly or intrahepatically as described in Materials and Methods. (a) Morphological examination of tumor nodules in different tissues from orthotopic HCC mice generated by intrasplenic inoculation of Hepa1-6 cells. The results showed the tumor formation in liver and spleen. (b) Histological assessment of liver tumor nodules in spleen, liver and lung (scale bar = 100 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C. (c) MRI analysis of the progression of liver tumors after intrahepatic inoculation of Hepa1-6 cells at different time-points. Arrows point to the tumor nodules. (d) Morphological examination of tumor nodules in liver from orthotopic HCC mice via intrahepatic injection of Hepa1-6 cells. The results showed both solitary and multinodular tumors formed in liver. (e) Histological assessment of liver tumor nodules in liver and lung (scale bar = 100 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C.

Fig 3. Intrahepatic tissue implantation in C57BL6 mice.

Hepa1-6 cells (3x10⁶) were suspended in 50μl PBS and injected into the left axilla of C57BL6 mice subcutaneously with 1 ml syringe. Subsequently, tumor tissues were cut into about 1mm³ pieces. Tumor pieces with a size about 1mm³ were implanted in the left liver lobe of C57BL6 mice. (a) MRI analysis of the progression of liver tumors after tissue implantation at different time-points. Arrows point to the tumor nodules. (b) Measurement of liver tumor sizes at different time-points after implantation. The data represents as mean±sem and significant difference was detected between different time-points (*p<0.05; **p<0.01, n = 10 for 2 week time points; n = 20 for 3 week time point; n = 19 for 7 week time point after implantation). (c) Assessment of survival rates of orthotopic HCC mice generated by intrahepatic tissue implantation (n = 19). (d) Morphological examination of tumor nodules in different tissues. The results showed the tumor formation in liver, peritoneum, mesenterium and diaphragm. (e) Histological assessment of liver tumor nodules in lung and liver (scale bar = 100 μm). A’, B’ or C’ represents the corresponding magnified boxed area from A, B or C. (f) Immunohistochemistry of CD3+ and Foxp3+ regulatory T cells in liver sections from orthotopic HCC mice and HCC patients to determine the extent of T cell infiltration (scale bar = 100 μm). Arrowhead points to CD3+ or Foxp3+ T cells. TI represents Tissue Implantation; CI denotes as Cell Inoculation. (g) Measurement of immune cytokines including IFN-γ and IL-10 in serum from orthotopic HCC mice (n = 5, *P<0.05; **P<0.01). The comparison was conducted between CI, TI and normal controls. TI represents Tissue Implantation; CI means Cell Inoculation.