Establishment of Highly Intrahepatic Metastatic Cell Line of HCC by In vivo Selection and Investigation of Mechanism by Integrated Microarray Analysis

Abstract

Introduction: Intrahepatic metastasis is a frequent pattern of tumor recurrence in hepatocellular carcinoma (HCC). The mechanism of intrahepatic metastasis was speculated as following; cancer cells invaded portal vein and spread via portal vein flow. However, the molecular mechanism of intrahepatic metastasis remains unclear. Here, we investigated the molecular mechanism of intrahepatic metastasis in HCC by establishing a novel highly metastatic cell line and analyzing the integrated microarray analysis. Methods: Highly intrahepatic metastatic HCC cell line (HuH7-M) was established by in vivo selection using trans-portal vein metastatic model. Briefly, parentalHuH-7-Luc cells (HuH7-P) were injected into the spleen of SCID Beige male mouse. After six weeks from the injection, liver metastatic tumors were dissociated and expanded in culture medium. The cell populations were subjected to the next cycle. This cycle was repeated in four cycles and the novel highly metastatic cell line (HuH7-M) was established. The characteristics of HuH7-P and HuH7-M were compared by MTS assay, transendothelial migration assay, sphere formation assay, and Annexin V analysis. Tumorigenesis was compared by simultaneously administering each cell to spleen followed by splenectomy. Subsequently, integrated microarray analysis was performed by combination with mRNA expression and miRNA expression analysis. Results: After four cycles, the metastatic tumor size and the tumorigenesis rate were gradually increased in HuH7-M (33% vs 88%, p=0.036). The microscopic finding revealed that the differentiation of tumor was not changed. Proliferative potential was increased in HuH7-M. But, invasive potential, migration potential, cancer stemness, and morphological change were not observed. The cell of HuH7-M showed the lower rate of apoptosis (3.4% vs 13.4%). Tumorigenesis assay showed that intrahepatic metastatic tumor was formed only in HuH7-M (67% vs 0%). Integrated microarray analysis demonstrated a specific combination of mRNA and miRNA related to apoptosis as a potential target. Conclusion: Highly intrahepatic metastatic HCC model was established by in vivo selection, and the result of integrated microarray analysis indicated a molecular mechanism of intrahepatic metastasis in HCC mediated by apoptosis.834_A Figure 1. The tumorigenesis rate were gradually increased by in vivo selection834_B Figure 2. HuH7-M showed the lower rate of apoptosis than HuH7-P