Experimental Research On The Change Of Liver Regeneration And Microscopic HCC Metastasis With Rats And Its Therapy Tactics

Background Objective:Hepatocellular carcinoma (HCC) ranks fifth in frequency worldwide among all malignancies and is the third most common cause of cancer mortality, with about 600 000 cancer related deaths annually. Liver surgery including liver resection and liver transplantation represents the best potential curative treatment modalities for HCC. However, tumour recurrence after surgery is the major problem influencing the clinical outcome. The liver is the most common site of recurrence of primary HCC, which often develops relatively soon after liver resection, presumably as a result of undetected, disseminated micrometastasis or multicentric tumor in the liver remnant. Both liver resection and partial liver transplantation implies a postoperative liver regeneration processes. The ability of the remnant liver to regenerate limits the extent of surgery and decides prognosis of the HCC patients. Clinical and experimental evidence suggests that the cellular and molecular changes resulting from hepatectomy, including surgical stress responses, ischemia reperfusion injury and liver regeneration may stimulate the growth of occult tumours or dormant micrometastasis and thereby contribute to recurrence. So it becomes a difficult clinic question on how to suppress tumor recurrence and to promote liver regeneration after liver surgery. The purpose of this investigation firstly was to establish the animal model of the microscopic HCC metastasis and to test the hypothesis that liver resection may influence the growth and malignant properties of experimental, microscopic HCC and that these changes are correspondent with the size of the partial hepatectomy. Under this background, we hypothesized further there are different expressions of cell signaling pathways in between growing hepatoma cells and regenerating hepatocytes with Sorafenib introduction, which establishes a rationale for targeting molecular signaling pathway for HCC recurrence after liver surgery. And in vivo and in vitro experiment in preclinical models of HCC recurrence are designed to verify the basis of the targeted therapy for micrometastasis in regenerating liver. In the end, based on the growing evidence about cancer stem cells and the notion that liver stem cell in HCC carcinogenesis which has been indicated, the aim of the last project was to test the hypothesis, hepatic stem cell may activate in the regenerating tissue with the effect of cancer nourishing and try to explore its effect. It may provide a new insight to solve the contradiction between suppressing tumor recurrence and promoting liver regeneration after surgery.Material and Method:Varying grades of hepatectomy were performed in Buffalo rats with concomitant implantation of a fixed number of hepatoma cells (1×106/0.2ml) in the liver remnant. In addition, the group received 70% resection without tumor implantation and was regarded as the control. Body weight (BW), tumor size and number were noted 21 days after hepatectomy. During the observation period of liver regeneration, liver regeneration was recorded by liver function tests and hepatocyte proliferation markers (Cyclin D1 and Ki-67) by Immunostaining. Cell markers including aFP, Ki-67, Cyclin D1, Calpain-small subunit 1 (CAPNS1), CD34, Vascular Endothelial Growth Factor (VEGF) and its receptor 2 (VEGFR-2) in the regenerating liver, lungs or tumors were evaluated by Immunostaining and compared. Rats were euthanatized at Day 3,7 and 21 to explore the OC status by the cell makers CK19 and aFP with double immunofluorescence staining. Sorafenib dissolved in DMSO and diluted with cell culture medium to the desired concentration with a final DMSO concentration of 0.1% for in vitro studies and Sorafenib is dissolved with Cremophor EL/ethanol for in vivo experiments. After Sorafenib compound was applied to cell culture systems for primary hepatocyte and hepatoma cell lines, the cell viability with MTT assay, DNA synthesis with Thymidine incorporation assay and MAKT/AKT kinase signaling pathways were investigated by Immunostaining. Rats with 70% hepatectomy with hepatoma cell implantation received a daily oral gavage of sorafenib at a dose of 2.5/10 mg/kg. At termination of the animal experiment, rats in 70% hepatectomy groups with tumor cell implantation were euthanatized at Day 21, tumors with and without Sorafenib incubation groups were compared with tumor size, number and tumor makers (Cyclin D1, Ki-67,αFP, CD34 and VEGF). Liver regeneration was recorded by liver function tests and hepatocyte proliferation markers (Cyclin Dl and Ki-67) by Immunostaining.Results:I. At 21 days after hepatectomy with HCC cells implantation, tumor size and tumor number increased significantly with the magnitude of the partial hepatectomy and liver regeneration (P<0.05). The largest resections were also associated with increased hepatoma cell infiltration in the lungs. Tumors from larger hepatectomy displayed significant upregulation of Cyclin Dl, aFP, Ki67, CAPNS1, CD34, VEGF and VEGFR-2. In addition, the level of CAPNS1 expression showed a strong correlation to tumor number, size and the concentrations of aFP, CD34 and VEGF.Ⅱ. Hepatocytes showed higher cell viability, proliferation and stronger active MAKT/AKT kinase compared with hepatoma cell lines in the medium with Sorafenib induction. And tumor volume, number and distant metastasis in rats after 70% partial hepatectomy and HCC cells implantation with Sorafenib were significant decreased (P<0.05) and no significant change in liver regeneration was found in vivo experiment (.P>0.05).Ⅲ. With the magnitude of the partial hepatectomy at 21 days after HCC cells implantation, BW decreased with liver function worse (P<0.05), tumor size and tumor number increased (P<0.05) significantly. Cyclin D1 and Ki67 in the regenerating livers showed liver regeneration increased with hepatectomy size and the maximum proliferation was at Day 3-7. Hepatic stem cell's activation was identified by CK.19 and aFP expression in regenerating livers with 70% and 80% hepatectomy at Day 7-21.Conclusion:The microscopic HCC metastasis model with the varying grades of hepatectomy and concomitant implantation of hepatoma cells in the remnant liver is a reliable animal model to mimic the clinical HCC recurrence of the undetected micrometastasis or multicentric tumor in the liver remnant. Liver regeneration after partial hepatectomy facilitates growth and malignant transformation of experimental microscopic HCC. These results are of significance for liver resection and partial liver transplantation strategies for HCC. RAF targeted inhibitor Sorafenib can inhibit tumor recurrence and metastasis without retarding normal liver regeneration as the different response of RAF signal pathway to Sorafenib in between hepatocytes and hepatoma cells, which justifies the adjunct therapy for preventing HCC recurrence after surgery. Liver stem cell can activate in the regenerating tissue after extended hepatectomy with tumor growth and its activation is associated with hepatectomy size and tumor aggressiveness, which is presumed as a result of parenchyma loss and tumor invasion to the regenerating liver. It may be a new and fundamental insight to solve the problem of inhibiting tumor recurrence and promoting liver regeneration after liver surgery.