FTY720 suppresses liver tumor metastasis by reducing the population of circulating endothelial progenitor cells

Abstract

Background: Surgical procedures such as liver resection and liver transplantation are the first-line treatments for hepatocellular carcinoma (HCC) patients. However, the high incidence of tumor recurrence and metastasis after liver surgery remains a major problem. Recent studies have shown that hepatic ischemia-reperfusion (I/R) injury and endothelial progenitor cells (EPCs) contribute to tumor growth and metastasis. We aim to investigate the mechanism of FTY720, which was originally applied as an immunomodulator, on suppression of liver tumor metastasis after liver resection and partial hepatic I/R injury.

Methodology/principal findings: An orthotopic liver tumor model in Buffalo rat was established using the hepatocellular carcinoma cell line McA-RH7777. Two weeks after orthotopic liver tumor implantation, the rats underwent liver resection for tumor-bearing lobe and partial hepatic I/R injury. FTY720 (2 mg/kg) was administered through the inferior caval vein before and after I/R injury. Blood samples were taken at days 0, 1, 3, 7, 14, 21 and 28 for detection of circulating EPCs (CD133+CD34+). Our results showed that intrahepatic and lung metastases were significantly inhibited together with less tumor angiogenesis by FTY720 treatment. The number of circulating EPCs was also significantly decreased by FTY720 treatment from day 7 to day 28. Hepatic gene expressions of CXCL10, VEGF, CXCR3, CXCR4 induced by hepatic I/R injury were down-regulated in the treatment group.

Conclusions/significance: FTY720 suppressed liver tumor metastasis after liver resection marred by hepatic I/R injury in a rat liver tumor model by attenuating hepatic I/R injury and reducing circulating EPCs.

Figure 1. FTY720 suppressed liver tumor intrahepatic and lung metastasis after hepatectomy and I/R injury.

(A) Orthotopic liver tumor model with metastatic potentials was established in buffalo rats. (B) intrahepatic and Lung metastasis were detected by Xenogen imaging and confirmed in specimen at four weeks after liver resection and I/R injury. (C) Histological features of liver tumors from buffalo rats at four weeks after hepatectomy and partial I/R injury with and without FTY720 treatment.

Figure 2. Comparison of circulating EPCs and bone marrow EPCs by Flow Cytometry.

(A) The number of circulating EPCs at different time points after the major hepatectomy and partial I/R injury. (B) The number of EPCs in bone-marrow was detected by Flow Cytometry at four weeks later after liver resection and I/R injury. Each group contained 16 rats, *P<0.05.

Figure 3. Comparison of tumor angiogenesis in metastatic tumor nodules.

(A) The expression of CD34 positive cells in intrahepatic and lung metastatic tumor nodules. (B) Comparison of MVD in metastatic tumor nodules between treatment group and control group. Each group contained 16 rats, *P<0.05.

Figure 4. Hepatic architecture and apoptosis at 6 hours after hepatectomy and hepatic I/R injury.

(A) Hepatic architecture was examined by H&E staining. (B) Hepatic apoptosis was detected by TUNEL staining. (C) Comparison of average apoptotic positive cells in 6 random fields (200×). Each group contained 6 rats, *P<0.05.

Figure 5. Hepatic gene and protein expressions at 6 hours after hepatectomy and hepatic I/R injury.

(A) Hepatic mRNA expressions by real time RT-PCR in different groups. (B) Hepatic protein expressions by Western blot. *P<0.05.