Anti-angiogenesis Gene Therapy Of Hepatocellular Carcinoma Via Systemic Injection Of Mesenchymal Stem Cells Engineered To Secrete Soluble Flt-1 In Nude Mice

Background: Hepatocellular carcinoma is a common malignant tumor in digestive system.Angiogenesis is crucial for tumor progression and metastasis.Recent studies demonstrated that s Flt-1(soluble Fms-like tyrosine kinase-1)could suppress tumor growth through anti-angiogenesis.Bone marrow mesenchymal stem cells(MSCs)are widely used as delivery vehicle for gene therapy due to their ability of homing to tumor sites.Therefore,our study was designed to investigate anti-angiogenesis effect of hepatocellular carcinoma via systemic injection of mesenchymal stem cells engineered to secrete soluble Flt-1.Methods:1.MSCs were cultured in vitro and were transfected with the lentiviral vectors expressing s Flt-1(LV-s Flt-1)and non-targeting control lentiviral vectors(LV-NC).PCR?Western blot and ELISA were used to detect the expression level of s Flt-1.2.MSCs engineered to secret s Flt-1(LV-s Flt-1-MSCs)were co-cultured with human umbilical vein endothelial cells(HUVECs)in a three dimensional co-culture system to evaluate the anti-angiogenesis effect of s Flt-1.3.SMMC-7721 cells were dyed with Di I before subcutaneously injecting into the rear of nude mice.Two weeks later,we intravenously injected MSCs labeled by green fluorescent protein(GFP)into tumor-bearing mice.After 7 days,the subcutaneous tumor tissue,heart,liver and lung were removed and cut into fresh-frozen sections.To visualize the distribution of MSCs in vivo,the sections were observed under a fluorescence microscope.4.SMMC-7721 cells were subcutaneously injected into the rear of nude mice.Two weeks later,tumor-bearing mice were randomized into four groups: PBS,LV-NC-MSCs,LV-s Flt-1 and LV-s Flt-1-MSCs.Tumor size was measured and immunohistochemistry assay was performed to analyse the expression of CD34 ?Ki-67 and VEGF.5.The comparison of overall mean of measurement data use Mann–Whitney U-test for statistical analysis(?= 0.05,P < 0.05 for a statistically significant difference).Results:1.Construction of LV-s Flt-1-MSCs and expression of s Flt-1 in vitro MSCs were transfected with LV-s Flt-1 and LV-NC.72 h after lentiviral transfection,the transfection rate was up to 90% under fluorescence microscopy.LV-s Flt-1-MSCs significantly overexpressed s Flt-1 at both m RNA and protein levels(*P<0.05).2.LV-s Flt-1-MSCs inhibited tube formation in vitro Tube formation significantly decreased in the LV-s Flt-1-MSCs group compared to the LV-NC-MSCs or MSCs group.The difference was statistically significant(*P<0.05).3.Tropism of MSCs towards HCC in vivo MSCs labeled by GFP mainly migrated to the subcutaneous tumor mass,rather than other normal organs.4.LV-s Flt-1-MSCs inhibited tumor growth and angiogenesis The tumor volume and microvessel density(MVD)in mice treated with LV-s Flt-1-MSCs were significantly smaller than that in PBS group.The median survival of mice treated with LV-s Flt-1-MSCs was higher than that in the control group treated with PBS.The difference was statistically significant(*P<0.05).Whereas there was no significant difference between the group treated with LV-NC-MSCs or LV-s Flt-1 and the control group.Conclusions:1.MSCs are capable of homing to the microenvironment of hepatocellular carcinoma after systemic injection.2.LV-s Flt-1-MSCs could secrete s Flt-1,inhibit growth and angiogenesis of hepatocellular carcinoma,and prolong the survival of tumor-bearing mice.