| Background and ObjectivesHepatocellular carcinoma (HCC) is one of the most common digestive cancer and the third most deadly malignancy worldwide, particularly in China where the mortality rate associated with HCC accounts for more than 50%. Although advancing in diagnosis, modalities, and surgical techniques, the long-term prognosis of patients with HCC remains poor, mainly because of frequent recurrence and metastasis after curative resection. HCC is a hypervascular tumor and tumor angiogenesis plays an important role in the progression of HCC. Therefore, the antiangiogenesis combined radioimmunotherapy is a hot research in the treatment of HCC, currently.Studies found that tumor angiogenesis is a complex process, regulated by a variety of factors, including Endoglin (ENG) plays an important role in the formation of new blood vessels, and several studies indicated that ENG represents a more specific and sensitive marker for tumor angiogenesis and/or tumor progression than the commonly used pan-endothelial markers such as CD34 and CD31 in various types of human malignancies. At present, studies have found that ENG expression of mRNA in HCC was significantly higher than that in normal liver tissue, ENG can be used as a micro vascular marker in HCC, and whether ENG can be used as a therapy target have not been reported.This study used SMMC7721 cells labled by green fluorescent protein to construct an animal model of HCC in nude mice. We realize in vivo noninvasive monitoring the growth and metastasis in HCC, and the effect of 131I-anti-ENG mAb combined with 5-FU in the treatment of HCC was observed and evaluated in real-time. The aim of this study is provide experimental evidence for the ENG-targeted radioimmunoassy combined therapy in HCC.Methods1. The HCC tumor models were established by subcutaneous injection SMMC7721-GFP cells into the BAlB/cA-nu mice.2. Using noninvasive fluorescence imaging to monitor the volume of the tumor and the number of the SMMC7721-GFP, we test the relathionship both between the tumor volume with fluorescent signal and the cell number with fluorescent signal.3. ENG expression on HCC cell line, HCC tumor tissue and normal liver tissue were detected with RT-PCR、Western blot experiments.4. The SMMC7721-GFP cell line and HUVECs were treated respectively with different concentration of anti-ENG mAb and then cell proliferation was evaluated by Cell Counting Kit-8 assay(CCK-8).5. Anti-ENG mAb was radioiodinated with Na131I by the Iodogen method, and then the radiotracer was separated from free iodine using Sephadex G-25 columns. We also investigate its radiochemical purity stability in vitro, and in vivo pharmacokinetics.6. After injection of 131I-anti-ENG mAb through tail vein in nude HCC model,tumor targeting imaging by wholebody autoradiography were analyzed at different time(1h,12h,24h,48h,and 72h).7. The HUVECs was treated with 131I-anti-ENG mAb, then we test the tube formation.8. The subcutaneous tumor-bearing mice were randomly divided into the 131I-anti-ENG mAb experimental group、PBS group、5-FU group and the group combine 131I-anti-ENG mAb with 5-FU. The treatment was administered intratumorally once a week for a month. At the end of the experiment, tumor tissue from each animal was excised and then weighed. The inhibition rate was calculated. Quantification of ENG protein levels in different treated groups by immunohistochemical staining.Results1. SMMC7721-GFP hepatocellular carcinoma model was successfully obtained.In vitro fluorescent signal is strongly correlated to cell number over a broad dynamic range (r=0.9247,P<0.01). we also show that fluorescent signal and tumor volume were highly correlative (r2=0.925,P<0.01).2. ENG expression in the SMMC7721-GFP group was markedly higher than that in the normal liver cell L-02 group. Between the two groups, there was a significant difference (P<0.01). Similarly, there was a significantly higher ENG mRNA level in HCC tissue than that in normal liver tissue (P<0.01).3. CCK-8 assay verified that anti-ENG mAb had both stronger growth inhibiting effect on SMMC7721-GFP cells and HUVECs in a dose and time dependent manner, (P<0.05).The proliferation inhibition ratio of the A8 (20ug/ml) group in SMMC7721-GFP cells and HUVECs was 39.9±0.96%,45.2±1.26%, respectively.4. Anti-ENG mAb was successfully labeled with 131I and maintained stable in vitro. The radiolabeling efficiency was 97.4%. The radiochemical purity of I31I-anti-ENG mAb was 92.8%. The specific activity of radioiodinated ENG is 69.7±6.26 MBq/umol. The pharmacokinetics of 131I-anti-ENG mAb was in accordance with the two-compartment model, with a rapid distribution phase and a slow decline phase T1/2α, T1/2P, and MRT were 7.1,144.5, and 10h, respectively.5. Whole body autoradiography showed that the subcutaneous SMMC7721-GFP tumors were all clearly visible, with good contrast to the background, at 24h after injection of 131I-anti-ENG mAb (T/NT=6.44±1.01).6. Tube formation assay show that HUVECs which were treated with 20 ug/ml131I-anti-ENG mAb was effectively inhibited to form extensive and enclosed tube networks as compared with the untreated ones (P<0.01).7. The results of NIFLI shows that during the treatment group of HCC nude model,a continuous increasing fluorescent signals in the control group of PBS-treated mice, whereas a significant reduction in tumor burden was seen in the mice treated with5-FU,131I-anti-ENG mAb, and the 131I-anti-ENG mAb and 5-FU combination therapy group,and the combination group has the lowest fluorescent signal.Compare the fluorescent signals in the combination group with 131I-anti-ENG mAb alone group or 5-FU, the combination group has lower fluorescent signal(P<0.05).8. After 4 weeks of treatment in HCC nude model, tumor weight was significantly decreased in131I-anti-ENG mAb treated group compared with PBS-treated group (P<0.01).The tumor volume, weight was 96±15mm3,0.4±0.19g, respectively in the 131I-anti-ENG mAb treated group, the inhibition rate was 61.95 ±0.27%.In the 131I-anti-ENG mAb and 5-FU combination therapy group,the inhibition rate was up to 76+0.62%.9. Compared with the PBS treated mice, there was significantly lower ENG protein level in 131I-anti-ENG mAb treated mice,5-FU treated mice and the combination group.Among the above four groups, the combination group has the lowest expression of ENG protein.Conclusion131-anti-ENG mAb easily obtained, biologically stable and can obviously inhibit proliferation of SMMC7721-GFP cell line and HUVECs.131I-anti-ENG mAb was effectively inhibited to form extensive and enclosed tube networks. Intratumorally inject of 131I-anti-ENG mAb in HCC nude model can significantly inhibited the growth of the HCC and the therapy combined the 131I-anti-ENG mAb with 5-FU has a higher tumor growth inhibition rate. Furthermore, NIFLI can monitor the growth of the tumor and the efficiency of the treatment in real time and NIFLI may plays an important role in cancer research. Our study provides the experimental evidence for the ENG-targeted radioimmunoassy combined therapy in HCC. |
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