Specific MR Imaging Of Hepatocellular Carcinoma With VEGF-C Targeted USPIO Probe

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumor, and more than50%of new patients occur in China. The mortality of HCC and its recurrence rate are quite high. Invasion and metastasis of HCC and the high recurrence rate after surgery are the main obstacles which hinder the survival and prognosis of patients. In recent years, some results have shown that the combination of vascular endothelial growth factor C (VEGF-C) and its ligand R3(VEGFR-3) could regulate the generation of tumor lymphatic vessels, which was the most important factor in the formation of tumor lymphatic metastases. Thus, VEGF-C has become a specific biomarker reflecting the invasion and metastasis of tumor and tumor lymphatic metastases. At present, imaging technology is the most principal approach in diagnose of the HCC. However, some small lesions or lesions lacking typical imaging characteristics are difficult to be diagnosed. And it is also difficult to give the metastasis tendency of tumor an accurate assessment. The aim of the study is to synthesize a molecular probe targeted to VEGF-C with ultrasmall superparamagnetic iron oxide particles (USPIO), to probe its clinical value in the specific molecular imaging of HCC by using cell and induced animal models of liver cancer, and also to reflect the metastasis of HCC.ObjectiveTo investigate the potential value of VEGF-C targeted USPIO molecular probe in specific detection of HCC by using MRI. MethodsThe targeted probe was synthesized by conjugating VEGF-C antibody with amino modified USPIO. Cell counting kit-8assay was conducted to ascertain the probe’s effect on the growth of the HUVEC and the HepG2cells. The HUVEC were respectively cultured with VEGF-C-USPIO or USPIO (100μg/ml of iron) by incubating4hours at20μl,40μl or80μl dose, and the following MR imaging of cell suspension in vitro was conducted. The signal intensities of the cell suspension on T2-weighted image (T2WI), R2*and T2*were measured. Rat models with HCC were divided into two groups (targeted group with VEGF-C-USPIO and a contrast with USPIO) with3rats for each group at random. Pre-and post-contrast enhanced MR imaging with time points of0.5,1and1.5h were performed with caudal vein injection at a dosage of20μmol/Kg. The signal intensities of the tumor on T2WI and T2*WI were measured, and the differences of the signal intensities between pre-and post-enhancements or between both groups were analyzed. The iron particles within cells or the tumors in two groups were confirmed by Prussian blue iron staining. The expression of VEGF-C in HUVEC was proved by Immunofluorescence technique. And the expression of VEGF-C in HCC was proved by immunohistochemistry.ResultsCytotoxicity experiment results showed that the cell vitality of HUVEC and HepG2cell were barely influenced by different concentration gradients or incubation time points of VEGF-C-USPIO. Magnetic resonance imaging results of cell suspension showed that the SI of T2WI and T2*in both groups was descended gradually with increasing dose of VEGF-C-USPIO or USPIO. While the SI of R2*in both groups was raised gradually with increasing dose of VEGF-C-USPIO or USPIO. A statistical difference between two groups at same dose (40μl,80μl) could be seen (P<0.05). Prussian blue iron staining of cell showed that the iron particles in cells were much more with the increasing dose of VEGF-C-USPIO. However, the iron particles were less in cells in USPIO group. The signal intensity of HCC on T2WI and T2*WI after VEGF-C-USPIO injection were decreased obviously with a minimum value at1h, indicating a significant difference (P<0.05). However, those in USPIO group were decreased less without statistical differences (P>0.05). The signal intensity on T2*WI after enhancement also showed statistical differences between both groups (P<0.05). Prussian blue staining results confirmed more iron particles within the tumor tissues in VEGF-C-USPIO group, whereas less ones in USPIO group. Immunohistochemical results showed that VEGF-C was expressed in cytoplasm and membrane.ConclusionVEGF-C-USPIO molecular probes have little influence on the cell vitality, and can initiatively target to HUVEC or the liver cancer in rat models expressing VEGF-C, which may help to achieve the specific MR imaging of HCC, indicating the metastasis potential of the tumor.