Application Of Glypican-3-targeted Fluorescent Probe In Hepatocellular Carcinoma Diagnosis

Objective:Based on the glypican-3 up-regulation in human hepatocellular carcinoma tissues,glypican-3-targeting fluorescent probe was constructed and the application of this probe was investigated in liver cancer cells, animal models, and hepatocellular carcinoma patients for the diagnosis of hepatocellular carcinoma.Methods:(1) Glypican-3 -targeting fluorescent probe was constructed and chemical characterization, safety, and stability of this probe were tested;(2) Western blot and RT-PCR analyses were performed to investigate glypican-3 expression in liver cancer cell lines.(3) Flow cytometry and cellular immunofluorescence testing were performed to investigate the cytoplasmic membrane expression of glypican-3 in HepG2 cells;(4) Cellular uptake experiment was performed to confirm the specific uptake of fluorescent probe by HepG2 cells and L02 cell lines at different time points;(5) Cutaneous HCC tumor model was constructed in nude mice using HepG2 cells.Cutaneous tumor model using U87 cell line was constructed in nude mice as a control.Fluorescent probe was injected into nude mice through cauda vein and in vivo uptake of this probe was calculated using xenogen IVIS spectrum in HCC tumor model and U87 tumor model, respectively. Mice xenografts pre-treated using glypican-3 antibody was constructed as a control;(6) The usage of this probe in the surgical navigation of HCC was investigated with a asana fluorescence microscope;(7) Ultrasound-guided biopsy was performed in patients with liver malignancies.Glypican-3-targeting fluorescent probe was sprayed to the specimen immediately after biopsy. The fluorescent intensity was examined and semi-quantitatively calculated using the near-infrared imaging system. The fluorescent intensity between liver metastases and HCC, tumor and adjacent normal tissues were compared.Additionally, the relationship between fluorescent intensity and pathological diagnosis and glypican-3 expression were analyzed;Results:(1) Glypican-3 -targeting fluorescent probe was successfully constructed.Additionally, this fluorescent probe is very stable in the serum. In vitro and in vivo experiments indicates that this probe is very safe;(2) Western blot and RT-PCR analyses confirmed the over-expression of glypican-3 in HepG2 cells (protein and mRNA levels).(3) Flow cytometry and cellular immunofluorescence analyses confirmed the cytoplasmic membrane expression of glypican-3 in HepG2 cells;(4) Compared with L02 cell line, HepG2 cells showed specific uptake of glypican-3 targeted fluorescent probe immediately after 30 minutes' incubation,while pre-treatment using glypican-3 antibody decreased the cellular uptake of fluorescent probe. Additionally, the specific uptake of fluorescent probe by HepG2 cells increased along with the prolongation of time;(5) The fluorescent probe was confirmed to specifically accumulate in cutaneous HepG2 models of nude mice through xenogen IVIS spectrum, and the peak intensity of Tumor/Background ratio was 6 hours after intravenous injection;(6) This probe can be used for the surgical navigation of HCC tumor models in nude mice;(7) The fluorescent intensity of the HCC specimens were significantly higher than that of the liver metastases and adjacent normal liver parenchyma. Additionally, the Tumor/Background ratio of 2.32 showed the best diagnostic efficacy with a sensitivity of 0.833 and specificity of 1.0. The fluorescent intensity of tumor tissues is correlated with glypican-3 expression. Additionally, no correlation between liver cirrhosis of fluorescent intensity was observed.Conclusion:The glypican-3-targeting fluorescent probe constructed in this research can be administered in the clinical diagnosis of HCC.