Constructing Molecular Probe Targeting Angiogenesis Of Gastric Cancer And MR Imaging In Vivo

Background: Most of solid malignant tumors are vascular dependent. Thenew-born blood vessels play an key role in the growth and metastasis of tumors. Thetumors’ blood vessel varies in the morphology, function, protein expression, reaction tocytokines and genetics compared with normal blood vessels. The conventional imagingmodality based on anatomy can not meet the needs of early diagnosis and therapeuticevaluation. However, the non-interventional dynamic and repeatable molecular imaginghas showed its potencials in the study of tumor angiogenesis and expected to improvethe specificity and sensitivity in the diagnosis, staging and evaluation of treatment oftumor. Our study, based on gastric cancer vascular endothelial cell special binding peptide（GEBP11）, has constructed a MR contrast media targeting gastric cancer vascularendothelial cell and investigated in its Biological activities, preparation, specificity andtoxity. Meanwhile, in vitro cell imaging as well as in vivo imaging labeled by thecontrast media was researched under1.5T and3.0T MR system to evaluate the tumorangiogenesis.Objective:1. To discuss the fisibility in making the MR probe with Fe₃O₄nanometer particles and GEBP11targeting gastric cancer vascular endothelial cell andtestify its ability to change MR signal, safety and specifity.2. This probe can tracegastric cancer vascular endothelial cell and make MR imaging possible, through a seriesof experiments.3. MR imaging of nude mice implanted with gastric cancer was used toevaluate the angiogenesis of tumor.Methods:1.SolGel method was used to synthesize Fe₃O₄nanometer particles.Coupled with gel, the particles were crosslinked with GEBP11, thus the MR probe-GEBP11@Fe₃O₄wasconstructed targeting gastric cancer vascular endothelial cell.Immobilized efficiency, as well as Acute toxicity tests in mice were tested. Short peptidelabeled by FITC was crosslinked with Fe₃O₄and this complex was used to evaluate itsspecificity.2. Human umbilical vein endothelial cells and human gastric adenal cencerline SGC7901was co-cultured and MTT method was used to identify the toxity of MRprobe GEBP11@Fe₃O₄to cultured cells.3. The specificity of GEBP11@Fe₃O₄bindingwith gastric cancer vascular endothelial cell was tested using Immune fluorescencetechnology and Transmission electron microscopy as well as non-relevant short peptideas contoll.4. The MR probe GEBP11@Fe₃O₄was tested to prove its ability to imagegastric cancer vascular endothelial cell. Several MR scanning sequences was set up todetect proper labell concentration and most sensitive imaging sequence in vitro.5. Thenude mice model implanted with gastric cancer was set up. GEBP11@Fe₃O₄wasinjected through caudal vein and MR signal change in mice organ and solid tumor wasabserved, as the proof for the fisibility of the probe to evaluate tumor angiogenesis invivo. Results:1. Fe₃O₄nanometer particles were successfully synthesized with thediameter20nm under Electron microscopy and after crosslinking with GEBP11, thediameter50nm. The probe solution can decrease significantly MR signal intensity inT2*WI, T2WI sequence and change nothing in T1WI sequences. This intensitydifference was confirmed by Statistics test. The acute toxicity tests showed0.5mg/per ofGEBP11@Fe₃O₄is the safe dose and the effects to mice are minimal, while among thegroup of1mg/per GEBP11@Fe₃O₄, three mouses was dead.2. Human umbilical veinendothelial cells were co-cultured with SGC7901. Immunohistochemical experiment,growth state observation, adhesion ability comparation proved the co-cultured cellsmimic the biological behavior of gastric cancer vascular endothelial cell. MTTColorimetric method created cell growth curve proved the cells labeled by50μg Fe/ml ofGEBP11@Fe₃O₄had smilar proliferation capacity to those unlabelled cells.3.Immunofluorescence stain showed intracellular green staining after incubation3omin-1h,while no staining in the control. TEM demonstrated many dense electronic densityparticles in cells.4.1.0×105/ml or more labeled endothelial cells can alter the MR signalin T2*images in vitro. These cells were labeled with50μg Fe/ml of GEBP11@Fe₃O₄.5.MR images of nude mice implanted with gastric cancer showed after30min the injectionof GEBP11@Fe₃O₄,the T2*-WI signal intensity of tumor tissue decreased comparedwith control, which means GEBP11@Fe₃O₄may come into a specific MR probe todetect the angiogenesis of the gastric cancer.Conclusions:1. The diameter of Fe₃O₄nanometer particles made by Solgel isabout20nm and superparamagnetic. Crosslinked with GEBP11, the MR probe can targetgastric cancer vascular endothelial cells and alter the MR signal.2. Immune fluorescencetechnology and in vitro experiments proved this probe can bind with gastric cancervascular endothelial cells and decrease MR signal in T2*sequence.3. Animal modelexperiments showed GEBP11@Fe₃O₄probe we constructed can change the signalintensity in tumor tissue, which would help in evaluation of gastric vancer angiogenesisin vivo.