Fe₃O₄-MG7 Target Probe Combined With White Light Endoscopy For Detection Of Early Gastric Carcinoma, A Basic And Translational Research

?Background? Gastric cancer is one of the malignant tumors that threaten the health of public severely. The incidence of gastric cancer account for 6.8% of total cancers, and cancer related morality rate of gastric cancer is 8.8%. A lot of patients who suffered gastric cancer are in condition of advanced stage when diagnosed, attributed to unconspicuous clinical symptoms as well as limit of medical conditions and technologies. For those early gastric cancer patients, 5-year survival rates can soarly reach 92.6% after endoscopic submucosal dissection. Therefore, discovering early gastric cancer is an effective method to reduce the mortality of gastric cancer. However, current methods are not relatively reliable and sensitive in diagnosis of early gastric cancer. MG7 is a specific monoclonal antibody for gastric cancer, established by our laboratory. It is not expressed in normal gastric mucosa, but increasing in intestinal metaplasia, dysplasia, and gastric cancer. Moreover, MG7-Ag could be secreted into gastral cavity, and the expression of MG7 in gastric juice among gastric cancer patients is 81.1%. We utilize the characteristic of MG7, combining with Fe₃O₄ to synthesize target molecular probe. The probe can identify gastric cancer area by immuno-dying which can be seen under endoscopy. Its immuno-dying mechanism is that Fe₃O₄ reacts with potassium ferrocyanide and generates blue sediment. This method overcomes general white light endoscopy's limitation, and could well target gastric cancer area is its advantage. Moreover, this novel imaging pattern is simpler and more convinient than probe imaging by fluorescence endoscopy and MRI.?Aims?We used iron oxide magnetic nanoparticles coverd with carboxyl which is combined with amidogen located in MG7, synthesizeing Fe₃O₄-MG7 molecular probe. We will utilize the new and low toxcity probe to verify its binding ability with gatric cell lines, then to verify in gastric cancer tissues. We aim to establish a new method combing molecular probe with endoscopy technology for the diagonosis of early gastric cancer, thus provide foundation for future clinical trials.?Methods?1. Preparation of MG7-Ab and identification of its staining in gastric cancer?1?Culturing MG7 hybridoma cells, planting in mouse abdominal cavity, collecting ascites and purification.?2?Using immunofluorescence to investigate the expression of MG7-Ag in gastric cell lines SGC-7901 and normal gastric mucosa epithelial cell lines GES.?3?Using immunohistochemistry to investigate the expression of MG7-Ag in gastric cancer tissues and normal gastric tissues.2. Synthesis and validation of Fe₃O₄-MG7 molecular probe?1?Using EDC and sulfo-NHS to excite Fe₃O₄ magnetic nanoparticles, then react with MG7.?2?Using dynamic light scattering to detect the diameter and Zeta potential.?3?Using XTT assay to evaluate probe's toxicity.3. Imaging study in gastric cancer cells?1?Fe₃O₄ can react with potassium ferrocyanide generating blue sediment.?2?Using inverted microscope to investigate the binding ability of probe target gastric cancer cells.4. In vitro gastric cancer tissues imaging studyUsing endoscopy to investigate the binding ability of probe target gastric cancer tissues.?Results?1. Preparation of MG7-Ab and identification of its staining?1?We succeeded in preparing MG7-Ab through collecting ascites.?2?MG7 is expressed in SGC-7901 cells, but not expressed in GES cells.?3?Among 20 specimens, the expression of MG7-Ag in gastric cancer tissues is 85%, and is negative in normal gastric tissues.2. Synthesis and validation of Fe₃O₄-MG7 molecular probeThe probe has a hydrosize of 87.69 ± 0.33 nm, its Zeta potential is-19.30 ± 0.47 m V. When the concentration of Fe is under 0.2mg/ml, the probe is relatively safe. It is 92.1 % of cells viability when the concentration of Fe is 0.2 mg/ml, 84.9% at 0.3mg/ml, only 53.4% at 0.5 mg/ml.3. Imaging of gastric cancer cellsAdding the probe into culture dishes covered with cells, then reacting with potassium ferrocyanide solution containg HCl, blue sediment was seen among SGC-7901 cells, and not seen among GES cells. It is proved that the molecular probe is targeted at gastric cancer cells.4. Ex vitro gastric cancer tissues imagingAmoung 22 cases of gatric tissues, 18 gastric tissues showed blue sediment. None of the normal gastric tissues or gastric cancer tissus added Fe₃O₄ showed blue sediment. The specificity of Fe₃O₄-MG7 probe is 100%, and the sensitivity is 81.82%.?Conclusions?We have succesfully synthesized a molecular probe utilizing Fe₃O₄ magnetic nanoparticles and MG7-Ab. The probe is stable and has low toxicity. Fe₃O₄-MG7 is validated to target gastric cancer cell lines and tissues. It is sensitive and specific. These results suggest that Fe₃O₄-MG7 probe has a potential clinical significance in diagnosing gastric cancer. The current work lays a foundation of future clinical trials.