Basic Screening And Analysis Of Human Esophageal Tumor Metestasis Associated Functional Antigen

Objective:Esophageal cancer is the eighth leading cause of cancer death in the word. China is one of the highest countries of esophageal cancer's incidence and mortality, the incidence of esophageal cancer is ranking forth in the common malignancy. The five-year survival rate is less than 10%. The metastasis of cancer is basic biological feature of malignancy and also is leading death cause of cancer. With development of genome project and protein project, human has realized that the development of metastasis of cancer is associated with metastatic gene and correlated protein.of metastasis. Blocking the espression of signal agent and correlated factor of metastasis may prevent metastasis of malignancy. Seeking specific functional antigen or gene of metastasis as to provid valuable target has becomed new popular topic. By a new technology of"The screening tumor-related antigen gene by functional monoclonal antibody library", developed by Department of Cell and Molecular Biology of Peking Union Medical College, our study is to research the monoclonal antibodies of anti-esophageal cancer cell, the screening and identification of correlated functional monoclonal antibody of metastasis in vitro, and primary determine some molecular weigh of functional proteins recognized by functional antibodies. This established an important ground on isolating and attaining sociated-functional-antigen-gene of human esophageal cancer metastasis.Method:Our experiments screened and checked the function of anti-metastasis of the established 485 hybridoma strains of anti-esophageal tumor monoclonal antibody library in vitro, which had been primary screened by ELISA. First, we detectedtheir antigen presentation on esophageal cancer cell by immunofluorescence assay; and then analyzed their inhibiting effects of metastasis function by adhere and migration assay. Based on these results, 7 strains mAbs with the obviously anti-invasion effecets were detected again from 22 mAbs that their inhibiting effects of adhere and migration had been identified. Subsequently, as for candidate antibody (3D11) which have best inhibition effect of metastasis on experiment in vitro, we purified the mAb from hybridoma supernatant, which purity quotient above 90%. Than we located the expression of the antigen recognized by 3D11 mAb on esophageal cancer cell by immunofluorescence and detected the different expression of human and normal esophageal tissue by IHC. Finally we extracted total protein of human esophageal cancer cell and xenografts, and analyzed it by Dot Blot and Western Blot. and attained primary result of functional protein's molecular weigh recognized by 3D11 monoclonal antibody.Results:1. The IF results of 485 mAbs realized antigen: it reveal that antigens of 109 mAbs recognized express in the esophageal cancer. In which 14 strains mAb recognized-antigen have a strong expression (+++), 448 strains mAb have a moderate expression (++), 47 strains mAb have an inferior expression (+).2. We analized the effects of the 485 mAbs on esophageal cancer adhering to normal vascular endothelial cell.Their results was that 40 mAbs significantly inhibited esophageal cancer cell adhering to the normal endothelial cell. Which had an inhibition rate range from 31% to 76% compared to control, and among them 32 antibodies have statistics significance. In addition, 8 strains mAbs promoted esophageal cancer cell adhere.3. The migration assay result: it was that 70 mAbs inhibit esophageal cancer cell magrition. In which 49 mAbs had an inhibition rate above 30%.compared to control, and had an inhibition rate.4. The invasion array result: it was that 7 mAbs from 22 mAbs that had been sieved and attained obviously inhibited invasion, which have a inhibition rate above 30%.compared to control, and have a statistics significance. This reveal 7 mAbs can prevent metastasis by block one step of migration,adhere or invasion.5. The collection and analysis of all data give us a scientific pick of 3D11monoclonal antibody for further purification, (inhibition rate of migration 46.53%,inhibition rate of adhere 13%,inhibition rate ofinvasion 73.0%) By cell immunofluorescence assay and IHC, the results indicate that 3D11 mAb associated antigen protein is a cell membrane antigen and express highly in esophgeal tumor cell but seldom found in normal esophageal tissue, we extracted total protein of esophageal cancer cell and xenografts, and analyzed it by Dot Blot and Western Blot., and attained primary result of functional protein's molecular weigh recognized by 3D11 mAb.Conclusion:1. 109 strains functional monoclonal antibody have been identified of its associated functional antigen expressed in esophageal cancer cell and attained from 485 hybridoma strains of anti-esophageal tumor functional monoclonal antibody library by IF.2. 40 strains functional monoclonal antibodies remarkably inhibited esophageal cancer adhering to normal vascular endothelial cell were screened and attained from 485 hybridoma strains of anti-esophageal tumor functional monoclonal antibody library by the adhesion assay in vitro.3. 49 strains functional monoclonal antibodies remarkably inhibited human esophageal cancer cell migration were screened and attained from 485 hybridoma strains of anti-esophageal tumor functional monoclonal antibody library by migration array.4. 7 strains functional monoclonal antibodies remarkably inhibited human esophageal cancer cell invasion were screened and attained from 22 hybridoma strains screened by adhere and migration array.5. One functional monoclonal antibody (3D11) have been attained identificated its associated functional antigen, which have a great capability of inhibiting migration,adhere and invasion in vitro. its protein's molecular weigh about 100kDa,300kDa. The functional antigen may be a potential target of blocking metastasis.