Experimental Study On Fusions Of Esophageal Carcinoma Cells With Dendritic Cells Derived From Cord Blood

Objective:To establish the cancer vaccine (EC109-DC) prepared by fusions of esophageal carcinomacell line with dendritic cell derived from cord blood and study the biological characteristics, and antitumor immunity.Methods:1) The CD34⁺ hematopoietic stem cells were isolated from mononuclear cells prepared with Ficoll-Hypaque density-gradients centrifugation from human cord blood using CD34⁺Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS);2) CD34⁺ hematopoietic stem cell were expended in RPMI 1640 medium supplemented with 10% fetal calf serum containing 300ng/ml rhGM-CSF (human Granulocyte-Macrophage Colony-Stimuting Factor) , 150μ/ml rhTNF- α (human Tumor Neucrosis Factor α )and 200ng/ml rhSCF (human Stem Cell Factor) at 37°C, 5%CO₂ for 2 weeks as mature DCs.3) Mature DCs and then were fused with esophageal carcinoma cell line by polyethyleneglycol (PEG-3600) at the ratio of 5:1. Selecting with MACS marked with HLA-DR MicroBeads generated the EC109-DC as tumor vaccine.4) The phenotypes and proliferation of EC109-DC were observed by light microscope, flowcytometry, immunocytochemistry and technique of cell culture in vitro. 5) Lymphocytes proliferation reaction and the cytotoxic T lymphocytes (CTL) cytotoxicity ofEC109-DC were examined with MTT assay. Results:1) Percentages of CD34+ hematopoietic stem cells purified from cord blood monocyte by magnetic ceil sorting system (MACS) were 0.8%—1.10%. The purity of CD34+ hematopoietic stem cells were greater than 98% stained by trypan blue, and suspend in culture medium with the micro round shape.2) Human cord blood CD34+ hematopoietic stem cells were expanded in the presence of rhGM-CSF, rhTNF- a and rhSCF. Cells with irregular-shaped and present cluster colony were occurred in the 3 th day, augmentation in cluster in 7 days and the cells had expended of nearly 20 fold after 2 weeks as mature DCs.3) After fusion of EC 109 and DCs successfully in the vitro by PEG, the resulting heterokaryons have the characteristic of adherent growth and irregular in shape. The cancer vaccine EC109-DC could divide and proliferate, which significantly decreased as compared to EC 109 cell line and the growth curve is more flat than that of EC 109.4) Analyzed by Immunohistochemistry and fluorescence-activated cell sorter analysis using FACScan, DCs express CD80, CD83 and CD86, the results were 84.7% ± 1.2%, 81.0% ± 5.4% and 66.4%±7.7% respectively. EC109-DC with expression of CD80, CD83 and CD86 were 90.2%±6.8%, 81.7%±4.3%and 49.2% ±2.2% respectively. The results showed thetwo groups have no statistic significance (P>0.05).5) Lymphocytes proliferation reaction and the specific cytotoxicity against EC 109 induced byEC109-DC cells were significantly higher than that of DCs, EC 109 groups and PBS groupsas the control (p<0.05).Conclusion:1) DCs derived from human cord blood using CD34+Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS) and cytokine-induced expansion cultured with rhGM-CSF, rhTNF- a and rhSCF were matural DCs with typical morphology and function.2) EC109-DC vaccine obtained by PEG fusion acquired the immuno-stimulating phenotype and could significantly stimulate the lymphocytes proliferation reaction, and the CTL activity.3) The results of this research provide the new materials for the DC based vaccine againstesophageal carcinoma.