Experimental Research On The Possibilities Of Human Osteosarcoma Cell-vaccine From Antigen Presenting Cell(APC)

AIM The research intended to investigate some effective methods to adopt activated APCs as new osteosarcoma-vaccine approaches ,to study the influence of activated APCs on the in vitro immunogenicity of apoptotic human osteosarcoma, the preparation of hybridoma of human osteosarcoma PL-11 with activated B lymphocytes, in vitro immunogenicity of those fused tumor cells, and their vaccine potentials.METHODS (1) To select appropriate H202 complete media concentration and working time to induced apoptosis, FCM was implied to analyze the DNA content and cell circle. Periphery blood mononuclear cells were separated from rurally separated white cells by density gradient centrifugation, monocytes separated by adhesion method. The lysates of apoptotic cells and unapoptotic cells were cultured with prepared lymphocytes and activated monocytes for 7 days (PL-11a group and pl-11u group) respectively. Lymphocytes with apoptotic cell lysates (Control group) and lymphocytes with activated APCs (MON group), were parallelly cultured as control, and the proliferation of each group was measured by count method. Lymphocytes from each group were harvested to measure the killing index in vitro by modified MTT method.(2) Sarcoma cells PL-11 was fused with B lymphocytes activated by anti-human Ig-M antibody, Staphylococcus aurous protein A, and morsel dose of IL-2 and phorbel-12-myristate-13-acetate by 500mg/ml PEG. The fused cells was separated from unfused B cells by adhesion method, and from unwanted sarcoma cells by B-lymphocyte-specific-antibody precoated plate, the content of fused cells were measured at 3 and 4 weeks, and hybridomas were cultured with ultraviolet-radiation inactivated lymphocytes. The unfused PL-11 cells were parallelly treated as control.The proliferation and lymphocytotoxicity of both groups were measured by count method and modified MTT method respectively.RESULTS (1)H202 was not able to induce apoptosis at 10-100mmol concentration in 7 hours, but 10 nmol/L, 50 nmol/L, 75nmol/L,100nmol/L H202 groups could induce apoptosis after 14 hours with dose-dependent and time-dependent manner, especially lOOnmol/L group after 36 hours(10.6%) .Lymphocytes of the PL-1 la group increased significantly, while that of the PL-1 lu group increased merely and the control group and MON group did not increase at all. The PL-llu group showed a low cytotoxicy to sarcoma cells(3.90%), the PL-1 la group a significant increase(9.18% ) (p<0.01),and the Control group and MON group no cytotoxicy (p>0.05).(2) Osteosarcoma fused-cells can be effectively produced by PEG fusion and selected by adhesion method and subsequently antibody precoated plates, and then properly proliferated. The hybridomas percentage at 3 and 4 week were 60.6% and 45.4% respectively, and inactivated hybridomas of human osteosarcoma significantly increased the proliferation of autologous lymphocytes (P<0.01) and induced their cytotoxicity(11.6%)to parental osteosarcoma.CONCLUSIONS Having been induced apoptosis by H202, human osteosarcoma cells could increase immunogenicity in vitro through the activated monocytes from periphery blood mononuclear cells, and it is also possible to rapidly prepare the hybridoma of human osteosarcoma with activated B-lymphocytes in vitro and to increase immunogenicity significantly. This indicates that both the activated monocytes phagocytosed lysates of apoptotic human osteosarcoma and the osteosarcoma fused-cell with activated B-lymphocytes could be new ways to induce effective anti-sarcoma immunoreaction, which have some important potentials for effective APC-derived individual human osteosarcoma vaccine.