The Study Of RAAV Carrying PSA Transfecting DC Activating Immune Effector Cell For Prostate Cancer Treatment

Ⅰ. ObjectiveWith the development of immunology and moleculer biology, people fund that the cancer is a kind of immune diseases related with the functional defection, which is related with the down-regulation of the mature dendritic cells by the body. So it is a feasible idea to culture the DC in vitro and than to induce and activate the immunity of a patient against cancer. One of the key processes in this idea is to provide the antigen to be presented by DC, the other one is to activate the CTL efficiently. Now the most efficient way is to transfecting antigen peptide to DC with virus vector, which can make the former acquire a constant and large amount of antigen peptide to stimulate T cells. Among these virus vectors, rAAV is the most suitable candidate. Prostate cancer, because of its special antigen named prostate specific antigen (PSA), provides a very suitable bull's-eye for the cancer therapy by utilizing DC to induce special CTL. On account of this reason our department of oncology cooperate with foreign unit and construct the rAAV/PSA. In this paper, we'll test its effect on LNCaP cells in vitro and LNCaP cells xenografts in nude mice.ⅡResearch contentThe first part: Study on effects of the CTL generated by rAAV/PSA transfecting DC in vitro 1 Methods1.1 rAAV/PSA transfecting the DC and generating the CTLPeripheral blood was derived from HLA-A2+ healthy donors. Ficoll gradient-purified PBMCs were inoculated into sixwell culture plates for 5 hours, and the adherent cells were selected following three gentle washes. Immediately after the removal of the nonadherent cells, the adherent cells were divided into 2 groups, one was gene transfer group, infected with rAAV/PSA on day 0; the other was control group, pulsed with LNCaP cells lysate on day 5 for 5 hours. After 5 hours of incubation, the medium/virus solution was removed, the fresh AIM-V medium added. GM-CSF at a final concentration of 800U/ml was included in medium in both groups throughout the culture. Human IL-4 was added at day 3 and 5, TNF-αadded at day 7. On day 8, non-adherent PBMCs from the same healthy donors were washed and added to rAAV/PSA loaded DC (ratio of 20:1, responders: dendritics) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF, IL-2, and IL-7. After 7 days of cocultrue (day 14), the CTL were got.1.2 DC and CTL surface marker analysis and test of cytokinesCell surface markers analysis of DCs and CTLs were done by flow cytometry on day 7 and on day 14. For the characterization of DCs, a panel of surface marker was used: CD40, CD80, CD86, HLA-DR, CD1a, CD83, PSA; for that of CTLs, CD8/CD4 and CD8/CD56. Meanwhile we tested the secreting of IL-12 of DC and IFN-γof CTL.1.3 Cytotoxicity of CTL to LNCaP cellsTo test the CTLs' cytotoxicity, MTT was used. Took the LNCaP cells on day 2 of passage as the target cells and then tested its activity with trypan blue. After that adjusted the cell concentration as 1×10~5/ml and put in the wells of 96 wells, 100μl/well. Following this, adjusted the activated CTL with F 12 to a concentration of 5×10~6/ml and put them in the wells with the LNCaP cells in different ratios of 5:1, 10:1, 20:1, and 40:1. After a period of culturing, 20μl MTT at 5mg/ml were put in every well. After 6 hours, removed the suspension, put 150μl dimethylsulfoxide in them and mix them. Then mensurated their OD value(A) in a HTS 7000 Bio Assay Reader at 570nm. Cytotoxicity%=[1- (experiment group A-effector cells A ) /targe cells A]×100%. Otherwise we do the same test of CTL cytotoxic effect on DU 145 cells.2 resultsrAAV/PSA was successfully transfected to former body of DC (monocyte), which were cultured very well and have a high expression of surface markers including CD40, CD80, CD86, HLA-DR, CD1a, CD83, PSA, PSA, whose IL-12 secreting was higher than the other two groups. Only a week, the CTLs induced by DCs loaded with rAAV/PSA had a obvious increasement in ratios of CD8/CD4 and CD8/CD56. Meanwhile the IFN-γexpression of CTLs increased and them had a well cytotoxity to LNCaP cells.3 conclusionsIt's freasible for the technology to transfect DCs with rAAV/PSA and induced specific CTLs, which had a high activity of anti-tumor.The second part: The study on effects of the CTL generated by rAAV/PSA transfecting DC on LNCaP cells xenograft in mice1 Methods1.1 The experiment of LNCaP cells xenograft in nude miceMale nude mice of 4-6 weeks age were divided into four groups randomly, three mice every group, 1×10~6 LNCaP cells were injected subcutaneously into mice of the first group; 5×10~6 LNCaP cells into mice of the second group; 1×10~6 LNCaP cells mixed with matrigel into mice of the third group; 5×10~6 LNCaP cells mixed with matrigel into mice of the forth group. After six weeks to eight weeks, no xenograft was observed in any mouse of the former three groups but in all mice of the last group. Then 5×10~6 LNCaP cells were injected subcutaneously into mice of the first group again, but no xenograft was observed in 6 weeks.1.2 Experiment of CTL in bodys of nude mice20 male nude mice were divided into 4 groups randomly. The mice of first group, as control, were injected subcutaneously only 5×10~6 LNCaP cells mixed with matrigel, the day before it 200μl AIM-V was injected subcutaneously into the same side, after the made of xenografts 200μl AIM-V were injected into xenografts in mice as the forth group; the mice of second group were injected subcutaneously 1×10~8 CTLs into the same side of tumor cells injecting site before the injection of 5×10~6 LNCaP cells mixed with matrigel in 1 day; the mice of third group were injected subcutaneously 1×10~8 CTLs before the injection of 5×10~6 LNCaP cells mixed with matrigel in 7 days; 1×10~8 CTLs the mice of forth group were injected into xenografts in mice after the xenograft were made. Time of xenograft coming up, volum of xenograft, and weight of xenograft after 8 weeks were obseved. Meanwhile 100μl blood of mice were acquired from the tails of them every 2 weeks.2 ResultsAlthough with different volum and in different aged mice, no xenograft was found in nude mice. Mixed with matrigel, we only found xenografts 100% with big volum tumor cells but in less volum tumor cells. Injecting CTLs before the injection of tumor cells delay the time of coming up of xenografts. After the coming up of xenografts, injecting CTLs into them inhibited their increasement.3 ConclusionsIt's very diffcult to make a xenograft of LNCaP in nude mice without matrigel. Even with matrigel, it's necessery to mixed with a big volum tumor cells to make a xenograft. CTLs induced by DCs loaded with rAAV/PSA can prevent the coming up and the increasement of tumors.ⅢConclusionsDCs infected by the rAAV can elicit the differentiation of the lymphocytes and the proliferation of CTLs, which had obvious killing effects on LNCaP cells. Meanwhile the functions and the surface markers of DCs remain unaffected after the infection. The tumor vaccine of this rAAV/PSA can do some defendence and treatment on LNCaP cell xenograft in nude mice. So DCs infected with rAAV may have the potential as an adjuvant immunotherapy for patiens with prostate cancer.