| The Her2/neu oncogene is frequently overexpressed in cancers of breast, ovary, uterus, lung, and gastrointestinal tract. Its overexpression in breast cancer is associated with a poorer prognosis. Dendritic cells (DCs) are the most powerful antigen presenting cells by far, and the initiators of immune response. Presently, transferring genetic engineered DC with genes encoding tumor associated antigens to cancer patients is a promising project for tumor immunotherapy. Our study practised it on pre-clinical level.We first utilized report gene to make a comparison among 4 gene delivery methods, including lipofection, DNA electroporation, recombinant adeno-associated virus type 2 (rAAV2) transduction and recombinant lentivirus (rLV) transduction. We found that rLV has the highest transduction efficiency on DC among these methods. Based on this, we made a further optimization on rLV transduction and finally improved the efficiency to 70%.Next, we prepared rLV containing a truncated rat neu gene (extracellular and transmembrane domains), and transduced DCs in vitro. Then we analysed the functions of transduced DCs using a series of experiments.We found that, the DCs modified with Her2/neu (named DCneu, the same below) are comparable to unmodified DCs(named DCctrl, the same below) in microscopic appearance, phenotype, antigen uptake capability and antigen presenting capability, suggesting that rLV transduction has few, if any, effects on DCs'main functions and it would not induce DC maturation.Animal experiments demonstrate that DCneu have both preventive and therapeutic effects on Her2/neu overexpressing tumors. In preventive experiments, only one single low dose of s.c. immunization (1.5×105/mouse) could sustain the five mice of that group in a healthy and tumor-free condition up to now (> 180 days); in contrast, all the mice in control group, DCctrl group and DCegfp group developed tumors in 31 days post tumor cells attack, respectively. When neu-negative tumor cells (B16-F1) were used,3 groups of mice (3 mice per group) all developed tumors between 12-16 day post attack, indicating the immunological preventive effects of DCneu is her2/neu specific. In therapeutic experiment, we first inoculated the mice with B16neu cells (2×105/mouse), then treated them respectively with DPBS (n=6), DCegfp (n=7) and DCneu (n=6) after 7 days. We found that, compared to that of DPBS and DCegfp groups, the tumor growth of DCneu group was significantly prohibited, the average tumor volumes in the end of observation period(19 days post tumor inoculation) are 976.64 mm3,592.63 mm3 and 132 mm3, respectively.For in vitro experiments, we detected an obviously elevated anti-neu antibody level in serum from mice immunized with DCneu, which declined gradually with time but still sustained in a relative higher level (101 day post DCneu immunization) in contrast to the basic level in DPBS-immunized mice. We also detected proliferation of both CD4+ and CD8+ T lymphocytes in spleens of mice immunized with DCneu-Further, compared with those from DCctrl immunized mice, splenic lymphocytes from DCneu immunized mice secreted higher level of IFN-ywhen stimulated by B16neu in vitro. These results show that DCneu can stimulate neu-specific humoral and cellular immunization effectively, supporting the results of our animal experiments. Finally, we found that tumors from DCneu-treated mice had a lower expression level of neu than DPBS-treated mice, suggesting a possible therapeutic effect. Moreover, both of the above-mentioned levels were much lower than that of B16neu tumor cells used for inoculation, suggesting a possible immune escape phenomenon.The conclusions are as follows:1. rLV can efficiently transduce murine bone marrow-derived DCs without the sacrifice of DC functions; 2. DCneu have considerable preventive and therapeutic effects on Her2/neu-overexpressed tumors.In addition, we found that the adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system, which are putatively discarded as macrophage or non-DC, are actually qualified dendritic cells.The originalities of our study lie in the following:1. for the first time (based on the present publications, the same below) compared efficiencies of 4 gene delivery methods in the same murine DC culture system; 2. for the first time proved spin transduction and repeated transduction could significantly improve the efficiency of rLV on murine DCs.3. for the first time utilized rLV containing Her2/neu to modify murine DC and used the genetic engineered DCs to treat Her2/neu-overexpressed tumor; 4. for the first time used single low dose immunization in similar pre-clinical studies on DCHer2/neu and gain effects not inferior to those with multiple high doses; 5. for the first time proved that adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells.Our study provides reference and evidence for clinical application of Her2/neu engineered DC vaccines.
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