Study On The Anti-Tumor Of Kidney Effects Of DC-RCC786-O Cell Line Fusion Cells In Vivo Of Hu-PBL-SCID Mice With Human Renal Cell Carcinoma Cells

Objective: To investigate the feasibility of establishing a human renal cell carcinoma model in human peripheral blood lymphocyte-engrafted to severe combined immunodeficient (huPBL-SCID) mice, and further to study the anti-tumor of kidney effects of DC-RCC786-O cell line fusion cells in vivo of huPBL-SCID mice with human renal cell carcinoma cells. It will provide the experimental foundation for renal cell carcinoma immunotherapy of DC fusion vaccine.Methods: Part 1. Establishment and identification of hu-PBL-SCID mice model with human renal cell carcinoma cells. Eighteen non-leakage SCID mice were randomly divided into three groups. Group RCC786-O-SCID (6 mice): subcutaneously injected with human renal cell carcinoma 786-O cells as a transplantation group. Group Hu-PBL-SCID (6 mice): intraperitoneally injected with huPBL as a humanized group. Group Hu-PBL-RCC786-0-SCID (6 mice): intraperitoneally injected with huPBL and subcutaneously injected with human RCC786-O cells as a humanized transplantation group. The biological and immunological features of mice were evaluated. Firstly observation of biological features including the latency period and the rate of tumor formation, general characteristic, tumor growth and the vital signs of SCID mice. Secondly human IgG level in SCID mice serum was measured by ELISA and human CD3+ T cells in murine peripheral blood were analyzed by FCM. Finally tumor tissues were stained with Haematoxylin/ Eosin, and infiltrating lymphocytes were counted under microscope; and human CD3+ T cells in spleen and tumor tissues were detected by SP immunohistochemistry.Part 2. Study the anti-tumor of kidney effects of DC-RCC786-O cell line fusion cells in vivo of Hu-PBL-RCC786-0-SCID mice. In the firs place, peripheral blood mononuclear cells were separated from the peripheral blood of health adult and co-cultured with recombinant human granulocyte-macrophage colony-stimulating factors, recombinant human Interleukin-4 and tumor necrosis factor-αto generate mature dendritic cells. In the second place, the dendritic cells and RCC786-O cells were fusioned by using of polyethylene glycol and the pure fusion cells were screened out by adherent incubating. Thirdly eighteen Hu-PBL-RCC786-0-SCID mice were randomly divided into three groups. Group 1 (6 mice): intravenously injected with fusion cells as the fusion cells group. Group 2 (6 mice): intravenously injected with DCs which were co-cultured with RCC786-O cells as the DC group. Group 3 (6 mice): intravenously injected with PBS as the control group. Then we evaluated the anti-tumor effects of fusion cells including the vital signs of SCID mice, tumor growth, tumor cell divide cycles by FCM, the proliferation index of tumor cells and the pathologic changes of the tumor tissues.Results: Part 1: 1.Subcutaneous tumors developed in all the group RCC786-O-SCID mice and group Hu-PBL-RCC786-O-SCID mice, but the latency period of group Hu-PBL- RCC786-O-SCID mice was significantly prolonged, and the tumor size was markedly depressed compared to the group RCC786-0-SCID mice(P<0.01). 2.In the 2nd, 4th and 6th week, human IgG in peripheral blood of the Hu-PBL-RCC786-O-SCID mice was (394.86±16.70)ug/ml, (629.83±35.03)ug/ml and (994.96±70.11)ug/ml, which was extensively higher than the group Hu-PBL-SCID mice(P<0.05); and in the 6th week the human CD3+ T lymphocytes was (12.31±0.86)% by FCM analysis, which was found to be greatly higher than the group Hu-PBL-SCID mice (9.02±0.64)%(P<0.01). 3. Haematoxylin/Eosin and SP immunohistochemical staining revealed presence of remarkable human CD3+T lymphocytes in the murine tumor and spleen tissues of Hu-PBL-RCC786-0-SCID mice and also in the spleen tissues of Hu-PBL-SCID mice.Part 2. Fusion cells could induce strongly anti-tumor biological effects in vivo: 1. tumor growth was remarkably inhibited and the tumor size in the fusion cells group was (257.12±7.01) mm3, which was extensively smaller than the size of control group (344.73±7.30)mm3(P<0.01). 2.The proliferation index of tumor cells in the fusion cells group was(25.04±3.55), which was found to be greatly restrained to the control group(66.74±3.89) (P<0.01) by detecting the cell divide cycles. 3.In the fusion cells group, the tumor tissues had more infiltrated lymphocytes than the control group. And the fusion cells group had more hemorrhage and necrotic tissues than the control group.Conclusion: 1. The Hu-PBL-SCID mice model with human renal cell carcinoma cells can be successfully established by combination of intraperitoneal injection of huPBL and subcutaneous injection of RCC786-O cells in SCID mice.2. The Hu-PBL-RCC786-O-SCID mice model can perfectly simulate biological feature of human renal cell carcinoma, and is an ideal animal for renal cell carcinoma immunotherapy.3. The fusion cells can inhibit the tumor growth and the proliferation index of tumor cells. By irritating the proliferation of T lymphocytes, fusion cells can induce more strong anti-tumor effects in vivo of Hu-PBL-RCC786-O-SCID mice.