Analysis of mechanism of HGF antagonist NK4 mediated anti-angiogenesis for cancer and assessment for human clinical trial

作为治疗肝癌的新策略，我们报道了NK4介导的基因治疗可以抑制肿瘤血管生成、转移和侵袭。在体外和体内均能明显抑制肿瘤的生长和侵袭。在此基础上，我们旨在阐明NK4介导的抗血管生成机制，并估计用于人体临床试验的基因转移方法。(1)虽然NK4能抑制小血管内皮细胞的生长和迁移，但尚未发现NK4对脑源性细胞的抑制作用。我们使用经致死性照射的荷瘤裸鼠移植LacZ +骨髓（BM）细胞，试图评估NK4是否抑制肿瘤血管中BM来源的内皮细胞的募集。裸鼠被致死性照射（4.5 Gray，以铯-137为源）。将转基因小鼠获得的LacZ^+骨髓（BM）细胞（5x10^6）注射到辐照裸鼠尾静脉。4周后，小鼠腹部注射10^7个HUH7细胞。在肿瘤细胞植入后14天（或肿瘤体积达到约500 mm3时），Ad；将NK4 （10^9 pfu）或PBS （50 μl）直接注射到生长中的肿瘤中。5 d后切除肿瘤，用抗lacz兔多克隆抗体和抗cd31大鼠单克隆抗体进行染色。在所有小鼠中，大部分原有的脾细胞群被供体细胞所取代。然而，在cd31阳性的肿瘤血管中，即使在pbs注射的对照小鼠中，免疫组化染色也没有细胞LacZ阳性。在nk4治疗的肿瘤中，cd31阳性血管明显少于lacz治疗的对照组。这些结果表明，NK4抑制肿瘤血管生成，尽管在目前的动物模型中，对脑转移源性内皮细胞的抑制作用尚不清楚。这一问题需要进一步研究，因为它对未来的抗血管生成治疗方法具有重要意义。(2)评价NK4对肝再生的影响。在肝切除70%的小鼠中感染Nk4。与对照组相比，感染Ad的小鼠肝脏再生明显延迟。肝脏重量、PCNA染色和BrDU结果显示为LacZ。(3)评估流体动力学转染介导的NK4转导替代腺病毒载体的有效性，以辅助人体临床试验。这种转染方法证实对肿瘤生长有抑制作用。少

As a new strategy for hepatocellular carcinoma, we have reported NK4 mediated gene therapy that could inhibit tumor angiogenesis, metastasis, and invasion. It markedly suppressed tumor growth and invasion not only in vitro but also in vivo. Based on this study, we aimed to elucidate the mechanism of NK4 mediated anti-angiogenesis, and estimated method of gene transfer for applying human clinical trial. (1) Although NK4 inhibits growth and migration of small-vessel endothelial cells, an inhibitory effect of NK4 in BM-derived cells has not been shown. We attempted to assess whether NK4 suppressed recruitment of BM-derived endothelial cells in tumor vessels using lethally irradiated, tumor-bearing athymic nude mice transplanted with LacZ^+ bone marrow (BM) cells. Nude mice were lethally irradiated (4.5 Gray, with cesium-137 as the source). LacZ^+ bone marrow (BM) cells (5x10^6) obtained from transgenic mice were injected into tail veins of irradiated nude mice. After 4 weeks, these mice r … More eceived injections in the flank of 10^7 HUH7 cells. At 14 days post tumor cell implantation ( or when tumor volume reached approximately 500 mm3 ), either Ad.NK4 (10^9 pfu) or PBS (50 μl) was injected directly into the growing tumor. After 5 days the tumor was removed and stained with anti-lacZ rabbit polyclonal antibody and anti-CD31 rat monoclonal antibody. In all mice most of the original splenic cell population was replaced by donor cells. However, no cells were positive for LacZ by immunohistochemical staining in CD31-positive tumor vessels, even in PBS-injected control mice. In NK4-treated tumors, CD31-positive vessels were significantly fewer than in LacZ-treated controls. These results indicate that NK4 suppressed tumor angiogenesis, although any s uppressive effect for BM-derived e ndothelial cells remains unknown in the present animal model. Further study of this issue is required, since it has important implications for future approaches to of anti-angiogenic therapy. (2) To assess the NK4 influence for liver regeneration, Ad.Nk4 was infected t o mice t hat were performed 70 % hepatectomy. Liver regeneration significantly delayed regarding to control that were infected Ad.LacZ, as a result of hepatic weight, PCNA staining and BrDU. (3) Efficacy of hydrodynamics transfection mediated NK4 transduction was assessed instead of adenoviral vector to aid human clinical trial. Suppression of tumor growth was confirmed by this transfection method. Less