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

作为一种新的治疗肝癌的策略，我们已经报道了NK 4介导的基因治疗，可以抑制肿瘤血管生成，转移和侵袭。它不仅在体外而且在体内都显著抑制肿瘤的生长和侵袭。在此基础上，我们的目的是阐明NK 4介导的抗血管生成的机制，并评估应用于人体临床试验的基因转移方法。(1)尽管NK 4抑制小血管内皮细胞的生长和迁移，但尚未显示出NK 4在BM来源的细胞中的抑制效果。我们尝试用移植LacZ^+骨髓（BM）细胞的无胸腺荷瘤裸鼠进行致死性照射，以评估NK 4是否抑制肿瘤血管中BM衍生内皮细胞的募集。对裸鼠进行致死性照射（4.5格雷，铯-137作为放射源）。将从转基因小鼠中获得的LacZ^+骨髓（BM）细胞（5 × 10^6）注射到辐照过的裸鼠的尾静脉中。4周后，这些小鼠 关于我们 在10^7个HUH 7细胞的侧腹中接受注射。在肿瘤细胞植入后14天（或当肿瘤体积达到约500 mm 3时），将Ad.NK4（10^9 pfu）或PBS（50 μl）直接注射到生长的肿瘤中。5天后，取出肿瘤并用抗lacZ兔多克隆抗体和抗CD 31大鼠单克隆抗体染色。在所有小鼠中，大部分原始脾细胞群被供体细胞取代。然而，在CD 31阳性肿瘤血管中，即使在PBS注射的对照小鼠中，也没有细胞通过免疫组织化学染色呈LacZ阳性。在NK 4处理的肿瘤中，CD 31阳性血管显著少于LacZ处理的对照。这些结果表明，NK 4抑制肿瘤血管生成，尽管在本动物模型中对BM衍生的内皮细胞的任何抑制作用仍然未知。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)为了评估NK 4对肝脏再生的影响，将Ad. NK 4感染到进行70%肝切除术的小鼠中。与对照组相比，Ad.LacZ感染组的肝再生明显延迟，这是肝重、PCNA染色和BrDU的结果。(3)评估流体动力学转染介导的NK 4转导代替腺病毒载体的功效以帮助人类临床试验。通过这种转染方法证实了肿瘤生长的抑制。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