Development of Organ-specific Treatment for Bone Metastasis in Neuroblastoma

神经母细胞瘤的骨转移即使在强化治疗的情况下也是一个不利的预后因素。我们先前已经报道，与单独培养骨髓细胞相比，人神经母细胞瘤细胞株与含有破骨细胞前体和基质细胞的小鼠骨髓细胞共培养时，核因子κB配体(RANKL)的表达被刺激。结果表明，在共培养条件下，破骨细胞的生成增加。在前列腺癌和多发性骨髓瘤等其他癌症中，RANKL信号也被认为发挥了作用。为了开发一种针对神经母细胞瘤的骨转移的器官特异性治疗方法，我们在一些溶骨性骨病动物模型中检测了使用骨保护素(OPG)表达载体进行基因治疗的效果。以含有CAG启动子的pCAGGS为表达载体。我们构建了pCAGGS-OPG-FLAG，并在体外破骨细胞实验中证实了OPG-FLAG的生物学活性。为了将pCAGGS-OPG-FLAG导入动物肌肉，我们采用裸DNA注射和电穿孔相结合的方法。在恶性体液高钙血症动物模型中，引入pCAGGS-OPG-FLAG可恢复高钙血症。在OPG基因敲除的小鼠中，使用pCAGGS-OPG-FLAG的治疗增加了骨密度。这些数据提示以RANKL信号为靶点的治疗对包含神经母细胞瘤中的骨转移的溶骨性骨病是有用的。

Bone metastasis in neuroblastoma is an unfavorable prognostic factor even with intensive therapy. We have previously reported that nuclear factor κB ligand (RANKL) expression is stimulated in the coculture of human neuroblastoma cell line and murine bone marrow cells containing osteoclast precursors and stromal cells, compared with the culture of bone marrow cells alone. As the result, osteoclastogenesis was increased in the coculture. In other cancers such as prostate cancer and multiple myeloma as well, RANKL signaling is suggested to play a role. In an attempt to develop an organ-specific treatment for bone metastasis in neuroblastoma, we examined the effect of gene therapy using osteoprotegerin (OPG) expression vector in some animal models of osteolytic bone diseases. As the expression vector, we utilized pCAGGS containing CAG promoter. We constructed pCAGGS-OPG-FLAG, and confirmed the biological activity of OPG-FLAG in an in vitro osteoclastogenesis assay. To introduce pCAGGS-OPG-FLAG into the muscle of animals, we utilized naked DNA injection method combined with electroporation. In animal models of humoral hypercalcemia of malignancy, introduction of pCAGGS-OPG-FLAG restored hypercalcemia. In OPG knockout mice, the treatment using pCAGGS-OPG-FLAG increased bone mineral density. These data suggested the usefulness of treatment targeting RANKL signaling in osteolytic bone diseases, containing bone metasatsis in neuroblastoma.