Mangieri Domenica, Nico Beatrice, Coluccia Addolorata M L, Vacca Angelo, Ponzoni Mirco, Ribatti Domenico
Department of Human Anatomy and Histology, University of Bari Medical School, Piazza G. Cesare 11, Policlinico, Bari, Italy.
Cancer Lett. 2009 May 18;277(2):199-204. doi: 10.1016/j.canlet.2008.12.014. Epub 2009 Jan 17.
In this study we purposed an alternative method to study the angiogenic and invasive potential of neuroblastoma cell suspensions implanted on the chick embryo chorioallantoic membrane (CAM) surface. Neuroblastoma cells were seeded in Matrigel and thereafter the suspension was pipetted onto the CAM surface at day 8 of incubation inside a silicon ring previously loaded onto the CAM surface. Four days after implantation, the silicon ring was removed and the angiogenic and invasive response were studied morphologically at macroscopic and microscopic levels and by reverse transcriptase-polymerase chain reaction (RT-PCR) by using human and chicken primers for several angiogenic cytokines, namely vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), hypoxia inducible factor-2alpha (HIF-2alpha), and for an endogenous angiostatic molecule, namely endostatin. Results showed that: (1) Neuroblastoma cells induced an angiogenic response in the CAM assay comparable to that induced by FGF-2; (2) neuroblastoma cells are packed inside Matrigel or are recognizable in the CAM mesenchyme; (3) Angiogenic activity of neuroblastoma cells is associated to an high expression of the transcripts of human VEGF-A, FGF-2, ANG-1 and HIF-2alpha and to a low expression in the transcript of a human endostatin while in the control specimens there is no expression of both angiogenic and angiostatic molecules; and (4) the expression of the transcripts of the same chicken angiogenesis stimulators and inhibitor is unmodified in treated and control specimens. Overall, these data indicate that neuroblastoma cells growth on the chick CAM express characteristics of the human disease. This experimental model could be employed for further research on human tumor progression and anti-angiogenic molecules screening.
在本研究中，我们提出了一种替代方法，用于研究植入鸡胚绒毛尿囊膜（CAM）表面的神经母细胞瘤细胞悬液的血管生成和侵袭潜力。将神经母细胞瘤细胞接种到基质胶中，然后在孵化第8天，将该悬液用移液管滴加到预先放置在CAM表面的硅环内的CAM表面上。植入后4天，取出硅环，通过宏观和微观水平的形态学研究以及使用针对几种血管生成细胞因子（即血管内皮生长因子-A（VEGF-A）、成纤维细胞生长因子-2（FGF-2）、血管生成素-1（ANG-1）、缺氧诱导因子-2α（HIF-2α））和一种内源性血管生成抑制分子（即内皮抑素）的人类和鸡引物进行逆转录聚合酶链反应（RT-PCR），来研究血管生成和侵袭反应。结果表明：（1）在CAM试验中，神经母细胞瘤细胞诱导的血管生成反应与FGF-2诱导的反应相当；（2）神经母细胞瘤细胞聚集在基质胶内或在CAM间充质中可识别；（3）神经母细胞瘤细胞的血管生成活性与人类VEGF-A、FGF-2、ANG-1和HIF-2α转录本的高表达以及人类内皮抑素转录本的低表达相关，而在对照标本中，血管生成和血管生成抑制分子均无表达；（4）在处理和对照标本中，相同的鸡血管生成刺激因子和抑制剂转录本的表达未发生改变。总体而言，这些数据表明神经母细胞瘤细胞在鸡CAM上生长表现出人类疾病的特征。该实验模型可用于人类肿瘤进展和抗血管生成分子筛选的进一步研究。
