title: An alternative in vivo system for testing angiogenic potential of human neuroblastoma cells.
abstract: 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.
authors.affiliation: Department of Human Anatomy and Histology, University of Bari Medical School, Piazza G. Cesare 11, Policlinico, Bari, Italy.
authors.forename: Domenica
authors.initials: D
authors.lastname: Mangieri
authors.forename: Beatrice
authors.initials: B
authors.lastname: Nico
authors.forename: Addolorata M L
authors.initials: AM
authors.lastname: Coluccia
authors.forename: Angelo
authors.initials: A
authors.lastname: Vacca
authors.forename: Mirco
authors.initials: M
authors.lastname: Ponzoni
authors.forename: Domenico
authors.initials: D
authors.lastname: Ribatti
mesh_terms: D042683:Angiopoietin-1 / Q000378:metabolism; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply* / Q000473:pathology; D043169:Endostatins / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D009361:Neoplasm Invasiveness; D009389:Neovascularization, Pathologic; D009447:Neuroblastoma / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D042683:Angiopoietin-1; D051792:Basic Helix-Loop-Helix Proteins; D043169:Endostatins; D016222:Fibroblast Growth Factor 2; D042461:Vascular Endothelial Growth Factor A; D000099162:Endothelial PAS Domain-Containing Protein 1