The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis : The CAM Assay in the Study of Angiogenesis and Metastasis

As pointed out by Auerbach in 1991 "Perhaps the most consistent limitation to progress in angiogenesis research has been the availability of simple, reliable, reproducible, quantitative assays of the angiogenesis response." In vitro angiogenesis assays, based on endothelial cell cultures or tissue explant, focus on isolated endothelial cell functions (e.g., endothelial cell proliferation, migration, or invasion) and do not examine the coordination of cell functions required for a successful angiogenic response (Jain et al., 1997; Auerbach et al., 2000). Although in vitro angiogenesis assays have been useful for identi cation of potential molecular targets to block endothelial cell responses and preliminary screening of novel pharmacological agents, they frequently cannot be correlated with in vivo angiogenesis measurements. This is most likely the result of the c- plex and multiple cellular mechanism evoked during new blood vessel formation in vivo. In vitro assays cannot be considered conclusive and the activity of a compound must be con rmed in other assays of increasing complexity, including in vivo assays of angiogenesis, angiogenic-dependent tumor growth, and metastasis. Acknowledgments Contents Introduction 1 Chorioallantoic Membrane Vasculature 1.1 Chorioallantoic Membrane and Its Embryological Origin 1.2 Morphology of Chorioallantoic Membrane Blood Vessels 1.3 Morphology of Chorioallantoic Membrane Lymphatic Vessels 1.4 A Single Blood Sinus or a Capillary Plexus Beneath the Chorionic Epithelium? 1.5 A New Model of Choriollantoic Membrane Vascular Growth, the Intussusceptive Mode 1.6 Chorioallantoic Membrane Vascular Growth 2 Chorioallantoic Membrane in the Study of Angiogenesis, Antiangiogenesis, and the Vascularization of Grafted Tissues 2.1 Use of Chorioallantoic Membrane in the Study of Angiogenic Molecules 2.2 Role of FGF-2 in Chorioallantoic Membrane Vascularization 2.3 Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules 2.4 Use of Chorioallantoic Membrane in the Study of Vascularization of Grafted Tissues 3 Chorioallantoic Membrane in the Study of Tumor Angiogenesis 3.1 Use of Chorioallantoic Membrane in the Study of Tumor Angiogenesis 3.2 Angiogenesis and Antiangiogenesis in Multiple Myeloma 3.2.1 Biological and Clinical Studies 3.2.2 Use of the Chorioallantoic Membrane 3.3 Angiogenesis and Antiangiogenesis in Human Neuroblastoma 3.3.1 Biological and Clinical Studies 3.3.2 Use of Chorioallantoic Membrane 4 Chorioallantoic Membrane in the Study of Tumor Metastasis 4.1 Use of Chorioallantoic Membrane in the Study of Tumor Metastasis 4.2 Spontaneous Metastasis Models 4.3 Experimental Metastasis Studies 5 Other Applications of Chorioallantoic Membrane 5.1 In Ovo and Ex Ovo Methods 5.2 Use of Chorioallantoic Membrane in the Study of Tumor Lymphangiogenesis 5.3 Use of Chorioallantoic Membrane in the Study of Angiogenesis Associated with Wound Healing and Inflammation 5.4 Use of Chorioallantoic Membrane in the Study of Angiogenic Properties of Biomaterials and of the Evaluation of Drug Delivery Systems 6 Different Morphological Techniques and Methods of Quantifying the Angiogenic Response Used in the Study of Vascularization in the Chorioallantoic Membrane 6.1 Different Morphological Techniques That Can be Used to Study Vascularization of the CAM and the Genes Involved 6.2 Methods of Quantifying the Angiogenic Response 7 Advantages and Limitations of Chorioallantoic Membrane in Comparison with Other Classical In Vivo Angiogenesis Assays 7.1 Advantages and Limitations of Chorioallantoic Membrane Assay 7.2 Other Classical In Vivo Assays in Comparison (Advantages, Disadvantages, and Limitations) with Chorioallantoic Membrane 7.2.1 The Corneal Micropocket Assay 7.2.2 The Sponge/Matrix Implant 7.2.3 The Disk Angiogenesis System (DAS) 7.2.4 The Matrigel Plug 7.2.5 The Dorsal Air Sac Model 7.2.6 The Chamber Assays 7.2.7 The Zebrafish 7.2.8 The Tumor Models Concluding Remarks References Annotation The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane which serves as a gas exchange surface and its function is supported by a dense capillary network. Because of its extensive vascularization and easy accessibility, the CAM has been broadly used to study the morpho-functional aspects of the angiogenesis process in vivo and to investigate the efficacy and mechanisms of action of pro-angiogenic and anti-angiogenic natural and synthetic molecules. The CAM is a suitable site for transplanting tissues, which can survive and develop in the CAM by peripheral anastomoses between graft and original CAM vasculature or by new angiogenic vessels grown from the CAM that invade the graft. While the formation of peripheral anastomoses between host and pre-existing donor vessels is the main, and the most common, mechanism involved in the revascularization of embryonic grafts, the growth of CAM-derived vessels into the graft is only stimulated in tumor grafts. The CAM has long been a favored system for the study of tumor angiogenesis and metastasis, because at this stage the chick immunocompetence system is not fully developed and the conditions for rejection have not been established. Tumors remain avascular for 72 h, after which they are penetrated by new blood vessels and begin a phase of rapid growth. Also, delivery of tumor cells onto the CAM allows the fine study of the effects of tumor derived angiogenic growth factors on blood vessel structure and functionality. The CAM may also used to verify the ability to inhibit the growth of capillaries by implanting tumors onto the CAM and by comparing tumor growth and vascularization with or without the administration of an anti-angiogenic molecule. Other studies using the tumor cells/CAM model have focused on the invasion of the chorionic epithelium and the blood vessels by tumor cells. The cells invade the epithelium and the mesenchymal connective tissue below, where they are found in the form of a dense bed of blood vessels, which is a target for intravasation