Chorioallantoic Membrane in the Study of Tumor Angiogenesis
The CAM has long been a favored system for the study of tumor angiogenesis (Dagg et al., 1956; Auerbach et al., 1976; Weiss et al., 1979; Ribatti et al., 1996a; Ribatti, 2004), because at this stage the chick’s immunocompetent system is not fully developed and the conditions for rejection have not been established (Leene et al., 1973). In birds, in fact, the immunocompetence only develops after hatching (Weber and Mausner, 1977). As other vertebrates, chickens are protected by a dual immune system comprised of B and T cells, controlling the antibody and cell-mediated immunity, respectively. The B cells are differentiated in the bursa of Fabricius, the organ equivalent to the bone marrow in mammals, whereas T cells are differentiated in the thymus (Funk and Thompson, 1996; Davison, 2003). Until day 10, the chick embryo immune system is not completely developed. The presence of T cells can be first detected at day 11 and of B cells at day 12 (Janse and Jeurissen, 1991). By day 12, mononuclear phagocytes are found in the yolk sac, spleen, bursa, gut, thymus, and liver (Janse and Jeurissen, 1991). The two major inflammatory cell types present in day 10–15 embryos are heterophils and monocytes. Heterophils functionally serve as an avian analogue of mammalian neutrophils and represent a main source of MMP-9 in the chick embryo. Therefore, chicken heterophils could be identified by staining with a specific anti-chicken MMP-9 antibody (Zijlstra et al., 2006). On the other hand, monocytes are the major source of MMP-13 in the chick embryo and could be identified by immunostaining with an anti-MMP-13 antibody (Zijlstra et al., 2004).