Metastasis is a complex process that includes several steps: neoplastic progression, angiogenesis, cell migration and invasion, intravasation into nearby blood vessels, survival in the circulatory system, extravasation followed by homing into distant tissues, the formation of micrometastases, and finally the growth into macroscopic secondary tumors. This complexity makes metastases difficult to investigate and quantify in animal models. The chick embryo is a unique in vivo model that overcomes many limitations for studying the metastatic process, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located under the eggshell, that is receptive to the xenografting of mammalian tumor cells, including human. Since the chick embryo is naturally immunodeficient at this stage, the CAM can support the engraftment of tumor cells, and their growth therein can faithfully recapitulate most of the characteristics of the carcinogenic process including: growth, invasion, angiogenesis and colonization of distant tissues (Deryugina and Quigley, 2008; Zijlstra et al., 2002). The CAM sustains rapid tumor formation within 5-7 days after cancer cell grafting. This feature provides a unique experimental model for a rapid study of the intravasation and colonization steps of the metastatic cascade. Furthermore, using quantitative PCR to detect species-specific sequences, such as Alu, the chick embryo CAM model can be used to monitor and quantify the presence of the xenografted, ectopic tumor cells in distant tissues. Thus, the chick embryo model has proved a valuable tool for cancer research, in particular for the investigation of molecules and pathways involved in cancer metastasis and to analyze the response of metastatic cancer to potential therapies (Herrero et al., 2015; Casar et al., 2014). In this respect, the use of the rapid and quantitative spontaneous metastasis chick embryo model can provide an alternative approach to conventional mouse model systems for screening anti-cancer agents.
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