In Ovo CAM-Based Xenograft Model for Investigating Tumor Developmental Biology in Breast Cancer

Breast cancer remains one of the most prevalent and deadly malignancies affecting women worldwide. Its progression and metastatic behavior are driven by complex mechanisms. To develop more effective therapeutic strategies, it is crucial to understand tumor growth, angiogenesis, and microenvironmental interactions. Although traditional in vivo models such as murine xenografts have long been used to study tumor biology, these approaches are often time-consuming, costly, and ethically constrained. In contrast, the chick embryo chorioallantoic membrane (CAM) assay offers a rapid, cost-effective, and ethically flexible alternative for evaluating tumor development and angiogenesis. This protocol describes an in ovo CAM-based xenograft model in which human breast cancer cells are implanted onto the vascularized CAM of chick embryos. This method enables real-time evaluation of tumor growth. Furthermore, the model allows for manipulation of experimental conditions, including pharmacological treatments or genetic modifications, to study specific molecular mechanisms involved in breast cancer progression. The major advantages of this protocol lie in its simplicity, reduced cost, and capacity for high-throughput screening, making it a valuable tool for translational cancer research. Key features • Enables rapid tumor formation (3-4 days) after implantation of breast cancer cells. • Ethical and low-cost alternative to rodent xenograft models; suitable for laboratories without animal facility infrastructure and aligned with the 3Rs principles. • Optimized for short-term studies of tumor development, angiogenesis, and early metastatic events. • Highly suitable for pharmacological testing and experimental manipulation of the tumor microenvironment.