Chorioallantoic Membrane Assay to Assess Primary and Metastatic Tumor Growth in Ewing Sarcoma

The chorioallantoic membrane (CAM) assay provides a rapid, cost-effective, and ethically advantageous in vivo model to investigate tumor growth, angiogenesis, invasion, and metastasis. The CAM of the developing chicken embryo forms a highly vascularized and naturally immunodeficient environment that supports the implantation and proliferation of human or murine cells without the need for immunosuppression. This chapter presents a detailed and standardized protocol for performing tumor xenografts and metastasis analyses on the CAM, including optimized conditions for egg handling, embryo viability, and aseptic manipulation. The described workflow covers the complete experimental timeline-from preincubation of fertilized eggs, windowing and antimicrobial treatment, cell inoculation and drug administration, to harvesting and molecular downstream analysis of Ewing sarcoma samples. The method allows for quantitative assessment of tumor size, vascularization, and metastatic spread through the extra-embryonal membrane and can be combined with histological-, molecular-, or imaging-based readouts. The CAM assay serves as a valuable intermediate system between in vitro culture and mammalian xenografts, enabling reproducible, fast, and physiologically relevant studies on cancer cell behavior and therapeutic response.