Simple SummaryAnimal testing is an important method in medical research and the development of new drugs (pharmaceuticals). Classic animal models feature mice or rats, but whenever possible, these animals should only be used for scientific purposes when absolutely necessary. Using less-developed forms, such as embryos not capable of pain perception, is considered an approach to reduce and replace adult animal testing. The use of chicken embryos for cancer research is well-known and features the implantation of cancer cells in order to form tumors (xenografts) on embryonic membranes (CAM). The tumor can be investigated via imaging methods, for example, nuclear medical imaging. However, chicken embryos are small and, therefore, require dedicated small animal imaging systems, which are expensive and require trained personnel. Therefore, we have investigated whether large ostrich embryos also are capable of growing tumors on the CAM. The large size of ostrich embryos would allow the use of routine imaging devices used for examinations in humans. We implanted breast cancer cells on the ostrich embryo CAM and successfully observed tumor growth. We suggest that the ostrich embryo is a suitable model for xenograft tumor imaging and cancer-related pharmaceutical research. This needs to be elucidated in further studies.AbstractThe avian chorioallantoic membrane (CAM) assay has attracted scientific attention in cancer research as an alternative or complementary method for in vivo animal models. Here, we present a xenograft model based on the ostrich (struthio camelus)CAM assay for the first time. The engraftment of 2 × 106breast cancer carcinoma MDA-MB-231 cells successfully lead to tumor formation. Tumor growth monitoring was evaluated in eight fertilized eggs after xenotransplantation. Cancer cells were injected directly onto the CAM surface, close to a well-vascularized area. Histological analysis confirmed the epithelial origin of tumors. The CAM of ostrich embryos provides a large experimental surface for the xenograft, while the comparably long developmental period allows for a long experimental window for tumor growth and treatment. These advantages could make the ostrich CAM assay an attractive alternative to the well-established chick embryo model. Additionally, the large size of ostrich embryos compared to mice and rats could help overcome the limitations of small animal models. The suggested ostrich model is promising for future applications, for example, in radiopharmaceutical research, the size of the embryonal organs may compensate for the loss in image resolution caused by physical limitations in small animal positron emission tomography (PET) imaging.