A Standard Protocol for the Production and Bioevaluation of Ethical In Vivo Models of HPV-Negative Head and Neck Squamous Cell Carcinoma

[Image: see text] Preclinical cancer research increasingly demands sophisticated models for the development and translation of efficient and safe cancer treatments to clinical practice. In this regard, tumor-grafted chorioallantoic membrane (CAM) models are biological platforms that account for the dynamic roles of the tumor microenvironment and cancer physiopathology, allowing straightforward investigations in agreement to the 3Rs concept (the concept of reduction, refinement, and replacement of animal models). CAM models are the next advanced model for tumor biological explorations as well as for reliable assessment regarding initial efficacy, toxicity, and systemic biokinetics of conventional and emerging neoplasm treatment modalities. Here we report a standardized and optimized protocol for the production and biocharacterization of human papillomavirus (HPV)-negative head and neck chick chorioallantoic membrane models from a commercial cell line (SCC-25). Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite the advancement in the available therapies. Thus, there is a persisting demand for new management approaches to establish more efficient strategies toward their treatment. Remarkably, the inclusion of CAM models in the preclinical research workflow is crucial to ethically foster both the basic and translational oncological research on oral malignancies as well as for the advancement of efficient cancer treatment approaches.