The Chicken Chorioallantoic Membrane Tumor Assay as a Relevant In Vivo Model to Study the Impact of Hypoxia on Tumor Progression and Metastasis

SIMPLE SUMMARY: Hypoxia is a negative prognostic factor known to be closely associated with tumor progression and metastasis. However, existing animal models with the ability to recreate the tumor hypoxic microenvironment have disadvantages that limit our ability to understand and target this pathological condition. The chicken ChorioAllantoic Membrane (CAM) assay is increasingly used as a rapid cost-effective drug-testing model that recapitulates many aspects of human cancers. Whether this model recreates the hypoxic environment of tumors remains understudied. Here, we demonstrate that the CAM model effectively supports the development of hypoxic zones in a variety of tumor types. Treatment of tumors with angiogenesis inhibitors or inducers significantly modulated the formation of hypoxic zones as well as tumor progression and metastasis. Our findings suggest that the CAM-based tumor model is a relevant in vivo platform to further understand the pathological responses to hypoxia and test therapeutic interventions aimed at targeting hypoxic cancers. ABSTRACT: Hypoxia in the tumor microenvironment is a negative prognostic factor associated with tumor progression and metastasis, and therefore represents an attractive therapeutic target for anti-tumor therapy. To test the effectiveness of novel hypoxia-targeting drugs, appropriate preclinical models that recreate tumor hypoxia are essential. The chicken ChorioAllantoic Membrane (CAM) assay is increasingly used as a rapid cost-effective in vivo drug-testing platform that recapitulates many aspects of human cancers. However, it remains to be determined whether this model recreates the hypoxic microenvironment of solid tumors. To detect hypoxia in the CAM model, the hypoxic marker pimonidazole was injected into the vasculature of tumor-bearing CAM, and hypoxia-dependent gene expression was analyzed. We observed that the CAM model effectively supports the development of hypoxic zones in a variety of human tumor cell line-derived and patient’s tumor fragment-derived xenografts. The treatment of both patient and cell line-derived CAM xenografts with modulators of angiogenesis significantly altered the formation of hypoxic zones within the xenografts. Furthermore, the changes in hypoxia translated into modulated levels of chick liver metastasis as measured by Alu-based assay. These findings demonstrate that the CAM xenograft model is a valuable in vivo platform for studying hypoxia that could facilitate the identification and testing of drugs targeting this tumor microenvironment.