The Chick Chorioallantoic Membrane Model: A New In Vivo Tool to Evaluate Breast Cancer Stem Cell Activity

The high plasticity of cancer stem-like cells (CSCs) allows them to differentiate and proliferate, specifically when xenotransplanted subcutaneously into immunocompromised mice. CSCs are highly tumorigenic, even when inoculated in small numbers. Thus, in vivo limiting dilution assays (LDA) in mice are the current gold standard method to evaluate CSC enrichment and activity. The chick embryo chorioallantoic membrane (CAM) is a low cost, naturally immune-incompetent and reproducible model widely used to evaluate the spontaneous growth of human tumor cells. Here, we established a CAM-LDA assay able to rapidly reproduce tumor specificities-in particular, the ability of the small population of CSCs to form tumors. We used a panel of organotropic metastatic breast cancer cells, which show an enrichment in a stem cell gene signature, enhanced CD44(+)/CD24(-/low) cell surface expression and increased mammosphere-forming efficiency (MFE). The size of CAM-xenografted tumors correlate with the number of inoculated cancer cells, following mice xenograft growth pattern. CAM and mice tumors are histologically comparable, displaying both breast CSC markers CD44 and CD49f. Therefore, we propose a new tool for studying CSC prevalence and function-the chick CAM-LDA-a model with easy handling, accessibility, rapid growth and the absence of ethical and regulatory constraints.

Automated summary

The high plasticity of CSCs allows them to differentiate and proliferate effectively in immunocompromised mice, making them highly tumorigenic even in small numbers. The CAM-LDA assay established here is a rapid and reproducible model for studying tumor specificities, particularly the ability of a small population of CSCs to form tumors. This model provides a convenient and accessible tool for studying CSC prevalence and function, with histologically comparable results to traditional mice xenograft models.