A New In Vivo Tool to Evaluate Breast Cancer Stem Cell Activity – the CAM

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A New In Vivo Tool to Evaluate Breast Cancer Stem Cell Activity – the CAM

Tuesday, 19.01.2021

We have established a in vivo limiting dilution assay (LDA) using the chick embryo chorioallantoic membrane (CAM), using a metastatic breast cancer cell model previously shown to be enriched for cancer stem cell (CSC) properties. This new tool was able to reproduce the same results found in mice, which is the gold standard method to evaluate CSC enrichment. Importantly, the chick model has several advantages, namely its easy handling, accessibility, rapid growth, lower costs and the absence of ethical and regulatory constraints. Thus, the chick CAM-LDA constitutes a new and valuable tool to study CSC activity in vivo.


Authors and Affiliations:

Marta Teixeira Pinto 1,2,† , Ana Sofia Ribeiro 1,2,† , Inês Conde 1,2, Rita Carvalho 1,2 and Joana Paredes 1,2,3

1 i3S—Institute of Investigation and Innovation in Health, 4200-135 Porto, Portugal

2 Ipatimup, Institute of Molecular Pathology and Immunology, University of Porto, 4200-135 Porto, Portugal

3 Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal

These authors contributed equally to this work.



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.


Journal: International Journal Molecular Sciences