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

Abstract

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.

Keywords: cancer stem cells; chicken chorioallantoic membrane; in vivo model; limiting dilution assay.

Figure 1

Organotropic breast cancer cell lines show an enriched stem cell-like signature. Enrichment analysis for stem cell gene ontology terms using the significantly deregulated gene (DEG) list between each organotropic breast cancer cell line and the parental MDA-MB-231 cell line. (a) Venn diagram showing the common and specific DEGs between the different organotropic variants. (b) Overall enrichment in stem cells gene ontology (GO) signature and (c) in stem cell-related GO terms after an analysis of the specific DEGs annotated with a stem cell function. SC: stem cell.

Figure 2

Organotropic breast cancer cells show increased in vitro cancer stem cell (CSC) properties. (a) Characterization of the cancer cell populations for CD44 and CD24 CSC markers by flow cytometry analysis in 231 breast cancer cells and its organotropic variants. (b) Percentage of the CSC population CD44⁺/CD24^−/low in 231 breast cancer cells and its organotropic variants. (c) Representative picture of monolayer (2D) and mammospheres (20× magnification). (d) Mammosphere-forming efficiency (MFE) in organotropic breast cancer cells (minimum of 4 biological replicates) compared with parental cells. (e) Fold change in cell viability and metabolic activity measured by the Presto blue assay. Five biological replicates; *—p < 0.05, **—p < 0.01 and ***—p < 0.001.

Figure 3

In vivo limiting dilution assay (LDA) workflow: a timeline of the chorioallantoic membrane (CAM) and mice experiments. Fertilized eggs are incubated for 3 days; at which time, a window in the shell is opened. At embryonic development day 9 (EDD9), breast cancer cells are inoculated on top of the CAM. At EDD16, eggs are sacrificed, and the tumor growth is examined. Mice with 6–8 weeks of age are subcutaneously injected with breast cancer cells. Tumor growth is monitored for 3 weeks. At weeks 9–11, mice are sacrificed and tumors are further examined.

Figure 4

Organotropic breast cancer cells display an increased tumorigenic ability in the choriallantoic membrane of the chick embryo (CAM). (a) Representative pictures of CAM xenografted tumors (4 cell lines × 4 cell dilutions); scale bar = 500 ¼m. (b) Tumor size evaluation in the LDA performed in the CAM by inoculations of 1-M, 100-K, 10-K and 1-K cells per egg. (c) Tumor size evaluation in a LDA performed in immunodeficient mice by subcutaneous injection of 1-M, 100-K and 10-K cells number per animal. *—p < 0.05, **—p < 0.01, ***—p < 0.001 and ****—p < 0.0001).

Figure 5

CAM and mice xenografts display similar citocellular organization and stem marker patterns. Each column regards a cell line (231, 231.LM2, 231.BoM and 231.BRMS). (C1–C4) Hematoxylin–eosin staining in the CAM xenografted tumors (63× magnification); m—Matrigel and inf—infiltrate. (M1–M4) Hematoxylin–eosin staining in the mice xenografted tumors (63× magnification). (C5–C8) CD44 labeling in CAM tumors (63 magnification). (M5–M8) CD44 labeling in mice tumors (63× magnification). (C9–C12) CD49f labeling in CAM tumors (63× magnification). (M9–M12) CD49f labeling in mice tumors (63× magnification).