Elsevier

Experimental Cell Research

Volume 328, Issue 2, 1 November 2014, Pages 314-324
Experimental Cell Research

Review Article
The chick embryo chorioallantoic membrane as a model for tumor biology

https://doi.org/10.1016/j.yexcr.2014.06.010Get rights and content

Highlights

  • •

    The chick embryo CAM is a model for tumor biology and metastasis.

  • •

    Being naturally immunodeficient, the chick embryo may receive transplantations from different tissues and species.

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    Compared with mammals models, tumor growth in the CAM is faster.

  • •

    In contrast to standard mouse models most cancer cells complete extravasation within 24 h after injections in the CAM.

Abstract

Among the in vivo models, the chick embryo chorioallantoic membrane (CAM) has been used to implant several tumor types as well as malignant cell lines to study their growth rate, angiogenic potential and metastatic capability. This review article is focused on the major compelling literature data on the use of the CAM to investigate tumor growth and the metastatic process.

Section snippets

Development and structure of the chick chorioallantoic membrane

The chicken is a well-known experimental model to study embryonic development. Chick embryos are readily accessible for visualization in ovo and experimental manipulation. Chick embryo development lasts 21 days before hatching [63].

Three extraembryonic membranes are formed during development: the yolk sac membrane, the amnion, and the chorioallantoic membrane (CAM). The CAM (Fig. 1) is formed on day 3–4 of incubation by the fusion of the chorion and the allantois and it consists of three

Natural immunodeficiency of the developing chick

Being naturally immunodeficient, the chick embryo accepts transplantation from various tissues and species without immune response. Chickens are protected by a dual immune system composed of B and T cells, controlling the antibody and cell mediated immunity, respectively. The B cells are differentiated in the bursa of Fabricius, whereas T cells are differentiated in the thymus [63], [12], [18]. The chick immune system does not begin to develop to function until about 2 weeks into its

Historical background

The CAM has long been used to study tumor growth because the chick׳s immunocompetent system is not fully developed and the conditions for rejection have not been yet established [34]. In 1911, Rous and Murphy demonstrated the growth of the Rous 45 chicken sarcoma transplanted onto the chick embryo CAM [64]. Murphy [44] in 1913 reported that mouse and rat tumors implanted onto the CAM could be maintained by continuous passage from egg to egg and described the effects of these transplantation on

Development of blood vessels in the tumor grafts and regulation of tumor angiogenesis in the CAM assay

Different tumors and cell suspensions derived from tumors have been implanted on the CAM (Table 1, Table 2) [55]. Between 2 and 5 days after tumor cell inoculation, the tumor xenografts become visible, are supplied with vessels of CAM origin, and begin a phase of rapid growth.

Knighton et al. [28] investigated the time-course of rat Walker 256 carcinoma specimens implanted on the CAM. Tumors did not exceed a mean diameter of 0.93±0.29 mm during the pre-vascular phase (approximately 72 h). Rapid

The CAM in the study of angiogenesis and anti-angiogenesis in human multiple myeloma and neuroblastoma

Plasma cell conditioned media of active multiple myeloma patients induced an angiogenic response in the CAM, inhibited by anti- fibroblast growth factor-2 (FGF-2) antibody [75]. The time-course of the angiogenic response induced by gelatin sponges soaked with multiple myeloma plasma cells on the CAM from day 8 to day 12 of incubation demonstrated that plasma cells from patients with active multiple myeloma induce a vasoproliferative response (Fig. 4), significantly higher as compared to that

Metastasis

Tumor cell metastasis involves a complex series of events that are linked both temporally and spatially (Fig. 8). The metastasis chick embryo model, based on the grafting of human tumor cells on the CAM has provided valuable information regarding tumor cell penetration of the chorionic epithelium, invasion of the mesenchyme (Fig. 9) below and the blood vessels, survival of tumor cells in the circulation, their arrest in the vasculature, extravasation, and proliferation in the distant organs [1]

Visualization and detection of tumor cells (Table 3)

Several methods for semiquantitative analysis of metastasis in the chick embryo have been developed including morphometric quantitation of individual fluorescent-labeled metastasized cells by videomicroscopy [49], [50], [39], detection of microscopic tumor colonies [39], detection of human urokinase plasminogen activator within secondary organs of the embryo [49], [50], the use of green fluorescent protein (GFP) and in vivo videomicroscopy [26], [31]. Because the human genome is uniquely

Advantages

Being naturally immunodeficient, the chick embryo may receive transplantations from different tissues and species, without immune responses. Moreover, CAM allows a rapid vascularization, development of tumor cells or bioptic specimens placed on its surface, and allows to observe and analyze the real time changes in morphology of cancer cells in its microcirculation.

In contrast to standard mouse models most cancer cells arrested in the CAM microcirculation survive without cell damage, and a

Concluding remarks

The experiments performed in the chick CAM have resulted in important progress in elucidating the mechanisms of tumor growth and metastatic spread. The main advantages of this assay are the low cost, reproducibility and reliability. The major drawback is the pre-existence of a rich vascular network and the neovascularization secondary to tumor implant or metastatic process is hardly distinguishable from a falsely increased vascular density due to a rearrangement of existing vessels or

Competing interest

The author declare that he has no competing interest.

Acknowledgments

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 278570.

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