Modeling Cancer in the chicken embryo The chicken embryo chorioallantoic membrane system is one of the earliest models used to grow xenografts, dating back to the early 1920's. Various human tissues have been grown in the CAM including skin, various tumor types and cornea. The CAM is a very efficient, timesaving and cost-effective system for the purpose of intital screening of in vivo properties of tumor cells. Further, due to the absence of a mature immune system and the presence of a rich vascular bed (see Fig6 and 7), the CAM provides a favorable mileu for the growth of several human tumor cell lines. Various assays have been designed to study specific steps of tumor progression on the CAM such as primary tumor growth, local invasion, intravasation, lung or liver metastasis and angiogenesis. To follow the growth of a primary tumor because the CAMs system only provides a window of ~7 days to monitor growth, tumors must be transplanted onto new CAMs to follow a single tumor cell population. Fig6.The image on the left shows an embryonated egg processed to prepare a CAM for tumor growth. Note the easy access that the “window” offers and the rich vasculature present in the CAM tissue. The image on the right shows an epidermoid human tumor (~200 mg) grown for 6 days on a CAM and imaged after opening the CAM. Note the rich vasculature surrounding and reaching into the tumor. Click on image to see enlarged image Fig7.Images obtained by fluorescence microscopy showing the vasculature of the CAM system 30 min. after injection of dextran-FITC into the blood stream of a CAM. The upper left image shows a large vessel used for injection site and the lymphatics surrounding this vessel are easily viewed. The upper right image shows the microvasculature of the CAM tissue a few cm away from the site of injection. Note the rich branching of the CAM tissue. The lower left image shown blood vessels in the chick embryo heart highlighted by the dextran-FITC. The lower right image shows the vasculature in the lower limbs of the chick embryo. Tumor cells that disseminate to the lungs for example and that are labeled with GFP can be easily detected with these techniques. The CAM system consists of 9-10 day old chick embryo CAMs that are inoculated with tumor cells. In a freshly prepared CAM the double epithelium that is part of the CAM is “wounded” exposing an ECM-rich (e.g., fibronectin, collagens, heparan sulphate) stromal tissue composed mostly of fibroblasts and both vascular and lymphatic endothelial cells that form the vast vascular network of the CAM (Fig 6 and 7). Upon inoculation, tumor cells encounter this microenvironment and growth can be monitored daily. Because of its rich vascular bed and due to the lack of a mature immune system, the growth of numerous human cell lines on CAMs is well supported and tumors develop quite efficiently. Early vascular dissemination (intravasation) can be monitored by recovering the CAM tissue from the “lower CAM” or by excising and inspecting livers and lungs. Cells can be detected by Alu-PCR amplification, regrowth in culture or on new CAMs or by imaging a live tag such as GFP. This system allows the assessment of the behavior of various cancer cell lines in vivo in a highly economical manner. Furthermore, due to the easy access to the tumor growth site (Fig3, 6 and 7) imaging can be readily performed. The CAM system allows to rapidly and efficiently test in vivo for example the function of genes that may promote or inhibit tumor growth. Our lab is using this system to readily screen for gene function using small interfering RNAs (siRNAs) that target known or unknown genes and determine their contribution to the malignant behavior of tumor cells.

The egg "farm"

Our lab is equipped with an incubator system that allows us to incubate embryonated chicken eggs for tumor xenograft and angiogenesis studies. We have a capacity to simultaneously incubate and test ~200 eggs for in vivo CAM experiments.