The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.
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Method Article
The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma
In This Article
Summary
Abstract
The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.
Introduction
Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer mortality in the world1. Currently only 30% of HCC patients are eligible for potentially curative surgical treatments2, and systemic chemotherapy is not efficacious3. Therefore, there is a pressing unmet clinical need for novel HCC therapies, and the development of model systems suitable for efficient testing of new agents. The chick chorioallantoic membrane (CAM) assay provides a reproducible, cost-effective, and fast medium-throughput method of testing potential anti-tumor drugs in vivo.
The CAM assay has been used extens....
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Protocol
1. Egg Incubation
- Obtain 8 day old specific pathogen-free embryonated eggs.
- Place eggs in rotating egg tray, stamped ends facing upwards, and place the rotating tray inside an egg incubator. Incubate eggs for 48 hr at 36 °C and 50% humidity.
2. Dropping the CAM and Opening the Eggs
- Gather eggs from incubator. Place eggs in egg rack, stamped side up, and bring to laminar flow hood.
- Turn off the room and hood lights.
- Hold the stamped end of egg lightly to the egg candler to expose the vasculature of the CAM as well as the air sac.
NOTE: The egg candler is a light so....
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Results
Representative pictures of key steps in the protocol are shown here. Figure 1A demonstrates the use of the candler to visualize the developing embryo, the air sac, and the vasculature of the CAM. Figure 1B-1C show the process of dropping the CAM by making the two holes and then applying negative pressure using the safety bulb, and Figure 1D shows a successfully dropped CAM with a large air bubble under the pencil-marked hole. Figure 1E and Figure.......
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Discussion
Several key steps in this protocol most likely account for the improved embryonic survival as well as increased reliability of tumor growth. Dropping the CAM away from the shell by applying suction to the air sac is less invasive than other existing methods (using a needle to remove albumin from the egg, blunt dissection, etc.). Using a sterile push pin to create the two small holes required for this method is the most challenging part of the protocol. Using too much force can result in damage to the CAM and its vasculat.......
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Disclosures
A.S. receives funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.
Acknowledgements
A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Premium incubated eggs | Charles River | N/A | http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx |
| Egg incubators | GQF | Hova-Bator 2362N | |
| Rotating egg trays | GQF | 1611 Automatic Egg Turner | |
| Egg candler | Lyon | Hi-Power 950-070 | |
| Dremel 100 rotary tool with 15/16 cut-off wheel | Dremel | 100-N/7 | |
| Sterile forceps, push pin, dissection scissors, Scotch tape | |||
| Matrigel | BD Biosciences | 356234 | |
| Cryogenic vials, external thread with silicone washer | Corning | 430659 | |
| Collagenase from Clostridium histolyticum | Sigma | C9891 |
References
- Sangiovanni, A., et al. Increased survival of cirrhotic patients with a hepatocellular carcinoma detected during surveillance. Gastroenterology. 126 (4), 1005-1014 (2004).
- Lopez, P. M., Villanueva, A., Llovet, J. M.
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