Chorionallantoic membrane model (CAM)
The chicken embryo metastasis (CEM) or chorionallantoic membrane model (CAM) as an attractive and unique in vivo model to study specific steps of the metastatic cascade in cancer
Metastasis is a complex process which involves several defined steps, i.e. local invasion, intravasation, dissemination within the systemic circulation, extravasation and, finally, the establishment of a metastatic lesion at a distant site. However, experimental models to specifically measure at least some of these specific steps are rare, and especially in vivo models to specifically model metastasis are still desperately needed.
Therefore, besides using widely applied methods to measure, e.g., local migration and invasion (e.g., Boyden chamber migration or Matrigel invasion assays), our department specifically has developed, and offers for collaborations, the chicken embryo metastasis (CEM) model or chorionallantoic membrane (CAM) model of the chicken embryo.
Advantage of this model
This powerful in vivo model, in contrast to most of the known mouse models, has the specific ability to differentiate the distinct step of in vivo intravasation into blood vessels, from local invasion and distant metastasis formation. At the same time, the model can measure each of these distinct steps within one in vivo model. Another advantage of this model is that it is very quick and can render first in vivo results after 7-9 days, in contrast to several weeks or months with conventional mouse or other animal models. Since, legally, bred chicken eggs or chicken embryos are not regarded as animals yet, an ethical animal experimentation approval is not necessary, again saving valuable time for the investigator. Along these lines, the method is a highly interesting in vivo model to replace animal experiments, enabling researchers to perform in vivo experiments in metastasis research, thereby actively supporting the moral concept of animal protection and sparing animals for experimentation.
Briefly, the tumor cells of interest are inoculated onto the upper CAM of a standardized fertilized chicken egg bred continuously at 37°C. For measuring in vivo invasion into the upper CAM, a quantitative highly sensitive PCR for human Alu sequences is performed to detect invaded tumor cell on the chicken genomic background. Also, primary tumor formation on the upper CAM can be measured if needed. For specific assessment of intravasation, we make use of the fact that only the fraction of tumor cells able to intravasate into the amniotic vessels will finally “arrive” at the lower CAM, which again can be specifically isolated after a defined incubation time. Again, the intravasated cells can selectively and quantitatively be measured with the aforementioned PCR for human Alu sequences. Finally, we can measure metastasis formation by harvesting the liver and lungs of the chicken embryos, and analyze them for the tumor cells that have metastasized to these particular organs.
A clear further advantage of the model is that it is a closed experimental system which also allows parallel application of, e.g., novel therapeutic compounds. Thus, rapid in vivo information can be generated as to whether a compound is able to affect specific metastatic steps in vivo. Furthermore, the model enables in vivo experiments using only small amounts of testing compound.
Our specific protocol of the CAM assay is based on original semiquantitative protocols of Ossowski et al., which we advanced and modified to a highly sensitive quantitative model which is able to detect up to 1 out of 1 million tumor cells applied to the system. For further information, please refer to our several publications in which we applied the model successfully.