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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.

Selected publications

  • Ossowski L, Reich E. Experimental model for quantitative study of metastasis. Cancer Res. 1980 Jul;40(7):2300-9.
  • Kim J, Yu W, Kovalski K, Ossowski L. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell. 1998 Aug 7;94(3):353-62.
  • Van der Horst EH, Leupold JH, Schubbert R, Ullrich A, Allgayer H: TaqMan-based quantification of invasive cells in the chick embryo metastasis assay. Biotechniques. 2004;37(6):940-2, 944, 946.
  • Leupold JH, Asangani I, Maurer GD, Lengyel E, Post S, Allgayer H: Src induces urokinase receptor gene expression and invasion/intravasation via activator protein-1/p-c-Jun in colorectal cancer. Mol Cancer Res. 2007;5(5):485-96.
  • Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, Post S, Allgayer H: MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion,intravasation and metastasis in colorectal cancer. Oncogene. 2008;27(15):2128-36.
  • Nikolova DA, Asangani IA, Nelson LD, Hughes DP, Siwak DR, Mills GB, Harms A, Buchholz E, Pilz LR, Manegold C, Allgayer H: Cetuximab attenuates metastasis and u-PAR expression in non-small cell lung cancer: u-PAR and E-cadherin are novel biomarkers of cetuximab sensitivity. Cancer Res. 2009;69(6):2461-70.
  • Ceppi P, Mudduluru G, Kumarswamy R, Rapa I, Scagliotti GV, Papotti M, Allgayer H: Loss of miR-200c expression induces an aggressive, invasive, and chemoresistant phenotype in nonsmall cell lung cancer. Mol Cancer Res. 2010;8(9):1207-16.
  • Körner A, Mudduluru G, Manegold C, Allgayer H: Enzastaurin inhibits invasion and metastasis in lung cancer by diverse molecules. Br J Cancer. 2010;103(6):802-11. Mudduluru G, Vajkoczy P, Allgayer H: Myeloid zinc finger 1 induces migration, invasion, and in vivo metastasis through Axl gene expression in solid cancer. Mol Cancer Res. 2010;8(2):159-69.
  • Rasheed SA, Efferth T, Asangani IA, Allgayer H: First evidence that the antimalarial drug artesunate inhibits invasion and in vivo metastasis in lung cancer by targeting essential extracellular proteases. Int J Cancer. 2010;127(6):1475-85.
  • Meseguer S, Mudduluru G, Escamilla JM, Allgayer H, Barettino D: MicroRNAs-10a and -10b contribute to retinoic acid-induced differentiation of neuroblastoma cells and target the alternative splicing regulatory factor SFRS1 (SF2/ASF). J Biol Chem. 2011;286(6):4150-64.
  • Mudduluru G, George-William JN, Muppala S, Asangani IA, Kumarswamy R, Nelson LD, Allgayer H: Curcumin regulates miR-21 expression and inhibits invasion and metastasis in colorectal cancer. Biosci Rep. 2011;31(3):185-97.
  • Kumarswamy R, Mudduluru G, Ceppi P, Muppala S, Kozlowski M, Niklinski J, Papotti M, Allgayer H: MicroRNA-30a inhibits epithelial-to-mesenchymal transition by targeting Snai1 and is downregulated in non-small cell lung cancer. Int J Cancer. 2012;130(9):2044-53.
  • Mudduluru G, Ceppi P, Kumarswamy R, Scagliotti GV, Papotti M, Allgayer H: Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer. Oncogene. 2011;30(25):2888-99.