We want to recognize Tyson Foods Inc. for generously providing the fertilized eggs. Shanna Arnold was instrumental during the filming of this protocol. 
Trenis Palmer was supported by the National Institutes of 
Health under the Ruth L. Kirschstein National Research Service Award (F31 National Cancer Institute). This work was partially supported by CCSRI Grant #700537 to JDL 
and NIH/NCI grant #CA120711-01A1 and CA120711-01A1 to AZ.

1. Preparing the eggs for xenografting (spontaneous metastasis)
<ol>
<li>Freshly laid fertilized chicken eggs are incubated in a rotating incubator for 10 days at 100&deg;F and 60% humidity. The eggs are rotated four times each hour.</li>
<li>On developmental day 10 the eggs are placed on their side in an egg rack. A goose-neck lamp or other suitable light source is used to candle the eggs by shining the light into the eggshell at the blunt end of the egg where the airsac is located.</li>
<li>The chorioallantoic vein ...

<ol>
	<li>Ghiso, A. g. u. i. r. r. e., Kovalski, J. A., K, ., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+dormancy+induced+by+downregulation+of+urokinase+receptor+in+human+carcinoma+involves+integrin+and+MAPK+signaling.">Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling.</a> J Cell Biol. 147, 89-104 (1999).</li><li>Chambers, A. F., Shafir, R., Ling, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+system+for+studying+metastasis+using+the+embryonic+chick.">A model system for studying metastasis using the embryonic chick.</a> Cancer research. 42, 4018-4025 (1982).</li><li>Chambers, A. F., Wilson, S. M., Tuck, A. B., Denhardt, G. H., Cairncross, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+metastatic+properties+of+a+variety+of+mouse,+rat,+and+human+cells+in+assays+in+nude+mice+and+chick+embryos.">Comparison of metastatic properties of a variety of mouse, rat, and human cells in assays in nude mice and chick embryos.</a> In Vivo. 4, 215-219 (1990).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+effect+of+matrix+metalloproteinase+down-regulation+on+vascular+intravasation+and+metastasis+of+human+fibrosarcoma+cells+selected+in+vivo+for+high+rates+of+dissemination.">Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.</a> Cancer Res. 65, 10959-10969 (2005).</li><li>Hahn-Dantona, E., Ramos-DeSimone, N., Sipley, J., Nagase, H., French, D. L., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+proMMP-9+by+a+plasmin/MMP-3+cascade+in+a+tumor+cell+model.+Regulation+by+tissue+inhibitors+of+metalloproteinases.">Activation of proMMP-9 by a plasmin/MMP-3 cascade in a tumor cell model. Regulation by tissue inhibitors of metalloproteinases.</a> Ann N Y Acad Sci. 878, 372-387 (1999).</li><li>Kim, J., Yu, W., Kovalski, K., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+specific+proteases+in+cancer+cell+intravasation+as+revealed+by+a+novel+semiquantitative+PCR-based+assay.">Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.</a> Cell. 94, 353-362 (1998).</li><li>Leong, H. S., Steinmetz, N. F., Ablack, A., Destito, G., Zijlstra, A., Stuhlmann, H., Manchester, M., Lewis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+embryonic+and+tumor+neovasculature+using+viral+nanoparticles.">Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles.</a> Nat Protoc. 5, 1406-1417 (2010).</li><li>Lewis, J. D., Destito, G., Zijlstra, A., Gonzalez, M. J., Quigley, J. P., Manchester, M., Stuhlmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+nanoparticles+as+tools+for+intravital+vascular+imaging.">Viral nanoparticles as tools for intravital vascular imaging.</a> Nat Med. 12, 354-360 (2006).</li><li>Ossowski, L., Reich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+malignant+phenotype+of+a+human+carcinoma+conditioned+by+growth+environment.">Changes in malignant phenotype of a human carcinoma conditioned by growth environment.</a> Cell. 33, 323-333 (1983).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nat Protoc. 3, 1101-1108 (2008).</li><li>Testa, J. E., Brooks, P. C., Lin, J. M., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+expression+cloning+with+an+antimetastatic+monoclonal+antibody+identifies+a+tetraspanin+(PETA-3/CD151)+as+an+effector+of+human+tumor+cell+migration+and+metastasis.">Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis.</a> Cancer Res. 59, 3812-3820 (1999).</li><li>Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Inhibition+of+Tumor+Cell+Intravasation+and+Subsequent+Metastasis+via+Regulation+of+In+Vivo+Tumor+Cell+Motility+by+the+Tetraspanin+CD151.">The Inhibition of Tumor Cell Intravasation and Subsequent Metastasis via Regulation of In Vivo Tumor Cell Motility by the Tetraspanin CD151.</a> Cancer Cell. 13, 221-234 (2008).</li><li>Zijlstra, A., Mellor, R., Panzarella, G., Aimes, R. T., Hooper, J. D., Marchenko, N. D., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+rate-limiting+steps+in+the+metastatic+cascade+using+human-specific+real-time+polymerase+chain+reaction.">A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction.</a> Cancer Res. 62, 7083-7092 (2002).</li></ol>

No conflicts of interest declared.

The chick CAM has been used for decades as a model system to study various aspects of cancer biology and metastatic progression. Principle evaluation of metastatic abilities have been performed in this model by a variety of groups including the analysis of dormancy (Aguirre Ghiso et al., 1999; Ossowski and Reich, 1983), tissue remodeling (Deryugina et al., 2005; Hahn-Dantona et al., 1999), and metastasis (Zijlstra et al., 2008; Zijlstra et al., 2002). It continues to be a very...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>1X Dulbecco's Modified Eagle Medium (DMEM)</td>
 <td>Invitrogen</td>
 <td>11995073</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X), liquid</td>
 <td>Invitrogen</td>
 <td>14190250</td>
 <td>pH 7.4</td>
 </tr>
 <tr>
 	<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
 <td>Invitrogen</td>
 <td>25300054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Sportsman hatcher</td>
 <td>Berry Hill</td>
 <td>1550HA</td>
 <td rowspan="2">These incubators are some of the most readily available. However, the video will show different equipment.</td>
 </tr>
 <tr>
 	<td>Sportsman incubator </td>
 <td>Berry Hill</td>
 <td>1502EA</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Dremel rotary tool</td>
 <td>Dremel</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Dremel cutoff wheels no. 36 </td>
 <td>Dremel</td>
 <td>409</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Portable Pipette Aid</td>
 <td>Fisher</td>
 <td>1368115E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Cotton Tipped Applicators</td>
 <td>Fisher</td>
 <td>23-400-001)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fertilized Eggs</td>
 <td>Various</td>
 <td>&nbsp;</td>
 <td>Many different vendor can be used. The eggs should be approximately 35-55 grams. The hens that lays them should be between 36 and 55 weeks of age.</td>
 </tr>
 <tr>
 	<td>Hemocytometer</td>
 <td>Hausser Scientific, VWR</td>
 <td>15170-090</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fiber-optic microscope illuminator</td>
 <td>Amscope</td>
 <td>HL250-AY</td>
 <td>150W</td>
 </tr>
 <tr>
 	<td>25 gauge needle</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Sybr Green Extract&#x2013;N-Amp Tissue PCR Kit</td>
 <td>Sigma-Aldrich</td>
 <td>XNATRG</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

quantitative analysis of cancer metastasis using an avian embryo model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Watch this Scientific Journal Video about Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model at JoVE.com

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

quantitative-analysis-of-cancer-metastasis-using-an-avian-embryo-model

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Medicine

Unit 1

Cancer

SCIENTISTS IN ACTION

18.9K visualizações.  Vanderbilt University. Usando PCR quantitativo, demonstramos como o bem-estabelecida pinto modelo CAM pode ser usado para analisar quantitativamente a metástase de células tumorais para órgãos distantes.