This work was supported by Canadian Cancer Society Research Institute Grant #702849 to JDL and KS. Dr. Lewis holds the Frank and Carla Sojonky Chair in Prostate Cancer Research supported by the Alberta Cancer Foundation.

All experiments were performed in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee at the University of Alberta. Avian embryos are not considered to be live animals by many research institutes and no animal protocols are required. It is, however, an accepted view that avian embryos can feel pain and, therefore, must be treated as humanely as possible. The local animal research authority must be contacted prior commencing of any research work to ensure that proper regulatio...

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+The+next+generation.">Hallmarks of cancer: The next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Van't Veer, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+predicts+clinical+outcome+of+breast+cancer.">Gene expression profiling predicts clinical outcome of breast cancer.</a> Nature. 415 (6871), 530-536 (2002).</li><li>Eccles, S. A., Welch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+recent+discoveries+and+novel+treatment+strategies.">Metastasis: recent discoveries and novel treatment strategies.</a> Lancet. 369 (9574), 1742-1757 (2007).</li><li>Weber, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+metastasis.">Molecular mechanisms of metastasis.</a> Cancer Letters. 270 (2), 181-190 (2008).</li><li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nature Reviews Cancer. 2 (8), 563-572 (2002).</li><li>Pantel, K., Brakenhoff, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4 (6), 448-456 (2004).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nature Reviews Cancer. 3 (5), 362-374 (2003).</li><li>Oudin, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell-driven+extracellular+matrix+remodeling+drives+haptotaxis+during+metastatic+progression.">Tumor cell-driven extracellular matrix remodeling drives haptotaxis during metastatic progression.</a> Cancer Discovery. 6 (5), 516-531 (2016).</li><li>Leong, H. S., et al. <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> Nature Protocols. 5 (8), 1406-1417 (2010).</li><li>Kain, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+embryo+as+an+expanding+experimental+model+for+cancer+and+cardiovascular+research.">The chick embryo as an expanding experimental model for cancer and cardiovascular research.</a> Developmental Dynamics : An official Publication of the American Association of Anatomists. 243 (2), 216-228 (2014).</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 (3), 221-234 (2008).</li><li>Palmer, T. D., Lewis, J., Zijlstra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cancer+metastasis+using+an+avian+embryo+model.">Quantitative analysis of cancer metastasis using an avian embryo model.</a> Journal of visualized experiments : JoVE. (51), e2815 (2011).</li><li>Stoletov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vivo+whole+genome+motility+screen+reveals+novel+therapeutic+targets+to+block+cancer+metastasis.">Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis.</a> Nature Communications. 9 (1), 2343 (2018).</li><li>Leong, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia+are+required+for+cancer+cell+extravasation+and+are+a+therapeutic+target+for+metastasis.">Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis.</a> Cell Reports. 8 (5), 1558-1570 (2014).</li><li>Willetts, L., Bond, D., Stoletov, K., Lewis, J. D., Ursini-Siegel, J., Beauchemin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Tumor Microenvironment: Methods and Protocols. , 27-37 (2016).</li></ol>

Here we describe a rapid fluorescence microscopy based intravital screening protocol that can be utilized for important applications such as genetic or drug candidate screens. Cancer cells that have been transduced with a genetic library of interest or transfected with individual expression constructs can be rapidly screened and quantified as to phenotype of interest using this chick CAM model. Since transduction or transfection protocols vary significantly, depending on the library type, they are not included in this pr...

Metastasis is the main cause of cancer patient death1,2,3. Metastatic cancer cells utilize distinct signaling pathways, dependent on the type of cancer, throughout the five steps of the metastatic cascade: local invasion, intravasation, survival in the circulation, extravasation, and colony expansion at distant metastatic sites. Current understanding of this metastatic process suggests that there are two bottleneck steps, one is directional invasion of the cancer cell from the primary tumor, and the second is the establishment of the distant site metastatic lesion4,5,6. Both steps require cancer cells to actively interact with collagen and vasculature at the sites of initial invasion or distant metastatic lesion formation. Therefore, metastatic cancer cells must be able to attach to cells, remodel collagen fibers, and directionally invade along vascular walls7. Screening models that can rapidly identify antimetastatic therapeutic targets to block cancer cells from completing these steps is of the highest importance. Existing in vitro screening models do not fully mimic the complex live tissue environment. Mouse models are costly and time consuming. Therefore, there is an urgent need for intravital screening platforms that provide complex live tissue environments and rapid identification of targets.
Within the last decade, the chicken embryo has been established as a robust and cost-effective model of human cancer metastasis8,9,10,11,12. The chick chorioallantoic membrane (CAM) tissue is thin and translucent which makes it ideal for intravital microscopic imaging of cell and colony behaviors in primary tumor and/or metastatic sites12. Primary tumor growth from multiple human cancer cell lines can be initiated and metastasized within a span of only several days after microinjection into the CAM tissue. Cancer cells can be administered into the CAM tissue in several ways, including intravenously, intra-CAM or as collagen onplants, this flexibility allows the researcher to focus on specific stages of cancer progression, for example, metastatic lesion formation, invasion out of the primary tumor or angiogenesis.
Here we describe a quantitative screening platform that can be used to measure the ability of cancer cells to establish invasive metastatic lesions. Cancer cells that have been transduced with an expression library are injected intravenously into the CAM vasculature at low density. Metastatic colonies form for 4-5 days, then the invasion capacity and vasculature interaction of the resulting colonies is evaluated. Individual colonies that fail to invade are excised, propagated and their phenotype is confirmed in the CAM by reinjection and quantification of colony compactness and cancer cell-blood vessel contacts. The expression library constructs responsible for the single metastatic colony mutant phenotype are identified from isolated colony genomic DNA via high throughput sequencing. The same platform may be further used to validate the causal link between a gene and the observed phenotype or to perform in-depth mechanistic studies on the observed phenotype.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL disposable syringes</td>
			<td>BD</td>
			<td>BD309659</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tubes.</td>
			<td>Corning</td>
			<td>CLS430791-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge x 1 1/2&#160; BD precision needle</td>
			<td>BD</td>
			<td>BD305196</td>
			<td>we use 1.2mm x 40mm, it is possible to use shorter needles if preferred</td>
		</tr>
		<tr>
			<td>2.5% Trypsin solution</td>
			<td>many sources are available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4T1 mouse breast cancer</td>
			<td>ATCC</td>
			<td>CRL-2539</td>
			<td></td>
		</tr>
		<tr>
			<td>B16F10 mouse melanoma cell line</td>
			<td>ATCC</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge.</td>
			<td>many sources are available</td>
			<td></td>
			<td>Any TC compatible centrifuge that can be used to spin down the cells is suitable</td>
		</tr>
		<tr>
			<td>Circular coverslips, 22 mm.</td>
			<td>Fisher Scientific</td>
			<td>12-545-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C0130-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td></td>
			<td></td>
			<td>We use Nikon A1r</td>
		</tr>
		<tr>
			<td>cotton swabs</td>
			<td>many sources are available</td>
			<td></td>
			<td>must be sterilized before use</td>
		</tr>
		<tr>
			<td>Culture media appropriate for the cell lines used</td>
			<td>many sources are available</td>
			<td></td>
			<td>We grow HT1080, HEp3 and b16 cell lines in DMEM, 10% FBS media</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>many sources are available</td>
			<td></td>
			<td>An exact model that is necessary depends on the scale of the screen. Available sources are MGF Company Inc., Savannah, GA, or&#160; Lyon Electric Company Inc., Chula Vista, CA</td>
		</tr>
		<tr>
			<td>eppendor tubes , 1.5ml</td>
			<td>Sigma</td>
			<td>T4816-250EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertilized White Leghorn eggs</td>
			<td>any local supplyer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>many sources are available</td>
			<td></td>
			<td>must be sterilized begfore use</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Millipore-Sigma</td>
			<td>MDH-4N1-50PK</td>
			<td></td>
		</tr>
		<tr>
			<td>HT1080 human fibrosarcoma cell line</td>
			<td>ATCC</td>
			<td>CCL-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td></td>
			<td></td>
			<td>We use Nikon Elements</td>
		</tr>
		<tr>
			<td>Lectin Lens Culinary Agglutinin (LCA) conjugated with Fluorescein or Rhodamine&#160;</td>
			<td>Vector Laboratories</td>
			<td>RL-1042, FL-1041</td>
			<td>Dilute stock (5mg/ml) 50-100x depending on the microscope sensetivity. Must be a different color from the color of cell line used for screening</td>
		</tr>
		<tr>
			<td>MDA-MB-468 human breast cancer</td>
			<td>ATCC</td>
			<td>HTB-132</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>many sources are available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic weighting dishes&#160;</td>
			<td>Simport</td>
			<td>CA11006-614</td>
			<td>dimensions are 78x78x25mm; many other sources are available</td>
		</tr>
		<tr>
			<td>small surgical scissors&#160;</td>
			<td>many sources are available</td>
			<td></td>
			<td>must be sterilized before use</td>
		</tr>
		<tr>
			<td>Sodium borosilicate glass capillary tubes, outer diameter 1.0 mm, inner diameter 0.58 mm, 10 cm length&#160;</td>
			<td>Sutter Instrument</td>
			<td>BF100-58-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Square petri dishes (used as lids for the weighting dishes).</td>
			<td>&#160;VWR&#160;</td>
			<td>CA25378-115&#160;</td>
			<td>dimensions are 100x100x15mm; many other sources are available</td>
		</tr>
		<tr>
			<td>Stereo fluorescent microscope&#160;</td>
			<td></td>
			<td></td>
			<td>We use Zeiss Lumar v12</td>
		</tr>
		<tr>
			<td>Tygon R-3603 laboratory tubing</td>
			<td>Cole-Parmer</td>
			<td>AAC00001</td>
			<td>1/32 in inner diameter, 3/32 in. outer diameter, 1/32 in. wall thickness</td>
		</tr>
		<tr>
			<td>U-118 MG human glioblastoma</td>
			<td>ATCC</td>
			<td>HTB-15</td>
			<td></td>
		</tr>
		<tr>
			<td>U-87 MG human glioblastoma</td>
			<td>ATCC</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical pipette puller&#160;</td>
			<td>many sources are available</td>
			<td></td>
			<td>we use David Kopf Instruments, Tujunga, CA; Model 720</td>
		</tr>
	</tbody>
</table>

discovery of metastatic regulators using a rapid and quantitative intravital chick chorioallantoic membrane model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

Cancer cell injection is considered to be successful if the majority of the cells that are lodged in the capillaries are single and located at a significant difference from each other (~0.05-0.1 cm) so the colonies will not overlap after 5-6 days of incubation period (Figure 3A). The injection was not successful if a buildup of cancer cells can be seen in most of capillaries, embryos displaying this should be discarded (Figure 2E...

Watch this Scientific Journal Video about Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model at JoVE.com

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Our protocol permits an efficient identification of gene or gene combinations that block cancer invasion in viewer. over culture does not require advanced animal facilities for maintenance. As a whole, as a generic or general special libraries can be screened in a matter of several weeks in viewer.This over screening system will help researchers discover novel therapeutic gene targets or gene targets combinations that it can be used to block cancer metastasis. For IV injection, concentrate cells to 0.5 to 1 million cells per milliliter, using ice cold 1X PBS to dilute or re-suspend the cells. Expect approximately one milliliter of cell suspension will be needed for every 10 embryos.To assemble the injection apparatus, mount a needle onto the syringe, and then extend the syringe needle with a three to five centimeter long piece of tubing. Break the tip of the borosilicate needle using fine forceps. Load the syringe with 50 to 200 microliters of the cancer cell suspension.And insert the borosilicate needle into the tubing. Inspect the needle for cell clogging and air bubbles. If cell clogging is observed within the capillary, remove the capillary, re-suspend the cells and replace it with a new capillary.Use the plunger to push out any bubbles. Remove the cover lid and transfer the embryo under the stereoscope. Identify the appropriate vein to be injected on the CAM surface;which is normally only slightly wider than the diameter of the borosilicate needle tip and located midway between the embryo and the weighing dish wall.It is generally easier to inject at the point immediately adjacent to the vein bifurcation. Press the tip of the needle against the blood vessel wall and apply gentle pressure in the same direction as the blood flow. If necessary, use a cotton swab to help anchor or stabilize the vessel that is being injected.Gently depress the syringe plunger for 2 to 10 seconds until the desired volume is injected. Discard the embryo if excessive bleeding or clear liquid accumulation appears at the injection site. Remove the needle from the CAM and gently dab the injection site with a cotton swab to remove any blood or excess cancer cells.Cover the injected embryo in the weighing dish with the lid and return it to the incubator. Repeat the procedure until all embryos are injected. Visually inspect the embryos for bacteria or mold contamination or death.And discard contaminated embryos according to laboratory disposal procedures. Ensure that metastatic colonies appear uniform in shape during the first one to three days post-injection. And identify invasive or noninvasive metastatic colonies at days four to five post-injection.At day five post-injection, remove the embryos from the incubator and inspect and locate the embryo CAMs for metastatic colony distribution. Under the dissection microscope, gently pull the CAM tissue that contains the metastatic colony of interest upwards using fine forceps, and cut it off with surgical scissors. Transfer the CAM tissue into an empty, sterile 1.5 milliliter tube and close the tube lid.Repeat the excision procedure until all the colonies of interest are collected into separate tubes. Gently mince the CAM tissue in a micro centrifuge tube, using a separate sterile 18 gauge needle for each colony. Add 100 microliters of 1X collagen-A solution and incubate for 30 minutes at 37 degrees Celsius.Spin down the cells and CAM tissue at 300 times G for five minutes at ambient temperature. Aspirate the collagen-A solution and re-suspend the cells incomplete media for the cell line of interest. Then spin the cells and tissue again.Re-suspend cells and tissue pieces in one milliliter of complete media and selection factor, if any. Then transfer into a single 12-well tissue culture dish well. For the next one to three weeks, monitor the cancer cells daily for growth and contamination.When the cells reach 70 to 80%of confluency transfer them into a larger volume culture dish. Proceed to sequencing or the next round of selection, as soon as adequate cancer cell numbers are reached. Embryos displaying a buildup of cancer cells due to unsuccessful injection, can be seen in most of the capillaries.When the optimal cell concentration and duration of injection is achieved, day five post-injection transduced cells should produce a wide variety of colony phenotypes, with the majority of the colonies invasive as determined by the cancer cells appearing scattered in the CAM tissue. Attention should be paid to the metastatic colonies that appear compact and are located far enough from neighboring colonies that they can be excised with forceps and scissors and one piece of CAM tissue. An isolated positive screen hit should display the compact colony phenotype upon re-injection.For measurement of the cancer cell blood vessel contacts, attention should be paid to the vascular wall stain brightness and the appropriate amount of lectins should be injected for the vascular wall signal. When attempting this protocol avoid under or over injection and remove excessive bleeding or liquid buildup. Several novel genes that are driving cancer cell invasion in viewer have been identified using this technique.

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Cancer Research

2.5K vistas.  University of Alberta, Edmonton, Alberta, Canada. Nuestro protocolo permite una identificación eficiente de genes o combinaciones de genes que bloquean la invasión del cáncer en el espectador. el sobreculto no requiere instalaciones avanzadas para el mantenimiento de los animales. En su conjunto, como un genérico o general, las bibliotecas especiales se pueden proyectar en cuestión de varias semanas en el visor.Este sistema de detección ayudará a los investigadores a descubrir nuevos objetivos genéticos terapéuticos o combina...