Name: discovery of metastatic regulators using a rapid and quantitative intravital chick chorioallantoic membrane model
Uploaded: 2021-02-03T13:30:00
Description: Watch this Scientific Journal Video about Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model at JoVE.com

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>
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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 ...

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