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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Isolation of Metastatic Colonies
<ol>
	<li>At day 5 post injection, remove embryos from the incubator and inspect the embryo CAMs for metastatic colony distribution. Identify the embryos with uniform colony distribution in which compact (or overly invasive) colonies are present. 
	NOTE: The process of culturing chicken embryos is not sterile, therefore, all the screening steps must be performed under highly clean conditions to avoid future tissue culture contamination. Contamination is quite rare and can be easily avoided by wearing gloves and a mask and using only sterile tools during cell injection and colony isolation procedures. It is recommended to inspect embryos one by one to decrease their exposure to the ambient laboratory temperature and to prevent contamination.</li>
	<li>Locate the metastatic colony of interest. A compact colony can be described as one with most of the cancer cells located within a limited area in the CAM tissue (cells appear &#34;clumped together&#34;). An invasive colony can be described as a colony where cancer cells &#34;scatter&#34; in the CAM tissue (Figure 1A, B). 
	NOTE: &#34;Compactness&#34; of the metastatic colony can be explained either by the inhibition of cancer cell invasion, inhibition of cancer cell proliferation, or both. Attention should be paid to all scenarios, and contact colonies should be isolated (Figure 1A, B). A simple fluorescent stereomicroscope can be used to discriminate between compact and invasive metastatic colony phenotypes.</li>
	<li>Under the dissection microscope, gently pull the CAM tissue that contains the metastatic colony of interest upwards using fine forceps (Figure 1C).</li>
	<li>Cut off the CAM tissue that contains the metastatic colony using surgical scissors.</li>
	<li>Transfer the CAM tissue that contains the metastatic colony into an empty, sterile 1.5 mL tube (on ice) and close the tube lid. 
	NOTE: Isolated colonies can be kept on ice for up to 3 h without loss of viability.</li>
	<li>Repeat the excision procedure until all the colonies of interest are collected into separate tubes. To avoid animal suffering, do not excise more than 2&#8211;3 colonies from one embryo. Euthanize embryos by freezing at -20 &#176;C (or another approved method) immediately after the colony excision.</li>
	<li>Gently mince the CAM tissue in a microcentrifuge tube using a sterile 18G&#160;needle. Use a separate needle for each colony.</li>
	<li>Add 100 &#181;L of 1x collagenase solution and incubate for 30 min at 37 &#176;C.</li>
	<li>Spin down the cells and CAM tissue at 300 x&#160;g&#160;for 5 min at ambient temperature.</li>
	<li>Aspirate the collagenase solution and resuspend cells in complete media used for the cell line of interest. 
	NOTE: Normally, CAM tissue is not completely dissociated after collagenase treatment, and cancer cells will first proliferate within the pieces of CAM tissue and only later migrate onto the tissue culture dish.</li>
	<li>Spin the cells and CAM tissue again at 300 x&#160;g&#160;for 5 min at ambient temperature.</li>
	<li>Resuspend cells and CAM tissue pieces in 1 mL of complete media plus selection factor (if any), then transfer into a single 12-well tissue culture dish well. 
	NOTE: Chicken CAM fibroblasts may persist in tissue culture for multiple passages inhibiting clonal expansion. The ability to expand the metastatic colonies in the presence of a selection factor (i.e., if a vector that was used to render cancer cells fluorescent also encodes a mammalian antibiotic resistance gene) may greatly speed up clonal expansion. A kill curve experiment should be performed before the screening to ensure that the proper concentration of antibiotics is used.</li>
	<li>For the next 1&#8211;3 weeks, monitor cancer cells daily for growth and contamination.</li>
	<li>When cells reach 70&#8211;80% of confluency, transfer the cells into a larger volume culture dish. 
	NOTE: It is recommended to expand cells till at least two cryogenic vials of each clone can be frozen. Maintaining large amounts of tissue culture can be laborious and unnecessary.</li>
	<li>Proceed to sequencing or next round of selection as soon as enough cancer cell numbers are reached. Generally, 1 x 106&#160;of cancer cells are sufficient for modern high-throughput sequencing techniques.</li>
	<li>Proceed to the clone reinjection and imaging and quantification of colony compactness or cancer cell-blood vessel contact (Figure 2A). Alternatively, proceed to high throughput sequencing or repeat the colony selection. 
	&#8203;NOTE: To decrease the number of false positives at least two rounds of selection are recommended. Two approaches have been successfully employed. 1) Expand each clone and reinject individually to confirm the colony phenotype. 2) Mix all the expanded clones in a 1:1 ratio each, reinject as a mixture, and repeat the selection cycle.</li>
</ol>

<table><tbody><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 BD precision&lt;br/&gt; needle</td><td>BD</td><td>BD305196</td><td>we use 1.2mm x 40mm, it is possible&lt;br/&gt; to use shorter needles if preferred</td></tr><tr><td>Benchtop centrifuge.</td><td>many sources are available</td><td></td><td>Any TC compatible centrifuge that&lt;br/&gt; can be used to spin down the cells is&lt;br/&gt; suitable</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C0130-100MG</td><td></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&lt;br/&gt; lines used</td><td>many sources are available</td><td></td><td>We grow HT1080, HEp3 and b16 cell&lt;br/&gt; 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&lt;br/&gt; depends on the scale of the screen.&lt;br/&gt; Available sources are MGF Company&lt;br/&gt; Inc., Savannah, GA, or Lyon Electric&lt;br/&gt; Company Inc., Chula Vista, CA</td></tr><tr><td>Eppendorf 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 before use</td></tr><tr><td>Image analysis software</td><td></td><td></td><td>We use Nikon Elements</td></tr><tr><td>PBS (1x)</td><td>many sources are available</td><td></td><td></td></tr><tr><td>Plastic weighting dishes</td><td>Simport</td><td>CA11006-614</td><td>dimensions are 78x78x25mm; many&lt;br/&gt; other sources are available</td></tr><tr><td>Small surgical scissors</td><td>many sources are available</td><td></td><td>must be sterilized before use</td></tr><tr><td>Stereo fluorescent microscope</td><td></td><td></td><td>We use Zeiss Lumar v12</td></tr></tbody></table>

metastatic cancer cell colony isolation from chicken cam: a procedure to isolate cancer cells from shell-less egg cultures

Source: Stoletov, K. et al. <a href="https://www.jove.com/video/62077" target="_blank">Discovery of Metastatic Regulators Using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model.</a> J. Vis. Exp. (2021)
In this video, we describe the isolation of metastatic cancer cell colonies from the chicken chorioallantoic membrane (CAM) using shell-less egg culture. This method helps screen for factors that may be responsible for cancer metastasis.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20470/20470fig1.jpg" /> 
Figure 1. Representative results for different steps of the screening protocol.&#160;(A) Metastatic colonies formed by heterogenous (library transduced cancer cell line HEp3 cells), 5 days post injection. Red arrow shows compact colony (potential positive hit) that should be excised. (B) Metastatic colonies formed by one of the isolated screen hits (<...

Metastatic Cancer Cell Colony Isolation from Chicken CAM: A Procedure to Isolate Cancer Cells from Shell-less Egg Cultures

Source: Stoletov, K. et al. Discovery of Metastatic Regulators Using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model. J. Vis. ...

The chicken chorioallantoic membrane or CAM is a tri-layered membrane located just underneath the eggshell. It consists of an outer ectoderm, a highly vascular mesoderm, and the innermost endoderm.
The CAM mesoderm comprises of blood vessels, fibroblasts, and stromal components, and thus provides a favorable environment for tumor cell colonization.
To isolate colonized cancer cells from CAM, begin by taking a shell-less egg culture pre-injected with fluorescently labeled cancer cells in a dish. Visualize the egg culture under a fluorescence microscope. Locate the fluorescent and compact metastatic cancer colonies.
Gently excise the CAM tissue containing metastatic colonies of interest. Transfer the excised CAM tissue into a microcentrifuge tube. Mince the tissue into small pieces to make the CAM accessible to enzymatic treatment in the subsequent steps.
Supplement the tube with collagenase enzyme and incubate. Collagenase degrades the collagen protein in CAM mesoderm and initiates cancer colony dissociation. Centrifuge the suspension to pelletize the cancer cells, remnants of CAM tissue, and CAM fibroblasts. Discard the collagenase-containing supernatant.&#160;
Add a selective growth media and transfer the suspension into a fresh culture plate.&#160; This media promotes the growth of suspended cancer cells over other cells. Aspirate the cancer cells and store them for further downstream analysis.

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Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

In vivo studies

1.1K Views. Source: Stoletov, K. et al. Discovery of Metastatic Regulators Using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model. J. Vis. Exp. (2021) In this video, we describe the isolation of metastatic cancer cell colonies from the chicken chorioallantoic membrane (CAM) using shell-less egg culture. This method helps screen for factors that may be responsible for cancer metastasis. 

Video: Metastatic Cancer Cell Colony Isolation from Chicken CAM: A Procedure to Isolate Cancer Cells from Shell-less Egg Cultures - Concept

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

JoVE Journal

Developmental Biology

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

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The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Medicine

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Metastatic Cancer Cell Colony Isolation from Chicken CAM: A Procedure to Isolate Cancer Cells from Shell-less Egg Cultures

Transcript

Metastatic Cancer Cell Colony Isolation from Chicken CAM: A Procedure to Isolate Cancer Cells from Shell-less Egg Cultures

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using Chick^GFPNeural Tube Grafting and Carbocyanine Dye DiI Injection

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer