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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. Preparation of the Cancer Cells for Injection
NOTE: Metastatic colony selection is based on a phenotype established by fluorescent cancer cells, derived from an expression library or vector tagged with fluorescent protein such as green or red fluorescent protein (see the whole screen flow in&#160;Figure 1A). It is not recommended to use cells transiently transfected with fluorescent proteins or labeled with cell permeable fluorescent dyes due to rapid fading of signal caused by cancer cell proliferation and uneven staining.
<ol>
	<li>Culture cancer cells to 60&#8211;70% confluency at the time of injection. Using cancer cells at higher levels of confluency will decrease their viability, resulting in low efficiency of metastatic colony formation. Ensure that the cancer cells used are mycoplasma-free since mycoplasma contamination may lead to decreased chicken embryo viability.</li>
	<li>Rinse cells twice with 1x PBS, pH 7.4. Remove the remaining PBS, then add 0.5% Trypsin-EDTA and incubate at 37 &#176;C for 2&#8211;5 min until all cells are lifted from the culture dish surface.</li>
	<li>Transfer the cell suspension into a 15 mL conical tube and centrifuge cells at room temperature at 200 x&#160;g&#160;for 5 min.</li>
	<li>Resuspend cells in 10 mL of PBS to and centrifuge the cells again as in step 1.3, to remove trypsin and other culture media components such as antibiotics. 
	NOTE: The presence of trypsin or cell culture components such as antibiotics in the cancer cell suspension may result in decreased chicken embryo viability.</li>
	<li>Carefully aspirate the supernatant and resuspend cells with 1 mL of ice-cold PBS.</li>
	<li>Count the number of cells using a hemocytometer or any other cell counting equipment available. 
	NOTE: This screening protocol requires that each metastatic colony be initiated by a single cell. When counting the cells make sure that the cells are fully trypsinized and exist as a single cell suspension (no cell clumps are present).</li>
	<li>For intravenous (IV) injection, concentrate cells to 0.5 x 106&#160;&#8211; 1.0 x 106&#160;cells/mL. Use ice-cold 1x PBS to dilute and/or resuspend cell concentrates. Expect that approximately 1 mL of cell suspension will be needed for every ten embryos. 
	NOTE: Necessary cell suspension concentration in suspension is mainly determined by the level of experimenter experience. Please see the next section for more details.</li>
</ol>
2. Intravenous Injection of Cancer Cells for Metastatic Colony Formation
NOTE: Following this protocol as many as hundred embryos can be injected within one day. It generally takes longer time to isolate the metastatic colonies than to inject the cancer cells and, therefore, it is recommended to perform an initial experiment to estimate the time needed to complete all the steps. Using differential cancer cell fluorescent labels helps to reduce animal numbers and the time required for each experiment. For example, control cells can be labeled with RFP (red fluorescent protein) while mutant tumor cells can be alternatively labeled with GFP (green fluorescent protein). In this case, each experiment has a built-in control that corrects for inter-embryo variability.
<ol>
	<li>Assemble the injection apparatus as shown in&#160;Figure 1B. First, mount a needle onto the syringe and then extend the syringe needle with a 3&#8211;5 cm long piece of tubing.</li>
	<li>Break the tip of the borosilicate needle off using the fine forceps (~20&#8211;60 &#181;L wide).</li>
	<li>Load the syringe with the cancer cell suspension (50&#8211;200 &#181;L) and insert the borosilicate needle into the tubing (Figure 1B).</li>
	<li>Inspect the borosilicate needle for cell clogging and air bubbles. It is critical to inject cells as a single cell suspension to ensure that each metastatic colony is initiated by a single cell. 
	NOTE: Cancer cells tend to aggregate in PBS within 1&#8211;2 h post trypsinization and, therefore, should be prepared immediately before injection. If necessary, cells can be resuspended periodically using the 1 mL syringe used for injections (without the borosilicate needle).</li>
	<li>If cell clogging is observed within the capillary, remove the capillary, resuspend the cells, and replace it with a new capillary. Use the plunger to push out any bubbles. If no cell clogging or bubbles are observed, proceed to the next step.</li>
	<li>Remove the cover lid and transfer the embryo under the stereoscope. Identify the appropriate vein to be injected on the CAM surface. Veins and arteries form the intricate network within the CAM tissue (Figure 1C). Veins are distinguished by the brighter red color because they carry oxygen-rich blood to the embryo. 
	NOTE: For general embryo manipulation, cancer cell injection and metastatic colony excision, use a fluorescent stereomicroscope equipped with 0.8x and 1.5x objectives and 10x eyepieces for visualization. Less advanced microscopes can also be successfully used for these procedures, depending on the users&#39; experience level. Readers can contact the authors for more detailed recommendations.</li>
	<li>Find the ideal vein to inject cells that is normally only slightly wider (10&#8211;20%) than the diameter of 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. 
	NOTE: Injection into a larger vein may appear easier but will lead to excessive bleeding and decreased embryo survival.</li>
	<li>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 (held in other hand) to help anchor or stabilize the vessel that is being injected.</li>
	<li>Gently depress the syringe plunger. One can visualize a successful injection by observing a &#34;clearing&#34; of the blood vessel when the cancer cell suspension enters the bloodstream.</li>
	<li>Continue depressing the syringe plunger for 2&#8211;10 s until the desired suspension volume is injected into the bloodstream of the vein. All together the injection of a single embryo may require 1&#8211;10 min depending on the experience level of the user. If excessive bleeding or clear liquid accumulation appears at the injection site, discard the embryo. 
	NOTE: It is easy to &#34;over-inject&#34; an embryo. The general consideration that should be kept in mind is that metastatic colonies should not touch each other after a 4&#8211;5 days growth period. Ideally, ~5&#8211;10% of terminal CAM vein capillaries should have cells immobilized in them after a successful injection (Figure 1D, E). As mentioned in the section 1, a range of cell concentration can be used (0.5 x 106&#160;&#8211; 1.0 x 106&#160;cells/mL). Lower concentrations will require longer injection time but will decrease the needle clogging. Higher concentrations will require shorter injection times but will increase the needle clogging. It is recommended to try several concentrations to find the one that is most comfortable for an operator. Generally, a higher concentration (1.0 x 106&#160;cells/mL) is preferred to decrease the injection time.</li>
	<li>Remove the needle from the CAM and gently dab the injection site with a cotton swab to remove any blood or excess cancer cells.</li>
	<li>Cover the embryo in the weighing dish with a lid and return the injected chicken embryo into the incubator.</li>
	<li>Repeat the procedure with the next embryo until all embryos are injected.</li>
</ol>

<table><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&amp;nbsp; 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>HT1080 human fibrosarcoma cell line</td><td>ATCC</td><td>CCL-121</td><td></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&amp;nbsp; 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 supplier</td><td></td><td></td></tr><tr><td>Plastic weighting dishes&amp;nbsp;&amp;nbsp;</td><td>Simport&amp;nbsp;</td><td>CA11006-614</td><td>Dimensions are 78x78x25mm; many other sources are available</td></tr><tr><td>Square petri dishes (used as lids for the weighting dishes)&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>CA25378-115&amp;nbsp;</td><td>Dimensions are 100x100x15mm; many other sources are available</td></tr><tr><td>Stereo fluorescent microscope&amp;nbsp;</td><td></td><td></td><td>We use Zeiss Lumar v12</td></tr><tr><td>Sodium borosilicate glass capillary tubes, outer diameter 1.0 mm, inner diameter 0.58 mm, 10 cm length&amp;nbsp;&amp;nbsp;</td><td>Sutter Instrument</td><td>BF100-58-10</td><td></td></tr><tr><td>Tygon R-3603 laboratory tubing&amp;nbsp;&amp;nbsp;</td><td>Cole-Parmer</td><td>AAC00001&amp;nbsp;</td><td>1/32 in inner diameter, 3/32 in. outer diameter, 1/32 in. wall thickness</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>Hemocytometer&amp;nbsp;</td><td>Millipore-Sigma</td><td>MDH-4N1-50PK</td><td></td></tr></tbody></table>

intravenous injection of cancer cells into chicken chorioallantoic membrane vasculature: a procedure to establish chorioallantoic membrane cancer model to study cancer invasion and metastasis

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)
This video demonstrates the procedure of intravenously injecting cancer cells into the chicken chorioallantoic membrane vasculature to generate a CAM cancer metastasis model. The injected cells eventually form scattered single-cancer cell colonies, allowing for the study of cancer invasion and metastasis.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20469/20469fig1.jpg" /> 
Figure 1: Outline of cancer cell injection and metastatic colony isolation.&#160;(A) Flowchart outlining the chicken embryo screening platform steps. (B) Cancer cell injection set up on the stereo fluorescent microscope stage. (C) Injection of the cancer cells into the CAM vasculature. (D) Image showing successful can...

Intravenous Injection of Cancer Cells into Chicken Chorioallantoic Membrane Vasculature: A Procedure to Establish Chorioallantoic Membrane Cancer Model to Study Cancer Invasion and Metastasis

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&#160;CAM, of a fertilized chicken egg is the extra-embryonic membrane supported by a rich vascular network. This highly vascularized membrane lining the non-vascular shell membrane facilitates the exchange of gases and nutrients with the developing embryo.
To generate a CAM cancer model in vitro, begin with a viable, fertilized, shell-less chicken embryo bearing a fully developed CAM in a dish.
Next, assemble the injection apparatus containing a tubing attached to a syringe carrying a single-cell suspension of transduced cancer cells in a suitable chilled buffer. Connect a customized glass needle to the tubing for high precision delivery during subsequent injection.&#160;
Now, transfer the embryo under a microscope. Identify a suitable vein close to the bifurcation point on the CAM surface. Inject the cancer cell suspension into the selected vein and ensure complete delivery. Gently retract the needle. Cover the embryo and incubate it under appropriate conditions.
The dense vasculature within the mesodermal layer harbors the injected tumor cells, facilitating primary tumor formation. Eventually, some of these cells migrate through the vasculature and extravasate, invading the surrounding stroma, where they proliferate to form metastatic foci.

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Head and Neck Cancer

In vivo studies

1.2K Views. Source: Stoletov, K. et al. Discovery of Metastatic Regulators Using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model. J. Vis. Exp. (2021) This video demonstrates the procedure of intravenously injecting cancer cells into the chicken chorioallantoic membrane vasculature to generate a CAM cancer metastasis model. The injected cells eventually form scattered single-cancer cell colonies, allowing for the study of cancer invasion and metastasis. 

Video: Intravenous Injection of Cancer Cells into Chicken Chorioallantoic Membrane Vasculature: A Procedure to Establish Chorioallantoic Membrane Cancer Model to Study Cancer Invasion and Metastasis - Concept

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

JoVE Journal

Medicine

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.

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

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 ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Intravenous Injection of Cancer Cells into Chicken Chorioallantoic Membrane Vasculature: A Procedure to Establish Chorioallantoic Membrane Cancer Model to Study Cancer Invasion and Metastasis

Transcript

Intravenous Injection of Cancer Cells into Chicken Chorioallantoic Membrane Vasculature: A Procedure to Establish Chorioallantoic Membrane Cancer Model to Study Cancer Invasion and Metastasis

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

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

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis