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EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparing the cancer cell suspension for transplantation (option 1)
NOTE: This is to be completed just prior to the implantation, which should ideally take place between days 7 and 10. Please see notes at the beginning of step 3 or 4 for further information concerning the implantation date. This approach was used for all the cell lines and cultured kidney cancer tumor digests.
<ol>
	<li>Thaw the extracellular matrix solution on ice.</li>
	<li>Using mechanical and/or enzymatic digestion, obtain a single cell suspension using a method appropriate for the cell type being implanted.</li>
	<li>Resuspend the total number of cells to be implanted in an appropriate medium for implantation. Implants of 1-2 x 106&#160;cells per egg are typical. 
	NOTE: The medium used for the implantation of the cell lines is typically the complete medium used for culturing the cells, containing FBS or serum replacement. The implantation of tumor digests typically uses the complete medium used for culturing cell lines of the same cancer type. However, adjustments to the medium formulation may be made if needed experimentally. The effects on tumor development and growth would need to be empirically determined.</li>
	<li>Pellet the cells using a centrifuge speed and time appropriate for the cells being used. Typical speeds are 250-300 x&#160;g, and times are 5-10 min.</li>
	<li>Remove the supernatant from the pelleted cells by pipetting. Mechanically resuspend the cells in the residual medium via flicking or pipetting. Place on ice to cool. Measure the volume of cells and medium using an appropriately sized pipette.</li>
	<li>Calculate implantation volumes such that 1-2 x 106&#160;cells are implanted in a volume of 20-100 &#956;L per egg with a final extracellular matrix concentration of 2.7-4 mg/mL protein. Add medium, any desired growth factors or additives, and extracellular matrix solution according to this calculation. Keep on ice until ready to implant. 
	NOTE: For the calculations in steps 1.3 and 1.6, an extra volume of at least one-half egg implant should be incorporated to ensure an adequate volume for all eggs in the group. For implantation of the ovarian cancer cell lines (i.e., SKOV3 and ID8), 106&#160;cells per egg were implanted. For implantation of the renal cell carcinoma cell line RENCA, cultured cells derived from digested primary human renal cell carcinoma, prostate cancer cell lines (i.e., CWR, C4-2, and MyC-CaP), and bladder cancer cell lines (i.e., HT-1376 and T24), 2 x 106&#160;cells were implanted.</li>
</ol>
2. Preparing tumor pieces for implantation (option 2)
NOTE: This is to be completed just prior to implantation, which should ideally take place between days 7 and 10. Please see notes at the beginning of step 3 or 4 for further information concerning the implantation date. Primary ovarian and bladder cancers were implanted as tumor pieces.
<ol>
	<li>Thaw extracellular matrix solution on ice. Dilute to a final protein concentration of 2.7-4 mg/mL in an appropriate medium containing any desired growth factors or additives. Keep on ice. 
	NOTE: Implantation of tumor pieces typically uses the complete medium used for culturing cell lines of the same cancer type. However, adjustments to the medium formulation may be done if needed experimentally. The effects on tumor engraftment and growth would need to be empirically determined.</li>
	<li>Using a scalpel or scissors, excise pieces from the fresh tumor. Ideal sizes range from 2-5 mm on each side. Keep tissues immersed in the medium until ready to implant.</li>
</ol>
3. Implantation using a nonstick ring (option 1)
NOTE: Cells may be implanted beginning on development day 7 if the CAM is fully developed. Implantation can occur any time prior to hatching that permits adequate time for tumor development and the desired experiment but note that the embryo&#39;s immune cells begin to be present around day 10 postfertilization. Tumor growth rate varies considerably by cell type and needs to be empirically determined for the cell type of interest. The ovarian cancer and the prostate cancer cells were implanted using the nonstick ring method. Note that when a nonstick ring is not available, a pipette tip may be cut to a similar size and used.
<ol>
	<li>Use 70% ethanol to disinfect a biosafety cabinet and all the required tools: an egg rack, curved iris forceps, nonstick rings (1/4 inch inner diameter), glass stir rod, appropriate volume pipettes and tips, 6 x 7 cm transparent film dressing, office scissors, and marker or pencil. Whenever possible, sterile, disposable or autoclaved tools should be used.</li>
	<li>Place eggs to be implanted on an egg rack in a biosafety cabinet. Select a manageable number of eggs. Up to six is typical. Avoid allowing the eggs to cool substantially while working with them.</li>
	<li>Remove transparent film dressing from the shell by rolling the edges towards the open window to avoid pulling away pieces of shell. Check that the eggs are viable and healthy. Ideal eggs will have a large vessel in the center of the opened area with smaller vessels branching from it.</li>
	<li>Using curved iris forceps, place a sterile, nonstick ring onto the CAM over the vessel, ideally over a branch point. Use a sterile glass stir rod to gently abrade the CAM.</li>
	<li>Pipet the cell suspension from step 1.6 into the center of the nonstick ring. Alternatively, use forceps to place a tumor piece from step 2.2 into the center of the nonstick ring and cover with 20-50 &#956;L of the extracellular matrix solution generated in step 2.1.</li>
	<li>Seal the opening with a one-quarter piece of 6 x 7 cm transparent film dressing. Label the eggs with an appropriate implant designation. 
	NOTE: Numbering the eggs within a group facilitates longitudinal observations.</li>
	<li>Return the egg to the incubator. Ensure that the opening of the shell sits upright and that the egg is secure. 
	NOTE: Eggs can be returned to an egg incubator without rotation. Alternatively, high postimplant viability can be obtained using a 37-38 &#176;C cell incubator with the CO2&#160;deactivated and a hygrometer to monitor the humidity, which should be 50%-80%.</li>
</ol>

<table><tbody><tr><td>010 Teflon (PTFE) White 55 Duro Shore D O-Rings</td><td>The O-Ring Store</td><td>TEF010</td><td>Nonstick ring for cell seeding. 1/4&quot;ID X 3/8&quot;OD X 1/16&quot;CS Polytetrafluoroethylene (PTFE).</td></tr><tr><td>Delicate Operating Scissors; Curved; Sharp-Sharp; 30mm Blade Length; 4-3/4 in. Overall Length</td><td>Roboz Surgical</td><td>RS6703</td><td>This is provided as an example. Any similar curved scissors would work as well.</td></tr><tr><td>Dremel 8050-N/18 Micro 8V Max Tool Kit</td><td>Dremel&amp;nbsp;</td><td>8050-N/18</td><td>This kit contains all necessary tools</td></tr><tr><td>Fertilized chicken eggs (Rhode Island Red - Brown, Lab Grade)</td><td>AA Lab Eggs Inc.</td><td>N/A</td><td>A local egg supplier would need to be identified, as this supplier only delivers regionally.</td></tr><tr><td>Hovabator Genesis 1588 Deluxe Egg Incubator Combo Kit</td><td>Incubator Warehouse</td><td>HB1588D-NONE-1102-1588-1357</td><td>Other egg incubators may be used, but their reliability would need to be verified. After implantation, a cell incubator with the CO2 disabled may also be used.</td></tr><tr><td>Incu-Bright Cool Light Egg Candler</td><td>Incubator Warehouse</td><td>1102</td><td>Other candlers may be used; however, this is preferred among those that we have tested. This candler is included in the aforementioned incubator kit.</td></tr><tr><td>Matrigel Membrane Matrix HC; LDEV-Free</td><td>Corning</td><td>354248</td><td>Extracellular matrix solution</td></tr><tr><td>SKOV3</td><td>ATCC</td><td>HTB-77</td><td>Human ovarian cancer cell line.</td></tr><tr><td>Stirring Rods with Rubber Policeman; 5mm diameter, 6 in. length</td><td>United Scientific Supplies</td><td>GRPL06</td><td>This is provided as an example. Any similar glass stir rods would work as well.</td></tr><tr><td>Specimen forceps</td><td>Electron Microscopy Sciences</td><td>72914</td><td>This is provided as an example. The forceps used for pulling away the shell for bioluminescence imaging are approximately 12.8 cm long with 3 mm-wide tips.</td></tr></tbody></table>

chicken chorioallantoic membrane-based cancer modeling: an in ovo model to study cancer cell tumorigenesis and metastasis

Source: Sharrow, A. C. et al. <a href="https://www.jove.com/t/60651">Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers.</a> J. Vis. Exp.&#160;(2020)
In this video, we describe the implantation of cancer cells onto a chicken chorioallantoic membrane to generate an in ovo model. The model successfully engrafts the ovarian cancer cells to study the progression of gynecological cancer.

Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

Source: Sharrow, A. C. et al. Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers. J. Vis. ...

The chicken chorioallantoic membrane, or CAM, is a highly vascularized membrane found in the eggs of certain vertebrates like chickens. This extra-embryonic membrane lines a non-vascular shell membrane and functions as the site of exchange of gaseous oxygen and carbon dioxide between the embryo and the environment.
To generate a CAM cancer model, begin with a viable, fertilized chicken egg bearing a fully developed CAM with a partially opened shell that exposes its branching vasculature. Confirm that the CAM is detached from the shell membrane.
Place a sterile non-stick ring over a blood vessel branch point. Using a glass rod, gently rub the membrane inside the ring to scrape its outer epithelial layer and expose the underlying mesodermal layer carrying the vasculature. Pipet a chilled suspension of cancer cells mixed with a suitable extracellular matrix into the ring. The ring restricts cellular dispersal.
Allow the matrix to solidify to secure the cells within. Seal the eggshell opening and incubate the egg under appropriate conditions. The dense vasculature within the mesodermal layer supplies oxygen and essential nutrients to the cancer cells, thus providing a suitable microenvironment for their growth and expansion.

chicken-chorioallantoic-membrane-based-cancer-modeling-an-ovo-model

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Animal model studies

1.4K Views. Source: Sharrow, A. C. et al. Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers. J. Vis. Exp. (2020) In this video, we describe the implantation of cancer cells onto a chicken chorioallantoic membrane to generate an in ovo model. The model successfully engrafts the ovarian cancer cells to study the progression of gynecological cancer. 

Video: Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis - Concept

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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 ...

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 ...

Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

Transcript

Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

The In ovo CAM-assay as a Xenograft Model for Sarcoma

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