eoe sub articles

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. Harvesting the CAM
<ol>
	<li>Prepare 6-well plates with 4% PFA (paraformaldehyde) pH 7.0; one well per egg, 2 mL per well.</li>
	<li>Bring the eggs to a laboratory bench. Using a needle attached to a syringe to perforate the film dressing, drop around 300 &#181;L of PFA over the CAM to slightly stiffen the CAM, thus facilitating ...

chick chorioallantoic membrane harvest: a method to isolate tumor graft-bearing chorioallantoic membrane from chicken egg

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
This video demonstrates the isolation of tumor-bearing chorioallantoic membrane from the chicken embryo. The harvested CAM can be used for further downstream applications.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20478/20478fig1.jpg" /> 
Figure 1: Grafting of DRG, cells, and harvesting of CAM: On day 8: A. CAM easily observed after paraffin wax membrane removal.&#160;B-C. With fine forceps, DRG is placed onto the CAM.&#160;D. Egg is covered with film dressing and put in the incubator; arrows point to the openings that are covered.&#160;On day 10: E-F. Film dressing is...

Chick Chorioallantoic Membrane Harvest: A Method to Isolate Tumor Graft-bearing Chorioallantoic Membrane from Chicken Egg

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

The chorioallantoic membrane or CAM is an extraembryonic membrane, adhering closely to the inner shell membrane of an intact egg. CAM is an ideal system for tumor implantation and modeling because it is a naturally immunodeficient and highly vascularized membrane.
To harvest a tumor-bearing CAM, first, take a viable fertilized chicken egg having a fully developed CAM previously transplanted with a tumor. Add paraformaldehyde to the CAM. Paraformaldehyde crosslinks the membrane proteins, making the membrane stiffer to facilitate its harvesting.
Next, cut around the egg to separate the upper portion of the eggshell from the remainder of the egg, with the tumor-bearing CAM still attached to the removed shell. Hold and lift the CAM at one edge to smoothly detach it from the eggshell.
Thereafter, place the harvested CAM in paraformaldehyde with the tumor graft facing upwards. Paraformaldehyde preserves the morphology of the CAM and tumor graft, thereby preparing the harvested membrane for various downstream analyses.

chick-chorioallantoic-membrane-harvest-method-to-isolate-tumor-graft

Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

In vivo studies

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

JoVE Journal

Medicine

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

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

To prepare a cancer cell suspension for transplantation, harvest the cells from the cancer cell culture of interest and sediment the cells by centrifugation. Aspirate the supernatant and mechanically resuspend the cells in the residual medium. Place the cell suspension on ice and use a pipette to measure the volume of cells in medium. Then, resuspend the cells at a concentration of 1 to 2 million cells in 20 to 100 microliters of medium, supplemented with 2.7 to 4 milligrams per milliliter extracellular matrix and any growth factors or other additives of interest.
For cancer cell implantation, using a non-stick ring, place up to 6 eggs to be implanted onto the egg rack and roll the edges of the transparent film dressing toward the open window to remove the film from the shells. Check whether the eggs are viable and healthy. Ideal eggs will have a large vessel with smaller branching vessels within the center of the opened area.
Using curved iris forceps, place a sterile non-stick ring onto the CAM over the vessel, ideally over a branch point. Use a sterile glass stir rod to gently abrade the CAM and pipet the cancer cell suspension into the center of the ring. When all of the cells have been delivered, seal the opening with a one quarter piece of 6 by 7 centimeter transparent film dressing and label the egg with an appropriate implant designation. Then, place the egg securely in the incubator, with the opening of the shell facing upright.

Chick Chorioallantoic Membrane Harvest: A Method to Isolate Tumor Graft-bearing Chorioallantoic Membrane from Chicken Egg

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