The authors wish to thank Dr. Fuyuhiko Tamanoi and Binh Vu for the initial training on this method. Discussions with Dr. Eva Koziolek have been instrumental in optimizing this approach and have been very much appreciated. This work would not have been possible without funding from the following sources: the Tobacco-Related Disease Research Program Postdoctoral Fellowship (27FT-0023, to ACS), the Department of Defense (DoD) Ovarian Cancer Research Program (W81XWH-17-1-0160), NCI/NIH (1R21CA216770), Tobacco-Related Disease Research Program High Impact Pilot Award (27IR-0016), and UCLA institutional support, including a JCCC Seed Grant (NCI/NIH P30CA016042) and a 3R Grant from Office of the Vice Chancellor for Research to LW.

<ol><li>Kersten, K., de Visser, K. E., van Miltenburg, M. H., Jonkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+engineered+mouse+models+in+oncology+research+and+cancer+medicine%5BTitle%5D)+AND+%22EMBO+Molecular+Medicine%22%5BJournal%5D)">Genetically engineered mouse models in oncology research and cancer medicine.</a> EMBO Molecular Medicine. 9 (2), 137-153 (2017).</li>
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<li>Lokman, N. A., Elder, A. S., Ricciardelli, C., Oehler, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chick+chorioallantoic+membrane+%28CAM%29+assay+as+an+in+vivo+model+to+study+the+effect+of+newly+identified+molecules+on+ovarian+cancer+invasion+and+metastasis%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Science%22%5BJournal%5D)">Chick chorioallantoic membrane (CAM) assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis.</a> International Journal of Molecular Science. 13 (8), 9959-9970 (2012).</li>
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<li>Majerník, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+Insights+into+the+Effect+of+Hyperforin+and+Photodynamic+Therapy+with+Hypericin+on+Chosen+Angiogenic+Factors+in+Colorectal+Micro-Tumors+Created+on+Chorioallantoic+Membrane%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Science%22%5BJournal%5D)">Novel Insights into the Effect of Hyperforin and Photodynamic Therapy with Hypericin on Chosen Angiogenic Factors in Colorectal Micro-Tumors Created on Chorioallantoic Membrane.</a> International Journal of Molecular Science. 20 (12), 3004(2019).</li>
<li>Swadi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimising+the+chick+chorioallantoic+membrane+xenograft+model+of+neuroblastoma+for+drug+delivery%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery.</a> BMC Cancer. 18 (1), 28(2018).</li>
<li>Klingenberg, M., Becker, J., Eberth, S., Kube, D., Wilting, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chick+chorioallantoic+membrane+as+an+in+vivo+xenograft+model+for+Burkitt+lymphoma%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">The chick chorioallantoic membrane as an in vivo xenograft model for Burkitt lymphoma.</a> BMC Cancer. 14 (1), 339(2014).</li>
<li>Avram, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardization+of+A375+human+melanoma+models+on+chicken+embryo+chorioallantoic+membrane+and+Balb%2Fc+nude+mice%5BTitle%5D)+AND+%22Oncology+Reports%22%5BJournal%5D)">Standardization of A375 human melanoma models on chicken embryo chorioallantoic membrane and Balb/c nude mice.</a> Oncology Reports. 38 (1), 89-99 (2017).</li>
<li>Zabielska-Koczywas, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+chick+embryo+chorioallantoic+membrane+model+as+an+in+vivo+model+to+study+morphological+and+histopathological+features+of+feline+fibrosarcomas%5BTitle%5D)+AND+%22BMC+Veterinary+Research%22%5BJournal%5D)">3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas.</a> BMC Veterinary Research. 13 (1), 201(2017).</li>
<li>Skowron, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applying+the+chicken+embryo+chorioallantoic+membrane+assay+to+study+treatment+approaches+in+urothelial+carcinoma%5BTitle%5D)+AND+%22Urologic+Oncology%3A+Seminars+and+Original+Investigations%22%5BJournal%5D)">Applying the chicken embryo chorioallantoic membrane assay to study treatment approaches in urothelial carcinoma.</a> Urologic Oncology: Seminars and Original Investigations. 35 (9), e511-e523 (2017).</li>
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<li>Strojnik, T., Kavalar, R., Barone, T. A., Plunkett, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+model+and+immunohistochemical+comparison+of+U87+human+glioblastoma+cell+xenografts+on+the+chicken+chorioallantoic+membrane+and+in+rat+brains%5BTitle%5D)+AND+%22Anticancer+Research%22%5BJournal%5D)">Experimental model and immunohistochemical comparison of U87 human glioblastoma cell xenografts on the chicken chorioallantoic membrane and in rat brains.</a> Anticancer Research. 30 (12), 4851-4860 (2010).</li>
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</ol>

The authors have nothing to disclose.

Tumor expansion and engraftment using the CAM model permits more rapid and directly observable tumor growth than existing in vivo animal models. In addition, costs are significantly lower once the initial purchase of equipment is complete, especially when compared to the cost of immunocompromised mice. The initial, immunocompromised state of chicken embryos readily permits engraftment of human and murine tissue. Even with these strengths, the CAM model does have limitations. The short time that can be a benefit could als...

Mice have served as the classic model organism for the study of human diseases, including malignancy. As mammals, they share many similarities with humans. Their high degree of genetic similarity has permitted transgenic manipulation of the mouse genome to provide enormous insight into the genetic control of human diseases1. Extensive experience in the handling of and experimentation with mice has resulted in their being the model of choice for biomedical research. However, in addition to the ethical and scientific concerns regarding murine models, they can also be quite costly and time consuming2,3. The development of tumors can take weeks or even months. The housing at a typical institution alone can run in the hundreds to thousands of dollars while tumors are developing. Ovarian cancer is an example of this drawback because its growth in murine models can easily take months. Delays in research progress potentially impact ovarian cancer patients&#39; persistently low 5-year survival rate of only 47% (i.e., an increase in survival of only 10% over 30 years)4. Similarly, urological cancers (kidney, prostate, and bladder cancers) constitute 19% of all cancer cases in the United States and 11% of cancer-related deaths4. Thus, a novel in vivo approach to study gynecological and urological cancers could save a laboratory considerable time, labor, and money, even if this model is only applied to initial screening experiments. Additionally, the resulting acceleration of research findings could significantly impact the 177,000 individuals diagnosed with these cancers annually.
The chicken CAM model offers many advantages that address the aforementioned issues. A popular model to study angiogenesis5,6, tumor cell invasion7,8, and metastasis7,9, the chick embryo CAM model has already been used to study many forms of cancers, including glioma10,11,12, head and neck squamous cell carcinoma13,14, leukemia15,16, pancreatic cancer17, and colorectal cancer18. Additionally, CAM models have been generated for neuroblastoma19, Burkitt lymphoma20, melanoma21, and feline fibrosarcoma22. Prior studies have also presented engraftment of bladder cancer23 and prostate cancer cell lines24, but with limited protocol details. Not only are eggs much cheaper than mice, but they also produce highly reproducible results25,26. They show fast vasculature development, and tumor engraftment can occur in as quickly as a few days and be visualized longitudinally through the open window. With the 21 day time frame between egg fertilization and hatching, experiments can be completed within a few weeks. Furthermore, the low cost, limited housing needs, and small size readily permit large-scale experiments that would be prohibitive for mouse studies.
Therefore, we sought to optimize the CAM model for the engraftment of gynecological and urological cancers. Due to the immunocompromised status of the early chicken embryo27, both mouse and human cells can be readily implanted. As such, we have successfully engrafted ovarian, kidney, prostate, and bladder cancers. For each of these tumor types, the CAM readily accepts established murine and/or human tumor cell lines. Importantly, freshly harvested primary human tumor tissues can also engraft from either digested cells or pieces of solid tissue with high rates of success. Each of these cancer types and cell sources requires optimization, which we share here.

<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>C4-2</td><td>ATCC</td><td>CRL-3314</td><td>Human prostate cancer cell line.</td></tr><tr><td>CWR22Rv1</td><td></td><td></td><td>CWR cells were the kind gift of Dr. David Agus (Keck Medicine of University of Southern California)</td></tr><tr><td>Cytokeratin 8/18 Antibody (C-51)</td><td>Novus Biologicals</td><td>NBP2-44929-0.02mg</td><td>Used at a dilution of 1:100 for immunohistochemical analysis of human ovarian CAM tumors.</td></tr><tr><td>D-Luciferin Firefly, potassium salt</td><td>Goldbio</td><td>LUCK-1G</td><td></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</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>HT-1376</td><td>ATCC</td><td>CRL-1472</td><td>Human bladder cancer cell line.</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>ID8</td><td></td><td></td><td>Not commercially available, please see PMID: 10753190.</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>Iris Forceps, 10cm, Curved, Serrated, 0.8mm tips</td><td>World Precision Instrument</td><td>15915</td><td>This is provided as an example. Any similar curved forceps would work as well. Multiple brands have been used for this method.</td></tr><tr><td>Isoflurane</td><td>Clipper Distributing</td><td>0010250</td><td></td></tr><tr><td>IVIS Lumina II In Vivo Imaging System</td><td>Perkin Elmer</td><td></td><td></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>MyC-CaP</td><td>ATCC</td><td>CRL-3255</td><td>Murine prostate cancer cell line.</td></tr><tr><td>Portable Pipet-Aid XP Pipette Controller</td><td>Drummond Scientific</td><td>4-000-101</td><td>Any similar pipet controller would be appropriate.</td></tr><tr><td>PrecisionGlide Hypodermic Needles</td><td>BD</td><td>305196</td><td>This is provided as an example. Any 18G needle would work similarly.</td></tr><tr><td>RENCA</td><td>ATCC</td><td>CRL-2947</td><td></td></tr><tr><td>Semken Forceps</td><td>Fine Science Tools</td><td>11008-13</td><td>This is provided as an example. Any similar forceps or another style that suits researcher preference would be appropriate.</td></tr><tr><td>SKOV3</td><td>ATCC</td><td>HTB-77</td><td>Human ovarian cancer cell line.</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><tr><td>Sterile Cotton Balls</td><td>Fisherbrand</td><td>22-456-885</td><td>This is provided as an example. Any sterile cotton balls would suffice.</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>T24</td><td>ATCC</td><td>HTB-4</td><td>Human bladder cancer cell line.</td></tr><tr><td>Tegaderm Transparent Dressing Original Frame Style 2 3/8&quot; x 2 3/4&quot;</td><td>Moore Medical</td><td>21272</td><td></td></tr><tr><td>Tissue Culture Dishes, 10 cm diameter</td><td>Corning</td><td>353803</td><td>This is provided as an example. Any similar, sterile 10-cm dish may be used. Tissue culture treatment is not necessary.</td></tr><tr><td>Tygon Clear Laboratory Tubing - 1/4 x 3/8 x 1/16 wall (50 feet)</td><td>Tygon</td><td>AACUN017</td><td>This is provided as an example. Any similarly sized tubing would work as well.</td></tr></tbody></table>

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.

Thus far, we have found this method of implantation to be successful for ovarian, kidney, prostate, and bladder cancers. Each was optimized to identify specific conditions for implantation, although there may be flexibility. Of the tested tumor types, ovarian cancer growth was much less pronounced and typically not visible without the assistance of bioluminescence imaging (Figure 1). However, a stiffening of the CAM could be felt with forceps in the area of i...

Watch this Scientific Journal Video about Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers at JoVE.com

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

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 ...

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Cancer Research

10.2K Views.  University of California Los Angeles. De engraftment van tumoren op de kip chorioallantoïsche membraan, of CAM, kan worden gebruikt voor studies van tumorheiciteit, werkzaamheid van de behandeling of cellulaire crosstalk. Deze methode maakt kosteneffectieve snelle tumorgroei mogelijk in een immunotolerante in vivo setting. Met behulp van het CAM-model kunnen nieuwe therapeutische benaderingen snel worden getest met behulp van cellijnen of zelfs tumormonsters van patiënten.De meest cruciale aspecten van dit protocol zijn ...

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

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

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 ...

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 Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

Bioengineering

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 ...

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining&#160;natural immunodeficiency up to day 17 postfertilization, making it ideal to study&#160;short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Here, we describe a protocol for developing a chick chorioallantoic membrane (CAM) xenografting model for human ovarian tissue and demonstrate the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle ...

Biology

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.

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

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Animal model studies

Chick Chorioallantoic Membrane or CAM Tumor Model: An In Ovo Method to Simulate Perineural Invasion of Tumor Cells

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)
In this video, we describe the method to co-culture dorsal root ganglia and cancer cells on chick chorioallantoic membrane or CAM to study perineural invasion.

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)
...

Head and Neck Cancer

In vivo studies

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.

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.

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

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

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