Name: discovery of metastatic regulators using a rapid and quantitative intravital chick chorioallantoic membrane model
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Description: Watch this Scientific Journal Video about Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model at JoVE.com

This work was supported by Canadian Cancer Society Research Institute Grant #702849 to JDL and KS. Dr. Lewis holds the Frank and Carla Sojonky Chair in Prostate Cancer Research supported by the Alberta Cancer Foundation.

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(51), e2815 (2011).</li><li>Stoletov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vivo+whole+genome+motility+screen+reveals+novel+therapeutic+targets+to+block+cancer+metastasis.">Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis.</a> Nature Communications. 9 (1), 2343 (2018).</li><li>Leong, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia+are+required+for+cancer+cell+extravasation+and+are+a+therapeutic+target+for+metastasis.">Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis.</a> Cell Reports. 8 (5), 1558-1570 (2014).</li><li>Willetts, L., Bond, D., Stoletov, K., Lewis, J. D., Ursini-Siegel, J., Beauchemin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Tumor Microenvironment: Methods and Protocols. , 27-37 (2016).</li></ol>

Nothing to disclose.

Here we describe a rapid fluorescence microscopy based intravital screening protocol that can be utilized for important applications such as genetic or drug candidate screens. Cancer cells that have been transduced with a genetic library of interest or transfected with individual expression constructs can be rapidly screened and quantified as to phenotype of interest using this chick CAM model. Since transduction or transfection protocols vary significantly, depending on the library type, they are not included in this procedure. Phenotypically relevant metastatic colonies are excised from the CAM, expanded, and DNA isolated for high throughput sequencing to identify the expression construct of interest. Generally, each genetic library is equipped with primer sequences and recommended methods of genomic DNA purification. We recommend using local facility guidelines for the best high throughput sequencing results.
It is recommended to determine the optimal multiplicity of infection (M.O.I.) for a library and cell line before screening. Ideally each cell (and therefore future metastatic colony) should contain only one gene expression construct, however in our experience that is not always achievable. We recommend following the library manufacturer&#39;s protocol and testing a wide range of M.O.I. (0.01-5) to achieve as close to 1 as possible. Presence of multiple library expression constructs within each metastatic colony may significantly complicate the colony phenotype analysis. We also recommend using the library manufacturer supplied negative control in your experiments (i.e. scramble shRNA expressing vector that is fluorescently tagged).
Our screening protocol is based on selection of non-invasive fluorescent colonies; it is critical to ensure that all cell lines used in the experiments have similar fluorescence intensity. Uneven fluorescence intensity among the cells (and metastatic colonies) may result in a biased colony selection due to cell or colony brightness instead of invasiveness.
Compared to existing methods our protocol provides several unique advantages such a speed, low cost and ability to complete the intravital screen cycle without need for sophisticated imaging equipment12,13,14. In addition, the whole screening cycle can be completed withing 3 to 6 weeks from the cell injection to sequencing stage. No high-resolution microscope is required for cancer cell injection or metastatic colony isolation as these steps can be performed using basic fluorescent stereo microscope that is available to most researchers. Finally, because shell-less embryo culturing is completely self-sustained there is no need for complicated research animal housing or feeding schedules. Most research institutions do not consider chicken embryos to be live animals which significantly decreases the cost and documentation burden associated with this model.
However, there are some limitations associated with the shell-less embryo model. First, not all cancer cell lines work in this model efficiently. In our laboratory we routinely use several cancer cell lines, such as HT1080 (human fibrosarcoma), HEp3 (head &#38; neck) and mouse b16 melanoma and 4T1 breast cancer cell lines, that robustly form metastatic colonies when injected into the chicken CAM vasculature. Other cell lines with different cancer types such as breast (MDA468), brain (U87 and U118) or ovarian (A2780s) readily form metastatic colonies when injected into the CAM and therefore can be used in this screening protocol as well12,14,15. Yet, in our experience commonly used cancer cell lines such as LnCaP and PC3 do not perform well in this model (unpublished observations). Another limitation is that a confocal microscope with a 100 to 200 &#181;m imaging range is required for high-resolution imaging, such as visualization of cancer cell blood vessel contacts, this equipment may not be available for many researchers.
Altogether, this protocol describes a rapid intravital screening platform that can be used for discovery of cancer metastasis suppressors and drivers. We strongly believe that the robustness and ease-of-use nature of this model will make it an essential screening model for many researchers.

Metastasis is the main cause of cancer patient death1,2,3. Metastatic cancer cells utilize distinct signaling pathways, dependent on the type of cancer, throughout the five steps of the metastatic cascade: local invasion, intravasation, survival in the circulation, extravasation, and colony expansion at distant metastatic sites. Current understanding of this metastatic process suggests that there are two bottleneck steps, one is directional invasion of the cancer cell from the primary tumor, and the second is the establishment of the distant site metastatic lesion4,5,6. Both steps require cancer cells to actively interact with collagen and vasculature at the sites of initial invasion or distant metastatic lesion formation. Therefore, metastatic cancer cells must be able to attach to cells, remodel collagen fibers, and directionally invade along vascular walls7. Screening models that can rapidly identify antimetastatic therapeutic targets to block cancer cells from completing these steps is of the highest importance. Existing in vitro screening models do not fully mimic the complex live tissue environment. Mouse models are costly and time consuming. Therefore, there is an urgent need for intravital screening platforms that provide complex live tissue environments and rapid identification of targets.
Within the last decade, the chicken embryo has been established as a robust and cost-effective model of human cancer metastasis8,9,10,11,12. The chick chorioallantoic membrane (CAM) tissue is thin and translucent which makes it ideal for intravital microscopic imaging of cell and colony behaviors in primary tumor and/or metastatic sites12. Primary tumor growth from multiple human cancer cell lines can be initiated and metastasized within a span of only several days after microinjection into the CAM tissue. Cancer cells can be administered into the CAM tissue in several ways, including intravenously, intra-CAM or as collagen onplants, this flexibility allows the researcher to focus on specific stages of cancer progression, for example, metastatic lesion formation, invasion out of the primary tumor or angiogenesis.
Here we describe a quantitative screening platform that can be used to measure the ability of cancer cells to establish invasive metastatic lesions. Cancer cells that have been transduced with an expression library are injected intravenously into the CAM vasculature at low density. Metastatic colonies form for 4-5 days, then the invasion capacity and vasculature interaction of the resulting colonies is evaluated. Individual colonies that fail to invade are excised, propagated and their phenotype is confirmed in the CAM by reinjection and quantification of colony compactness and cancer cell-blood vessel contacts. The expression library constructs responsible for the single metastatic colony mutant phenotype are identified from isolated colony genomic DNA via high throughput sequencing. The same platform may be further used to validate the causal link between a gene and the observed phenotype or to perform in-depth mechanistic studies on the observed phenotype.

<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>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 supplyer</td><td></td><td></td></tr><tr><td>fine forceps</td><td>many sources are available</td><td></td><td>must be sterilized begfore use</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Millipore-Sigma</td><td>MDH-4N1-50PK</td><td></td></tr><tr><td>HT1080 human fibrosarcoma cell line</td><td>ATCC</td><td>CCL-121</td><td></td></tr><tr><td>Image analysis software</td><td></td><td></td><td>We use Nikon Elements</td></tr><tr><td>Lectin &lt;em&gt;Lens Culinary&lt;/em&gt; Agglutinin (LCA) conjugated with Fluorescein or Rhodamine&amp;nbsp;</td><td>Vector Laboratories</td><td>RL-1042, FL-1041</td><td>Dilute stock (5mg/ml) 50-100x depending on the microscope sensetivity. Must be a different color from the color of cell line used for screening</td></tr><tr><td>MDA-MB-468 human breast cancer</td><td>ATCC</td><td>HTB-132</td><td></td></tr><tr><td>PBS (1x)</td><td>many sources are available</td><td></td><td></td></tr><tr><td>Plastic weighting dishes&amp;nbsp;</td><td>Simport</td><td>CA11006-614</td><td>dimensions are 78x78x25mm; many other sources are available</td></tr><tr><td>small surgical scissors&amp;nbsp;</td><td>many sources are available</td><td></td><td>must be sterilized before use</td></tr><tr><td>Sodium borosilicate glass capillary tubes, outer diameter 1.0 mm, inner diameter 0.58 mm, 10 cm length&amp;nbsp;</td><td>Sutter Instrument</td><td>BF100-58-10</td><td></td></tr><tr><td>Square petri dishes (used as lids for the weighting dishes).</td><td>&amp;nbsp;VWR&amp;nbsp;</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>Tygon R-3603 laboratory tubing</td><td>Cole-Parmer</td><td>AAC00001</td><td>1/32 in inner diameter, 3/32 in. outer diameter, 1/32 in. wall thickness</td></tr><tr><td>U-118 MG human glioblastoma</td><td>ATCC</td><td>HTB-15</td><td></td></tr><tr><td>U-87 MG human glioblastoma</td><td>ATCC</td><td>HTB-14</td><td></td></tr><tr><td>Vertical pipette puller&amp;nbsp;</td><td>many sources are available</td><td></td><td>we use David Kopf Instruments, Tujunga, CA; Model 720</td></tr></tbody></table>

discovery of metastatic regulators using a rapid and quantitative intravital chick chorioallantoic membrane model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

Cancer cell injection is considered to be successful if the majority of the cells that are lodged in the capillaries are single and located at a significant difference from each other (~0.05-0.1 cm) so the colonies will not overlap after 5-6 days of incubation period (Figure 3A). The injection was not successful if a buildup of cancer cells can be seen in most of capillaries, embryos displaying this should be discarded (Figure 2E). A significant number of injected cancer cells perish within 24 h of injection making some embryos appear to reject all the cancer cells. Cancer cell survival will vary depending on the local egg supplier (i.e., amount of cells to be injected may vary) and we strongly recommend that optimal injection conditions (cancer cell concentration versus injection duration) are determined before proceeding with the experiment. We recommend cell concentration in the range between 0.5 x 106 to 1.0 x 106 cells/m. When the optimal cell concentration and duration of injection is achieved, the day 5 post injection transduced cells should produce a wide variety of colony phenotypes with the majority of the colonies appearing invasive as determined by the cancer cells appearing scattered in the CAM tissue (Figure 3A). Attention should be paid to the metastatic colonies that appear &#34;compact&#34; and are located far enough from neighboring colonies that they can be excised with forceps and scissors in one piece of CAM tissue (Figure 3C).&#160;An isolated positive screen hit (i.e., a compact colony) should display the compact colony phenotype upon reinjection (Figure 3B). A decrease in cell survival (or cell proliferation rate within the colony) may be observed as well. Therefore, it is possible that higher cell numbers will need to be injected for some of the positive screen hits to obtain enough colony numbers. For measurement of the cancer cell-blood vessel contacts attention should be paid to the vascular wall stain brightness (fluorescent lectin; Figure 3D,E) and the appropriate amount of lectin should be injected for the bright/contrast vascular wall signal. Any available image analysis software can be used during the quantification steps after microscope-software calibration has been performed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62077/62077fig01.jpg" /> 
Figure 1: Chicken embryo shell-less culture overview. (A) A weighing dish with lid prepared for shell-less culture. Arrow points to the cut corner. (B) Weighing dishes arranged in rows in the flow hood. (C) Cutting an eggshell with an electric rotary tool. (D) Cracking an egg into a weighing dish. (E) Embryos cultured in the humidified incubator. Scale bars =1 cm (A-D) or 5 cm (E). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62077/62077fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62077/62077fig02.jpg" /> 
Figure 2: Outline of cancer cell injection and metastatic colony isolation. (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 cancer cell injection (acceptable cancer cell density), taken immediately after injection. (E) Image showing poor cancer cell injection, seen as an over-injected embryo. Note cancer cell build up in the blood capillaries (white arrows). Scale bars = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62077/62077fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62077/62077fig03.jpg" /> 
Figure 3. Representative results for different steps of the screening protocol. (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 (KIF3B) after reinjection, 5 days post injection. Insets show digitally cut out metastatic colony images from the dashed squares. This is acceptable image quality for C.I quantification. Average C.I. value for hit reinjection (KIF3B) is shown. (C) Isolation of the metastatic colony of interest from the CAM tissue. Representative optical sections of (D) a control colony, and (E) a KIF3B shRNA overexpressing colony, both showing cancer cell-blood vessel contact measurements. CAM vasculature is labeled with fluorescent lectin-649. Scale bars =1 cm (A-C) or 50 &#181;m (D, E).&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62077/62077fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model at JoVE.com

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

discovery-metastatic-regulators-using-rapid-quantitative-intravital

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Cancer Research

2.6K Views.  University of Alberta, Edmonton, Alberta, Canada. Ons protocol maakt een efficiënte identificatie mogelijk van gen- of gencombinaties die kankerinvasie in de kijker blokkeren. over cultuur vereist geen geavanceerde dierlijke faciliteiten voor onderhoud. Als geheel kunnen als generieke of algemene speciale bibliotheken binnen enkele weken in de kijker worden gescreend.Dit over screeningssysteem zal onderzoekers helpen nieuwe therapeutische gendoelen of gendoelencombinaties te ontdekken die kunnen worden gebruikt om kankermetastasen te...

Video: Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

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 ...

Medicine

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, ...

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 ...

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 ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Biology

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 ...

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 ...

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.remotexs.ntu.edu.sg/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.

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

JoVE Encyclopedia of Experiments

Head and Neck Cancer

In vivo studies

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.remotexs.ntu.edu.sg/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.

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

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