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>
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F., 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=In+vivo+Wnt+pathway+inhibition+of+human+squamous+cell+carcinoma+growth+and+metastasis+in+the+chick+chorioallantoic+model.">In vivo Wnt pathway inhibition of human squamous cell carcinoma growth and metastasis in the chick chorioallantoic model.</a> Journal of Otolaryngology - Head &amp; Neck Surgery. 45 (1), 26 (2016).</li><li>Canale, 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=Interleukin-27+inhibits+pediatric+B-acute+lymphoblastic+leukemia+cell+spreading+in+a+preclinical+model.">Interleukin-27 inhibits pediatric B-acute lymphoblastic leukemia cell spreading in a preclinical model.</a> Leukemia. 25, 1815 (2011).</li><li>Loos, C., 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=Amino-functionalized+nanoparticles+as+inhibitors+of+mTOR+and+inducers+of+cell+cycle+arrest+in+leukemia+cells.">Amino-functionalized nanoparticles as inhibitors of mTOR and inducers of cell cycle arrest in leukemia cells.</a> Biomaterials. 35 (6), 1944-1953 (2014).</li><li>Rovithi, M., 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=Development+of+bioluminescent+chick+chorioallantoic+membrane+(CAM)+models+for+primary+pancreatic+cancer+cells:+a+platform+for+drug+testing.">Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing.</a> Scientific Reports. 7, 44686 (2017).</li><li>Majern&#237;k, M., 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=Novel+Insights+into+the+Effect+of+Hyperforin+and+Photodynamic+Therapy+with+Hypericin+on+Chosen+Angiogenic+Factors+in+Colorectal+Micro-Tumors+Created+on+Chorioallantoic+Membrane.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+the+chick+chorioallantoic+membrane+xenograft+model+of+neuroblastoma+for+drug+delivery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+chorioallantoic+membrane+as+an+in+vivo+xenograft+model+for+Burkitt+lymphoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+A375+human+melanoma+models+on+chicken+embryo+chorioallantoic+membrane+and+Balb/c+nude+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+chick+embryo+chorioallantoic+membrane+model+as+an+in+vivo+model+to+study+morphological+and+histopathological+features+of+feline+fibrosarcomas.">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. 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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 also be a detriment if long-term treatment studies are warranted. The immunocompromised/immune tolerant status of the CAM model could complicate studies of tumor-immune interactions. For these studies, coimplantation of immune cells of interest may be necessary, possibly with replenishment of the immune compartment. Furthermore, although all the tumor types we presented engraft into the CAM model, they do so with varying degrees of growth. For example, renal cell carcinoma rapidly forms large tumors, but ovarian cancer tumors are difficult to visualize without the assistance of bioluminescence imaging. Tumor growth and speed would need to be assessed for a particular tumor type to determine if the CAM would be a suitable experimental model.
Successful tumor engraftment requires the careful completion of specific steps of the protocol. First, eggs need to be incubated under ideal conditions to have optimal embryo survival and CAM formation prior to engraftment. Due to the natural variability in batches, we suggest obtaining excess eggs from the specific egg supplier. We have also found that cooler, rainier weather can lead to fungal growth in the eggs. During wetter weather, more eggs may need to be engrafted to obtain adequate yields at the endpoint. This seasonal variation is likely to be region-specific. Adequate CAM formation is essential for successful tumor engraftment. Any indications of the CAM remaining attached to the shell when opening should not be ignored. If several eggs fail to have intact CAM when opening the first batch, then we recommend delaying the opening of the remaining eggs for at least 1 additional day.
If poor viability or engraftment are obtained, several steps could be at fault. First, the embryo viability from the supplier may be poor. Absence of an air cell and visible vasculature indicates a nonviable egg. Sometimes, embryo movement can be visualized to confirm viability. First, though, ensure that fresh batteries are in the egg candler, because a strong light is needed for visualization. The float test may also be done to assess viability (a variety of instructions and videos are available online). Another possible explanation for poor viability is the incubator. Using an independent hygrometer and thermometer, ensure that the settings are accurate and stable. Manufacturer&#39;s instructions for the incubator should contain detailed troubleshooting instructions to assess proper settings. Improper incubator settings could also compromise CAM development. Using tape and/or a marker, determine if the egg rotator is actually spinning the eggs. If nonstick rings sink into the albumin in a high proportion of the engrafted eggs, then the CAM did not adequately develop. Finally, we have found that frequent checking of the engrafted egg, leading to temperature and humidity fluctuations, may decrease postimplant viability. If implanted eggs are initially viable, but the viability decreases throughout the engraftment period, the eggs should be checked less frequently. This decrease in viability could also be due to inaccurate or inappropriate incubator settings after implantation.
When optimizing this method for a new cell type, several factors can be controlled. The first is cell number. We typically implant 5 x 105-2 x 106 cells from cell lines. Implanted tumor pieces are typically 2-5 mm on each side. These amounts may be adjusted to improve engraftment or size. However, we have found that tumor growth lessens above a certain threshold, which is cell type dependent. Additional engraftment parameters include the presence or absence of a nonstick ring. This choice is typically made based on whether the ring will hinder the endpoint analysis, although it may also influence successful engraftment. The presence of the nonstick ring may also permit covering the nascent graft with medium at desired intervals to improve survival prior to vascular recruitment, if so desired. The choice of extracellular matrix type, concentration, and medium in which the matrix is diluted may also impact engraftment. The addition of growth factors or hormones may further improve tumor take rate or size. The selection of additives and concentrations would need to be done based on what would be appropriate for the specific cell type and not interfere with the experiment. These would also have the potential to be replenished at intervals if a nonstick ring is used.
Future applications of this model system depend on the hypothesis to be tested. For example, these tumor grafts could be used to test novel therapeutic approaches to reduce tumor size or deplete a subpopulation of the tumor. Co-implantation with cells from the host tumor microenvironment could permit studies of the influence of these cells on a variety of parameters, including tumor growth and treatment resistance. This model could also serve as an opportunity to incrementally expand primary human tumors prior to establishing xenografts in immunocompromised animal models. The initial adaptation to CAM engraftment could perhaps facilitate tumor take rate in murine models. These applications have yet to be tested, but warrant further exploration.

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 implantation. This may help identify possible tumor growth in the absence of bioluminescence imaging, though further validation would be required via histology or another suitable method. We achieved successful engraftment of both human and murine cell lines that show morphologies consistent with those seen in other in vivo models (Figure 1A and 1B). In addition, the implantation of tumor pieces was possible (Figure 1C, blue arrow indicates tumor). The implanted tumor in this case came from a patient with high-grade, metastatic, ovarian serous carcinoma. The resulting tumors morphologically resembled their tumors of origin and expressed human-specific proteins, such as cytokeratin 8/18, that readily permit their differentiation from CAM and chicken embryonic tissues.
When optimizing tumor engraftment and growth, appropriate growth factors or hormones may be added at the time of implantation. For example, a subtle, nonsignificant increase in tumor size (as measured by total radiance flux) was observed in ID8 cells when supplemented with three units (U) of human follicle stimulating hormone (FSH) at the time of implantation (Figure 1D). The selection of FSH for ovarian cancer growth stemmed from the expression of the FSH receptor in ID8 cells and the high levels of FSH typically found in postmenopausal women, who constitute the majority of ovarian cancer cases. Similar approaches may be adopted for other difficult-to-grow cancer types to enhance the utility of the CAM model. Furthermore, FSH was only added at the time of cell implantation. Replenishing the growth factor or hormone could be accomplished by pipetting an appropriate amount of the additive in the medium into the nonstick ring at appropriate intervals, which could enhance the effect.
For kidney cancer, implantation of 2 x 106 clear cell renal cell carcinoma cells from established cell lines, such as RENCA, produced rapid, robust tumor formation, though lower cell doses may also be used (Figure 2A, 10 days postimplantation). Resulting tumors morphologically resembled those obtained through standard mouse in vivo models28. Implantation of RENCA cells marked with firefly luciferase permitted bioluminescence imaging. Primary human tumors may also be implanted. The pieces of tumor persisted and recruited vasculature without showing significant growth. Implantation of digested cells originating from a primary, clear cell renal cell carcinoma that were expanded in vitro, however, grew similarly to the established cell lines (Figure 2B). Renal cell carcinoma cells may be seeded using either the nonstick ring method or the method without the nonstick ring.
Multiple human and murine prostate cancer cell lines were tested for CAM engraftment (Figure 3). Each grew well when 2 x 106 cells were implanted using the nonstick ring. Histological evaluation of the resulting tumors was consistent with expectations for each cell line. Additionally, implantation of cells stably marked with firefly luciferase permitted tumor identification with bioluminescence imaging (Figure 3A).
Bladder cancer was established in the CAM model from established cell lines and pieces of primary human tissue (Figure 4). Cell lines grew well when implanted with 2 x 106 cells without the nonstick ring (Figure 4A and 4B), although implantation with the ring was successful. Primary human tumor may be implanted from pieces of tissue (Figure 4C). The presented case originated from a patient with high grade, non-muscle-invasive, urothelial carcinoma. Implantation of digested cells has not yet been attempted due to ongoing tumor digestion optimization. Cancer cells of the resulting CAM tumors retained the morphology of the original tumor, but with an altered stromal component. Tumor growth was less than for renal cell carcinoma and prostate cancer, though still readily visible.
To ensure a high number of viable, assayable eggs at the endpoint, care must be taken when incubating and opening the eggs. If the incubator conditions are suboptimal either before or after implantation, then embryo viability may be compromised. If significant embryonic death occurs, troubleshoot the incubator conditions according to the manufacturer&#39;s instructions. In our experience, temperature and humidity stability appear to be more crucial than their exact values. Therefore, we found that incubating the inoculated eggs in a modified, cell-culture, CO2 incubator achieved superior viability over retaining the eggs in small, dedicated egg incubators that require more frequent opening to replenish the water that controls the humidity. Minimizing the frequency with which the inoculated eggs are removed for tumor examination may also enhance embryo viability.
An additional concern of CAM implantation is the integrity of the CAM at inoculation. If the CAM is not intact after opening the shell, then the nonstick ring and implanted cells will sink into the albumin of the embryo (see the note after step 2.12). This causes cancer cell dispersal. Some tumors may still form, but cancers that receive significant growth and survival signaling from neighboring cancer cells, such as ovarian cancer, will not reliably form tumors under such conditions. If nonstick rings are used for implantation, the failure of the CAM can be identified by the sinking of the ring into the albumin. This must be distinguished from cases in which the ring and implanted cells were moved to the bottom of the egg by embryonic movement. In those cases, the ring will be found between the CAM and the underside of the shell. Embryonic movement cannot be controlled. However, ring placement can make it more difficult for the embryo to disrupt the implanted cells. Rings placed at the edges of the air pocket generated in step 2.7 are more likely to be moved by the embryo than those placed at the center of the field.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60651/60651fig1a.jpg" /> 
Figure 1: Representative tumor development from ovarian cancer. Ovarian cancer cells successfully implanted include: (A) the human SKOV3 cell line, (B) the murine ID8 cell line, and (C) primary tumors. Representative hematoxylin and eosin staining is presented along with bioluminescence imaging, as appropriate. For the CAM tumor resulting from the implantation of a primary tumor piece (C) hematoxylin and eosin staining of both the CAM tumor and primary tumor are included, along with cytokeratin 8/18 (CK 8/18) staining of the resulting CAM tumor. The blue arrow in the first panel indicates the tumor. (D) Tumor size of ID8 implants with and without 3U FSH, calculated as the total flux resulting from bioluminescence imaging (n = 3 for untreated and n = 4 for FSH-supplemented). <a href="https://www.jove.com/files/ftp_upload/60651/60651fig1large.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/60651/60651fig2.jpg" /> 
Figure 2: Representative tumor development from clear cell renal cell carcinoma. Tumors resulting from implantation of (A) the murine renal cell carcinoma cell line, RENCA, or (B) cultured cells derived from a digested human primary tumor. The measurement scale adjacent to the excised tumor in (B) shows 1 mm markings. Corresponding histology in (A) and (B) shows hematoxylin and eosin staining. In (A), the representative bioluminescence imaging of firefly-luciferase-marked RENCA cells is also shown. <a href="https://www.jove.com/files/ftp_upload/60651/60651fig2large.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/60651/60651fig3.jpg" /> 
Figure 3: Representative tumor development from prostate cancer cell lines. Representative tumors and hematoxylin and eosin staining resulting from the implantation of the human prostate cancer cell lines (A) CWR and (B) C4-2 along with the murine cell line (C) MyC-CaP. Bioluminescence imaging of the resulting tumors from firefly luciferase marked CWR cells is shown in (A). The measurement scale adjacent to the excised tumors shows 1 mm markings. <a href="https://www.jove.com/files/ftp_upload/60651/60651fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60651/60651fig4a.jpg" /> 
Figure 4: Representative tumor development from bladder cancer. Representative tumors resulting from the implantation of the established human bladder cancer cell lines (A) HT-1376, (B) T24, and (C) primary human bladder tumor. In (C), hematoxylin and eosin staining of the resulting CAM tumor and the primary tumor are shown. The measurement scale adjacent to the excised tumors shows 1 mm markings. <a href="https://www.jove.com/files/ftp_upload/60651/60651fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Universidad San Sebastian

Research

JoVE Journal

Cancer Research

10.1K Views.  University of California Los Angeles. 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.

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

Summary

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Chapters in this video

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

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 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 Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

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

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

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

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

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