This work was funded by the UCLA JCCC seed grant, UCLA 3R grant, UCLA CTSI, and UC TRDRP (LW). We thank the Crump Institute&#39;s Preclinical Imaging Facility, the TPCL, and UCLA&#39;s Department of Laboratory Animal Medicine (DLAM) for their help with experimental methods. Flow cytometry was performed in the UCLA Johnson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by National Institutes of Health awards P30 CA016042 and 5P30 AI028697, and by the JCCC, the UCLA AIDS Institute, the David Geffen School of Medicine at UCLA, the UCLA Chancellor&#39;s Office, and the UCLA Vice Chancellor&#39;s Office of Research. Statistics consulting and data analysis services were provided by the UCLA CTSI Biostatistics, Epidemiology, and Research Design (BERD) Program that is supported by NIH/National Center for Advancing Translational Science UCLA CTSI Grant Number UL1TR001881.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC), designated as UCLA Chancellor&#39;s Animal Research Committee (ARC) (ARC 2002-049-53 and ARC 2017-102-01A). The 2002-049-53 protocol is optimized for the implantation of ccRCC tumor cells into the kidney capsule of Nude or BALB/c mice. Tumor implantation experiments in fertilized chicken eggs prior to hatching does not require IACUC approval. To extend the time for establishment of lung metastasis, the embryos with CAM tumor are allowed to hatch and grow into chickens. The 2017-102-01A protocol covers these animal experiments.
1. Orthotopic tumor studies in mice
NOTE: The timeline for this experiment is shown in Figure 1A. These procedures were adapted from previous publications11,12.
<ol>
	<li>Preparing single cell suspension for grafting
	<ol>
		<li>Detach the RENCA VHL-WT and VHL-KO cells from the culture dishes using trypsin/EDTA.</li>
		<li>Count the cells with a hemocytometer and resuspend in a precooled 1:1 mixture of PBS and extracellular matrix solution at a concentration of 1 x 105 cells/&#181;L. 
		NOTE: Use a 1:4 ratio of VHL-WT:VHL-KO cells for heterogeneous implants and VHL-WT alone for homogeneous implants.</li>
		<li>Transfer the resuspended cells into a 1.5 mL microcentrifuge tube and keep on ice until implantation.</li>
	</ol>
	</li>
	<li>Implantation to renal capsule
	<ol>
		<li>Anesthesia: Preheat a warm pad to 37 &#176;C and cover it with a piece of thick sterile drape. Anesthetize the mouse by either isoflurane inhalation via an induction chamber or intraperitoneal (IP) injection of 1% pentobarbital sodium at the dosage of 10 mL/kg.</li>
		<li>Shave the hair from the surgical site. This step can be skipped for nude mice.</li>
		<li>Disinfection and surgical draping: Disinfect the back of the mouse entirely with povidone-iodine 3x followed by 70% ethanol 1x and wipe it dry with sterile cotton swabs. Then apply three sterile medical dressings sequentially, covering the whole back in order to create a surgical field as well as to immobilize the mouse.</li>
		<li>Incision and kidney exteriorization: Before the operation, disinfect the operator&#39;s fingers with povidone-iodine or use a pair of sterile gloves. Place the mouse in the prone position and use the fingers to locate the left kidney right under the left flank. Use a pair of blunt forceps and scissors to cut the skin open and the muscle layer under the specified location. Use sterile cotton swabs and forceps to partially exteriorize the left kidney out of the abdomen.</li>
		<li>Tumor cell implantation: Load the cell suspension prepared in step 1.1 in either insulin syringes or customized Hamilton syringes (see Table of Materials for specifications). Inject 20 &#181;L of resuspended cells under the kidney capsule. 
		NOTE: Successful injection is determined by the formation of a translucent bulge on the surface of the kidney. Accidental injection into the renal parenchyma results in bleeding and a post-operative mortality rate as high as 90% due to fatal hemorrhage at the injection site.</li>
		<li>Slowly pull out the needle in order to allow the extracellular matrix solution to solidify and prevent hemorrhage or tumor cell leakage. Then, using a sterile cotton swab, push the kidney back into the abdomen.</li>
		<li>Wound stitching: Use a 5-0 coated VICTRYL suture to stitch the muscle layer. Disinfect the skin with povidone-iodine once and close the skin with wound autoclips.</li>
		<li>Recovery: Place the mouse on the warm pad until it wakes up. If the mouse was anesthetized by inhalation, withdraw the isoflurane and keep the mouse on the warm pad until it is awake.</li>
	</ol>
	</li>
	<li>Bioluminescence imaging (BLI) and tissue collection
	<ol>
		<li>Six weeks after tumor implantation, take firefly-luciferase-based BLI images. Then euthanize mice with isoflurane inhalation followed by cervical dislocation.</li>
		<li>Collect blood for circulating tumor cell (CTC) detection by flow cytometry.</li>
		<li>Harvest tumor and organs of interest (kidneys, lungs, liver, intestines, and spleen) using a sterile tissue harvesting technique22. Fix them in 4% paraformaldehyde overnight for paraffin-wax embedding.</li>
	</ol>
	</li>
</ol>
2. CAM tumor xenograft model
NOTE: These procedures were adapted and modified from previously published protocols23,24. The timeline for this procedure is shown in Figure 1B. This article presents only the streamlined protocol. For detailed protocols, please refer to another JoVE article published by our group25.
<ol>
	<li>Preincubation: Incubate freshly laid, fertilized chicken eggs in a rotating egg incubator at 37 &#176;C and 55-65% humidity for 7 days.</li>
	<li>Drop the CAM and open window (developmental day 7):
	<ol>
		<li>Locate and mark the air sac and veins. 
		NOTE: Usually 10-15% of the eggs are removed because they are either unfertilized or die within 7 days of fertilization.</li>
		<li>Create a new air sac on top of a marked vein.</li>
		<li>Delineate the new air sac, apply packing tape, and put the eggs back to the incubator. 
		NOTE: The procedure can be paused here. It is recommended to resume the procedures within the same day because the air sac may move.</li>
		<li>Open a window: Using a pair of curved microdissecting scissors and a pair of needle-nose forceps, cut a 1.5 x 1.5 cm circular window in the shell. 
		NOTE: Disruption of CAM is indicated by the blood and a piece of CAM present on the cut shell piece.</li>
		<li>Seal the hole with transparent medical dressing and place the eggs in a stationary incubator at 37 &#176;C and 55%-65% humidity. 
		NOTE: Use the same egg incubator as in step 2.1. Turn off the rotator to make it stationary.</li>
	</ol>
	</li>
	<li>Health check (developmental day 9): Remove dead eggs and then randomly group the rest of the eggs for tumor cell implantation. 
	NOTE: Ideally, the survival rate at this point is approximately 80% of developmental day 0.</li>
	<li>Grafting the tumor cells onto the newly exposed CAM (developmental day 10)
	<ol>
		<li>Dilute the extracellular matrix solution in double the volume of precooled RPMI 1640 (with L-glutamine). Detach the RENCA cells and resuspend in the above solution to reach a concentration of 2 x 104 cells/&#181;L. 
		NOTE: Use a 1:1 ratio of VHL-WT:VHL-KO cells for heterogeneous implants and VHL-WT alone for homogeneous implantation.</li>
		<li>To presolidify the cell suspension, fill 100 &#181;L of each cell mix in 200 &#181;L pipette tips and place them in a cell incubator for 15 mins.</li>
		<li>Implant 100 &#181;L of cell suspension for each egg on the CAM surface through the window. 
		NOTE: Some protocols require scratching the CAM before implantation23. This is not necessary for RENCA cells, because they grow very quickly.</li>
	</ol>
	</li>
	<li>Grow cells on the CAM for 10 days and photograph every 2 days.</li>
	<li>Euthanize and harvest the tumor, blood, and organs.
	<ol>
		<li>On developmental day 20 (tumor day 10), collect blood via the chorioallantoic vein with a heparinized 10 mL syringe.</li>
		<li>Euthanize the embryos by putting them on ice for 15 min.</li>
		<li>Harvest and weigh tumors. Dissect lungs and livers using a sterile tissue harvesting technique similar to that used for mice22. 
		NOTE: Chickens livers have two lobes, which are the first organs seen in the abdominal cavity. Do not confuse these with the lungs. The chicken lungs are located under the heart and septum26. The successful collection of the lungs can be confirmed by exposed ribs.</li>
		<li>Fix the tumors and the dissected organs in 4% paraformaldehyde overnight for paraffin-wax embedding.</li>
	</ol>
	</li>
	<li>Hatch the chickens.
	<ol>
		<li>To allow an extended period of tumor growth, continue the incubation through day 21 and let the chickens hatch at 37 &#176;C and at least 60% humidity. 
		NOTE: Chickens naturally hatch after day 21 over a 24 h time period but occasionally have trouble hatching by themselves. In this case, cracking the eggshells some helps. It is important for chickens to complete the hatching process within 24 h because they will die from lack of nutrients after then.</li>
		<li>Grow the chickens in an animal facility (2017-102-01A) for 2 weeks.</li>
		<li>On developmental day 34 (tumor day 24), euthanize the chicks with isoflurane inhalation followed by cervical dislocation.</li>
		<li>Dissect the lungs using a sterile tissue harvesting technique similar to those used for mice22. Then, fix them in 4% paraformaldehyde overnight for paraffin-wax embedding.</li>
	</ol>
	</li>
</ol>
3. Immunohistochemistry
NOTE: All tissue sectioning and H&#38;E staining was done by the Translational Pathology Core Laboratory (TPCL) at the University of California, Los Angeles.
<ol>
	<li>Bake slides at 65 &#176;C for 20 min and deparaffinize 3x using xylene and rehydrate serially from 100% ethanol to water.</li>
	<li>Retrieve the antigens in a citrate buffer boiled in a vegetable steamer for 25 min.</li>
	<li>Apply 1% BSA for blocking. Then apply the primary antibodies (anti-VHL, anti-HA, anti-flag) prepared at a 1:200 dilution ratio in PBS. Incubate overnight at 4 &#176;C.</li>
	<li>After washing 3x with TBST (7 min each), incubate slides with the secondary antibody at 1:200 dilution. Wash 3x with TBST (7 min each) and apply DAB reagents followed by hematoxylin counterstaining.</li>
</ol>
4. Flow Cytometry
<ol>
	<li>Process the mouse or chicken blood with red blood cell (RBC) lysis buffer according to the manufacturer&#39;s protocol. 
	NOTE: Sufficient RBC lysis is especially important when analyzing CAM blood because chicken RBCs are nucleated and cannot be easily distinguished in flow cytometry using the forward and side scatter.</li>
	<li>Run flow cytometry on the blood lysate and analyze the data for mStrawberry and EGFP expression.</li>
	<li>Set the primary gates based on the forward and side scatter excluding debris, dead cells, and unlysed RBCs.</li>
	<li>Set the fluorescence gates based on the unstained samples and single stained controls. Use blood lysate primed with VHL-WT, VHL-KO, or unlabeled RENCA cells as the single stained controls and unstained controls, respectively.</li>
</ol>

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The authors have nothing to disclose.

For many patients with epithelial malignancies, metastasis to vital organs is the primary cause of mortality. Therefore, it is essential to find the underlying mechanism and a new avenue of therapy for metastatic disease. Unfortunately, there is a lack of relevant metastatic ccRCC animal models. The challenge in large part is due to the inability to recreate ccRCC in mice despite the generation of numerous transgenic kidney epithelial-targeted VHL knockout mouse models9,<sup class="xref...

Renal cell carcinoma (RCC) is the 7th most common cancer in the United States. Annually, 74,000 Americans are estimated to be newly diagnosed, accounting for more than 14,000 deaths (Clear-cell histological subtype, or ccRCC, is the most common subtype, accounting for approximately 80% of RCC cases. Patients with localized malignancy are treated with nephrectomy and have a favorable 5-year survival rate of 73%1. However, 25%-30% of patients develop distant metastases to vital organs such as the lungs, resulting in a poor mean survival of 13 months and 5-year survival rate of only 11%1,2,3. Further understanding of the metastatic mechanism is needed to improve the deadly outcome for metastatic ccRCC.
The loss of the VHL tumor suppressor gene is a hallmark genetic lesion observed in a majority of human ccRCC cases4,5,6,7. However, the precise oncogenic mechanism of VHL loss in ccRCC is unknown. Also, VHL expression status is not predictive of outcome in ccRCC8. Notably, despite numerous attempts at renal-epithelial-targeted VHL knockout, scientists have failed to generate renal abnormality beyond the preneoplastic cystic lesions observed in mice9, even when combined with deletion of other tumor suppressors such as PTEN and p5310. These findings support the idea that VHL loss alone is insufficient for tumorigenesis or the subsequent spontaneous metastasis.
Recently, our laboratory created a new VHL knockout (VHL-KO) cell line using CRISPR/Cas9 mediated deletion of the VHL gene in the murine VHL+ ccRCC cell line (RENCA, or VHL-WT)11,12. We showed that VHL-KO is not only mesenchymal, but also promotes epithelial to mesenchymal transition (EMT) of VHL-WT cells12. EMT is known to play an important role in the metastatic process13. Our work further showed that distant lung metastasis occurs only with co-implantation of VHL-KO and VHL-WT cells in the kidney, supporting a cooperative mechanism of metastasis. Importantly, our orthotopically implanted VHL-KO and VHL-WT model leads to robust lung metastases, recapitulating the clinical ccRCC cases. This spontaneous metastatic ccRCC model compensates for the lack of a transgenic metastatic mouse model, especially in the development of novel anti-metastasis drugs. This protocol demonstrates the renal capsule implantation of the heterogeneous cell populations of genetic engineered RENCA cells.
Chicken CAM models have a long history in research for angiogenesis and tumor biology due to their numerous advantages, as summarized in Table 114,15,16,17,18. Briefly, the time window for CAM tumor growth is short, allowing a maximum of 11 days until the CAM is destroyed upon hatching of the chicken16. Despite the short growth time, the rich nutrition supply and immunodeficient state of the chicken embryo enable very efficient tumor engraftment16,19,20,21. Finally, the cost of each fertilized egg is ~$1, compared to over $100 for a SCID mouse. Together, the CAM model can serve as a valuable alternative animal model in establishing new PDXs at a great saving in time and cost in comparison to the mouse. In this protocol, we assessed whether the model was able to recapitulate the biology of metastatic ccRCC observed in the mouse orthotopic model.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>(SCID) Mouse</td>
			<td>CAM</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>Cost</td>
			<td>&#62;$100 each</td>
			<td>~$1 each</td>
			<td>Viability ranging from 50-75%</td>
		</tr>
		<tr>
			<td>Need for barrier housing</td>
			<td>Yes</td>
			<td>No</td>
			<td>Further reduces cost &#38; simplifies serial monitoring of the tumors</td>
		</tr>
		<tr>
			<td>Tumor directly visible</td>
			<td>No</td>
			<td>Yes</td>
			<td>Figure 3A</td>
		</tr>
		<tr>
			<td>Time to first engraftment (RENCA)</td>
			<td>2 weeks</td>
			<td>2-4 days</td>
			<td>ref 14, 15</td>
		</tr>
		<tr>
			<td>Endpoint of growth (RENCA)</td>
			<td>3-6 weeks</td>
			<td>10 days</td>
			<td>ref 14, 15</td>
		</tr>
		<tr>
			<td>Metastasis (RENCA) observed</td>
			<td>Yes</td>
			<td>Yes in chicks</td>
			<td>Figure 3D</td>
		</tr>
		<tr>
			<td>Serial passages</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>ref 16-18</td>
		</tr>
		<tr>
			<td>Passage to mice (RENCA)</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>Hu, J., et al. under review (2019)</td>
		</tr>
		<tr>
			<td>Maintain tumor heterogeneity</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>Hu, J., et al. under review (2019)</td>
		</tr>
	</tbody>
</table>
Table 1: Advantages and limitations of the mouse and CAM models. This table compares the two models for their advantages and limitations in terms of required time, cost, labor, as well as the biology. The CAM model has advantages in efficiency, but it also has its own unique limitations due to the different morphology between birds and mammals. Therefore, it is important to confirm that the model can retain the biology of the xenografts.

<table><tbody><tr><td>0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate</td><td>Corning</td><td>25053CI</td><td></td></tr><tr><td>8050-N/18 Micro 8V Max Tool Kit</td><td>Dremel</td><td>8050-N/18</td><td></td></tr><tr><td>anti-VHL antibody</td><td>Abcam</td><td>ab135576</td><td></td></tr><tr><td>BD Lo-Dose U-100 Insulin Syringes</td><td>BD Biosciences</td><td>14-826-79</td><td></td></tr><tr><td>BD Pharm Lyse</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>BDGeneral Use and PrecisionGlide Hypodermic Needles</td><td>Fisher Scientific</td><td>14-826-5D</td><td></td></tr><tr><td>DAB Chromogen Kit</td><td>Biocare Medical</td><td>DB801R</td><td></td></tr><tr><td>D-Luciferin Firefly, potassium salt</td><td>Goldbio</td><td>LUCK-1G</td><td></td></tr><tr><td>DPBS without Calcium and Magnesium</td><td>Gibco</td><td>LS14190250</td><td></td></tr><tr><td>DYKDDDDK Tag Monoclonal Antibody (FG4R)</td><td>eBioscience</td><td>14-6681-82</td><td></td></tr><tr><td>Ethanol 200 Proof</td><td>Cylinders Management</td><td>43196-11</td><td>Prepare 70% in water</td></tr><tr><td>Fetal Bovine Serum, Qualified, USDA-approved Regions</td><td>Fisher Scientific</td><td>10-437-028</td><td></td></tr><tr><td>Fisherbrand Sharp-Pointed Dissecting Scissors</td><td>Fisher Scientific</td><td>08-940</td><td></td></tr><tr><td>Fisherbrand Sterile Cotton Balls</td><td>Fisher Scientific</td><td>22-456-885</td><td></td></tr><tr><td>FisherbrandHigh Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td><td>Fisher Scientific</td><td>12-000-122</td><td></td></tr><tr><td>FisherbrandPremium Microcentrifuge Tubes: 1.5mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Formaldehyde Soln., 4%, Buffered, pH 6.9 (approx. 10% Formalin soln.), For Histology</td><td>MilliporeSigma</td><td>1.00496.5000</td><td></td></tr><tr><td>Hamilton customized syringe</td><td>Hamilton</td><td>80408</td><td>25 &amp;micro;L, Model 702 SN, Gauge: 30, Point Style: 4, Angle: 30, Needle Length: 17 mm</td></tr><tr><td>HA-probe Antibody (Y-11)</td><td>Santa Cruz Biotechnology</td><td>sc805</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>3100</td><td></td></tr><tr><td>Hovabator Genesis 1588 Deluxe Egg Incubator Combo Kit</td><td>Incubator Warehouse</td><td>HB1588D</td><td></td></tr><tr><td>Isothesia (Isoflurane) solution</td><td>Henry Schein Animal Health</td><td>1169567762</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 GFR Membrane Matrix</td><td>Corning</td><td>C354230</td><td></td></tr><tr><td>Medline Surgical Instrument Drape, Clear Adhesive, 24&quot; x 18&quot;</td><td>Medex Supply</td><td>MED-DYNJSD2158</td><td></td></tr><tr><td>OmniPur BSA, Fraction V [Bovine Serum Albumin] Heat Shock Isolation</td><td>MilliporeSigma</td><td>2910-25GM</td><td></td></tr><tr><td>Penicillin-Streptomycin Sollution, 100X, 10,000 IU Penicillin, 10,000ug/mL Streptomycin</td><td>Fisher Scientific</td><td>MT-30-002-CI</td><td></td></tr><tr><td>Pentobarbital Sodium</td><td>Sigma Aldrich</td><td>57-33-0</td><td>Prepare 1% in saline</td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>115-035-062</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>111-035-045</td><td></td></tr><tr><td>Povidone-Iodine Solution USP, 10% (w/v), 1% (w/v) Available Iodine, for Laboratory Use</td><td>Ricca Chemical</td><td>395516</td><td></td></tr><tr><td>pSicoR</td><td>Addgene</td><td>11579</td><td></td></tr><tr><td>Puromycin dihydrochloride hydrate, 99%, ACROS Organics</td><td>Fisher Scientific</td><td>AC227420500</td><td></td></tr><tr><td>Renca</td><td>ATCC</td><td>CRL-2947</td><td></td></tr><tr><td>RPMI 1640 Medium (Mod.) 1X with L-Glutamine</td><td>Corning</td><td>10040CV</td><td></td></tr><tr><td>Scientific 96-Well Non-Skirted Plates, Low Profile</td><td>Fisher Scientific</td><td>AB-0700</td><td></td></tr><tr><td>SHARP Precision Barrier Tips, For P-200, 200 &amp;micro;l, 960 (10 racks of 96)</td><td>Thomas Scientific</td><td>1159M40</td><td></td></tr><tr><td>Shipping Tape, Multipurpose, 1.89&quot; x 109.4 Yd., Tan, Pack Of 6 Rolls</td><td>Office Depot</td><td>220717</td><td></td></tr><tr><td>Suture</td><td>Ethicon</td><td>J385H</td><td></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>1634</td><td></td></tr><tr><td>Thermo-Chicken Heated Pad</td><td>K&amp;amp;H manufacturing</td><td>1000</td><td></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></td></tr><tr><td>VHL-KO</td><td></td><td></td><td>CRISPR/Cas9-mediated knockout of VHL, then lentivirally labeled with flag-tagged EGFP &amp;amp; firefly luciferase</td></tr><tr><td>VHL-WT</td><td></td><td></td><td>Lentivirally labeled with HA-tagged mStrawberry fluorescent protein &amp;amp; firefly luciferase</td></tr><tr><td>World Precision Instrument FORCEPS IRIS 10CM CVD SERR</td><td>Fisher Scientific</td><td>50-822-331</td><td></td></tr><tr><td>Wound autoclips kit</td><td>Braintree scientific, inc.</td><td>ACS KIT</td><td></td></tr><tr><td>Xylenes (Histological), Fisher Chemical</td><td>Fisher Scientific</td><td>X3S-4</td><td></td></tr></tbody></table>

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.

Each experiment was performed at least 3x unless otherwise stated. Data are presented as mean &#177; standard deviation (SD). Significance was determined by a paired, Student&#39;s T-test when there were two groups or by a one-way ANOVA when there were three or more groups. A p-value cutoff of 0.05 was used to establish significance.
Orthotopically implanted RENCA cells successfully grew on the mice kidneys, as confirmed by BLI ...

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

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, ...

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Research

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Cancer Research

8.8K Views.  David Geffen School of Medicine, University of California, Los Angeles. Clear cell niercelcarcinoom of CCRCC, is de meest voorkomende subtype van nierkanker. Localize CCRCC heeft gunstige uitkomst met een operatie. Echter, een derde van de CCRCC patiënten zal ontwikkelen verre metastase naar de long.Wat leidt tot een zeer slechte uitkomst voor patiënten. Helaas is er geen therapie beschikbaar voor deze dodelijke fase. Aangezien het moleculaire mechanisme van metastase zinspeling blijft.Het doel van dit protocol is het introduceren en vergelijken v...

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

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

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

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 ...

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.

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 ...

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

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

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 ...

Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells

Source: Murphy, K. A. et al. <a href="https://www.jove.com/video/55080" target="_blank">A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies.</a> J. Vis. Exp. (2017)
In this video, we develop an orthotopic mouse model of renal cell carcinoma. This model results in reproducible primary tumors in the injected kidney and metastatic tumors in the lung, allowing the evaluation of disease progression and treatment.

Source: Murphy, K. A. et al. A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical ...

JoVE Encyclopedia of Experiments

Urinary Tract 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/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.

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

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