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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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&#34; x 18&#34;</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 &#181;l, 960 (10 racks of 96)</td>
			<td>Thomas Scientific</td>
			<td>1159M40</td>
			<td></td>
		</tr>
		<tr>
			<td>Shipping Tape, Multipurpose, 1.89&#34; 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&#34; x 2 3/4&#34;</td>
			<td>Moore Medical</td>
			<td>1634</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo-Chicken Heated Pad</td>
			<td>K&#38;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 &#38; firefly luciferase</td>
		</tr>
		<tr>
			<td>VHL-WT</td>
			<td></td>
			<td></td>
			<td>Lentivirally labeled with HA-tagged mStrawberry fluorescent protein &#38; 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 ...

Watch this Scientific Journal Video about Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane at JoVE.com

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, ...

comparing-metastatic-clear-cell-renal-cell-carcinoma-model

Research

JoVE Journal

Cancer Research

8.8K Views.  David Geffen School of Medicine, University of California, Los Angeles. 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.

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

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

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 ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

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 ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (&#181;mBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model &#181;mBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

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 ...