Name: comparing metastatic clear cell renal cell carcinoma model established in mouse kidney and on chicken chorioallantoic membrane
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Description: 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

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 C...

<ol>
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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>
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			<td>Scientific 96-Well Non-Skirted Plates, Low Profile</td>
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			<td>Office Depot</td>
			<td>220717</td>
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			<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>
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			<td>Thermo-Chicken Heated Pad</td>
			<td>K&#38;H manufacturing</td>
			<td>1000</td>
			<td></td>
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		<tr>
			<td>Tygon Clear Laboratory Tubing - 1/4 x 3/8 x 1/16 wall (50 feet)</td>
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			<td>AACUN017</td>
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			<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>
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			<td>VHL-WT</td>
			<td></td>
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			<td>Lentivirally labeled with HA-tagged mStrawberry fluorescent protein &#38; firefly luciferase</td>
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			<td>World Precision Instrument FORCEPS IRIS 10CM CVD SERR</td>
			<td>Fisher Scientific</td>
			<td>50-822-331</td>
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			<td>Braintree scientific, inc.</td>
			<td>ACS KIT</td>
			<td></td>
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		<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, ...

Clear cell renal cell carcinoma or CCRCC, is the most common subtype of kidney cancer. Localize CCRCC has favorable outcome with surgery. However, one third of CCRCC patients will develop distant metastasis to the lung.Which leads to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage. As the molecular mechanism of metastasis remains allusive.The purpose of this protocol is to introduce and compare the two animal models recently established for metastatic CCRCC. The first model is the mouse of the topic implantation. In this mouse model, heterogeneous CCRCC is implanted to the renal capsule of the mouse.Tumor growth and the metastasis are asset after 10 weeks. The second model is the chicken chorioallantoic membrane model, or the CAM model. In this chicken model the heterogeneous CCRCC is implanted in the egg.Tumor growth and circulating tumor cells are asset after 10 days of implantation before the embryo hatches. Lung metastasis is asset after 24 days of the implantation. Which is two weeks after hatching.These two novel models will be useful to further study the molecular mechanism behind the metastasis in CCRCC. Please note that the CAM procedure is not featured in this video. The detailed protocol for the CAM model is described and filmed in another Trove article by Sharrow et al.After anesthetizing a the mouse, disinfect the back of the mouse entirely with povidone-iodine for three times. Follow by 70%ethnol once, and wipe it dry with sterile cotton swabs. Then apply three sterile medical dressing sequentially covering the whole back in order to create a surgical field as well as to immobilize the mouse.Before the operation, the operators should disinfect their fingers with povidone-iodine. Place the mouse in the prone position. And, use fingers to locate the left kidney just under the left flank.Use a pair of blunt forceps and scissors to cut open the skin and muscle layer under the specified location. Use sterile cotton swabs and forceps to partially exteriorize the left kidney out of the abdomen. Load either an insulin syringe or a customized Hamilton syringe with the tumor cell suspension prepared separately.Inject 20 microliters of cell suspension under the kidney capsule. Slowly pull out needle in order to solidify the extra cellular matrix solution. As well as to prevent hemorrhage or tumor cell leakage.And then, use a sterile cotton swab to push the kidney back into the abdomen. Use suture to stitch the muscle layer. Disinfect the skin with povidone-iodine.And, close the skin with wound auto-clips. Orthotopic laying planted tumor cells successfully grew on mice kidney as confirmed by BLI. Although there was no difference in the primary growth, only the heterogeneous metastatic tumor had robust metastasis to the lung.The metastasis was also confirmed by immunohistochemistry. Similarly, the circulating tumor cell counts were higher in the mice bearing metastatic tumor than those bearing non-metastatic tumor. In concordance with the mouse model the CAM model also showed no growth difference between metastatic and non-metastatic tumors.On the other hand, the number of circulating tumor cells was significantly higher in the eggs with metastatic tumors. Histologically detectable metastasis was observed in the lungs of hatched chicken. In conclusion, this protocol illustrated two novel animal models for metastatic CCRCC.Both the mouse and the chicken models faithfully recapitulated the lung metastasis observed in the clinic. These two pre-clinical models will be useful to expand the understanding of the molecular mechanism behind the metastasis. As well as to discover novel treatments for metastatic CCRCC.

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