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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		<tr>
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			<td>Fisher Scientific</td>
			<td>AC227420500</td>
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			<td></td>
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		<tr>
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			<td>Corning</td>
			<td>10040CV</td>
			<td></td>
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			<td>Scientific 96-Well Non-Skirted Plates, Low Profile</td>
			<td>Fisher Scientific</td>
			<td>AB-0700</td>
			<td></td>
		</tr>
		<tr>
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	</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, ...

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