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

Summary

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.

Abstract

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.

Introduction

Renal cell carcinoma (RCC) is the 7^th 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,

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC), designated as UCLA Chancellor'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. Orthotop....

Results

Each experiment was performed at least 3x unless otherwise stated. Data are presented as mean ± standard deviation (SD). Significance was determined by a paired, Student'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 and H&.......

Discussion

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 models^9,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate	Corning	25053CI
8050-N/18 Micro 8V Max Tool Kit	Dremel	8050-N/18
anti-VHL antibody	Abcam	ab135576
BD Lo-Dose U-100 Insulin Syringes	BD Biosciences	14-826-79
BD Pharm Lyse	BD Biosciences	555899
BDGeneral Use and PrecisionGlide Hypodermic Needles	Fisher Scientific	14-826-5D
DAB Chromogen Kit	Biocare Medical	DB801R
D-Luciferin Firefly, potassium salt	Goldbio	LUCK-1G
DPBS without Calcium and Magnesium	Gibco	LS14190250
DYKDDDDK Tag Monoclonal Antibody (FG4R)	eBioscience	14-6681-82
Ethanol 200 Proof	Cylinders Management	43196-11	Prepare 70% in water
Fetal Bovine Serum, Qualified, USDA-approved Regions	Fisher Scientific	10-437-028
Fisherbrand Sharp-Pointed Dissecting Scissors	Fisher Scientific	08-940
Fisherbrand Sterile Cotton Balls	Fisher Scientific	22-456-885
FisherbrandHigh Precision Straight Tapered Ultra Fine Point Tweezers/Forceps	Fisher Scientific	12-000-122
FisherbrandPremium Microcentrifuge Tubes: 1.5mL	Fisher Scientific	05-408-129
Formaldehyde Soln., 4%, Buffered, pH 6.9 (approx. 10% Formalin soln.), For Histology	MilliporeSigma	1.00496.5000
Hamilton customized syringe	Hamilton	80408	25 µL, Model 702 SN, Gauge: 30, Point Style: 4, Angle: 30, Needle Length: 17 mm
HA-probe Antibody (Y-11)	Santa Cruz Biotechnology	sc805
Hemocytometer	Hausser Scientific	3100
Hovabator Genesis 1588 Deluxe Egg Incubator Combo Kit	Incubator Warehouse	HB1588D
Isothesia (Isoflurane) solution	Henry Schein Animal Health	1169567762
IVIS Lumina II In Vivo Imaging System	Perkin Elmer
Matrigel GFR Membrane Matrix	Corning	C354230
Medline Surgical Instrument Drape, Clear Adhesive, 24" x 18"	Medex Supply	MED-DYNJSD2158
OmniPur BSA, Fraction V [Bovine Serum Albumin] Heat Shock Isolation	MilliporeSigma	2910-25GM
Penicillin-Streptomycin Sollution, 100X, 10,000 IU Penicillin, 10,000ug/mL Streptomycin	Fisher Scientific	MT-30-002-CI
Pentobarbital Sodium	Sigma Aldrich	57-33-0	Prepare 1% in saline
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)	Jackson ImmunoResearch Laboratories	115-035-062
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)	Jackson ImmunoResearch Laboratories	111-035-045
Povidone-Iodine Solution USP, 10% (w/v), 1% (w/v) Available Iodine, for Laboratory Use	Ricca Chemical	395516
pSicoR	Addgene	11579
Puromycin dihydrochloride hydrate, 99%, ACROS Organics	Fisher Scientific	AC227420500
Renca	ATCC	CRL-2947
RPMI 1640 Medium (Mod.) 1X with L-Glutamine	Corning	10040CV
Scientific 96-Well Non-Skirted Plates, Low Profile	Fisher Scientific	AB-0700
SHARP Precision Barrier Tips, For P-200, 200 µl, 960 (10 racks of 96)	Thomas Scientific	1159M40
Shipping Tape, Multipurpose, 1.89" x 109.4 Yd., Tan, Pack Of 6 Rolls	Office Depot	220717
Suture	Ethicon	J385H
Tegaderm Transparent Dressing Original Frame Style 2 3/8" x 2 3/4"	Moore Medical	1634
Thermo-Chicken Heated Pad	K&H manufacturing	1000
Tygon Clear Laboratory Tubing - 1/4 x 3/8 x 1/16 wall (50 feet)	Tygon	AACUN017
VHL-KO			CRISPR/Cas9-mediated knockout of VHL, then lentivirally labeled with flag-tagged EGFP & firefly luciferase
VHL-WT			Lentivirally labeled with HA-tagged mStrawberry fluorescent protein & firefly luciferase
World Precision Instrument FORCEPS IRIS 10CM CVD SERR	Fisher Scientific	50-822-331
Wound autoclips kit	Braintree scientific, inc.	ACS KIT
Xylenes (Histological), Fisher Chemical	Fisher Scientific	X3S-4