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%^1,2,3. 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 cases^4,5,6,7. 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 1^{14,15,16,17,18}. 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 engraftment^16,19,20,21. 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.

	(SCID) Mouse	CAM	Note
Cost	>$100 each	~$1 each	Viability ranging from 50-75%
Need for barrier housing	Yes	No	Further reduces cost & simplifies serial monitoring of the tumors
Tumor directly visible	No	Yes	Figure 3A
Time to first engraftment (RENCA)	2 weeks	2-4 days	ref 14, 15
Endpoint of growth (RENCA)	3-6 weeks	10 days	ref 14, 15
Metastasis (RENCA) observed	Yes	Yes in chicks	Figure 3D
Serial passages	Yes	Yes	ref 16-18
Passage to mice (RENCA)	Yes	Yes	Hu, J., et al. under review (2019)
Maintain tumor heterogeneity	Yes	Yes	Hu, J., et al. under review (2019)

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.

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. Orthotopic tumor studies in mice

NOTE: The timeline for this experiment is shown in Figure 1A. These procedures were adapted from previous publications^11,12.

1. Preparing single cell suspension for grafting
   1. Detach the RENCA VHL-WT and VHL-KO cells from the culture dishes using trypsin/EDTA.
   2. 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/µL.
      NOTE: Use a 1:4 ratio of VHL-WT:VHL-KO cells for heterogeneous implants and VHL-WT alone for homogeneous implants.
   3. Transfer the resuspended cells into a 1.5 mL microcentrifuge tube and keep on ice until implantation.
2. Implantation to renal capsule
   1. Anesthesia: Preheat a warm pad to 37 °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.
   2. Shave the hair from the surgical site. This step can be skipped for nude mice.
   3. 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.
   4. Incision and kidney exteriorization: Before the operation, disinfect the operator'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.
   5. 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 µL 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.
   6. 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.
   7. 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.
   8. 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.
3. Bioluminescence imaging (BLI) and tissue collection
   1. Six weeks after tumor implantation, take firefly-luciferase-based BLI images. Then euthanize mice with isoflurane inhalation followed by cervical dislocation.
   2. Collect blood for circulating tumor cell (CTC) detection by flow cytometry.
   3. 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.

2. CAM tumor xenograft model

NOTE: These procedures were adapted and modified from previously published protocols^23,24. 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.

1. Preincubation: Incubate freshly laid, fertilized chicken eggs in a rotating egg incubator at 37 °C and 55-65% humidity for 7 days.
2. Drop the CAM and open window (developmental day 7):
   1. 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.
   2. Create a new air sac on top of a marked vein.
   3. 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.
   4. 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.
   5. Seal the hole with transparent medical dressing and place the eggs in a stationary incubator at 37 °C and 55%-65% humidity.
      NOTE: Use the same egg incubator as in step 2.1. Turn off the rotator to make it stationary.
3. 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.
4. Grafting the tumor cells onto the newly exposed CAM (developmental day 10)
   1. 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/µL.
      NOTE: Use a 1:1 ratio of VHL-WT:VHL-KO cells for heterogeneous implants and VHL-WT alone for homogeneous implantation.
   2. To presolidify the cell suspension, fill 100 µL of each cell mix in 200 µL pipette tips and place them in a cell incubator for 15 mins.
   3. Implant 100 µL 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.
5. Grow cells on the CAM for 10 days and photograph every 2 days.
6. Euthanize and harvest the tumor, blood, and organs.
   1. On developmental day 20 (tumor day 10), collect blood via the chorioallantoic vein with a heparinized 10 mL syringe.
   2. Euthanize the embryos by putting them on ice for 15 min.
   3. 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.
   4. Fix the tumors and the dissected organs in 4% paraformaldehyde overnight for paraffin-wax embedding.
7. Hatch the chickens.
   1. To allow an extended period of tumor growth, continue the incubation through day 21 and let the chickens hatch at 37 °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.
   2. Grow the chickens in an animal facility (2017-102-01A) for 2 weeks.
   3. On developmental day 34 (tumor day 24), euthanize the chicks with isoflurane inhalation followed by cervical dislocation.
   4. 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.

3. Immunohistochemistry

NOTE: All tissue sectioning and H&E staining was done by the Translational Pathology Core Laboratory (TPCL) at the University of California, Los Angeles.

1. Bake slides at 65 °C for 20 min and deparaffinize 3x using xylene and rehydrate serially from 100% ethanol to water.
2. Retrieve the antigens in a citrate buffer boiled in a vegetable steamer for 25 min.
3. 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 °C.
4. 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.

4. Flow Cytometry

1. Process the mouse or chicken blood with red blood cell (RBC) lysis buffer according to the manufacturer'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.
2. Run flow cytometry on the blood lysate and analyze the data for mStrawberry and EGFP expression.
3. Set the primary gates based on the forward and side scatter excluding debris, dead cells, and unlysed RBCs.
4. 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.

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 ...

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

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