An Orthotopic Murine Model of Human Prostate Cancer Metastasis

For all procedures involving animals, protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University. Surgical techniques and animal care conditions were observed by veterinary staff and modified to minimize animal stress or mortality. Individual institutions may have different requirements and it is important to work with IACUC and animal staff when developing and executing this surgical technique.

1. Preparation of Cells for Injection

1. This step should be performed as close as possible to starting surgeries. Trypsinize cells being used for the experiment, remove from plate, and neutralize with media. The PC3-M human PCa cell line stably transfected with GFP has been successfully used for these experiments, due to rapid growth conditions of the PC3-M cells. GFP was added for additional ease of detection of the lung metastasis in lung tissue samples, and rapid determination of cancer cells versus immune cells in cultures obtained from blood and bone marrow to identify circulating tumor cells. If modifying a specific gene of interest, then creation of stable cell lines with this gene alteration is performed first, followed by transfection with GFP. If GFP will alter the function of this gene of interest, GFP it can be omitted. This will make detection of lung metastasis more difficult, but still achievable. Additionally, if using specific imaging technology using fluorescent markers, such as luciferase-based IVIS, other fluorescent proteins can be used instead.
2. Centrifuge for 5 min at 225 x g.
3. Isolate 2.5 x 10⁵ cells, and centrifuge for 5 min at 225 x g.
4. Remove supernatant and resuspend cells in 20 μl of sterile saline.
5. Aspirate cell suspension into a 0.5 ml syringe with a permanent 28 ½ G needle (recommended: Kendall Monojet, as other brands have caused issues), ensuring no air enters the needle along with the suspension.
6. Wrap syringe in sterile foil and store on ice until used.

2. Orthotopic Implantation of Human Prostate Cancer Cells

1. Male 6-8 week old Balb/c athymic mice are used. The ideal mouse body weight for surgery is 19-21 g, and mice should be allowed to grow to at least 18 g before surgery. Smaller animals are technically more difficult to operate on. Larger animals tend to experience slower kinetics of tumor growth and metastasis. Animals should be housed in the animal facility at least one week prior to surgery to minimize animal stress. Depending on the experimental design, drug treatment may begin one week prior to surgery.
2. Inject pre-surgery pain medication according to animal facility’s instructions. 0.1 mg/kg body weight subcutaneous Buprenex using a 30 ½ G needle attached to a 1 ml syringe is suggested.
3. Place animals into the isoflurane chamber and wait until animals are fully anesthetized. No toe reflex of muscle tone should be present at this point. This method is recommended, but if unavailable, other methods of anesthesia can be used according to animal facility’s instructions.
4. Move animal out of the isoflurane chamber into a sterile procedure hood. Place animal into the nose cone apparatus, and re-ensure that animal is under full anesthetization before proceeding.
5. Disinfect lower abdominal region with a Betadine scrub with sterile cotton balls, wipe with alcohol wipe, and final spray with Betadine solution. Allow to dry.
6. Using either a sterile scalpel or sharpened sterile surgical scissors, a low midline abdominal incision of approximately 3-4 mm is made. Gently lift the bladder using forceps and identify the ventral lobe of the prostate. These two lobes are located directly beneath the bladder. Some mice will have a small layer of fat covering the prostate which can be gently moved aside using a sterile cotton swab. Minimize all movement of organs and musculature if possible.
7. Inject 20 ul volume cell solution into the prostate gland, minimizing leakage and ensuring a small bubble is observed.
8. Replace bladder and close the muscle layer using 4.0 absorbable vicryl monofilament sutures in a simple interrupted pattern.
9. Close the skin layer using sterile 9 mm staples.
10. Remove animal from isoflurane anesthesia and monitor until awake and moving normally. Place on heating pad during recovery period.
11. If multiple surgeries are being performed, between surgeries, clean all tools with 70% ethanol, and sterilize using a glass bead sterilizer. Allow tools to cool fully before next animal. Do not reuse sutures, cotton balls, or sterile swabs.

3. Monitoring Animals

1. Administer pain medication based on animal facility’s instructions. 4 hr post-surgery administering of one dose of subcutaneous Meloxicam at 1 mg/kg body weight using a 30 ½ G needle attached to a 1 ml syringe is suggested, followed by an additional dose every 12-24 hr for the next 48 hr.
2. Staples can be removed from animals 7-10 days post-surgery once the wound has healed.
3. Monitor animal weight, food consumption, and palpate mice for tumors twice a week until termination of experiment. Increase frequency up to every other day when tumors become visible to check for animals under significant tumor burden or duress. Early death from this model is due to the large primary tumor burden, as primary tumors can block urinary flow, so any animals with a loss of greater than 15% body weight or a primary tumor reaching a diameter of 1.5 cm should be necropsied immediately to prevent urinary obstruction and animal death.
4. Monitor for need for additional enrichment of the animal environment. The stress of surgery increases animal infighting. The addition of two plastic huts per cage instead of one and additional behavioral enrichment can decrease these incidents. Discuss with veterinary staff options available at the facility the animals are housed at. Given the lack of eyelashes on this strain of mice, huts and enrichment made of paper are not recommended as they can irritate eyes.

4. Necropsy Procedures

1. After 4-6 weeks, tumors are fully evident and animals begin to lose weight due to increased tumor burden. Tumors can typically be palpated and/or are visually apparent. Necropsy should be performed at this point. Perform an intraperitoneal injection of Nembutal at 260 mg/kg body weight using a 30 ½ gauge needle attached to a 1 ml syringe, and allow animals to become fully unconscious, with no toe reflex or muscle tone present.
2. Once the animal is anesthetized, using sterile surgical scissors or a scalpel, cut horizontally across the torso of the animal directly under the rib cage, then vertically to the armpit along the side of the animal, expose the heart. Perform a terminal cardiac puncture using a 30 ½ G needle attached to a 1 ml syringe flushed with 4% sodium citrate in DPBS to prevent coagulation, obtaining as much blood as possible from the animal.
3. Remove the lungs from the animal by cutting the trachea with surgical scissors or a scalpel and immediately place into a tissue culture cassette and into 10% formalin.
4. Remove the primary prostate tumor from the animal by individually cutting any blood vessels attached to the tumor and ensuring no additional organs such as the vas deferens or seminal vesicles are attached.
5. Record the weight and size of the tumor. Measure the weight of the tumor in grams on a laboratory scale. Using calipers, measure the length of the longest diameter of the tumor in centimeters, and the corresponding perpendicular axis. Multiply these values to obtain tumor size. Depending on intended usage, immediately snap freeze in liquid nitrogen, and/or place into a tissue cassette into 10% formalin.
6. Expose the hip and knee joints with surgical scissors or a scalpel, and disconnect leg joints, being careful to keep the femur intact. Remove the femurs from the animal and place into sterile saline.
7. Obtain any other organs or materials of interest, and dispose of animal according to your institute’s instructions. In particular, regional lymph nodes tend to have metastasis, tend to be enlarged if harboring them, and can be harvested. However, care should be taken as this can be affected by changes in hydrostatic pressure caused by the surgical procedure.

5. Processing and Immunostaining of Lung Tissue Samples

1. Within 24-48 hr of placing tissue into 10% formalin in PBS, embed the lungs into paraffin.
2. Section the lungs at 45 μm step sections, taking 2-3 adjacent 4 μm lung tissue sections.
3. If GFP-positive cells were used (recommended), perform immunostaining for GFP. If not, perform standard Hematoxylin and eosin (H&E) staining.

NOTE: The Dako Envision+ Kit combined with the GFP antibody from Invitrogen has been used successfully according to manufacturer’s instructions, but this can be modified to any immunohistochemistry procedure.

4. Score metastasis in a blinded fashion. If GFP-positive, tumor cells will stain brown in addition to having large and distinctive nuclei. When first scoring metastasis, consult a pathologist to ensure correct scoring, and use H&E stained lung tissue to confirm presence of tumor cells, and not infiltrating immune cells.

6. Identification of Circulating Tumor Cells from Blood and Bone Marrow

1. Add all blood collected from the necropsy to a 1.5 ml centrifuge tube. Centrifuge 5 min at 800 x g.
2. Remove the plasma and add 1 ml ACK Lysis Buffer (154.95 mM Ammonium Chloride, 9.99 mM Potassium Bicarbonate, 0.0995 mM EDTA,). Allow the blood to sit at room temperature 3-5 min.
3. Centrifuge 5 min at 800 x g.
4. Remove the supernatant and add 1 ml of cell culture media containing antibiotics. Add cells to a T75 flask containing 9 ml of cell culture media.
5. Culture cells at 37 °C in a humidified atmosphere containing 5% CO₂ for 10 days, checking for the presence of CTCs every 2-3 days.
6. For bone marrow, remove all muscle tissue from femurs using a pair of scissors, a razor blade, or scalpel. Remove the ends of the bone, as close to the ends as possible.
7. Using a 23 ¾ G needle, gently eject the bone marrow from the femur into a 1.5 ml centrifuge tube.
8. Add to 1 ml of cell culture media and gently pipette well to mix.
9. Add cells to a T75 flask containing 9 ml of cell culture media.

7. Molecular Characterization of Tumors

1. For polymerase chain reaction (PCR) assays, tumor samples that were snap frozen in liquid nitrogen are first pulverized on dry ice and then homogenized using a tissue homogenizer for 3-5 min with 1 ml TRIzol. TRIzol extraction is performed per manufacturer’s instructions. Purify RNA using the RNeasy RNA isolation kit according to manufacturer’s instructions. Performing cDNA synthesis and quantitative real time PCR (qRT/PCR) using TaqMan reagents according to manufacturer’s instructions is recommended but can be modified for any PCR method currently employed.
2. For Western blot assays, homogenize tissues using a tissue homogenizer for 3-5 min with 1 ml of lysis buffer: PBS (137 mM NaCl, 10 mM Na phosphate, 2.7 mM KCl), 0.5% Triton X-100, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, with addition of protease inhibitor cocktail, phosphate inhibitor cocktails 2 and 3, 10 mM sodium fluoride, and 1 mM sodium orthovanadate. The cell lysate mixture is placed in a 1.5 ml centrifuge tube.
3. Centrifuge cell lysate mixture for 10 min at 13,000 x g.
4. Remove supernatant and place in a new 1.5 ml centrifuge tube and centrifuged again for 10 min at 13,000 x g. If cell debris is still present, an additional centrifugation step can be performed.
5. Remove supernatant and perform a Western blot as per normal laboratory conditions.