A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

All of the animal procedures outlined in this protocol are to be performed in compliance with ethical regulations and the approval of the appropriate university Institutional Animal Care and Use Committee (IACUC).

1. Preparation of Surgical Materials and Cancer Cells

1. Before the day of surgery, autoclave the micro-dissecting scissors, Graefe forceps, Graefe tissue forceps, needle holder with suture cutters, and the appropriate number of drapes. Sterilize a 50 µL syringe and 28-gauge needles by ethylene oxide gas (Figure 1C).
2. Before the day of surgery, culture Myc-CaP cells in 10 cm dishes in RPMI supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) in a 37 °C 5% CO₂ incubator. For bioluminescent and/or fluorescent tumor monitoring, transfect 293T cells with the appropriate vectors and subsequently transduce Myc-CaP cells with 0.45 µm filtered lentivirus³¹. Sort cells to generate a stable cell line with 100% transduction positivity.
   NOTE: Perform all tissue culture under sterile conditions, and verify that all cell lines are mycoplasma-free. The murine prostate cancer cell line utilized in this study, Myc-CaP³², is transduced to stably express both firefly luciferase and mCherry. The Myc-CaP cell line iss isolated from a c-myc-overexpressing Hi-myc mouse, and these cells contain an amplified androgen receptor (AR), which are androgen-dependent³² and form CRPC tumors after murine castration³³. All mice in this study are 6-8 week old male FVB/NJ mice. Alternative murine prostate cancer cell lines can be used with the mice of appropriate genetic background, as well as human prostate cancer cell lines or primary patient-derived prostate cancer cells with immunodeficient mice. The optimal number of cells per injection should be determined empirically for alternative cell lines. In addition, alternative imaging modalities can be utilized to visualize tumors, including magnetic resonance imaging (MRI), positron emission tomography (PET), or computed tomography (CT), as well as GFP as an alternative to mCherry.
3. The night before surgery, place matrigel basement membrane matrix and phenol red-free on ice at 4 °C to thaw.
4. On the day of surgery, collect the cells by washing them with phosphate buffered saline (PBS) and detaching with 0.25% trypsin-EDTA. Neutralize trypsin with RPMI with 10% FBS and 1% P/S.
5. Centrifuge the cells at 400 x g for 5 min.
6. Wash the cells with 10 mL of RPMI without FBS or P/S.
7. Count the cells and resuspend the cells in PBS at a concentration of 6.67×10⁷ cells/mL (1×10⁶ cells/15 µL).
8. Add an equal volume of matrigel to create a 1:1 PBS/matrigel cell suspension at a final concentration of 1×10⁶ cells/30 µL, and keep the cells on ice until injection to prevent solidification.
   NOTE: Perform intra-prostatic injections within 3 h of preparing matrigel cell suspensions. If performing intra-prostatic injections on more than 5 mice, repeat the above steps to prepare a fresh matrigel cell suspension.

2. Pre-operative mouse preparation

NOTE: House mice in the university animal facility for at least one week before surgery to allow for adequate adaptation and to minimize animal stress. Steps 2.1-2.6 are performed by the surgeon assistant. All subsequent surgical steps are performed by the surgeon using sterile gloves and surgical tools with sterile technique.

1. Anesthetize the mice with isoflurane (2-5% for induction via chamber, 1-3% for maintenance via nose cone). Verify full induction by the loss of toe pinch reflex.
2. Weigh the mice and administer at least 0.05 mg/kg of the analgesic buprenorphine s.c.
   NOTE: Mouse weight ≥20 g is ideal for successful intra-prostatic injections.
3. Apply ophthalmic ointment lubrication to both eyes to prevent corneal drying.
4. Shave all fur from the abdomen.
5. Sterilize the abdomen by three rounds of circular application of surgical scrub using sterile non-adhering pads followed by sterile alcohol wipes. Allow the abdomen to dry.
   NOTE: For the rest of the surgical procedure, only sterile gloves and surgical instruments can contact the sterilized mouse abdomen.
6. Transfer the mouse supine onto a clean surface on a heating pad directly under the objective of a clean surgical microscope.
7. Cover the mouse with a sterile drape with a small hole cut over the abdomen.

3. Intra-prostatic injection

NOTE: Perform all surgical steps under sterile conditions. Adjusting the microscope, the mouse placement, or any other non-sterile objects must be done by the surgeon assistant.

1. Perform an approximately 1 cm incision of the outer skin along the midline of the abdomen superior to the penis and the preputial glands (Figure 1D).
2. Separate the connective tissue between the outer abdominal skin and the inner abdominal musculature by opening the scissors between the layers.
3. Perform a similar incision of the inner abdominal musculature while using forceps to raise the musculature to avoid puncturing the intestines or bladder (Figure 1D).
4. Locate one of the bilateral seminal vesicles (Figure 1A, white)/anterior prostate lobes (Figure 1A, black), which are often posterior, lateral, and slightly superior to the bladder (Figure 1A, yellow), but this may vary between mice. The anterior prostate lobes are translucent and attached to the lesser curvature of the white seminal vesicles.
5. Gently raise the tip of the seminal vesicle using the Graefe tissue forceps to create tension on the tissue without puncturing the seminal vesicle (Figure 1E).
6. Mix the Myc-CaP matrigel solution by gentile pipetting (performed by the surgeon assistant), as cells may have pelleted, and slowly aspirate 30 µL (1×10⁶ cells) into the needle to avoid air bubbles.
7. Carefully insert the bevel of the needle parallel to the long axis of the anterior prostate lobe. Slowly inject 30 µL into the lobe and slowly retract the needle to prevent leakage (Figure 1F). Verify adequate injection by engorgement of the anterior prostate lobe (Figure 1G).
8. Carefully hold the seminal vesicle and injected prostate lobe outside of the mouse for approximately 30 s to allow matrigel to partially solidify within the lobe. During this time, collect any cell solution leakage in the abdomen using a sterile polyester tipped applicator to prevent non-orthotopic tumor development, without pressing on the injected prostate lobe.
9. Carefully return the seminal vesicle and injected prostate lobe into the abdomen without putting pressure on the lobe. Replace any externalized tissues.
10. Perform continuous sutures to close the inner abdominal musculature with 5-0 vicryl absorbable reverse cutting needle sutures.
11. Perform interrupted sutures to close the outer abdominal skin with 4-0 nylon monofilament non-absorbable reverse cutting needle sutures.
    NOTE: Sterilized 9 mm staples can also be used to close the outer abdominal skin. However, in contrast to sutures, they interfere with any subsequent bioluminescent and fluorescent tumor imaging signal.
12. Clean all tools with sterile ethanol wipes (opened by the surgeon assistant) and place them in a glass bead sterilizer for 30 s. Allow the tools to dry before use on the next mouse.
13. Flush the syringe and needle in sterile saline (opened by the surgeon assistant) to prevent clogging by remnant matrigel.
    NOTE: A surgeon assistant should be present for all operations, performing all non-sterile pre-operative mouse preparation and post-operative mouse care, as well as to mix the Myc-CaP matrigel solution before injections. To minimize time, as each intra-prostatic mouse procedure will require 20-30 min, the surgeon assistant can begin preparing the next mouse as the surgeon is suturing the current mouse.

4. Post-operative Mouse Care

1. Administer 1 mg/kg of the analgesic meloxicam s.c. immediately, 24 h, and 48 h after surgery.
2. Allow mice to recover in a cage with no bedding placed halfway on a heating pad. Monitor mice for at least 30 min after surgery until they regain normal mobility and activity, after which place them in a clean cage with food on the floor of the cage.
3. Further monitor mice daily for proper wound healing, body weight, grooming, and ambulation after surgery. Separate mice into individual cages upon any evidence of fighting.
4. Remove any remaining sutures within 14 days after surgery.

5. Bioluminescent Tumor Imaging

1. Prepare sterile-filtered Na⁺ or K⁺ D-luciferin, as described by the manufacturer and protected from light.
2. Inject mice intra-peritoneally with 10 µL/g of body weight of luciferin.
3. At least 10 min later, image mice with an IVIS Spectrum Imaging System, as previously described^34,35.
4. Analyze images using Living Image Software, as previously described^34,35.
   NOTE: IVIS imaging analysis is useful for determining successful intra-prostatic injections and initial tumor development to normalize tumor burden among experimental groups. However, depending on the intensity of the bioluminescent or fluorescent signal, saturation may cause a plateau in the image quantification despite an increase in actual tumor size. Therefore, in the later stages of tumor growth, IVIS imaging may be more useful to determine decreases in tumor size upon successful treatment rather than increases in size after image saturation.

6. Tumor Collection, Tumor Analysis, Survival Endpoint

1. If collecting tumor for analysis, humanely euthanize mice by CO₂ exposure and secondary cervical dislocation, or by alternative IACUC approved methods, and dissect tumors from the abdomen. Tumors should be located orthotopically at the prostate.
   NOTE: Tumors attached to the anterior abdominal wall or seeded throughout the abdomen indicate poor or leaky intra-prostatic injection, and should not be considered orthotopic tumors.
2. Weigh the tumors.
3. Calculate tumor volume as π/6×L×W×H (L=length of the longest axis of the tumor, W=perpendicular width, H=perpendicular height).
4. Tumors can be analyzed by histology (fix in 10% neutral buffered formalin), flow cytometry (create single cell suspensions), protein (prepare tissue lysate in RIPA buffer), or RNA (immediately place tissue in RNAlater).
5. For immunological analysis, also collect the prostate tumor-draining para-aortic lymph nodes (Figure 1B, orange) and the spleen.
   NOTE: If following mice for survival, the endpoint of this model is defined as the appearance of hemorrhagic abdominal ascites³⁶ and/or decreased ambulation, grooming, and/or piloerection³⁷. Mice for survival analysis receive pre- and post-operative analgesia (protocol steps 2.2, 4.1) and must be monitored regularly. Mice should be separated into individual cages if any fighting occurs, and should be humanely euthanized upon the appearance of any of the above endpoint readouts.

7. Surgical Castration to Model CRPC

1. To model CRPC, perform the above intra-prostatic injection protocol (protocol steps 1-5).
2. At least one week later, after tumor development, perform surgical castration via cauterization, as previously described³⁸.
3. Monitor tumor regression and recurrence by bioluminescent imaging. After castration, tumor regression will occur within 3 days and tumor recurrence will occur within approximately 30 days, representing CRPC.