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

The overall goal of this procedure is to perform an intra-prostatic injection of murine cancer cells to induce the formation of syngeneic orthotopic tumors that can model both androgen-dependent and castration-resistant prostate cancer. This method can help answer key questions in the prostate cancer field, such as what drives prostate cancer development and what treatments work best in eliminating these tumors. The main advantages of this technique are that orthotopic tumors are genetically more complex, they develop in a clinically representative tumor microenvironment, and they can be genetically modified before injection.

The implications of this technique extend toward the therapy of human prostate cancer as orthotopic tumors respond to therapies in a similar manner as that observed in human patients. After confirming a lack of response to toe pinch, apply ointment to the mouse's eyes and shave all of the fur from the animal's abdomen. Sterilize the exposed skin with three rounds of surgical scrub using sterile non-adhering pads followed by sterile alcohol wipes.

When the skin is dry, place the mouse in the supine position on a clean surface over a heating pad directly under the objective of a clean surgical microscope. And cover the animal with a sterile drape with a small hole cut out over the abdomen. Make a one-centimeter incision in the outer skin and the inner abdominal musculature along the midline of the abdomen superior to the penis and the preputial glands.

Locate one of the bilateral seminal vesicles and attached anterior prostate lobes that are typically posterior, lateral and slightly superior to the bladder Ask an assistant to mix the MIC-CAP Matrigel cancer cell solution with gentle pipetting and to slowly aspirate 30 microliters of cells into a 28-gauge needle attached to a 50-microliter syringe. Then, using Graefe Tissue Forceps, gently raise the tip of one seminal vesicle to create tension on the tissue without puncturing the vesicle and carefully insert the bevel of the needle parallel to the long axis of the anterior prostate lobe. Slowly inject the entire volume of cells into the lobe followed by slow retraction of the needle to prevent leakage.

A successful injection will be apparent by an engorgement of the anterior prostate lobe. Carefully and steadily maintain the seminal vesicle and injected prostate lobe outside of the mouse for approximately 30 seconds to allow the Matrigel to partially solidify within the lobe. Use a sterile polyester-tipped applicator to collect any cell solution leakage into the abdomen to prevent nonorthotopic tumor development.

An accurate intra-prosthetic injection is critical for nonorthotopic tumor development. Taking extra care to avoid puncturing the seminal vesicle, to slowly inject the cancer cell suspension and to collect any cell leakage will increase the success of this procedure. Now, gently return the seminal vesicle and injected lobe to the abdomen without putting pressure on the lobe and replace any externalized tissues.

Using 5-0 vicryl absorbable reverse cutting needle sutures, close the inner abdominal musculature with continuous stitches followed by closure of the outer abdominal skin with interrupted stitches using 4-0 nylon monofilament non-absorbable reverse cutting needle sutures. Then, administer post-operative analgesia and place the mouse in a cage with no bedding halfway on a heating pad with monitoring until full recovery. Stable transfection of Myc-CaP cells for firefly luciferase and mCherry expression allows the prostate tumors to be followed by non-invasive, in vivo bioluminescence and fluorescence.

Here, representative tumors dissected from the abdomen on day 30 after intra-prostatic injection are shown. With proper technique, tumor volume and weight can be recorded with relatively small standard error, although some variability is expected between small and large tumor masses. Intra-prostatically injected murine cancer cells can also be analyzed by immunohistochemistry for the presence of tumor-infiltrating CD3-positive T-cells or other immune cells of interest.

More, this model provides an objective survival endpoint as the large primary tumor mass causes hemorrhagic abdominal ascites and/or decreased ambulation grooming and/or piloerection. Castration three days after intra-prostatic injection results in robust tumor regression followed by eventual tumor recurrence after approximately 30 days representative of castration-resistant prostate cancer. Dissection and histological analysis of castration-resistant prostate cancer tumors reveals no neuroendocrine differentiation as the tumors maintain high androgen receptor levels and are negative for the neuroendocrine marker synaptophysin.

Once mastered, this technique can be completed in 20 to 30 minutes if performed properly. While attempting this procedure, it's important to remember to administer pre and postoperative analgesics and to perform all of the surgeries using sterile technique and with the aid of a surgical assistant. Following this procedure, other methods like immunohistochemistry, flow cytometry and assessment of tumor volume and weight and animal survival can be performed to answer additional questions about responses to immunotherapies and other experimental therapeutas.

After watching this video, you should have a good understanding of how to inject prostate cancer cells into the anterior prostate lobe of mice for eventual surgical castration and tumor regression and recurrence analysis. Don't forget that working with cancer cells can be extremely hazardous and there are precautions such as wearing the correct personal protective equipment should always be taken while performing this procedure.