The overall goal of the following experiment is to quantify the primary tumor size, the formation of lung metastases, and the number of circulating tumor cells in a mouse model of human prostate cancer. This is achieved by direct implantation of human prostate cancer cells into the ventral lobe of the prostate of a babsi athymic mouse. After the tumors have developed, the femurs and cardiac blood are removed from the animal, allowing identification of the circulating tumor cells.
Finally, primary tumors and lungs are removed for analysis. Ultimately changes in the primary tumor size, weight, and molecular characteristics, as well as lung metastasis formation can be measured. The main advantage of this technique over other techniques, such as a tail vein or inter cardiac injection, is that with this technique, the full extent of metastasis can be measured from formation of the primary tumor extravasation and survival in the bloodstream and arrival and formation of a secondary metastasis in a new site.
The implications of this technique relate to the treatment of human prostate cancer metastasis. We have used this model to test the effects of several small molecule therapeutics upon inhibiting human prostate cancer metastasis. In addition, the model can be used to validate potential therapeutic targets for inhibiting human prostate cancer metastasis Begin by using sterile cotton balls and Betadine to scrub the lower abdominal region of an anesthetized six to eight week old athymic bopsy mouse.
Next, swab the area with an alcohol wipe, and then wipe the animal with a Betadine solution. After allowing the area to dry, use a pair of sharpened sterile surgical scissors to make a three to four millimeter low midline abdominal incision. Then use forceps to gently lift the bladder without moving any other organs or muscles, and identify the ventral lobe of the prostate located directly beneath the bladder.
Now inject the tumor cell population of interest with a 0.5 cc syringe equipped with a 28 and a half gauge needle into the ventral prostate gland, minimizing leakage and ensuring that a small bubble is observed within the tissue. Then replace the bladder and use 4.0 absorbable Vicryl monofilament sutures to close the muscle layer in a simple interrupted pattern. Close the skin layer with sterile nine millimeter staples.
After four to six weeks, tumors are visually apparent or can be palpated following euthanasia. Use a pair of surgical scissors to cut horizontally directly under the rib cage, then vertically to the armpit to expose the heart, remove blood from the animal using a terminal cardiac puncture using surgical scissors, cut the trachea and then remove the lungs and immediately place lungs into a tissue cassette and then into a container containing 10%formalin. Next, individually, cut any blood vessels attached to the primary prostate tumor being careful to ensure that no additional organs are attached, record the weight and size of the tumor.
Then immediately snap, freeze the tissue in liquid nitrogen or place tissue into a tissue cassette, and then into a container containing 10%formin. Finally, use a pair of surgical scissors to expose the hip and knee joints. Then disconnect the legs, being careful to keep the femur intact before placing them into sterile saline.
In this first figure, the changes in body weight and food consumption of mice six weeks after tumor inoculation are shown. Note that there is a small dip in body weight and food consumption around the date of surgery due to the anesthesia. During the course of the experiment, the body weight and food consumption slowly increase post-surgery and then begin to decline towards the end of the experiment.
As the tumor burden reaches a critical level in these graphs, representative tumor sizes are shown. Individual tumor sizes vary, but on average, tumors of approximately one gram in weight and one centimeter cubed in size are achieved as illustrated. Here, it is important to consider the changes in tumor weight and tumor size that occur between four and six weeks.
When planning the endpoint of the experiment in this representative experiment during the last two weeks, the average tumor weight increased 2.7 fold, and the tumor size grew 1.9 fold dramatically. Influencing the experimental results changes in the total metastatic cells per lung and mice necropsy at four versus six weeks can be observed as well as illustrated in this graph. Mice sacrificed at four weeks, shown no metastatic development while mice at six weeks post tumor inoculation showed metastatic cells in all mice.
Evaluated further underscoring the importance that mice necropsies be performed at a late stage endpoint to ensure metastasis has occurred. These times may vary by cell line or by gene modifications. The number of metastasis can be quantified three different ways.
For example, the total number of GFP positive human prostate cancer cells can simply be counted using either immunohistochemical staining for GFP or using h and e staining as in these images where an individual cell at 40 x magnitude is highlighted with an arrow. Note, the brown staining in the GFP stained lung section and large distinct nuclei. Another way to quantify the number of metastases is to consider the number of locations where metastatic deposits are present.
For example, in this image at 10 x magnitude, several loci of varying cell numbers each highlighted with an arrow can be observed in this increased magnitude image, one loci with three distinct cells is also highlighted. Finally, the number of distinct metastases as defined by a clearly bound group of cells showing five or more GFP positive human prostate cancer cells also can be quantified as in this image where one metastatic deposit of 10 cells is shown in these last three graphs, representative QR TPCR R experiments measuring the expression of three genes of interest in metastatic progression matrix metalloproteinase type two, matrix metalloproteinase type nine, and heat shock protein 27 in individual tumor samples collected from mice at six weeks are shown. When initially developing this technique, it's important to work with your animal facility and veterinary staff in order to properly administer anesthesia, pain medication, and minimize surgical duress.
