Pré-clínica Ortotópico Modelo murino de cancro da próstata humano

The overall goal of this procedure is to establish a mouse orthotopic prostate cancer xenograft model that provides a useful approach to studying the molecular events of primary tumor development in the early events of the metastatic cascade. This model can help answer key questions regarding the spastic interactions between genetically and molecularly altered tumor cells in the micro-environment of the growing tumor. It can also help in the pre-clinical evaluation of new therapeutic approaches against the human prostate cancer.

The main advantage of this technique over the spontaneous metastatic models are that it promotes the study of the molecular events of primary tumor development and also the recapitulation of the initial phase of metastasis prior to entry and embolism of tumor cells into the circulation. Orthotopic model of prostate cancer is one of the best pre-clinical models to understand how prostate cancer cells metastasize to different organs. Most prostate cancer patients die of metastasis, and therefore this model is very important to investigate the molecular mechanisms of metastasis.

Using this model we can design better strategies for the treatment of metastasis by using neural compounds, those are specific to metastatic pathways. Demonstrating this procedure will be Melissa Colden, a grad student and Dr.Varahram Shahryari, a research associate from our laboratory. Before beginning the procedure, wash the tumor cell culture of interest with PBS and incubate the cells with 2 milliliters of 05 percent trypsin for 3 to 5 minutes in the cell culture incubator.

When the cells have detached, stop the reaction with 5 milliliters of complete medium and count the cells by trypan blue exclusion. Dilute the cells to a 2.5 times 10 to the 5 cells per 10 microliters of medium concentration, and mix the cell suspension with 10 microliters of basement membrane-like extracellular matrix extract. Hold the cells on ice, then place an anesthetized six to eight week old immunocompromised male experimental mouse in the supine position on a sterile heating pad and confirm a lack of response to toe pinch.

Apply ointment to the animals eyes and remove the hair from the lower abdomen. Disinfect the surgical area with 10 percent povidone iodine scrub, followed by 70 percent ethanol about 1 to 2 centimeters above the penis sheath, and about 2 to 3 centimeters below the bottom of the rib cage. Use a pair of fine forceps to lift 2 millimeters of skin above the preputial gland.

Using a scalpel, make a 1 centimeter mid-line incision through the skin, followed by a 1 centimeter incision through the muscle layer. Locate the yellow, light brown spherical bladder directly below the incision. Then use the fine forceps to grip the bladder and to lift the organ up and out of the body cavity toward the penis sheath to expose the seminal vesicles.

With a wet cotton swab in each hand, carefully externalize the vesicles one at a time, and lay them top down on the outer surface of the abdomen with the bladder in the middle. Next, use the cotton swabs to gently tilt back the seminal vesicles at the point of insertion near the bladder neck towards the penis sheath so that the two dorsal prostate lobes are clearly visible. Now use a micro-pipette to re-suspend the cells in a single-cell suspension and label them with luciferin.

Aspirate 25 microliters of the cells into a 0.5 cc syringe equipped with a 28.5 gauge needle and transfer the mouse under a dissecting microscope. Then, insert the syringe needle into a dorsal prostatic lobe and slowly inject 20 microliters of the cell suspension until a bulla formation is observed. When all of the cells have been administered, retract the needle, pressing lightly on the injection site with a cotton swab for a few seconds to prevent leakage.

Then use the swabs to carefully return the vesicles to the body cavity one at a time, followed by the bladder. After placing the organs back into the body cavity, suture the muscle layer first in an interrupted pattern with absorbable 4-0 chromic cat gut sutures, followed by the skin closure with non-absorbable 4-0 nylon surgical sutures. The skin can also be pulled together and closed with surgical clamps to close the incision completely.

Then, immediately image the animal, and return the mouse to a clean cage under a warming lamp with monitoring until it is fully recovered. Following the orthotopic implantation of luciferin-labeled prostate cancer cells into the posterior prostatic lobe, as just demonstrated, weekly imaging using live animal bioluminescence imaging reveals a successful colonization of the prostate lobes by the tumor cells. Indeed, an increase in the bioluminescence over time is indicative of the enlargement of the primary tumor over the course of the experiment, confirming the effectiveness of this technique in non-invasive, radiographic, fluorescent or luminescent imaging for tumor growth and metastatic lesion monitoring.

While attempting this procedure, it is important to remember that intra-prostatic injection requires open abdominal surgery. So, it should be performed in a pathogen-free environment using proper aseptic surgical techniques. Working with human cancer cells can be extremely hazardous and precautions, such as wearing personal protective equipment, and working within biosafety level two containment should always be taken while performing this procedure.

This model paved the way for the transplant of histologically intact tumor fragments to examine the effects of potential drug therapies on tumor growth or original lymph node metastasis. After watching this video, you should have a good understanding of how to implant cancer cells into the dorsal prostatic lobes for the establishment of an orthotopic human prostate cancer xenograft mouse model.