Ovarian cancer is generally diagnosed at an advanced stage when the case to fatality ratio is high. Thus, ovarian cancer remains the most lethal of all gynecologic malignancies among women in the United States. Here we describe a syngeneic mouse model of serous ovarian cancer permitting, both in vivo imaging and studying the tumor microenvironment.
We stably transduced with the C nmy derived fluorescent protein kaka and ovarian cancer cell line derived from a DR 26 mouse transgenic for MIS two R tag. These cells named MOV one cat are injected into the ovary of a non-tumor prone TG MIS two R tag transgenic mouse to grow and develop into a fluorescent serous ovarian cancer. In vivo imaging is performed to monitor tumor progression.
The ovary is then dissected and immunohistochemistry is performed to study the tumor microenvironment. This animal model present three main advantages over existence in genetic models. First, the mice developed tumor similar to human zeros ovarian cancer, which is the most frequent histological type in patients.
Second tumor cells are fluorescent labeled. Thus tumor progression can be followed by inbivo imaging. Third, the mice are immunocompetent and thus you can study the tumor infiltration by the host leukocytes.
Altogether, these characteristics made the animal model ideal for preclinical studies. Mice transgenic for the SB 40 large tumor antigen under the transcriptional control of the MU and inhibiting substance type two receptor or tgm MIS S two R tag were developed by doctors Connelley and Hamilton at the Fox Chase Cancer Center. Female tgm IS two R tag mice spontaneously developed bilateral epithelial ovarian cancer that resembles human serous ovarian cancer in Tgm IS two R tag mice.
The IS two R promoter targets the expression of the SV 40 T antigen to the epithelium of the female mouse reproductive tract, including the ovaries. Although transgene expression can be detected in the uterus and fallopian tubes, tumors arise only in the ovaries. Independent lines of TG MIS two R tag mice were isolated.
That exhibit distinct phenotypes specifically tag transgene expression in tgm IS two R tag DR 26. MICE is transforming leading to the development of bilateral ovarian tumors with 100%penetrance. The DR 26 mice live six months, but due to early ovarian tumor development are infertile.
Additional transgenic lines have been identified that express the SB 40 tag transgene, but do not develop ovarian tumors. These mice exhibit typical lifespan for C 57 black six mice and are fertile. These mice can be used as SYNGENEIC allograft recipients for tumor cells isolated from TG MIS S two R tag DR 26 mice.
The advantages of this new orthotopic model of serous ovarian cancer in syngeneic mice is that it permits first to follow the tumor development by in vivo imaging. Second to control tumor development without any confounding factors such as spontaneous tumor growth. Third, to study tumor infiltration by host leukocytes and fourth to easily set up large experimental groups due to fecundity of the non-tumor prone tgm IS two R tagged mice before orthotopic injection culture MOV one cat cells derived from DR 26 tumors in a T 1 75 flask until they are 90%confluent.
Plan to use one to 5 million cells per injection, which will require one to two T 1 75 flasks on the day of the injection. Harvest the cells and determine the cell number using a hemo cytometer. Once the cell concentration has been determined, pellet the cells by centrifugation for five minutes at 300 G at room temperature.
Following the spin Reese suspend the cells to have 1 million in 10 microliters of sterile PBS with 0.002 molar EDTA prior to surgery. Fill up a three 10 cubic centimeter insulin syringe with 1 million mov one cat cells in 10 microliters of P-B-S-E-D-T-A. Transfer an isof fluorine anesthetized mouse to a heating pad.
Add eye ointment to prevent I dehydration. Then immediately insert the animal's head into a nose cone system connected to an isof fluorine vaporizer to deliver anesthesia throughout the surgery on a disinfected site. Subcutaneously inject five milligrams per kilogram of ketoprofen, a presurgical analgesic with a three 10 cubic centimeter insulin syringe.
Using clippers, shave the left caral portion of the dorsum from the thorac columbar junction to the base of the animal tail. Apply hair removal cream to completely remove the hair. Then remove the excess with a wet paper towel.
Once the hair has been removed, sterilize the shaved area with povidone iodine and alcohol swabs. Then place a surgical drape around the area of incision just before surgery. Adjust the isof fluorine vaporizer levels to 1.5%Verify that the animal is completely anesthetized by pinching the footpad.
Next, locate the spleen under the skin. Then using surgical scissors, make a dorsal lateral incision one to two centimeters long. On the top right of the spleen, dissect the retroperitoneum.
The pad surrounding the mouse ovary will be observed. Used curved forceps to grasp and expose the fat pad surrounding the mouse ovary. Hydrate the organ with some drops of sterile PBS used curved forceps to grasp, retract position, and then secure the ovary for injection while firmly grasping the ovary with the forceps.
Inject 10 microliters of mov one cat tumor cells into the ovary. A firm grasp will prevent fluid regurgitation or leakage immediately following injection. Release the tension exerted by the forceps.
The perforation in the ovary should spontaneously retract and close using an absorbable polyglycolic acid suture, close the retroperitoneum wound, release the animal from the nose cone, stretch the skin and seal the dorsal lateral wound edges with a few drops of tissue adhesive. Place the animal back in its cage and monitor for recovery. Once the animal has recovered orally, administer a hundred microliters of antibiotics.
Keep the animal on antibiotics in the drinking water for one week. One week after the orthotopic injection of mov one cat tumor cells perform in vivo imaging. Begin by transferring an isof fluorine anesthetized mouse to the imaging chamber.
Turn the isof fluorine vaporizer level down to 2%Perform in vivo imaging according to the imaging system manufacturer's instructions. In this video, the Lumina system will be used to image. Click the living image software desktop icon.
Then on the IVIS acquisition control panel, select initialize. The instrument settings are analogous to a camera settings on the acquisition control panel. Set up the instrument's acquisition parameters for fluorescence check fluorescent.
Click the photograph box to acquire a photograph with each image. Next, set the exposure time as auto under pixel bidding or CCD resolution, check medium. Then under F-stop or aperture, check the value of two.
Next, select the 5 35 excitation filter and the DS red emission filter under field of view. Click on view B to image a single mouse. Then click on acquire to begin image acquisition.
Once the image acquisition is complete, use the region of interest or ROI tool to measure the signal. Click on the icon to release the signal values and area. Finally, click on save to save the image in the user folder.
After the images have been saved, stop the administration of isof fluorine and return the mouse to its cage. The mouse should wake up immediately using this protocol. The in vivo growth of an ortho topic.
Ovarian cancer can be monitored for at least three weeks. Using a non-invasive procedure, MOV one cat cells or PBS as negative control or ortho topically injected into the ovary of non-tumor prone recipient. Mice in vivo imaging was performed two weeks later.
The fluorescence emission generated by mve one cat cells engrafted in the ovary was measured and compared with that from negative control mouse. Frozen sections of mve one cat ovarian tumors were stained with biotinylated anti CD four monoclonal antibody, followed by DAB substrate. To detect tumor infiltrating lymphocytes cells were counter stained with methyl green to visualize cell nuclei.
Tumor cells were morphologically distinct from T cells. After watching this video, you should have a good understanding of how to perform orthotopic injections in the mouse ovary. Also, this technique paved the way for researchers in the field of ovarian cancer to study tumor interaction with the host microenvironment in an immunocompetent mouse model.
