An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

1. Cell Culture

1. Before orthotopic injection, culture MOV1^Kat cells, derived from DR26 tumors, in a T175 flask until they are 90% confluent. Plan to use 1 to 5 million cells per injection, which will require 1 or 2 T175 flasks.
2. On the day of the injection, harvest the cells and determine the cell number using a hemocytometer.
3. Once the cell concentration has been determined, pellet the cells by centrifugation for 5 minutes at 300 X g at room temperature.
4. Following the spin, resuspend the cells to have 1 million in 10 microliters of sterile PBS with 0.002 M EDTA

2. Pre-Surgery

1. Prior to surgery, fill up a 3/10cc insulin syringe with 1 million MOV1-^Kat cells in 10 microliters of PBS EDTA.
2. Transfer an isofluorane anesthetized mouse to a heating pad. Add eye ointment to prevent eye dehydration. Then, immediately insert the animal’s head into a nose cone system connected to an isoflurane vaporizer to deliver anesthesia throughout the surgery.
3. After disinfection of the injection site with alcohol swabs, subcutaneously inject 5 mg/kg of Ketoprofen, a pre-surgical analgesic, with a 3/10cc Insulin syringe.
4. Using clippers, shave the left caudal portion of the dorsum from the thoracolumbar 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.
5. 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.

3. Surgery

1. Just before surgery, adjust the isoflurane vaporizer levels to 1.5%.
2. Verify that the animal is completely anesthetized by pinching the foot pad.
3. Next, locate the spleen under the skin. Then using surgical scissors, make a dorsolateral incision 1-2 cm long on the top right of the spleen.
4. Dissect the retroperitoneum. The pad surrounding the mouse ovary will be observed. Use curved forceps to grasp and expose the fat pad surrounding the mouse ovary.
5. Hydrate the organ with some drops of sterile PBS.
6. Use curved forceps to grasp, retract…position…and then secure the ovary for injection
7. While firmly grasping the ovary with the forceps, inject 10 microliters of MOV1^Kat tumor cells into the ovary. A firm grasp will prevent fluid regurgitation or leakage.
8. Immediately following injection, release the tension exerted by the forceps. The perforation in the ovary should spontaneously retract and close.
9. Using an absorbable Polyglycolic Acid suture attached to a needle, close the retro-peritoneum wound.
10. Release the animal from the nose cone.
11. Stretch the skin and seal the dorsolateral wound edges with few drops of tissue adhesive.
12. Finally, orally administer 100 microliters of antibiotics to the animal. Then place it back in its cage and monitor for recovery. Keep the animal on antibiotics in the drinking water for one week.

4. In vivo Imaging

1. One week after the orthotopic injection of MOV1^Kat tumor cells, perform in vivo imaging. Begin by transferring an isofluorane anesthetized mouse to the imaging chamber.
2. Turn the isoflurane vaporizer level down to 2%.
3. Perform in vivo imaging according to the imaging system manufacturer’s instructions. In this video, the Lumina system will be used.
4. 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
5. 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.
6. Next, set the exposure time as Auto. Under “Pixel binning or CCD resolution”, check “Medium”. Then Under F/stop or aperture, check the value of 2. Next, select the 535 excitation filter and the DsRed emission filter.
7. Under field of view, click on View B to image a single mouse.
8. Then, click on acquire to begin the image acquisition.
9. Once the image acquisition is complete, use the Region of Interest, or ROI, tool to measure of the signal. Click on the measurement icon to release the signal values and area.
10. Finally, click on “save” to save the image in the user folder
11. After the images have been saved, stop the administration of isoflurane and return the mouse to its cage. The mouse should wake up immediately.

5. Representative Results

-Using this protocol, the in vivo growth of an orthotopic ovarian cancer can be monitored for at least 3 weeks using a non-invasive procedure.

Figure 1. MOV1^Kat cells, or PBS as negative control, were orthotopically injected into the ovary of non-tumor prone mice (right animal and left animal and respectively). In vivo imaging was performed 2 weeks later. The fluorescence emission generated by MOV1^Kat cells engrafted in the ovary was measured and compared with that from negative control mouse.

Figure 2. Frozen sections of MOV1^Kat ovarian tumors were stained with biotinylated anti-CD4 mAb followed by DAB substrate (dark brown) to detect tumor infiltrating lymphocytes (black arrow). Cells were counterstained with methyl-green to visualize cell nuclei (blue). Tumor cells (red arrows) were morphologically distinct from T cells. The slide appears magnified 40x.