Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

The overall goal of this procedure is to establish an intraperitoneal ovarian cancer xenograft model that mimics the clinical profile observed in patients with ovarian cancer. This is achieved by intrauterine injection of mCherry fluorescence labeled ovarian cancer stem cells in nude mice. Next, the establishment of tumor tumor progression and the response to therapy are determined by measuring the fluorescence as a surrogate for tumor burden.

This allows real-time monitoring of tumor progression or the response to treatment without the need to sacrifice the mice. The model that we show here. B recapitulates the clinical profile observed in patients with ovarian cancer, and therefore it allows us to not only study the biology of the disease, but also to identify novel therapies, especially for recurrent ovarian cancer.

The model allows us to monitor the progression of the tumor to determine in real time if they respond to chemotherapy, and also to determine if the disease would recur if chemotherapy is discontinued. To begin this procedure, anesthetize a seven to eight week old, a thymic nude mouse with 2%isof fluorine. Then verify that it is completely anesthetized by pinching its foot pad.

Next, place the animal on its right side on it, sterile gauze pad with its head away from the experimenter. Apply ointment to both eyes to prevent dryness while under anesthesia afterward. Quickly insert the animal's head in a nose cone system connected to an isoflurane vaporizer.

Disinfect the right side of the abdomen using alcohol pads, followed by iodine. Then place the sterile surgical drape over it using sterile surgical scissors and forceps. Make a one to two centimeter skin incision on the lower left quadrant of the mouse.

After that, lift the muscle and make an incision in order to reach the peritoneum. Subsequently, dissect the oblique muscle to expose the abdominal cavity. Then locate the left uterine horn using a sterile hemostat clamp, both the anterior and posterior sides of the horn.

Place the anterior clamp right below the fallopian tube and the posterior clamp right above the cervix. At this point, have the second experimenter. Inject the cell suspension by placing the cells containing needle at a 45 degree angle perpendicular to the horn.

Then slowly inject 50 microliters of cell suspension into the lumen of the uterine horn. Next, release the anterior clamp followed by the posterior clamp. After that, place the uterine horn back in the abdominal cavity.

Close the peritoneum using a synthetic absorbable suture. Then close the skin using a tissue adhesive. When finished, remove the mouse from the nose cone and place it back in its cage.

Make sure the animal is awake and active before leaving it unattended. Provide ibuprofen in the drinking water for the first 48 hours after surgery. Now open the molecular imaging software, then open the Mars Capture software.

This module provides coordinated control of the Mars capture settings while leveraging the imaging systems capture and analysis capabilities. Next, input the predetermined capture values. Subsequently save the parameters as individual session files.

Then create a rotation sequence protocol by selecting the create edit protocols button From the system interface acquire window, select the previously saved fluorescent session file and save as step one. Next, select the corresponding x-ray session file and save as step two. With step one selected.

Use the set rotation series from the before image capture pop-up menu to set the desired starting angle range and increment. To ensure seamless visualization of features, the specific values include a starting angle of minus 180 degrees, a range of 375 degrees and an increment of 15 degrees. Save the protocol and click the done button to initialize the Mars.

Then start the alignment by selecting the preview button in the acquire menu to bring up the rotator tab, use the fluorescence capture setting as specified for M cherry imaging. Keep the door open during preview to speed up positioning, which also allows for visual inspection of the mouse positioning during the alignment procedure. Next, select the load mouse option and proceed through the series of positioning menus.

Ensure that the tubed end of the collapsible nose cone is in the nose cone recess. Then place the anesthetized mouse in the prone orientation with its head in the nose cone. Begin the calibration at the zero degree position with the ventral side of the animal facing down.

Use the two knobs on the rotation system to position the animal so that it appears centered in the preview window. Repeat the process as prompted for minus 180, minus 90 plus 90 and plus 180 degree positions and click done. Here is an example of a movie generated from the images acquired.

This figure shows the correlation between the intraperitoneal tumor burden and the fluorescence x-ray overlay image. About 32 days post-injection of F two M cherry ovarian cancer cells 2D imaging was performed and the mice were sacrificed to correlate the acquired image with the actual tumor burden. And shown here are the rotation data sets that allow multi-angle imaging and the detection of tumors in control, paclitaxel treated, and the recurrent mice.

The image of a single mouse per panel is shown as it is rotated using the Mars system. Note that even in control mice with significant tumor burden, tumor size could be underestimated depending on the angle the image was taken from. After watching this video, you should be able to establish an intraperitoneal ovarian cancer xenograft model using the intrauterine route, as well as be able to perform live animal imaging using a multimodal rotation system.